CN116917472A - amylase variants - Google Patents

Abstract

The present invention provides novel amylases. More specifically, genetically engineered amylases, compositions comprising the enzymes, and methods of making and using the enzymes or compositions comprising the enzymes are provided.

Description

Amylase variants

Technical Field

The present invention provides novel amylases. More specifically, genetically engineered amylases, compositions comprising the enzymes, and methods of making and using the enzymes or compositions comprising the enzymes are provided.

Background

Enzymes are increasingly being used in a variety of applications as sustainable alternatives to petrochemical industry. Enzymes are biodegradable and may already have catalytic activity at lower temperatures, which results in reduced energy consumption. In particular, in the detergent industry enzymes are used in detergent formulations to improve cleaning efficiency and reduce energy consumption in the washing step.

Amylase is an enzyme capable of hydrolyzing starch. Accordingly, amylases have been used to remove starch stains and have been added to cleaning compositions for this purpose. In these detergent applications, the amylase should remain stable at high temperatures and/or under denaturing conditions of the detergent and wash liquor. Thus, there is a need for new amylases with improved properties, in particular with improved stability and improved performance.

Summary of The Invention

The present invention relates to an alpha-amylase variant of a parent alpha-amylase, wherein the variant comprises:

(i) Amino acid changes, preferably insertions, deletions, substitutions or combinations thereof, preferably substitutions, at one or more positions corresponding to a position selected from the group consisting of: 1. 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 47, 48, 51, 54, 59, 60, 63, 70, 71, 72, 73, 75, 76, 81, 82, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 103, 106 108, 109, 113, 114, 115, 116, 117, 119, 120, 123, 125, 126, 128, 129, 131, 132, 133, 134, 135, 136, 139, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 154, 155, 156, 158, 160, 161, 162, 163, 164, 165, 169, 170, 172, 173, 174, 175, 176, 177, 178, 180, 182, 184, 185, 187, 188, 189, 191, 192, 193, 203, and 208, 210, 211, 212, 213, 214, 215, 216, 218, 219, 221, 222, 223, 224, 225, 226, 227, 230, 231, 233, 234, 235, 236, 237, 238, 240, 242, 243, 245, 249, 250, 251, 252, 253, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 301, 302, 303, 304, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 333, 334, 336, 337, 338, 341, 342, 343, 344, 345, 346, 348, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 362, 363. 364, 365, 366, 367, 368, 370, 372, 375, 376, 378, 379, 381, 382, 383, 384, 385, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 405, 406, 407, 408, 410, 413, 414, 415, 416, 418, 421, 424, 426, 427, 429, 431, 432, 434, 436, 438, 439, 440, 442, 443, 445, 446, 447, 448, 449, 451, 453, 455, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 470, 471, 474, 478, 480 and 482;

(ii) The variant has at least 80% but less than 100% sequence identity with any one of SEQ ID NO. 1, 3, 4 or SEQ ID NO. 15-41, preferably the amino acid sequence shown in SEQ ID NO. 1, and

(iii) The variant has alpha-amylase activity.

Furthermore, the present invention relates to polynucleotides encoding the alpha-amylase variants and nucleic acid constructs or expression vectors comprising the polynucleotides, as well as host cells comprising the polynucleotides, the nucleic acid constructs or the expression vectors.

Furthermore, the present invention relates to compositions, preferably detergent compositions (e.g. laundry detergent compositions or Automatic Dishwashing (ADW) detergent compositions) comprising the alpha-amylase variant and at least one other ingredient, and the use of the alpha-amylase variant in such compositions.

The invention also relates to a method of producing an alpha-amylase variant comprising culturing a host cell under conditions suitable for expression of the alpha-amylase mutant; and recovering the alpha-amylase variant.

Detailed Description

The present invention may be understood more readily by reference to the following detailed description of embodiments of the invention and the examples included herein.

Although the invention will be described with reference to specific embodiments, these descriptions should not be construed in a limiting sense.

Definition of the definition

Unless otherwise indicated, the terms used herein will be understood according to conventional usage by those of ordinary skill in the relevant art.

Before describing in detail exemplary embodiments of the present application, definitions that are important to an understanding of the present application are provided. Unless otherwise indicated or apparent from the nature of the definitions, these definitions apply to all compounds, methods and uses described herein.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

In the context of the present application, the terms "about" and "approximately" mean the exact interval within which the technical effect of the feature in question can still be ensured, as will be understood by the person skilled in the art. The term generally means a deviation from the indicated values of + -20%, preferably + -15%, more preferably + -10%, even more preferably + -5%.

Furthermore, the terms "first," "second," "three," or "(a)", "(b)", "(c)", "(d)", and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operation in other sequences than described or illustrated herein. Where the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii", etc. relate to steps of a method or use or assay, there is no time interval or consistency of time intervals between the steps, i.e., the steps may be performed simultaneously, or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between the steps, unless otherwise stated in the application as set forth above or below.

Throughout the present application, various publications are referenced. The disclosures of all of these publications and those references cited in these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this application pertains.

It should be understood that the term "include" is not limiting. For the purposes of the present application, the term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising at least a certain number of embodiments, this is meant to also cover groups which preferably consist of only these embodiments.

A "parent" sequence (also referred to as a "parent enzyme" or "parent protein") is a starting sequence for introducing sequence changes (e.g., by introducing one or more amino acid substitutions) that will result in the production of a "variant" of the parent sequence. Thus, the terms "enzyme variant", "sequence variant" or "protein variant" are used with respect to the parent enzyme from which the respective variant enzyme is derived. Thus, parent enzymes include wild-type enzymes and variants of wild-type enzymes used to develop other variants. Variant enzymes differ to some extent in their amino acid sequence from the parent enzyme.

In describing variants of the invention, the abbreviations are used to designate individual amino acids according to accepted IUPAC single letter or three letter amino acid abbreviations.

As used herein, "amino acid change" refers to an amino acid substitution, deletion or insertion.

"substitution" is described by: the original amino acid is followed by numbering of the positions within the amino acid sequence, followed by the amino acid for substitution. For example, substitution of alanine for histidine at position 120 is designated "His120Ala" or "H120A". Substitutions may also be described by referring to only the amino acids obtained in the variant and not to the amino acid of the parent at that position, e.g. "X120A" or "Xaa120Ala" or "120Ala".

"deletions" are described by providing the original amino acid, then providing the position number within the amino acid sequence, then providing. Thus, the deletion of glycine at position 150 is named "Gly150 x" or "G150 x". Alternatively, the deletion is expressed as, for example, "deletion of D183 and G184".

"insertion" is described by providing the original amino acid, then providing the position number within the amino acid sequence, then providing the original amino acid and the added amino acid. For example, insertion of lysine immediately adjacent glycine at position 180 may be designated "Gly180GlyLys" or "G180GK". When more than one amino acid residue is inserted, e.g. Lys and Ala are inserted after Gly180, this can be expressed as: gly180 GlyLysla or G195GKA.

In the case where substitution and insertion occur at the same position, this may be denoted as s99sd+s99a, or abbreviated as S99AD. In the case of insertion of amino acid residues identical to existing amino acid residues, a named degeneracy appears to be apparent. For example, if glycine is inserted after glycine in the above example, this will be denoted G180GG. Variants containing multiple changes are separated by "+" e.g. "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, the multiple changes may be separated by spaces or commas, e.g., denoted R170Y G195E or R170Y, G195E, respectively. Where different alternative changes can be introduced at one position, the different changes are separated by commas, e.g., "Arg170Tyr, glu" and R170T, E respectively represent an arginine substituted at position 170 with tyrosine or glutamic acid. Alternative substitutions at specific positions may also be denoted as X120A, G, H, X120A/G/H or 120A/G/H. Alternatively, different changes or optional substitutions may be given in brackets, such as Arg170[ Tyr, gly ] or Arg170{ Tyr, gly }, or abbreviated as R170[ Y, G ] or R170{ Y, G }.

"synthetic" or "artificial" compounds are produced by in vitro chemical and/or enzymatic synthesis. The term "naturally" (or naturally occurring or wild-type or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or peptide that is present in nature (i.e., without any human intervention). For the purposes of the present invention, "recombinant" (or transgenic) means, for a cell or organism, that the cell or organism contains a heterologous polynucleotide artificially introduced by genetic techniques, whereas for a polynucleotide it includes all constructs artificially produced by genetic/recombinant DNA techniques, wherein

(a) The sequence of the polynucleotide or a portion thereof, or

(b) One or more genetic control sequences operably linked to the polynucleotide, including but not limited to a promoter, or

(c) a) and b)

Not located in its wild-type genetic environment or has been artificially modified.

The term "heterologous" (or exogenous or foreign or recombinant or non-native) polypeptide is defined herein as: a polypeptide native to the non-host cell; a polypeptide native to the host cell but wherein structural modifications (e.g., deletions, substitutions and/or insertions) have been made thereto by recombinant DNA techniques to alter the native polypeptide; or a polypeptide native to the host cell but whose expression is quantitatively altered or whose expression proceeds from a genomic location other than that in the native host cell as a result of manipulation of the host cell's DNA (e.g., a stronger promoter) by recombinant DNA techniques. Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-natural) polynucleotide refers to: polynucleotides native to the non-host cell; a polynucleotide native to the host cell but wherein structural modifications (e.g., deletions, substitutions and/or insertions) have been made thereto by recombinant DNA techniques to alter the native polynucleotide; or a polynucleotide native to the host cell but whose expression is quantitatively altered by manipulation of regulatory elements (e.g., a stronger promoter) of the polynucleotide by recombinant DNA techniques; or polynucleotides native to the host cell but integrated in a non-native genetic environment as a result of genetic manipulation by recombinant DNA techniques. For two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences do not naturally occur in this particular combination with each other.

Variant polynucleotide and variant polypeptide sequences may be defined by their sequence identity compared to the parent sequence. Sequence identity is typically provided as "% sequence identity" or "% identity". To calculate sequence identity, a sequence alignment is generated in a first step. According to the invention, a pairwise global alignment is produced, which means that two sequences are aligned over their full length, which is typically produced by using a mathematical method called an alignment algorithm.

According to the invention, the alignment is generated by using algorithms of Needleman and Wunsch (j.mol. Biol. (1979) 48, pages 443-453). Preferably, the program "NEEDLE" (open software suite for European Molecular Biology (EMBOSS)) is used for the purposes of the present invention, using the program default parameters (polynucleotide: vacancy open = 10.0, vacancy extension = 0.5 and matrix = EDNAFULL; polypeptide: vacancy open = 10.0, vacancy extension = 0.5 and matrix = EBLOSUM 62). After aligning the two sequences, in a second step, an identity value is determined from the alignment produced. For this purpose, the% identity is calculated by dividing the number of identical residues by the length of the alignment region showing the corresponding sequence of the invention over its complete length, and multiplying by 100: % identity= (identical residues/length of the alignment region showing the corresponding sequence of the invention over its complete length) ×100.

For calculating the percent identity of two nucleic acid sequences, the same method as well as some specifications for the calculation of percent identity of two amino acid sequences apply. For nucleic acid sequences encoding proteins, the coding regions of the sequences of the invention should be aligned over the entire length from the start codon to the stop codon (excluding introns). For alignment purposes, introns present in another sequence compared to the sequences of the invention may also be removed. Percent identity was then calculated by = (identical residues/length of aligned region of the sequence of the invention showing from start codon to stop codon over its full length and excluding introns) ×100. After aligning the two sequences, in a second step, an identity value is determined from the alignment produced.

Furthermore, the preferred nucleic acid sequence alignment program for performing Needleman and Wunsch algorithms (j.mol. Biol. (1979) 48, pages 443-453) is "NEEDLE" (european open software suite of molecular biology (EMBOSS)), using program default parameters (gap open = 10.0, gap extension = 0.5 and matrix = EDNAFULL).

Sequences having regions identical or similar to the sequences of the present invention and to be compared to the sequences of the present invention to determine% identity can be readily identified by a variety of means within the skill of the art, e.g., using publicly available computer methods and programs, such as BLAST, BLAST-2, e.g., available at NCBI.

Variant polypeptides may be defined by their sequence similarity when compared to a parent sequence. Sequence similarity is typically provided as "% sequence similarity" or "% similarity". % sequence similarity allows for amino acids of a given group to have similar properties, such as size, hydrophobicity, charge, or other properties. The exchange of one amino acid for a similar amino acid may be referred to herein as a "conservative mutation". According to the invention, similar amino acids are defined as follows, which shall also be applicable for the determination of the% similarity of the invention, which also corresponds to the BLOSUM62 matrix used for example by the procedure "NEEDLE", which is one of the most used amino acid similarity matrices for database searches and sequence alignments:

amino acid A is similar to amino acid S

Amino acid D is similar to amino acid E.N

Amino acid E is similar to amino acid D, K: Q

Amino acid F is similar to amino acid W:Y

Amino acid H is similar to amino acid N:Y

Amino acid I is similar to amino acid L.M.V

Amino acid K is similar to amino acid E, Q, R

Amino acid L is similar to amino acid I: M: V

Amino acid M is similar to amino acid I:L:V

Amino acid N is similar to amino acid D, H, S

Amino acid Q is similar to amino acid E:K:R

Amino acid R is similar to amino acid K: Q

Amino acid S is similar to amino acid A, N, T

Amino acid T is similar to amino acid S

Amino acid V is similar to amino acid I, L, M

Amino acid W is similar to amino acid F:Y

Amino acid Y is similar to amino acid F: H: W

Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein, such as an enzyme. In one embodiment, such mutations are unrelated to the functional domain of the enzyme. In one embodiment, the conservative mutation is independent of the catalytic center of the enzyme. To calculate sequence similarity, in a first step, sequence alignments are generated as described above. After aligning the two sequences, in a second step, a similarity value is determined from the alignment produced. For this purpose, the% similarity is calculated by dividing the number of identical residues by the number of similar residues, by the length of the alignment region showing the sequence of the invention over its entire length, and multiplying by 100: % similarity = [ (identical residue + similar residue)/length of the alignment region showing the sequence of the invention over its entire length ] ×100.

For nucleic acids, similar sequences can also be determined by hybridization using correspondingly stringent conditions. The term "high stringency conditions" refers to prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200. Mu.g/ml sheared salmon sperm DNA, and 50% formamide at 42℃for 12 to 24 hours following standard Southern blotting procedures for probes at least 100 nucleotides in length. The support material was finally washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 65 ℃. The term "very high stringency conditions" means that for probes of at least 100 nucleotides in length, prehybridization and hybridization is performed in 5 XSSPE, 0.3% SDS, 200. Mu.g/ml sheared salmon sperm DNA, and 50% formamide at 42℃for 12 to 24 hours following standard Southern blotting procedures. The support material was finally washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 70 ℃.

The terms "a and B domain" of an amylase as used herein refer to the two domains as one unit, while the C domain is the other unit of an alpha amylase. Thus, the amino acid sequence of the "a and B domains" is understood to be a contiguous sequence or portion of a sequence of an alpha-amylase comprising the "a and B domains" and other additional domains, such as the C domain.

As used herein, a "fragment" or "subsequence" is a portion of a polynucleotide or amino acid sequence. The term "functional fragment" refers to any nucleic acid or amino acid sequence that comprises only a portion of the full-length amino acid sequence, but still has the same or similar activity and/or function. Preferably, the functional fragment is at least 75% identical, at least 76% identical, at least 77% identical, at least 78% identical, at least 79% identical, at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5% identical to the original full-length amino acid sequence. Functional fragments comprise contiguous nucleic acids or amino acids as compared to the original nucleic acid or original amino acid sequence.

As used herein, a "genetic construct" or "expression cassette" is a nucleic acid molecule consisting of at least one sequence of interest to be expressed operably linked to one or more control sequences described herein, at least to a promoter.

The term "vector" as used herein includes any type of construct suitable for carrying a foreign polynucleotide sequence for transfer to another cell or stable or transient expression within a given cell. The term "vector" as used herein encompasses any type of cloning vector, such as, but not limited to, plasmids, phagemids, viral vectors (e.g., phage), phages, baculoviruses, cosmids, F-cosmids, artificial chromosomes, and any other vector that is particularly useful for a particular host of interest. The foreign polynucleotide sequence typically comprises a coding sequence, which may be referred to herein as a "gene of interest". The gene of interest may comprise introns and exons, depending on the type of source or destination of the host cell.

The term "introducing" or "transforming" as used herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. That is, the term "transformation" as used herein is independent of the vector, shuttle system or host cell, and it relates not only to transformation by polynucleotide transfer methods known in the art (see, e.g., sambrook, J. Et al (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY), but also encompasses any other type of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

Polynucleotides encoding polypeptides may be "expressed". The term "expression" or "gene expression" may refer to the transcription of one or more specific genes or specific nucleic acid constructs. The term "expression" or "gene expression" may refer to the transcription of one or more genes or genetic constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, followed by translation of the latter into protein or not. This process involves transcription of DNA and processing of the resulting mRNA product.

Recombinant cells may exhibit "increased" or "decreased" expression compared to corresponding wild-type cells. The terms "increased expression", "enhanced expression" or "overexpression" as used herein refer to any form of expression above the original wild-type expression level (which may also be non-expressed or non-measurable expression). Reference herein to "increased expression", "enhanced expression" or "overexpression" means that the gene expression is increased and/or the polypeptide level is increased (when referring to a polypeptide) and/or the polypeptide activity is increased relative to a control organism. The increase in expression may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or 100% or even more in a preferred ascending order compared to the expression of a control organism.

The term "purification" refers to a process in which at least one component (e.g., a protein of interest) is separated from at least another component (e.g., particulate matter of a fermentation broth) and transferred to a different compartment or phase, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filter membrane or a filter cloth, namely filtrate and retentate; examples of such different phases may be precipitation and supernatant or filter cake and filtrate. The solution obtained after purification of the enzyme of interest from the fermentation broth is referred to herein as "purified enzyme solution".

"protein formulation" refers to any non-complex formulation (non-complex formulation) comprising minor amounts of components for stabilizing the proteins contained in the protein formulation and/or stabilizing the protein formulation itself.

"enzymatic properties" include, but are not limited to, catalytic activity, substrate/cofactor specificity, product specificity, time course stability, thermostability, pH stability, and chemical stability. "enzymatic activity" or "catalytic activity" refers to the catalytic action exerted by an enzyme, expressed as units per milligram of enzyme (specific activity) or as the number of substrate molecules converted per molecule of enzyme per minute (molecular activity). The enzymatic activity may be specified by the actual function of the enzyme, for example, protease exerts proteolytic activity by catalyzing hydrolytic cleavage of peptide bonds, lipase exerts lipolytic activity by hydrolytic cleavage of ester bonds, amylase activity involves hydrolyzing glycosidic bonds in polysaccharides, and the like.

The term "enzyme stability" according to the invention relates to the retention of enzyme activity over time during storage or handling. The retention of enzyme activity over time during storage is referred to as "storage stability" and is preferred in the present invention.

To determine and quantify the change over time of the catalytic activity of an enzyme stored or used under certain conditions, the "initial enzyme activity" was measured under defined conditions at time zero (100%) and at a later point in time (x%). By comparing the measured values, the potential degree of loss of enzyme activity can be determined. The stability or instability of the enzyme can be determined by the degree of loss of enzyme activity. The "enzyme activity half-life" is the time required for the enzyme activity to decay to half (50%) of its initial value. The "half-life of an enzyme activity" may be expressed in terms of the challenging factors of the study, e.g., for the thermal stability of the enzyme, T50 may represent the temperature at which 50% residual enzyme activity remains after a certain time of thermal inactivation when compared to a reference sample that has not been heat treated.

"pH stability" or "pH dependent activity" refers to the ability of an enzyme to exert its activity upon exposure to a certain pH.

The term "thermostable" or "temperature dependent activity" refers to the ability of an enzyme to exert catalytic activity or wash performance upon exposure to high temperatures, preferably at a temperature of 40 ℃ for 14 days, preferably in a detergent composition (preferably in an ES1-C detergent), or at 92 ℃ for at least 10 minutes.

The term "detergent stability" or "stability in storage in a detergent composition" refers to the ability of an enzyme to exert catalytic activity or wash performance after storage in a detergent composition, preferably in a detergent composition (preferably in an ESI-C detergent) for 14 days, preferably at a temperature of 40 ℃ or 50 ℃.

An "improvement factor" (IF) is the degree of improvement in an enzyme variant over the corresponding parent enzyme in a property. The improvement of an enzyme variant relative to the corresponding parent enzyme may be characterized by an Improvement Factor (IF) >1.0. Alternatively, the improvement factor may be expressed as a percentage, for example, IF 1.1 equals 110%.

As used herein, the "wash performance" (also referred to herein as "cleaning performance") of an enzyme refers to the contribution of the enzyme to the cleaning performance of a detergent composition, i.e., the cleaning performance that is added to the detergent composition by the performance of the enzyme. The term "wash performance" is used similarly herein for laundry and hard surface cleaning. The wash performance can be compared under relevant wash conditions. The term "relevant washing conditions" is used herein to denote the conditions actually used by the household in the detergent market segment, in particular the washing temperature, time, washing equipment, suds concentration, detergent type and water hardness. The term "improved wash performance" is used to indicate that better end results are obtained in stain removal under relevant wash conditions or that less enzyme (based on heavy chain) is required to obtain the same end results relative to corresponding control conditions.

The term "specific performance" as used herein refers to the cleaning and removal of a particular stain or soil per unit of active enzyme. In some embodiments, the specific property is determined using stains or soils such as egg, egg yolk, milk, grass, meat emulsion, chocolate paste, baby food, sebum, and the like.

"detergent composition" or "detergent formulation" or "cleaning formulation" or "detergent solution" refers to a composition designated for cleaning contaminated materials. The detergent compositions and/or detergent solutions of the present invention include those used in different applications such as laundry and hard surface cleaning. The term "detergent ingredients" is defined herein to mean the types of chemicals that can be used in detergent compositions and/or detergent solutions.

The detergent formulations of the present invention may comprise one or more surfactants. "surfactant" (synonymous herein with "surfactant") refers to an organic chemical that, when added to a liquid, alters the properties of the liquid at the interface. Surfactants are referred to as nonionic, anionic, cationic or amphoteric surfactants, depending on their ionic charge.

The term "effective amount of detergent ingredients" includes amounts of certain ingredients that provide effective stain removal and effective cleaning conditions (e.g., pH, sudsing amount), amounts of certain ingredients that provide optical benefits (e.g., optical brightness enhancement, dye transfer inhibition), and amounts of certain ingredients that are effective to aid in processing (maintaining physical characteristics during processing, storage, and use; e.g., rheology modifiers, hydrotropes, desiccants).

The term "laundry" or "laundry" refers to both home laundry and industrial laundry and refers to the process of treating textiles and/or fabrics with a solution containing the detergent composition of the present invention. The laundry washing course may be performed by using technical equipment such as a domestic or industrial washing machine. Alternatively, the laundry washing process may also be performed by hand washing.

The term "textile" refers to any textile material, including yarns (threads made of natural or synthetic fibers for knitting or braiding), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics made from such materials, such as clothing, cloths, and other articles. The term "fabric" (textile made by braiding, knitting or felting fibers) or "garment" (any garment made from textile) as used herein is also intended to be included in the broader term textile.

The term "hard surface cleaning" is defined herein as the cleaning of hard surfaces, wherein hard surfaces may include any hard surface in the household, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces, including tableware or dishes. One particular form of hard surface cleaning is dishwashing, especially Automatic Dishwashing (ADW).

The term "dishwashing" refers to all forms of dishwashing, such as manual or automatic dishwashing. Dish washing includes, but is not limited to, cleaning all forms of ceramic dishes, such as plates, cups, glasses, bowls, all forms of dishes, such as spoons, knives, forks and serving utensils, and ceramics, plastics, such as melamine, metals, porcelain, glass, and acrylics.

Cleaning performance was evaluated under relevant cleaning conditions. "relevant cleaning conditions" in this context means the conditions actually used in a washing machine, an automatic dishwasher or a manual cleaning process, in particular the cleaning temperature, time, cleaning equipment, foam concentration, detergent type and water hardness.

In the technical field of the present invention, the term "stains" is generally used in connection with laundry washing, such as cleaning of textiles, fabrics or fibres, whereas the term "soils" is generally used in connection with hard surface cleaning, such as cleaning of dishes and cutlery. However, the terms "stain" and "soil" should be used interchangeably herein.

As used herein, a "chelating builder" differs from a precipitating builder in that no significant amount of precipitate is formed when the builder is used in an amount sufficient to bind all calcium ions in an aqueous solution of 7°dh (german hardness) of the initial neutral pH. A "strong builder" may be a highly efficient chelator capable of strongly binding divalent cations such as ca2+, wherein the cation/chelator complex has a Log stability constant (Log-KCa) higher than 4, in particular higher than 5, higher than 6 or higher than 7. The stability constant was determined at an ionic strength of 0.1M and a temperature of 25 ℃. "Strong chelating builder" combines both of the above properties.

Detailed Description

The present invention provides novel amylases. More specifically, variants of a parent alpha-amylase, methods of making the variant alpha-amylase, compositions comprising the variant alpha-amylase, and methods of using the variant alpha-amylase or compositions comprising the variant alpha-amylase are provided.

Alpha-amylase variants

The present invention relates to an alpha-amylase variant of a parent alpha-amylase, wherein the variant comprises:

(i) Amino acid changes, preferably insertions, deletions, substitutions or combinations thereof, preferably substitutions, at one or more positions corresponding to a position selected from the group consisting of: 1. 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 47, 48, 51, 54, 59, 60, 63, 70, 71, 72, 73, 75, 76, 81, 82, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 103, 106 108, 109, 113, 114, 115, 116, 117, 119, 120, 123, 125, 126, 128, 129, 131, 132, 133, 134, 135, 136, 139, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 154, 155, 156, 158, 160, 161, 162, 163, 164, 165, 169, 170, 172, 173, 174, 175, 176, 177, 178, 180, 182, 184, 185, 187, 188, 189, 191, 192, 193, 203, and 208, 210, 211, 212, 213, 214, 215, 216, 218, 219, 221, 222, 223, 224, 225, 226, 227, 230, 231, 233, 234, 235, 236, 237, 238, 240, 242, 243, 245, 249, 250, 251, 252, 253, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 301, 302, 303, 304, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 333, 334, 336, 337, 338, 341, 342, 343, 344, 345, 346, 348, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 362, 363. 364, 365, 366, 367, 368, 370, 372, 375, 376, 378, 379, 381, 382, 383, 384, 385, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 405, 406, 407, 408, 410, 413, 414, 415, 416, 418, 421, 424, 426, 427, 429, 431, 432, 434, 436, 438, 439, 440, 442, 443, 445, 446, 447, 448, 449, 451, 453, 455, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 470, 471, 474, 478, 480 and 482;

(ii) The variant has at least 60% but less than 100% sequence identity with any one of SEQ ID NO. 1, 3, 4 or SEQ ID NO. 15-41, preferably the amino acid sequence shown in SEQ ID NO. 1, and

(iii) The variant has alpha-amylase activity.

The alpha-amylase variants of the invention are non-naturally occurring amylases. Preferably, the alpha-amylase variants of the invention are isolated, synthetic and/or recombinant alpha-amylase variants.

The amylase of the invention has "amylolytic activity" or "amylase activity". "amylolytic activity" or "amylase activity" describes the ability to hydrolyze glycosidic linkages in polysaccharides. Alpha-amylase activity may be determined by assays known to those of skill in the art for measuring alpha-amylase activity. Examples of assays for measuring alpha-enzyme activity are Phadebas assay or EPS assay ("affinity reagent"). In the Phadebas assay, alpha-amylase activity was determined by using Phadebas tablets as substrate (Phadebas Amylase Test, provided by Magle Life Science). Alpha-amylase hydrolyzes starch to give soluble blue fragments. The absorbance of the resulting blue solution was measured spectrophotometrically at 620nm as a function of alpha-amylase activity. The absorbance measured is proportional to the specific activity of the alpha-amylase in question (activity per mg pure alpha-amylase protein) under a given set of conditions.

Alternatively, alpha-amylase activity may also be determined by a method using ethylene-4-nitrophenyl-alpha-D-maltoheptaoside (EPS). D-maltoheptaoside is a blocked oligosaccharide that can be cleaved by endo-amylase. After cleavage, the α -glucosidase contained in the kit digests the substrate, releasing a yellow free PNP molecule, which can be measured by visible spectrophotometry at 405 nm. Kits containing EPS substrate and alpha-glucosidase are manufactured by, for example, roche Costum Biotech (catalog No. 10880078103). The slope of the time-dependent absorbance curve is proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under a given set of conditions.

In one embodiment, an alpha-amylase variant of the invention exhibits at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200% amylolytic activity of a parent amylase. In one embodiment, the alpha-amylase variants of the invention exhibit the same or increased amylolytic activity as compared to the parent amylase. Preferably, the alpha-amylase variants of the invention exhibit increased amylolytic activity compared to the parent amylase.

Preferably, the parent alpha-amylase of the alpha-amylase variant of the invention is an amylase according to SEQ ID NO. 1 or SEQ ID NO. 3, or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1.

The present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at one or more positions corresponding to positions selected from the group consisting of: 1. 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 47, 48, 51, 54, 59, 60, 63, 70, 71, 72, 73, 75, 76, 81, 82, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 103, 106 108, 109, 113, 114, 115, 116, 117, 119, 120, 123, 125, 126, 128, 129, 131, 132, 133, 134, 135, 136, 139, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 154, 155, 156, 158, 160, 161, 162, 163, 164, 165, 169, 170, 172, 173, 174, 175, 176, 177, 178, 180, 182, 184, 185, 187, 188, 189, 191, 192, 193, 203, and 208, 210, 211, 212, 213, 214, 215, 216, 218, 219, 221, 222, 223, 224, 225, 226, 227, 230, 231, 233, 234, 235, 236, 237, 238, 240, 242, 243, 245, 249, 250, 251, 252, 253, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 301, 302, 303, 304, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 333, 334, 336, 337, 338, 341, 342, 343, 344, 345, 346, 348, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 362, 363. 364, 365, 366, 367, 368, 370, 372, 375, 376, 378, 379, 381, 382, 383, 384, 385, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 405, 406, 407, 408, 410, 413, 414, 415, 416, 418, 421, 424, 426, 427, 429, 431, 432, 434, 436, 438, 439, 440, 442, 443, 445, 446, 447, 448, 449, 451, 453, 455, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 470, 471, 474, 478, 480 and 482.

Preferably, the parent alpha-amylase of the alpha-amylase variant of the invention is an amylase according to any of SEQ ID NO. 1, 3, 4 or SEQ ID NO. 15-41, preferably SEQ ID NO. 1, or any alpha-amylase having at least 60% sequence identity to any of SEQ ID NO. 1, 3, 4 or SEQ ID NO. 15-41, preferably SEQ ID NO. 1. More preferably, the parent alpha-amylase of the alpha-amylase variant of the invention is an amylase according to SEQ ID NO. 1 or SEQ ID NO. 3, or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1.

In a preferred embodiment, the resulting amino acid residues of the amino acid substitutions are not identical to the amino acid residues in the corresponding positions in SEQ ID NO. 3 or 5.

Particularly preferably, according to the numbering of the amino acid sequence shown in SEQ ID NO. 3, an alpha-amylase variant having alpha-amylase activity compared to the parent alpha-amylase comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, at one or more amino acid positions selected from the group consisting of 4, 7, 25, 37, 70, 100, 118, 135, 160, 176, 193, 210, 251, 281, 258, 323, 361, 363, 368, 405, 434, 441, 459, 460, 451 and 482, and wherein the variant has alpha-amylase activity. More particularly preferably, an alpha-amylase variant having alpha-amylase activity comprises an amino acid change, preferably an insertion, deletion, substitution or combination thereof, at one or more amino acid positions selected from 25, 100, 135, 176, 193 and 460 compared to the parent alpha-amylase according to the numbering of the amino acid sequence set forth in SEQ ID NO. 3, and wherein the variant has alpha-amylase activity.

Preferably, the present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at one or more positions corresponding to positions selected from the group consisting of 4, 25, 100, 135, 160, 176, 193, 251, 258, 276, 299, 323, 363, 382, 405, 460 and 482 compared to the parent alpha-amylase according to the numbering of the amino acid sequence shown in SEQ ID NO. 3.

Preferably, the present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of: 1. 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 47, 48, 51, 54, 59, 60, 63, 70, 71, 72, 73, 75, 76, 81, 82, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 103, 106 108, 109, 113, 114, 115, 116, 117, 119, 120, 123, 125, 126, 128, 129, 131, 132, 133, 134, 135, 136, 139, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 154, 155, 156, 158, 160, 161, 162, 163, 164, 165, 169, 170, 172, 173, 174, 175, 176, 177, 178, 180, 182, 184, 185, 187, 188, 189, 191, 192, 193, 203, and 208, 210, 211, 212, 213, 214, 215, 216, 218, 219, 221, 222, 223, 224, 225, 226, 227, 230, 231, 233, 234, 235, 236, 237, 238, 240, 242, 243, 245, 249, 250, 251, 252, 253, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 301, 302, 303, 304, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 333, 334, 336, 337, 338, 341, 342, 343, 344, 345, 346, 348, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 362, 363. 364, 365, 366, 367, 368, 370, 372, 375, 376, 378, 379, 381, 382, 383, 384, 385, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 405, 406, 407, 408, 410, 413, 414, 415, 416, 418, 421, 424, 426, 427, 429, 431, 432, 434, 436, 438, 439, 440, 442, 443, 445, 446, 447, 448, 449, 451, 453, 455, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 470, 471, 474, 478, 480 and 482.

Particularly preferably, according to the numbering of the amino acid sequence shown in SEQ ID NO. 3, an alpha-amylase variant having alpha-amylase activity comprises amino acid substitutions at one or more amino acid positions selected from the group consisting of 4, 7, 25, 37, 70, 100, 118, 135, 160, 176, 193, 210, 251, 281, 258, 323, 361, 363, 368, 405, 434, 441, 459, 460, 451 and 482 compared to the parent alpha-amylase, and wherein the variant has alpha-amylase activity. More particularly preferably, an alpha-amylase variant having alpha-amylase activity comprises amino acid substitutions at one or more amino acid positions selected from the group consisting of 25, 100, 135, 176, 193 and 460 compared to the parent alpha-amylase according to the numbering of the amino acid sequence set forth in SEQ ID NO. 3, and wherein the variant has alpha-amylase activity.

Preferably, the present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of 4, 25, 100, 135, 160, 176, 193, 251, 258, 276, 299, 323, 363, 382, 405, 460 and 482 compared to the parent alpha-amylase according to the numbering of the amino acid sequence shown in SEQ ID NO 3.

In a preferred embodiment, the present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of 4, 429 and 459 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity to any of SEQ ID nos. 1, 3, 4, or 15-41, preferably SEQ ID No. 1, 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably SEQ ID No. 1; preferably, a deletion is comprised at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably the deletion of amino acids at positions 183 and 184, preferably D183 and G184, wherein numbering is according to the amino acid sequence shown in SEQ ID No. 3.

In another preferred embodiment, the invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the alpha-amylase variant has an alpha-amylase activity according to SEQ ID NO:3, which variant comprises an amino acid substitution at one or more positions corresponding to positions selected from 4, 25, 116, 176, 225, 251, 320, 400, 405, 408, 410, 418, 429, 446, 449, 458, 459, 460, 471 and 482 compared to the parent α -amylase, preferably wherein the α -amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably comprises at least 91% or at least 95% but less than 100% sequence identity to any one of SEQ ID NOs 1, 3, 4 or 15-41, preferably SEQ ID NO 1, 3 or 4, more preferably SEQ ID NO 1 or 3, most preferably SEQ ID NO 1, amino acid sequence shown in SEQ ID NO 1, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably comprises at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably comprises at least 183, amino acids corresponding to positions 181, 183 and 183, and preferably amino acids 183, 182, 183, and amino acid sequence numbers shown in sequence numbers which are deleted according to the preferred sequence numbers.

In another preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from 4, 25, 176, 251, 405 and 482 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably wherein the amino acid sequence corresponding to one or more positions selected from 181, 182, 183 and 184 comprises amino acid deletions at any one of positions selected from positions 1, 3, 4, or 15-41, preferably SEQ ID No. 1, 3 or 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1 has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 183, preferably at one position corresponding to positions selected from 181, 183 and 184, preferably comprising amino acid deletions at one or more than 182, 183, according to amino acid sequence shown in SEQ ID No. 1, and preferably comprising amino acid numbers shown in SEQ ID No. 182, or.

In another preferred embodiment, the invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the alpha-amylase variant has an alpha-amylase activity according to SEQ ID NO:3, the variant comprising a substitution of one or more of the amino acids at positions corresponding to positions selected from the group consisting of 4, 6, 10, 12, 13, 14, 15, 17, 20, 21, 22, 23, 24, 27, 28, 32, 34, 36, 38, 39, 42, 45, 47, 51, 70, 75, 76, 83, 89, 92, 95, 96, 99, 100, 108, 115, 154, 156, 164, 191, 192, 213, 215, 221, 222, 223, 226, 233, 234, 236, 237, 240, 245, 249, 268, 271, 277, 282, 285, 288, 289, 290, 297, 301, 308, 309, 312, 326, 327, 333, 336, 338, 341, 343, 348, 351, 352, 353, 355, 356, 358, 366, 367, 370, 379, 381, 389, 392, 413, 424, 426, 432, 436, 440, 442, 443, 448, 453, 451, 463, and the amino acid sequence of the variant, as compared to the parent α -amylase, wherein the variant comprises an amino acid at positions of one or more than one of the amino acid positions of SEQ ID; 1, 3, 4, or any of SEQ ID NO 15-41, preferably SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 4, more preferably SEQ ID NO 1 or 3, most preferably SEQ ID NO 1 has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably comprising a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably corresponding to the deletion of amino acids at positions 181 and 182, 182 and 183 or 183 and 184, preferably d183 and g184, wherein numbering is according to the amino acid sequence shown in SEQ ID No. 3.

In another preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from 4, 429 and 459 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity to any one of SEQ ID nos. 1, 3, 4 or 15-41, preferably SEQ ID No. 1, 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1, has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 184, preferably at one or more amino acid corresponding to positions selected from 181, 182, 183 and 184, preferably at least 91% and 183, amino acid sequence numbers corresponding to positions selected from 181, 182, 183 and 183, preferably comprising deletions, amino acids shown in SEQ ID nos. 182, and 183, and preferably according to amino acid sequences shown in SEQ ID nos. 1.

In another preferred embodiment, the invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the alpha-amylase variant has an alpha-amylase activity according to SEQ ID NO:3, which variant comprises an amino acid substitution at one or more positions corresponding to positions selected from the group consisting of 4, 10, 12, 13, 14, 15, 17, 20, 21, 23, 24, 27, 34, 36, 38, 39, 42, 45, 47, 51, 89, 92, 99, 100, 108, 115, 164, 191, 192, 213, 221, 222, 223, 233, 234, 236, 237, 240, 245, 249, 268, 271, 282, 288, 289, 290, 297, 301, 308, 309, 312, 326, 327, 333, 336, 338, 341, 343, 348, 351, 352, 353, 355, 356, 358, 366, 367, 370, 376, 379, 381, 389, 392, 413, 424, 426, 429, 432, 440, 442, 443, 448, 451, 453, 456, and 480, preferably wherein the alpha-amylase and SEQ ID NO:1, 3, 4, or any of SEQ ID NOs 15-41, preferably SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 4, more preferably SEQ ID NO 1 or 3, most preferably the amino acid sequence shown in SEQ ID NO 1 has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably comprises a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183 and G184, wherein numbering is according to the amino acid sequence shown in SEQ ID No. 3.

In another preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises an amino acid substitution at one or more positions corresponding to positions selected from 4 and 429 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity to any one of SEQ ID nos. 1, 3, 4, or 15-41, preferably SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably amino acid sequence shown in SEQ ID No. 1, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 183, at one or more amino acids corresponding to positions selected from 181, 183 and 184, preferably comprising deletions of amino acids 181, 183, and 184, preferably according to amino acid sequence numbers shown in SEQ ID nos. 183, and 3.

In another particularly preferred embodiment, the present invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises an amino acid substitution at one or more positions corresponding to positions selected from 25 and 176 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity to any one of SEQ ID nos. 1, 3, 4, or 15-41, preferably SEQ ID No. 1, 3 or 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1, preferably at least 184, at least 91%, at least 93%, at least 94%, at least 95% but less than 100% sequence identity to one or more amino acids corresponding to positions selected from 181, 183 and 184, preferably comprises deletion of amino acids 184, 182 and 183, preferably amino acids corresponding to positions 181, 183, and 183, according to the preferred amino acid sequence shown in SEQ ID No. 1.

In another particularly preferred embodiment, the present invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from 25, 176 and 186 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity to any one of SEQ ID nos. 1, 3, 4 or 15-41, preferably SEQ ID No. 1, 3 or 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1, preferably has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 184, preferably at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably at least 91% and 183, amino acid deletions corresponding to positions selected from 181, 182, 183 and 183, preferably amino acids shown in SEQ ID nos. 182, 183, and 183.

In another particularly preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of 4, 25, 176, 186, 251, 405, 439 and 482 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably at least 183, preferably at one or more positions corresponding to positions selected from the group consisting of 181, 182, 184, and 183, amino acid substitutions preferably at any of SEQ ID No. 1, 3, 4, or SEQ ID No. 15-41, preferably SEQ ID No. 1, 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1 has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% but less than 183% sequence identity, preferably at one position 183, preferably at one or more than 183, amino acid sequence corresponding to positions selected from the amino acid sequence shown in SEQ ID No. 181, 183, and preferably comprising amino acid numbers shown in SEQ ID No. 182, and 183, and preferably comprising amino acid sequences shown.

In another particularly preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of 4, 25, 176, 251, 405, 439 and 482 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID NO:3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably at least 183 position corresponding to one or more positions selected from the group consisting of 181, 182, 183 and 184, preferably amino acid substitutions at any one of SEQ ID NO:1, 3, 4, or SEQ ID NO: 15-41, preferably SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, more preferably SEQ ID NO:1 or 3, most preferably the amino acid sequence shown in SEQ ID NO:1, has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% but less than 183% sequence identity, preferably at least 183% or at one position corresponding to one or more positions selected from the amino acid sequence selected from the group consisting of 181, 183, and preferably comprising amino acid substitutions shown in SEQ ID NO: 182, and amino acid sequence shown in SEQ ID NO: 183, and preferably.

In another particularly preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from 4, 25, 176, 186, 251, 405 and 482 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably at least 183 position corresponding to one or more positions selected from 181, 182, 183 and 184, preferably any of SEQ ID nos. 15-41, preferably SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 183% sequence identity, preferably at one or more positions corresponding to positions selected from 181, 182, 184, and 184, preferably comprising amino acid deletions shown in SEQ ID No. 182, 183, and preferably according to amino acid sequence numbers shown in SEQ ID No. 1.

In another particularly preferred embodiment, the present invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of 4, 25, 176, 251, 405 and 482 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably at least 183, amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably amino acid substitutions at any one of SEQ ID No. 1, 3, 4 or SEQ ID No. 15-41, preferably SEQ ID No. 1, 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1 has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% but less than 183% sequence identity, preferably at one or more than 183, amino acid substitutions selected from the group consisting of positions selected from the group consisting of 181, 183 and 183, and preferably amino acid substitutions shown in SEQ ID No. 182, and preferably comprising amino acid sequences shown in SEQ ID No. 1.

In another particularly preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises an amino acid substitution at positions 4 and 25, or 25 and 176, or 176 and 186, or 186 and 251, or 251 and 405, or 405 and 482, preferably at positions 4 and 25 and 176, or 25 and 176 and 186, or 176 and 251, or 186 and 405, or 251 and 405 and 482, compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID NO:3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably at positions 4 and 25 and 176, or 25 and 186 and 251, or 186 and 251 and or 251 and 405 and 482, or 251 and 482, preferably wherein the amino acid sequence shown in SEQ ID NO:1 or 3, most preferably the amino acid sequence shown in SEQ ID NO:1, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% but less than 100% but less than 183% and 183, preferably at least 183, position 182.

In another particularly preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from 116, 181, 225 and 320 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity to any one of SEQ ID nos. 1, 3, 4 or 15-41, preferably SEQ ID No. 1, 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 183 one or more positions corresponding to positions selected from 181, 182, 184 and 184, preferably at least 91% or 183, preferably comprising amino acid deletions corresponding to positions selected from 182, 184, and 183, according to the preferred amino acid sequence numbers shown in SEQ ID nos. 182, and 183.

In another particularly preferred embodiment, the invention relates to an alpha-amylase variant having alpha-amylase activity, wherein the variant comprises an amino acid substitution at positions 116 and 181, or 181 and 225, or 225 and 320, preferably at positions 116 and 181 and 225, or 181 and 225 and 320, compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID NO:3, preferably wherein the alpha-amylase variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, sequence identity to the amino acid sequence shown in SEQ ID NO:1, most preferably wherein the alpha-amylase variant comprises a deletion at one or more amino acids corresponding to positions 116 and 181 and 225, or 181 and 225 and preferably at positions 183, 183 and 183, preferably 183 and 184, preferably amino acid sequence numbers shown in SEQ ID NO:1, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 95% but less than 100%, sequence identity to the amino acid sequence shown in SEQ ID NO: 3.

In an alternative embodiment, the invention relates to an alpha-amylase variant, wherein said variant comprises one or more amino acid substitutions compared to the parent sequence according to the numbering of the amino acid sequence set forth in SEQ ID NO:3 selected from the group consisting of: X430F, X430G, X430L, X430P, X430Q, X430S, X430T, X430V, X435K, X435P, X435S, X435A, X435D, X437L, X437T, X437W, X441C, X441K, X441L, X441M, X441S, X444H, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450L, X450M, X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X452F, X452I, X452K, X452M, X430F, X430G, X430L, X430P, X430Q, X430S, X430T, X430V, X435K, X435P, X435S, X435A, X435D, X437L, X437T, X437W, X441C, X441K, X441L, X441M, X441S, X444H, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450L, X450M, X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X452F, X452I, X452K, X452M, X452N, X452T, X454A, X454E, X454K, X454L, X454P, X454S, X454T, X466E, X466W, X469F, X469L, X469Y, X475A, X475K, X475E, X475L, X479I, X479K, X479M, X483F, X483L, X483Q, and X483R, wherein the variant has alpha-amylase activity, preferably wherein the parent alpha-amylase of the alpha-amylase variant of the invention is according to SEQ ID NO:1 or SEQ ID NO. 3, or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of said alpha-amylase variant is an amylase according to SEQ ID NO. 1.

Preferably, the variant alpha-amylase comprises amino acid substitutions at one or more of the following amino acid positions (numbering according to the amino acid sequence shown in SEQ ID NO: 3) compared to the parent alpha-amylase. Preferably, the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1 or SEQ ID NO. 3 or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1. Preferably, the amino acid residue (i.e.X) of the parent alpha-amylase at the indicated position corresponds to the amino acid residue shown in SEQ ID NO. 1 or 3, preferably at the corresponding position in SEQ ID NO. 1 (numbering according to SEQ ID NO. 3).

In one embodiment, the variant alpha-amylase comprises an amino acid substitution at position 9 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X9D, X9F, X9K, X9N, X9P, X9Q, X9S, X9T or X9Y.

In one embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X179G, at position 179 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 181 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X181D, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X W or X181Y, preferably X181T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 186 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X186A, X186C, X186D, X186F, X186H, X186I, X186K, X186L, X186M, X186R, X186V, X W or X186Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X190H, at position 190 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 195 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X195H, X195K, X195L, X195W or X195Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 206 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X206C, X206H, X M or X206Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 244 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X244A, X244C, X244D, X244E, X244F, X244G, X244H, X244M, X N or X244V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X402T, at position 402 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X419C, at position 419 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 420 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X420D, X420E, X420G, X420H, X420K, X420L or X420Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 422 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X422C, X422N or X422H.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 423 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X423F, X423M, X423Q or X423S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 428 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X428C, X428D, X428E, X428G, X428I, X428K, X428L, X428M, X N, X428R, X428S, X428V, X W or X428Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 430 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X430A, X430C, X D, X430E, X430F, X430G, X430L, X430P, X430Q, X430S, X T or X430V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 435 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X435K, X435P, X435S, X435A or X435D.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 437 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X437L, X437T or X437W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 441 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X441C, X441K, X441L, X441M or X441S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 444 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X444H, X444M, X444N, X R or X444T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 450 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X450C, X450D, X450E, X450H, X450L, X450M, X450P, X450Q, X450R or X450T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 452 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X452A, X452C, X452E, X452F, X452I, X452K, X452M, X N or X452T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 454 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X454A, X454E, X454K, X454L, X454P, X S or X454T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 466 (numbering according to SEQ ID NO: 3), preferably substitution X466E or X466W, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 469 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X469F, X469L or X469Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 475 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X475A, X475K, X475E or X475L.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 479 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X479I, X479K or X479M.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 483 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X483F, X483L, X483Q or X483R.

Preferably, in this alternative embodiment, the amino acid residue at the above position (i.e.X) in the parent alpha-amylase corresponds to the amino acid residue shown in SEQ ID NO. 1 or 3, preferably at the corresponding position in SEQ ID NO. 1 (numbering according to SEQ ID NO. 3). Preferably, the present invention relates to an alpha-amylase variant, wherein said variant comprises one or more amino acid substitutions compared to the parent sequence according to the numbering of the amino acid sequence set forth in SEQ ID NO:3 selected from the group consisting of: the parent alpha-amylase of the alpha-amylase variant of the invention is preferably an amylase according to SEQ ID NO. 1 or SEQ ID NO. 3 or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1, and X181 181 181 181 181 186 186 186 186 186 186 186 186 186 186 186 186 186 195 195 195 195 195 195 195 195 195 195 206 206 441 441 441 441 441 441 441M and X441S. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Particularly preferably, the present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of 4, 25, 100, 135, 160, 176, 193, 251, 258, 276, 299, 323, 363, 382, 405, 460 and 482 compared to the parent alpha-amylase according to the numbering of the amino acid sequence shown in SEQ ID NO: 3.

Preferably, the present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of: h1, H2, N3, G4, T5, N6, G7, T8, M10, Y12, F13, E14, W15, Y16, L17, P18, N19, D20, G21, N22, H23, W24, N25, R26, L27, R28, S29, D30, A31, S32, N33, L34, K35, D36, K37, G38, I39, T40, A41, V42, P45, A47, W48, A51, G59, A60, L63, N70, Q71, K72, G73, V75, R76, T81, R82, N83, Q86, V89, T90, A91, L92, K93, S94, N95, G96, I97, Q98, V99, Y100, V103, N106K 108, G109, A113, T114, E115, W116, V117, A119, S125, N126, N128, Q129, V131, S132, G133, D134, Y135, T136, A139, T141, K142, F143, D144, F145, P146, G147, G149, N150, T151, N154, F155, K156, Y160, H161, F162, G164, V165, Q169, S170, Q172, L173, Q174, N175, R176, G184, D188, V191, Y203, H210, P211, E212, V213, V214, N215, E216, R218, N219, G221, V222, W223, Y224, T225N 226, T227, L230, D231, F233, R234, I235, D236, A237, V238, H240, K242, Y243, F245, W249, L250, T251, H252, V253, N255, T256, T257, K259, M261, F262, A263, V264, A265, W268, N270, I272, G273, A274, E276, L279, S280, W284, N285, H286, S287, V288, F289, D290, V291, P292, L293, H294, Y295, N296, L297, N299, S301, R302, S303, G304, N306, Y307, D308, M309, R310, Q311, I312F 313, N314, G315, V318, Q319, R320, H321, T323, H324, V326, T327, D333, P336, E337, E338, E341, S342, F343, V344, E345, E346, F348, P350, L351, A352, Y353, A354, L355, L357, T358, R359, D360, G362, Y363, P364, V366, F367, Y368, D370, Y372, P375, T376, G378, V379, A381, M382, K383, S384, K385, D387, P388, I389, L390, E391, A392, R393, Q394, K395, Y396, A397, Y398, G399, T400, Q401, D403, L405, D406, H407, P408, V410, W413, T414, R415, E416, D418, H421, S424, L426, A427, L429, S431, G436, K438, W439, M440, V442, G443, N445, N446, A447, G448, E449, W451, D453, T455, G456, N457, Q458, T459, N460, T461, V462, T463, N465, D467, G468, G470, V474, S478, S480, Y482, A119, D163, D271, F180, G182, G258, I177, I275, K185, K269, K281, N224, N260, N277, P322, P434, R118, S365, T193, T282, T356, V120, V317, W187, W189, Y178, and Y298.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of G4, N25, K37, N70, Y100, R118, Y135, Y160, R176, T193, H210, T251, K281, G258, T323, Q361, Y363, Y368, L405, P434, E441, T459, N460, T451 and Y482 compared to the parent alpha-amylase according to the numbering of the amino acid sequence shown in SEQ ID No. 3, and wherein the variant has alpha-amylase activity.

It is particularly preferred that the alpha-amylase variant having alpha-amylase activity comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of N25, Y100, Y135, R176, T193 and N460, according to the numbering of the amino acid sequence shown in SEQ ID No. 3, compared to the parent alpha-amylase, and wherein the variant has alpha-amylase activity.

Particularly preferred, the present invention relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises an amino acid substitution at one or more amino acid positions corresponding to positions selected from the group consisting of G4, N25, Y100, Y135, Y160, R176, T193, T251, G258, E276, N299, T323, Y363, M382, L405, N460 and Y482 compared to the parent alpha-amylase according to numbering of the amino acid sequence shown in SEQ ID No. 3.

Preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO 3, as compared to the parent alpha-amylase, and wherein said variant has alpha-amylase activity: X100D, X100F, X100K, X100L, X103A, X106D, X108A, X109A, X10A, X10C, X10E, X10F, X10L, X10W, X10Y, X113F, X113H, X113M, X113R, X113V, X113W, X113Y, X114A, X114C, X114D, X114E, X114F, X114G, X114H, X114I, X114K, X114L, X114M, X114N, X114Q, X114R, X114S, X114V, X114W, X114Y, X115C, X115D, X117K, X115M, X115N, X115Q, X115R, X115S, X115T, X115V, X116I, X116K, X116M, X116R, X116T, X117A, X117C, X116D, X117N, X117X 116D, X117W, X117X 116R, X117Y, X118A, X118D, X118E, X119H, X119P, X119R, X119S, X119Y, X120E, X120G, X120M, X120S, X120W, X120Y, X123D, X123S, X125A, X125D, X125E, X125F, X125G, X125H, X125K, X125L, X125M, X125N, X125Q, X125R, X125T, X125V, X125W, X125Y, X126D, X128C, X128E, X128L, X128M, X128W, X128Y, X129E, X12N, X12S, X131C, X131I, X131L, X131Q, X131W, X132T, X133A, X133C, X133D, X133E, X133H, X133K, X133N, X134L, X134E, X134L, X134M, X134P, X134T, X134V, X134W, X134Y, X135D, X135E, X135G, X135M, X135N, X135P, X135S, X135T, X135W, X136A, X136C, X136D, X136E, X136F, X136H, X136L, X136M, X136N, X136P, X136W, X139C, X139S, X13A, X13Y, X141V, X142C, X142E, X142F, X142L, X142M, X142Q, X142R, X142W, X142Y, X143F, X144C, X144E, X144G, X144K, X144N, X144Q, X144R, X144S, X144T, X144V, X144Y, X145A, X146C, X146D, X146E, X146F, X146G, X146H, X146K, X146L, X146S, X146T, X146W, X147M, X149E, X14N, X150C, X150E, X150Q, X151C, X151E, X154A, X154Y, X155Y, X156E, X156V, X158H, X158N, X158Y, X15R, X160D, X160E, X160W, X161T, X162V, X163A, X163Q, X163T, X164V, X165S, X165T, X165W, X169A, X169D, X169E, X169S, X169V, X16F, X16R, X170C, X170F, X172A, X172C, X172D, X172K, X172N, X173I, X173Y, X174D, X174E, X174G, X174N, X174P, X174S, X174T, X175A, X175G, X175H, X175K 176, X176K 176A, X177G, X176K 176G, X177G, X177N, X177P, X177R, X177S, X177W, X178F, X17K, X17V, X180M, X180N, X180T, X180W, X182D, X182E, X182N, X182Q, X182S, X185E, X185M, X185N, X187A, X187D, X187M, X187V, X188H, X188T, X188V, X189I, X18F, X18I, X18K, X18M, X18R, X18T, X191F, X191H, X191K, X191M, X191W, X191Y, X192A, X192T, X193D, X193E, X193G, X193V, X19A, X19C, X19D, X19F, X19G, X19H, X19I, X19K, X19L, X19M, X19P, X19Q, X19S, X19T, X19Y, X1R, X1V, X203E, X203R, X208F, X208I, X208Y, X20A, X20C, X20D, X20E, X20F, X20G, X20H, X20K, X20L, X20M, X20N, X20P, X20Q, X20S, X20T, X20V, X20W, X20Y, X210A, X210C, X210D, X210E, X210F, X210M, X210N, X210Q, X210S, X210Y, X211A, X211C, X211D, X211E, X211G, X211H, X211L, X211N, X211Q, X211S, X211T, X211V, X212C, X212D, X212I, X212L, X212W, X213A, X213C, X213M, X213R, X213S, X214E, X214P, X215A, X215D, X215E, X215H, X215G, X216W 218G, X218G, X218E, X218F, X218G, X218H, X218I, X218K, X218L, X218N, X218Q, X218S, X218T, X218V, X218W, X218Y, X219A, X219C, X219D, X219E, X219F, X219G, X219H, X219K, X219M, X219Q, X219R, X219S, X219T, X219W, X219Y, X21E, X21S, X221F, X221N, X222D, X222K, X222S, X222T, X223C, X223L, X223V, X223Y, X224F, X224V, X225A, X225C, X225E, X225F, X225H, X225I, X225N, X225R, X225S, X225Y, X226A, X226C, X226D, X226E, X226G, X226H, X226I, X226L, X226M, X226Q, X226R, X226S, X226T, X226V, X226W, X226Y, X227C, X227F, X227G, X227H, X227I, X227K, X227L, X227M, X227R, X227T, X227V, X227W, X227Y, X22A, X22D, X22E, X22F, X22G, X22K, X22L, X22M, X22Q, X22R, X22T, X22W, X22Y, X230A, X230F, X231C, X231D, X231N, X233C, X233H, X233I, X233M, X233T, X233W, X234C, X235L, X235M, X235V, X236Y, X237C, X237I, X238A, X238F, X238T, X23N, X23Q, X23W, X240M, X243D, X245H, X245M, X249D, X249I, X24G, X250V, X251A, X251E, X251F, X251L, X251M, X251S, X251T, X252C, X252I, X252S, X253C, X253G, X253Y, X255D, X255F, X255I, X255T, X255V, X256C, X257L, X257V, X257Y, X258F, X258Q, X258R, X259L, X25A, X25C, X25D, X25F, X25G, X25H, X25K, X25L, X25M, X25Q, X25S, X25W, X25Y, X260H, X261R, X262C, X262D, X262P, X262Y, X263I, X H, X264T, X264W, X268S, X268F, X26G, X26M 26F, X26M, X26P, X26S, X26V, X26Y, X270A, X270Q, X270Y, X271S, X272F, X272G, X272L, X272S, X273A, X273C, X273D, X273E, X273F, X273H, X273I, X273L, X273M, X273P, X273Q, X273R, X273V, X273W, X273Y, X274F, X274S, X275V, X276D, X276K, X276L, X276N, X276R, X276Y, X277D, X277E, X277T, X272A, X279P, X27A, X27D, X27F, X27G, X27H, X27I, X27Q, X27V, X280D, X280F, X280G, X280H, X280I, X280K, X280N, X280D, X280V, X280Y, X281E, X281H, X284A, X284F, X284H, X284L, X284M, X284N, X284Y, X285G, X285L, X285N, X285P, X286Q, X287A, X287D, X287E, X287H, X287T, X288A, X288K, X288P, X288Y, X289F, X289G, X289R, X289T, X28C, X28D, X28E, X28F, X28G, X28I, X28K, X28N, X28Q, X28S, X28T, X28V, X290D, X290M, X290N, X290Q, X290W, X291D, X291K, X291T, X292D, X292F, X292I, X292L, X292T, X292W, X293Y, X293D, X293E, X293F, X294T, X294F, X296A, X296C, X296L, X296Y, X297E, X297F, X297H, X297K, X297M, X297S, X297V, X299G, X299I, X299K, X299L, X299S, X299Y, X29D, X29E, X29F, X29G, X29H, X29I, X29K, X29L, X29N, X29P, X29Q, X29V, X29W, X29Y, X2I, X2S, X301F, X302H, X302I, X302Q, X302V, X302Y, X303E, X303H, X303I, X303K, X303L, X303M, X303N, X303P, X303R, X303T, X304A, X304D, X304E, X304H, X304K, X304M, X304N, X304P, X304R, X304T, X304W, X304Y, X306A, X306D, X306E, X306G, X306H, X306I, X306M, X306Q, X306R, X306S, X306T, X306V, X306W, X306Y, X307F, X307M, X308S, X309H, X309L, X309Q, X30A, X30E, X30F, X30G, X30H, X30I, X30K, X30L, X30M, X30Q, X30T, X30W, X30Y, X310A, X310Q, X311A, X311E, X311G, X311H, X311K, X311N, X311R, X311T, X311Y, X312L, X312M, X313V, X314C, X314E, X314K, X314Q, X315A, X315C, X315E, X315H, X315K, X315T, X318I, X318S, X318T, X319A, X319D, X319H, X319I, X319K, X319M, X319N, X319P, X319S, X319T, X319W, X31N, X31Q, X31S, X31T, X31V, X31W, X320A, X320C, X320D, X320E, X320G, X320H, X320K, X320L, X320N, X320Q, X320S, X320Y, X321A, X321E, X321K, X321N, X321T, X321V, X321W, X323A, X323G, X323K, X323L, X323V, X324K, X324L, X324M, X324W, X324Y, X326G, X326N, X326S, X326Y, X327C, X327L, X327M, X32A, X32D, X32E, X32F, X32I, X32L, X32M, X32N, X32P, X32Q, X32K, X337K, X32T, X337W, X32K, X337W, X337C, X32X 337C, X337X 32X 337X 32X 37X 32X, X337G, X337I, X337K, X337L, X337M, X337N, X337Q, X337R, X337S, X337T, X337V, X337Y, X338G, X338S, X338T, X33D, X33E, X33H, X33K, X33M, X33Q, X33R, X33Y, X341V, X342P, X343L, X343T, X343W, X343Y, X344I, X344Q, X344V, X345D, X345G, X345M, X345N, X345Q, X345S, X345T, X346A, X346C, X346D, X346G, X346H, X346N, X346Q, X348T, X34H, X34I, X34V, X350H, X350K, X350P, X351A, X351M, X354S, X354I, X354T, X355M, X355I, X356V, X357A, X358I, X358L, X358N, X358P, X358V, X359E, X35A, X35C, X35D, X35G, X35H, X35I, X35L, X35M, X35N, X35P, X35Q, X35R, X35S, X35T, X35V, X35Y, X360A, X360F, X360G, X360I, X360L, X360N, X360Q, X360R, X360S, X360T, X360V, X360Y, X362F, X362K, X362M, X362N, X362T, X362V, X362Y, X363A, X363C, X363D, X363E, X363G, X363H, X363K, X363L, X363M, X363P, X363Q, X363R, X363S, X363T, X363V, X363W, X363Y, X364A, X364C, X364G, X364K, X364L, X364N, X364S, X364T, X364V, X366I, X366L, X366T, X367E, X367S, X368A, X368F, X368L, X368N, X36A, X36E, X36G, X36I, X36K, X36M, X36N, X36P, X36Q, X36R, X36S, X36T, X36V, X370E, X370I, X372A, X372C, X372E, X372F, X372H, X372M, X372N, X372Q, X375A, X375D, X375E, X375I, X375K, X375Q, X375R, X376T, X375W, X375Y, X376G, X376I, X376M, X376Q, X376R, X376S, X376V, X377, X37C, X379A, X379L, X37L and X37L. X37G, X37M, X37P, X37T, X37V, X37W, X381E, X381V, X382A, X382H, X382K, X382L, X382N, X382Q, X382S, X383C, X383D, X383E, X383H, X383I, X383M, X383N, X383Q, X383R, X383S, X383V, X383Y, X384A, X384C, X384D, X384E, X384F, X384I, X384L, X384M, X384N, X384Q, X384R, X384T, X385V, X385W, X385Y, X385A, X385C, X385D, X385E, X385F, X385G, X385H, X385I, X385L, X385M, X385N, X385P, X385Q, X385R, X385S, X385V, X385W, X385C, X385 387E, X387N, X388E, X388F, X388H, X388I, X388M, X388R, X388V, X389G, X389H, X389K, X38N, X390D, X390F, X390M, X390N, X390P, X390R, X391A, X391F, X391G, X391K, X391M, X391N, X391Q, X391S, X391T, X391Y, X392C, X392V, X393E, X393H, X393P, X393S, X393V, X394A, X394C, X394E, X394H, X394I, X394L, X394M, X394N, X394R, X394S, X395A, X395H, X395V, X396H, X396P, X397D, X397H, X397P, X397S, X39M, X39K, X39G, X3G, X39G 3G, X3Q, X3V, X400A, X400D, X400E, X400G, X400H, X400I, X400K, X400L, X400M, X400N, X400P, X400Q, X400R, X400S, X400V, X400W, X401I, X401K, X401M, X401T, X403N, X405C, X405H, X405M, X405T, X405V, X406P, X407D, X407R, X407S, X408E, X408I, X408Q, X40I, X40S, X410H, X410I, X410K, X410L, X410P, X410R, X410Y, X413S, X414C, X414E, X414S, X415I, X416S, X418C, X418N, X418P, X41C, X41D, X41E, X41G, X41G 41S, X41S 41T 41S, X41S 41T 41A, X41S, X426D, X426W, X427C, X427F, X427G, X427K, X427Q, X427R, X427S, X427T, X427V, X429A, X429D, X429E, X429F, X429G, X429I, X429M, X429N, X429P, X429Q, X429S, X429T, X429V, X429W, X42C, X42I, X42Q, X42V, X431I, X434S, X436E, X438F, X438P, X438Y, X439K, X439C, X439P, X440V, X442Q, X443H, X445T, X445A, X445C, X445D, X445T, X445V, X446F, X446I, X446L, X446R, X446S, X446W, X447V, X448D, X448E, X448H, X448N, X449A, X449F, X449G, X449K, X449L, X449M, X449N, X449P, X449Q, X449R, X449S, X449V, X449W, X449Y, X451L, X453G, X453I, X453N, X453P, X453Y, X455L, X456L, X456M, X456R, X456S, X456V, X456W, X456Y, X457A, X457C, X457E, X457F, X457G, X457K, X457L, X457M, X457R, X457S, X457T, X457V, X457W, X458I, X458K, X458V, X458W, X459C, X459D, X459E, X459I, X459K, X459N, X459Q, X459R, X459V, X459Y, X45G, X45N, X460A, X460D, X460E, X460G, X460Q, X460R, X460S, X460T, X460V, X461A, X461E, X461F, X461K, X461L, X461M, X461N, X461Q, X461R, X461S, X461V, X462K, X463L, X465H, X465P, X465R, X465V, X467A, X467E, X467H, X468D, X468H, X470A, X470Q, X470T, X471E, X474F, X474H, X474P, X478A, X478E, X47S, X480D, X480E, X480M, X480R, X480Y, X482C, X482T, X482W, X48F, X48I, X48M, X48Y, X4A, X4C, X4K, X4M, X4Q, X4R, X4S, X51Q, X51T, X51V, X54D, X54G, X54Q, X59T, X5A, X5C, X5D, X5E, X5F, X5H, X5I, X5K, X5L, X5M, X5N, X5P, X5Q, X5R, X5V, X5Y, X60T, X63C, X63V, X6A, X6C, X6E, X6F, X6G, X6H, X6K, X6L, X6M, X6P, X6Q, X6S, X6T, X6V, X6W, X6Y, X70F, X70H, X70L, X70M, X70N, X70Y, X71D, X72C, X72D, X72E, X72N, X72T, X73L, X73N, X73Q, X75A, X75G 75L, X75G, X75T 76C, X75G, X75T 76T, X76G, X75T, X75M 76T, X76G, X75M and X76T, X7F, X7H, X7K, X7N, X7P, X7Q, X7R, X7S, X7V, X7W, X7Y, X81H, X81L, X82K, X82M, X83A, X83D, X83E, X83G, X83R, X83S, X86K, X87D, X87R, X89A, X89C, X89F, X89G, X89L, X89M, X89R, X89S, X8A, X8C, X8F, X8I, X8M, X8P, X8S, X8V, X8W, X8Y, X90A, X90D, X90E, X90F, X90G, X90I, X90M, X90N, X90Q, X90R, X90S, X90V, X90Y, X91C, X91D, X91E, X91F, X91G, X91H, X91I, X91K, X91L, X91M, X91N, X91Q, X91S, X91T, X91V, X91W, X91Y, X92D, X92M, X92V, X93K, X93Q, X94A, X94D, X94E, X94K, X94M, X94V, X94Y, X95E, X95I, X95L, X95V, X96K, X96N, X96Q, X97V, X98E, X98G, X98N, X99H, X99K, X99N, X100W, and X1G. Preferably, the amino acid residue at the above position (i.e., X) corresponds to the amino acid residue of SEQ ID NO:1 or 3, preferably SEQ ID NO:1 at the corresponding position (numbered according to SEQ ID NO: 3).

Preferably, the variant alpha-amylase comprises amino acid substitutions at one or more of the following amino acid positions (numbering according to the amino acid sequence shown in SEQ ID NO: 3) compared to the parent alpha-amylase. Preferably, the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1 or SEQ ID NO. 3 or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1. Preferably, the amino acid residue (i.e.X) of the parent alpha-amylase at the indicated position corresponds to the amino acid residue shown in SEQ ID NO. 1 or 3, preferably at the corresponding position in SEQ ID NO. 1 (numbering according to SEQ ID NO. 3).

In one embodiment, the variant alpha-amylase comprises an amino acid substitution at position 100 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X100D, X100F, X100K, X100L or X100W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X103A, at position 103 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X106D, at position 106 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X108A, at position 108 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X109A, at position 109 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 10 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X10A, X10C, X10E, X10F, X10L, X10W or X10Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 113 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X113F, X113H, X113M, X113R, X113V, X113W or X113Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 114 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X114A, X114C, X114D, X114E, X114F, X114G, X114H, X114I, X K, X114L, X114M, X N, X114Q, X114R, X114S, X114V, X114W or X114Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 115 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X115C, X115D, X115K, X115M, X115N, X115Q, X115R, X115S, X T or X115V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 116 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X116I, X116K, X116L, X116M, X R or X116T, preferably X116K.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 117 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X117A, X117C, X117D, X117F, X117I, X117N, X117P, X117R, X W or X117Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 118 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X118A, X118D or X118E.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 119 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X119H, X119P, X119R, X119S or X119Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 120 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X120E, X120G, X120M, X120S, X W or X120Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X123D or X123S, at position 123 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 125 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X125A, X125D, X125E, X125F, X125G, X125H, X125K, X125L, X125M, X125N, X125Q, X125R, X125T, X125V, X W or X125Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X126D, at position 126 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 128 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X128C, X128E, X128L, X128M, X W or X128Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X129E, at position 129 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X12N or X12S, at position 12 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 131 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X131C, X131I, X131L, X131Q or X131W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X132T, at position 132 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 133 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X133A, X133C, X133D, X133E, X133H, X133K, X133N, X133P, X Q or X133S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 134 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X134C, X134E, X134F, X134I, X134L, X134M, X134P, X134T, X134V, X W or X134Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 135 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X135D, X135E, X135G, X135M, X135N, X135P, X135S, X T or X135W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 136 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X136A, X136C, X136D, X136E, X136F, X136H, X136L, X136M, X136N, X P or X136W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X139C or X139S, at position 139 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X13A or X13Y, at position 13 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X141V, at position 141 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 142 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X142C, X142E, X142F, X142L, X142M, X142Q, X142R, X W or X142Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X143F, at position 143 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 144 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X144C, X144E, X144G, X144K, X144N, X144Q, X144R, X144S, X144T, X V or X144Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X145A, at position 145 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 146 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X146C, X146D, X146E, X146F, X146G, X146H, X146K, X146L, X146S, X T or X146W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X147M, at position 147 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X149E, at position 149 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X14N, at position 14 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X150C, X150E or X150Q, at position 150 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 151 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X151C, X151E, X154A or X154Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X155Y, at position 155 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X156E or X156V, at position 156 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 158 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X158H, X N or X158Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X15R, at position 15 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 160 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X160D, X E or X160W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X161T, at position 161 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X162V, at position 162 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 163 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X163A, X163Q or X163T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X164V, at position 164 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 165 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X165S, X T or X165W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 169 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X169A, X169D, X169E, X S or X169V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X16F or X16R, at position 16 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X170C or X170F, at position 170 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 172 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X172A, X172C, X172D, X172K or X172N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X173I or X173Y, at position 173 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 174 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X174D, X174E, X174G, X174H, X174M, X174N, X174P, X S or X174T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 175 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X175A, X175G, X H or X175Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X176K, X S or X176T, preferably X176K, at position 176 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 177 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X177A, X177G, X177K, X177N, X177P, X177R, X S or X177W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 178 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X178F.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X17K or X17V, at position 17 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 180 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X180M, X180N, X180T or X180W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 182 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X182D, X182E, X182N, X182Q or X182S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 185 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X185E, X185M or X185N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 187 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X187A, X187D, X187M or X187V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 188 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X188H, X T or X188V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X189I, at position 189 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 18 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X18F, X18I, X18K, X18M, X R or X18T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 191 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X191F, X191H, X191K, X191M, X W or X191Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 192 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X192A or X192T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 193 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X193D, X193E, X193G or X193V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 19 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X19A, X19C, X19D, X19F, X19G, X19H, X19I, X19K, X L, X19M, X19P, X Q, X19S, X T or X19Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X1R, X V or X1G, at position 1 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X203E or X203R, at position 203 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 208 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X208F, X208I or X208Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 20 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X20A, X20C, X20D, X20E, X20F, X20G, X20H, X20K, X20L, X20M, X20N, X20P, X20Q, X20S, X20T, X20V, X20W or X20Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 210 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X210A, X210C, X210D, X210E, X210F, X210M, X210N, X210Q, X S or X210Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 211 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X211A, X211C, X211D, X211E, X211G, X211H, X211L, X211N, X211Q, X211S, X T or X211V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 212 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X212C, X212D, X212I, X212L or X212W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 213 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X213A, X213C, X213M, X213R or X213S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X214E or X214P, at position 214 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 215 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X215A, X215D, X215E, X215H or X215W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X216G or X216H, at position 216 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 218 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X218A, X218C, X218D, X218E, X218F, X218 823 218H, X218I, X218K, X218L, X218Q, X218S, X218T, X218V, X W or X218Y.

In another embodiment, the variant alpha-amylase is at position 219 compared to the parent alpha-amylase (according to SEQ ID NO: 3), preferably the substitution X219A, X219C, X219D, X219E, X219F, X219G, X219H, X K, X219M, X219Q, X219S, X219T, X W or X219Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X21E or X21S, at position 21 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X221F or X221N, at position 221 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 222 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X222D, X222K, X222S or X222T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 223 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X223C, X223L, X V or X223Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X224F or X224V, at position 224 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 225 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X225A, X225C, X225E, X225F, X225H, X225I, X225N, X225R, X S or X225Y, preferably X225A.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 226 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X226A, X226C, X226D, X226E, X226G, X226H, X226I, X226L, X226M, X Q, X226R, X226S, X226T, X226V, X226W or X226Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 227 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X227C, X227F, X227G, X227H, X227I, X227K, X227L, X227M, X227R, X227T, X227V, X W or X227Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 22 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X22A, X22D, X22E, X22F, X22G, X22K, X22L, X22M, X Q, X22R, X22T, X W or X22Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X230A or X230F, at position 230 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 231 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X231C, X231D or X231N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 233 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X233C, X233H, X233I, X233M, X233T, X233W or X233Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X234C, at position 234 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 235 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X235L, X235M or X235V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X236Y, at position 236 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X237C or X237I, at position 237 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 238 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X238A, X238F or X238T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X23N, X Q or X23W, at position 23 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X240M, at position 240 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 242 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X242M or X242N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X243D or X243F, at position 243 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 245 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X245E, X H or X245M.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 249 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably substitution X249D or X249I.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X24G, at position 24 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X250V, at position 250 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X251A, X251E, X251F, X251L, X251M, X251S or X251T, preferably X251E, at position 251 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 252 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X252C, X I or X252S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 253 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X253C, X253G or X253Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 255 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X255D, X255F, X255I, X255T or X255V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X256C, at position 256 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 257 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X257L, X257V or X257Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 258 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X258F, X258Q or X258R.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X259L, at position 259 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 25 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X25A, X25C, X25D, X25F, X25G, X25H, X25K, X25L, X25M, X25Q, X25S, X W or X25Y, preferably X25H.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X260H, at position 260 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 261 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably substitution X261R.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 262 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X262C, X262D, X262E, X262H, X P or X262Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X263I, at position 263 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 264 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X264H, X264T or X264W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X265S, at position 265 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 268 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X268F or X268G.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 269 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X269M.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 26 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X26A, X26D, X26E, X26F, X26L, X26M, X26P, X26S, X V or X26Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 270 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X270A, X270Q or X270Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X271S, at position 271 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 272 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X272F, X272G, X L or X272S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 273 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X273A, X273C, X273D, X273E, X273F, X273H, X273I, X273L, X273M, X273P, X273Q, X273R, X273V, X W or X273Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X274F or X274S, at position 274 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X275V, at position 275 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 276 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X276D, X276K, X276L, X276N, X R or X276Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 277 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X277D, X277E or X277T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X279A or X279P, at position 279 (according to the numbering of SEQ ID NO: 3) compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 27 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X27A, X27D, X27F, X27G, X27H, X27I, X27Q, X27R, X T or X27V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 280 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X280D, X280F, X280G, X280H, X280I, X280K, X280N, X280R, X V or X280Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 281 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X281A, X281D, X281E or X281H.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 284 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X284A, X284F, X284H, X284L, X284M, X284N or X284Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 285 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X285G, X285L, X N or X285P.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X286Q, at position 286 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 287 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X287A, X287D, X287E, X287H or X287T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 288 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X288A, X288K, X288P or X288Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 289 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X289F, X289G, X289R or X289T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 28 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X28C, X28D, X28E, X28F, X28G, X28I, X28K, X28N, X28Q, X28S, X T or X28V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 290 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X290D, X290M, X290N, X290Q or X290W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 291 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X291D, X291K, X291T or X291Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 292 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X292C, X292D, X292F, X292I, X292L, X292T, X W or X292Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 293 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X293D, X293E, X293F, X293K or X293R.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X294G or X294T, at position 294 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X295F, at position 295 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 296 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X296A, X296C, X296L or X296Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 297 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X297E, X297F, X297H, X297K, X297M, X297S or X297V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 299 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X299G, X299I, X299K, X299L, X S or X299Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 29 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X29D, X29E, X29F, X29G, X29H, X29I, X29K, X29L, X N, X29P, X29 5629V, X W or X29Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X2I or X2S, at position 2 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X301F, at position 301 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 302 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X302H, X302I, X302Q, X V or X302Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 303 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X303E, X303H, X303I, X303K, X303L, X303M, X N, X303P, X R or X303T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 304 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X304A, X304D, X304E, X304H, X304 67304 304M, X304N, X304P, X9795 304T, X W or X304Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 306 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X306A, X306D, X306E, X306G, X306H, X I, X306M, X306Q, X306R, X306S, X306T, X306V, X W or X306Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X307F or X307M, at position 307 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X308S, at position 308 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 309 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X309H, X309L or X309Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 30 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X30A, X30E, X30F, X30G, X30H, X30I, X30K, X30L, X M, X30Q, X30T, X W or X30Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X310A or X310Q, at position 310 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 311 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X311A, X311E, X311G, X311H, X311K, X311N, X311R, X T or X311Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X312L or X312M, at position 312 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X313V, at position 313 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 314 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X314C, X314E, X314K or X314Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 315 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X315A, X315C, X315E, X315H, X315K or X315T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 318 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X318I, X318S or X318T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 319 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X319A, X319D, X319H, X319I, X319K, X319M, X319N, X319P, X319S, X319T or X319W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 31 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X31N, X31Q, X31S, X31T, X V or X31W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 320 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X320A, X320C, X320D, X320E, X320G, X320H, X K, X320L, X320N, X320Q, X S or X320Y, preferably X320K.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 321 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X321A, X321E, X321K, X321N, X321T, X321V or X321W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 323 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X323A, X323G, X323K, X323L or X323V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 324 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X324K, X324L, X324M, X324W or X324Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 326 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X326G, X326N, X326S or X326Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 327 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X327C, X327L or X327M.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 32 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X32A, X32D, X32E, X32F, X32H, X32I, X32L, X32M, X N, X32P, X32 3232 32Q, X32T or X32W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X333I, at position 333 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X334T, at position 334 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X336K, at position 336 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 337 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X337A, X337C, X337F, X337G, X337I, X K, X337L, X337M, X N, X337Q, X337R, X337S, X337T, X V or X337Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 338 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X338G, X S or X338T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 33 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X33D, X33E, X33H, X33K, X33M, X33Q, X R or X33Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 341 (numbering according to SEQ ID NO: 3), preferably substitution X341V, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X342P, at position 342 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 343 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X343L, X343T, X343W or X343Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 344 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X344I, X Q or X344V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 345 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X345D, X345G, X345M, X345N, X345Q, X S or X345T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 346 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X346A, X346C, X346D, X346G, X346H, X N or X346Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X348T, at position 348 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 34 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X34H, X34I or X34V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 350 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X350H, X350K or X350P.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X351A or X351M, at position 351 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X352S, at position 352 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X353H, at position 353 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 354 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X354I, X354N, X T or X354Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 355 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X355I or X355M.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X356I or X356V, at position 356 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 357 (numbered according to SEQ ID NO: 3), preferably substitution X357A, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 358 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X358I, X358L, X358N, X358P or X358V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X359E, at position 359 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 35 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X35A, X35 82348 35D, X35G, X35H, X35I, X35 35L, X35M, X35N, X35 8235 35P, X35Q, X35R, X35S, X35T, X V or X35Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 360 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X360A, X360F, X360G, X360I, X360L, X360Q, X360R, X360S, X360T, X V or X360Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 362 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X362F, X362K, X858 362K, X362N, X T, X362V or X362Y.

In another embodiment, the variant alpha-amylase is at position 363 compared to the parent alpha-amylase (according to SEQ ID NO: 3) comprises an amino acid substitution at position(s), preferably, the substitution of X363A, X363C, X363D, X363E, X363G, X363 823 363K, X363L, X363M, X363P, X363Q, X363R, X363S, X363T, X363V, X363W or X363Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 364 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X364A, X364C, X364G, X364K, X364L, X364N, X S, X T or X364V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 366 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X366I, X366L or X366T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 367 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X367E or X367S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 368 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X368A, X368F, X L or X368N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 36 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X36A, X36E, X36G, X36I, X36K, X36M, X36N, X36P, X36Q, X36R, X36S, X T or X36V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X370E or X370I, at position 370 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 372 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X372A, X372C, X372E, X372F, X372H, X372M, X372N or X372Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 375 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X375A, X375D, X375E, X375I, X375K, X375Q, X375R, X375T, X W or X375Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 376 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X376G, X376I, X376K, X376L, X376M, X376Q, X376R, X376S or X376V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 377 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably substitution X377Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 378 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X378C, X378D, X378E or X378R.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 379 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X379A, X379L or X379S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 37 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X37A, X37G, X37M, X37P, X37T, X37V or X37W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X381E or X381V, at position 381 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 382 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X382A, X382H, X382K, X382L, X382N, X Q or X382S.

In another embodiment, the variant alpha-amylase is in position 383 (according to SEQ ID NO: 3), preferably the substitution X383C, X383D, X383H, X383I, X383M, X383N, X383Q, X383R, X383S, X383V or X383Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 384 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X384A, X384C, X384D, X384E, X384F, X384I, X384L, X384M, X N, X384Q, X384R, X384T, X384V, X384W or X384Y.

In another embodiment, the variant alpha-amylase is at position 385 compared to the parent alpha-amylase (according to SEQ ID NO: 3) comprises an amino acid substitution at position(s), preferably replaces X385A, X385C, X385D, X385E, X385G, X385H, X385I, X385L, X385M, X385N, X385P, X385Q, X385R, X385T, X385 4639 385W or X385Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 387 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X387C, X387E or X387N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 388 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X388E, X388F, X388H, X388I, X388M, X R or X388V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 389 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X389G, X389H, X389K or X389N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 390 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X390D, X390F, X390M, X390N, X P or X390R.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 391 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X391A, X391F, X391G, X391K, X391M, X391N, X391Q, X391S, X391T or X391Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X392C or X392V, at position 392 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 393 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X393E, X393H, X393P, X S or X393V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 394 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X394A, X394C, X394E, X394H, X394I, X394L, X394M, X394N, X R or X394S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 395 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X395A, X H, X395M or X395V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 396 (numbering according to SEQ ID NO: 3), preferably a substitution of X396H or X396P, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 397 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X397D, X397H, X397P or X397S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 398 (numbering according to SEQ ID NO: 3), preferably substitution X398M, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X399P, at position 399 (numbered according to SEQ ID NO: 3) compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X39E or X39K, at position 39 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 3 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X3A, X3F, X3G, X3I, X3K, X3L, X3Q or X3V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 400 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X400A, X400D, X400E, X400G, X400H, X400I, X400K, X400L, X400M, X400N, X400P, X400Q, X400R, X400S, X400V or X400W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 401 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X401I, X401K, X M or X401T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X403N, at position 403 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 405 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X405C, X405H, X405M, X T or X405V, preferably X405M.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X406P, at position 406 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 407 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X407D, X R or X407S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 408 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X408E, X I or X408Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X40I or X40S, at position 40 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 410 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X410H, X410I, X410K, X410L, X410P, X410R or X410Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X413S, at position 413 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 414 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably substitution X414C, X E or X414S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X415E or X415I, at position 415 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X416S, at position 416 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 418 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X418C, X418N or X418P.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 41 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X41C, X41D, X41E, X41G, X41Q, X S or X41T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X421N or X421P, at position 421 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X424A, at position 424 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X426D or X426W, at position 426 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 427 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X427C, X427F, X427G, X427K, X427Q, X427R, X427S, X427T or X427V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 429 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X429A, X429D, X429E, X429F, X429G, X429I, X429M, X429N, X429P, X429Q, X429S, X429T, X V or X429W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 42 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X42C, X42I, X42Q or X42V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X431I, at position 431 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X434S, at position 434 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X436E, at position 436 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 438 (numbering according to SEQ ID NO: 3), preferably the substitution X438F, X438P or X438Y, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 439 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably substitution X439K, X439C or X439P.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X440V, at position 440 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X442Q, at position 442 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X443H or X443T, at position 443 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 445 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X445A, X445C, X445D, X445F, X445G, X445H, X445K, X445M, X445Q, X445R, X445S, X445T or X445V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 446 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X446F, X446I, X446L, X446P, X446Q, X446R, X446V or X446W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 447 (numbering according to SEQ ID NO: 3), preferably the substitution X447V, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 448 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X448D, X448E, X448H or X448N.

In another embodiment, the variant alpha-amylase is in position 449 (according to SEQ ID NO: 3), preferably the substitution X449A, X449F, X449G, X449K, X449L, X449M, X449N, X449P, X449Q, X449R, X449S, X449V, X449W or X449Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X451L, at position 451 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 453 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X453G, X453I, X453N, X453P or X453Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X455L, at position 455 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 456 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X456L, X456M, X456R, X456S, X456V, X W or X456Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 457 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X457A, X457C, X457E, X457F, X457G, X457K, X457L, X457M, X457R, X457S, X457T, X457V or X457W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 458 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X458I, X458K, X458V or X458W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 459 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X459C, X459D, X459E, X459I, X459K, X459N, X459Q, X459R, X459V or X459Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X45G or X45N, at position 45 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 460 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X460A, X460D, X460E, X460G, X460Q, X460R, X460S, X T or X460V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 461 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X461A, X461E, X461F, X K, X461L, X461M, X461N, X461Q, X461R, X461S or X461V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X462K, at position 462 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X463L, at position 463 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 465 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X465H, X465P, X R or X465V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 467 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X467A, X467E or X467H.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 468 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X468D or X468H.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 470 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X470A, X470Q or X470T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X471E, at position 471 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 474 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X474F, X474H or X474P.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 478 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably substitution X478A or X478E.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X47S, at position 47 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 480 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X480D, X480E, X480M, X480R or X480Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 482 (numbering according to SEQ ID NO: 3), preferably substitution X482C, X482T or X482W, preferably X482W, as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 48 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X48F, X48I, X M or X48Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X4A, X4C, X4K, X4M, X4Q, X4R or X4S, preferably X4Q, at position 4 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 51 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X51Q, X T or X51V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 54 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X54D, X G or X54Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X59T, at position 59 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 5 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X5A, X5C, X5D, X5E, X5F, X5H, X5I, X5K, X5L, X5M, X5N, X5P, X5Q, X5R, X5V or X5Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X60T, at position 60 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X63C or X63V, at position 63 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 6 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably the substitution X6A, X6C, X6E, X6F, X6G, X6H, X6K, X6L, X6M, X6P, X6Q, X6S, X6T, X6V, X6W or X6Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 70 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X70F, X70H, X70L, X70M, X N or X70Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X71D, at position 71 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 72 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X72C, X72D, X72E, X N or X72T.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 73 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X73L, X N or X73Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 75 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X75A, X75G, X75I, X75L, X75P, X T or X75W.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 76 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X76C, X76E, X76G, X76L, X76T or X76V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 7 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X7C, X7E, X7F, X7H, X7K, X7N, X7P, X7Q, X7R, X7S, X7V, X W or X7Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X81H or X81L, at position 81 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X82K or X82M, at position 82 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 83 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X83A, X83D, X E, X83G, X R or X83S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X86K, at position 86 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X87D or X87R, at position 87 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 89 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X89A, X89C, X89F, X89G, X L, X89M, X R or X89S.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 8 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X8A, X8C, X8F, X8I, X8M, X8P, X8S, X8V, X W or X8Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 90 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X90A, X90D, X90E, X90F, X90G, X90I, X90M, X90N, X90Q, X90R, X90S, X V or X90Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 91 (numbering according to SEQ ID NO: 3) compared to the parent alpha-amylase, preferably a substitution of X91C, X91D, X E, X91F, X91G, X91H, X91I, X91K, X91L, X M, X91N, X91Q, X91S, X91T, X91V, X W or X91Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 92 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X92D, X M or X92V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably a substitution of X93K or X93Q, at position 93 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 94 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X94A, X94D, X94E, X94K, X94M, X94V or X94Y.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 95 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X95E, X95I, X95L or X95V.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 96 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X96K, X N or X96Q.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution, preferably substitution X97V, at position 97 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 98 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably a substitution of X98E, X G or X98N.

In another embodiment, the variant alpha-amylase comprises an amino acid substitution at position 99 (numbering according to SEQ ID NO: 3) as compared to the parent alpha-amylase, preferably the substitution X99H, X K or X99N.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: X4Q, X7H, X7W, X25H, X Y, X5483H, X70 100 79118D, X135T, X160W, X35176K, X193E, X C, X251E, X281N, X258N, X323N, X361N, X363N, X363N, X363N, X368N, X405N, X434N, X441N, X459N, X460N, X451L and X482W, and wherein the variant has alpha-amylase activity. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: X25H, X Y, X100W, X135T, X176K, X193E and X460G, and wherein the variant has alpha-amylase activity. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: X4Q, X25H, X100 3839E, X T, X135W, X135D, X160 38324 176K, X193E, X251E, X Q, X276R, X S, X323G, X363E, X363H, X382Q, X M, X460G and X482W and wherein the variant has alpha-amylase activity. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: X4Q, X H, X176K, X186E, X E, X405M and X482W, and wherein the variant has alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at any one of SEQ ID NOs 1, 3, 4, or 15-41, preferably SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 4, more preferably SEQ ID NO 1 or 3, most preferably the amino acid sequence shown in SEQ ID NO 1, having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably a deletion at one or more amino acids corresponding to positions 181, 182, 183 and 184, preferably corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183 and g.184, wherein the amino acid sequence shown in SEQ ID NO 3 is numbered according to the amino acid sequence shown in SEQ ID NO 3. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: X4Q, X H, X176K, X E, X M and X482W, and wherein the variant has alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion with any one of SEQ ID NOs 1, 3, 4, or 15-41, preferably any one of SEQ ID NOs 1, 3, or 4, more preferably the amino acid sequence shown in SEQ ID NO 1 or 3, most preferably SEQ ID NO 1, having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably a deletion at one or more amino acids corresponding to positions 181, 182, 183, and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183 and G184, wherein the amino acid sequence numbers according to SEQ ID NO 3 are numbered. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: X116K, X181T, X a and X320K, and wherein the variant has alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at any one of SEQ ID NOs 1, 3, 4, or 15-41, preferably at any one of SEQ ID NOs 1, 3, or 4, more preferably at any one of SEQ ID NOs 1 or 3, most preferably at any one of SEQ ID NOs 1, having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, of the sequence identity, preferably at one or more amino acids corresponding to positions selected from 181, 182, 183, and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183X and G184, wherein the amino acid sequences set forth in accordance with SEQ ID NO 3 are numbered. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase, and wherein the variant has alpha-amylase activity: H1G, H1V, H1R, H2S, H2I, N3Q, N3K, N3F, N3G, N3L, G4Q, G4R, G4S, G4K, G4M, G4A, G4C, T5F, T5E, T5M, T5Y, T5R, T5Q, T5K, T5L, T5A, T5V, T5C, T5N, T5H, T5D, T5P, T5I, N6T, N6E, N6A, N6K, N6Q, N6V, N6M, N6G, N6L, N6H, N6W, N6F, N6P, N6S, N6C, N6Y, G7Y, G7S, G7H, G7C, G7Q, G7F, G7P, G7W, G7R, G7K, G7E, G7V, G7N, T8C, T8V, T8F, T8Y, T8P, T8W, T8A, T8S, T8M, T8I, M10W, M10Y, M10L, M10C, M10E, M10F, M10A, Y12N, Y12S, F13A, F13Y, E14N, W15R, Y16F, Y16R, L17K, L17V, P18R, P18F, P18K, P18T, P18I, N19P, N19L, N19F, N19Y, N19T, N19I, N19Q, N19C, N19H, N19S, N19M, N19K, N19G, N19D, N19A, D20C, D20S, D20T, D20D, D20Q, D20F, D20P, D20G, D20V, D20W, D20A, D20Y, D20H, D20K, D20E, G21S, G21E 22E, N22E, N22M, N22T, N22K, N22Y, N22G, N22R, N22Q, N22W, N22L, H23N, H23Q, H23W, W24G, N25H, N25Y, N25K, N25F, N25S, N25C, N25D, N25A, N25G, N25W, N25Q, N25M, N25L, R26S, R26V, R26M, R26A, R26D, R26E, R26F, R26P, R26Y, R26L, L27D, L27I, L27F, L27R, L27G, L27Q, L27V, L27T, L27A, L27H, R28N, R28S, R28F, R28C, R28E, R28T, R28G, R28K, R28D, R28V, R28I, R28Y 29S 29W, S29S and S29W, S29K, S29F, S29N, S29H, S29D, S29V, S29G, S29Q, S29I, D30F, D30I, D30A, D30K, D30W, D30Y, D30M, D30L, D30H, D30Q, D30G, D30E, D30T, A31S, A31V, A31Q, A31T, A31N, A31W, S32T, S32H, S32W, S32I, S32A, S32P, S32E, S32M, S32N, S32D, S32L, N33Y, N33M, N33Q, N33K, N33R, L34H, L34I, K35C, K35Y, K35P, K35T, K35M, K35H, K35A, K35V 35K 35G 35M, K35G 35M, K35N, K35R, K35S, K35L, D36V, D36I, D36T, D36K, D36A, D36S, D36R, D36M, D36E, D36P, D36G, D36N, D36Q, K37P, K37V, K37W, K37A, K37G, G38N, I39E, I39K, T40I, T40S, A41G, A41Q, A41C, A41D, A41E, A41S, A41T, V42Q, V42V, V42I, V42C, P45N, P45G, A47S, W48F, W48I, W48M, A51T, G59T, A60T, L63V, L63C, N70N, N70L, N70M, N70F, N70Y, Q71D, K72T, K72E, K72D, K72N, K72C, G73Q, G73L, G73N, V75A, V75I, V75W, V75P, V75G, V42Q, V42V, V42I, V42C, P45N, P45G, A47S, W48F, W48I, W48M, A51T, G59T, A60T, L63V, L63C, N70N, N70L, N70M, N70F, N70Y, Q71D, K72T, K72E, K72D, K72N, K72C, G73Q, G73L, G73N, V75A, V75I, V75W, V75P, V75G, G96N, G96K, I97V, Q98G, Q98E, Q98N, V99K, V99H, V99N, Y100D, Y100K, Y100L, Y100F, V103A, N106D, K108A, G109A, A113Y, A113H, A113M, A113V, A113F, A113W, A113R, T114D, T114H, T114N, T114W, T114E, T114C, T114G, T114L, T114M, T114Q, T114F, T114A, T114V, T114K, T114S, T114Y, T114R, T114I, E115C, E115K, E115N, E115T, E115M, E115S, E115Q, E115R, E115D, E115V, W116I, W K, W116M, W116R, X116T 116L, W116R, W116V 117, V116C, V117, V117R, V117W, V117A, V117I, V117P, V117F, A119Y, A119R, A119P, A119H, S125A, S125K, S125L, S125T, S125R, S125F, S125W, S125Q, S125V, S125Y, S125H, S125N, S125E, S125G, S125M, N126D, N128C, N128L, N128Y, N128E, N128W, N128M, Q129E, V131I, V131C, S132T, G133E, G133P, G133C, G133D, G133Q, G133S, G133H, G133A, G133N, G133K, D134Y, D134W, D134F, D134P, D134E, D134V, D134L, D134M, D134C, D134I, Y135M 136T 136, T136F, T136M, T136P, T136H, T136C, T136A, T136W, T136L, T136N, A139C, T141V, K142C, K142W, K142Y, K142L, K142M, K142Q, K142F, K142E, K142R, F143F, D144Q, D144C, D144V, D144K, D144T, D144Y, D144E, D144G, D144N, D144S, D144R, F145A, P146C, P146H, P146K, P146T, P146E, P146S, P146W, P146F, P146L, P146G, G147M, G149E, N150Q, N150E, N150C, T151E, N154Y, N154A, F155Y, K156V, Y160W, H161T 162, F170V, V170C 170W, Q170S, Q170C 170F 170V, Q170C 170E, Q172K, Q172A, L173I, L173Y, Q174P, Q174M, Q174E, Q174S, N175G, N175Q, N175H, R176K, R181D, R181F, R181H, R181I, R181N, R181Q, R181S, R181T, R181V, R181W, R181Y, D188V, D188H, V191K, V191H, V191F, V191W, V191Y, Y203E, Y203R, H210D, H210C, H210E, H210N, H210F, H210Y, H210M, H210Q, H210S, H210A, P211N, P211C, P211T, P211E, P211G, P211V, P211Q, P211A, P211H, P211L, P211S, E212W, E212L, E212I, E212D, E212C, V213A, V213S, V213M, V213R, V213C, V214P, V214E, N215E, N215D, N215H, N215W, N215A, E216H, E216G, R218Q, R218E, R218C, R218N, R218F, R218S, R218G, R218K, R218I, R218T, R218D, R218Y, R218H, R218L, R218V, R218A, R218W, N219E, N219Q, N219K, N219T, N219M, N219D, N219F, N219S, N219R, N219A, N219Y, N219H, N219W, N219G, N219C, G221N, G221F, V222S, V222T, V222D, W223V, W223Y, W223L, W223C, Y224V, Y224F, T225A, T225R, T225N, T225E, T225H, T225F, T225C, T225Y, T225I, N226C, N226W, N226I, N226R, N226Y, N226D, N226L, N226M, N226G, N226T, N226Q, N226S, N226A, N226V, N226E, N226H, T227W, T227C, T227T, T227R, T227M, T227K, T227Y, T227V, T227F, T227I, T227H, T227L, T227G, L230A, L230F, D231N, D231C, F233T, F233W, F233H, F233C, F233I, F233Y, F233M, R234C, I235M, I235L, D236Y, A237C, A237I, V238T, H240M, K242M, Y243F, F245E, F245M, F245H, W249I, L250V, T251E, T251A, T251L 251M 251S 251M, F251M 252F 252V 253G, N255T, N255V, N255D, N255F, N255I, T256C, T257Y, T257L, T257V, K259L, M261R, F262D, F262Y, F262C, F262P, F262E, F262H, A263I, V264T, V264W, V264H, A265S, W268G, N270A, N270Y, I272L, I272G, G273H, G273V, G273L, G273M, G273C, G273D, G273W, G273F, G273A, G273E, G273P, G273Y, G273R, G273I, A274S, E276Y, E276K, E276L, E276D, L279P, S280I, S280V, S280N, S280H, S280Y, S280K, S280R, W284Y, W284A, W284L, W284F, W284H, W284N, W284M, N285N, N285L, N285G, N285P, G273W, G273F, G273A, G273E, G273P, G273Y, G273R, G273I, A274S, E276Y, E276K, E276L, E276D, L279P, S280I, S280V, S280N, S280H, S280Y, S280K, S280R, W284Y, W284A, W284L, W284F, W284H, W284N, W284M, N285N, N285L, N285G, N285P, S303R, S303K, S303N, S303L, S303H, S303T, G304H, G304A, G304R, G304Y, G304D, G304N, G304M, G304E, G304P, G304W, G304K, G304T, N306M, N306E, N306A, N306G, N306W, N306V, N306S, N306I, N306H, N306T, N306D, N306Q, N306Y, N306R, Y307F, Y307M, D308S, M309H, M309L, M309Q, R310Q, R310A, Q311G, Q311E, Q311H, Q311A, Q311K, Q311T, Q311N, Q311R, Q311Y, I312M, I312L, F313V, N314C, N314Q, G315C, G315A, G315H, G315T, G315E, G315K, V318T, V318S, V318I, Q319K, Q319H, Q319P, Q319A, Q319I, Q319D, Q319M, Q319S, Q319N, Q319T, Q319W, R320G, R320Y, R320L, R320N, R320A, R320D, R320K, R320Q, R320E, R320H 320C, R320S, H321E, H321W, H321T, H321A, H321N, H321V, H321K, T323K, T323G, H324W, H324K, H324Y, V326Y, V326N, V326G, V326S, T327M, T327C, T327L, D333I, P336K, E337N, E337K, E337M, E337T, E337V, E337Q 337S, E337I, E337Y, E337G, E337F, E337A, E337L, E338G 338, T338S, E338F 338S, T37V 37C, T338F 37W, T338S, W37F 37S, V344I, V344Q, V344V, E345N, E345T, E345M, E345G, E345S, E345D, E345Q, E346D, E346C, E346H, E346A, E346G, E346Q, E346N, F348T, P350P, P350H, P350K, L351M, L351A, A352S, Y353H, A354Y, A354N, A354T, A354I, L355I, L355M, L357A, T358P, R359E, D360N, D360R, D360S, D360Y, D360V, D360L, D360A, D360I, D360T, D360G, D360F, G362T, G362M, G362V, G362N, G362Y, G362F, G362K, Y363M, Y363G, Y363S, Y363V, Y363C, Y363T, Y363H, Y363P, Y363D, Y363L, Y363R, Y363Q, Y363E, R359E, D360N, D360R, D360S, D360Y, D360V, D360L, D360A, D360I, D360T, D360G, D360F, G362T, G362M, G362V, G362N, G362Y, G362F, G362K, Y363M, Y363G, Y363S, Y363V, Y363C, Y363T, Y363H, Y363P, Y363D, Y363L, Y363R, Y363Q, Y363E, K383V, K383M, K383E, K383N, K383S, K383Q, K383Y, K383C, K383H, S384W, S384Y, S384V, S384R, S384N, S384E, S384A, S384D, S384T, S384C, S384F, S384L, S384I, S384M, S384Q, K385G, K385V, K385T, K385Y, K385I, K385D, K385C, K385Q, K385A, K385W, K385L, K385H, K385M, K385N, K385S, K385F, K385E, K385R, K385P, D387N, D387C, D387E, P388R, P388F, P388H, P388V, P388E, P388I, I389G, I389K, I389H, L390P, L390N, L390R, L390F, L390M, L390D, E391T, E391F, E391S, E391A, E391G, E391M, E391N, E391Y, E391Q, E391K, A392C, A392V, R393V, R393E, R393H, R393S, R393P, Q394C, Q394L, Q394R, Q394S, Q394A, Q394E, Q394N, Q394M, Q394H, Q394I, K395V, K395M, K395A, K395H, Y396P, Y396H, A397P, A397D, A397S, A397H, Y398M, G399P, T400L, T400P, T400I, T400S, T400A, T400R, T400Q, T400D, T400K, T400N, T400V, T400W, T400H, T400E, T400G, T400M, Q401M, Q401K, Q401T, Q401L, Q401D, A397S, A397H, Y398M, G, T400M, T400R 400M, T400L 405L, T400L 405H, T405L 407H 407, H407D, P408Q, P408E, P408I, V410R, V410K, V410I, V410L, V410H, W413S, T414E, T414C, T414S, R415I, R415E, E416S, D418C, D418P, D418N, H421P, H421N, S424A, L426D, L426W, A427Q, A427C, A427F, A427T, A427V, A427G, A427S, A427R, A427K, L429M, L429D, L429E, L429N, L429T, L429A, L429P, L429W, L429F, L429G, L429I, L429V, S431I, G436E, K438P, K439F, K439C, W445P, M440V, V442Q, G442M, N445N, N445G, N445N, N445F, N445R, N445Q, N445C, N445D, N445K, N445H, N445V, N446P, N446I, N446V, N446F, N446L, N446R, N446Q, N446W, A447V, G448E, G448H, G448N, G448D, E449V, E449Y, E449F, E449L, E449P, E449G, E449W, E449M, E449R, E449N, E449K, E449Q, E449S, E449A, W451L, D453G, D453I, D453P, D453Y, D453N, T455L, G456M, G456S, G456Y, G456V, G456L, G456W, G456R, N457K, N457C, N457L, N457S, N457R, N457F, N457T, N457A, N457V, N457W, N457E, N457M, N457G, Q458V, Q458W, Q458I, Q458K, T459Q, T459D, T459I, T459K, T459C, T459V, T459E, T459Y, N460E, N460Q, N460S, N460R, N460A, N460T, N460D, N460V, T461A, T461R, T461F, T461E, T461S, T461V, T461M, T461K, T461N, T461Q, V462K, T463L, N465P, N465V, N465H, N465R, D467A, D467E, D467H, G468H, G468D, G470Q, G470T, G470A, V474P, V474H, V474F, S478A, S478E, S480Y, S480M, S480E, S480D, S480R, Y482T, Y482C, A119S, A139S, A274F, D163A, D163Q, D163T, D188T, D20N, D231D, D271S, D370S, D36H, E276N, F180M, F180N, F180T, F180W, G182D, G182E, G182N, G182Q, G182S, G258F, G258Q, G258R, G273Q, G362C, G73C, G96Q, I177A, I177G, I177K, I177N, I177P, I177R, I177S, I177W, I272F, I272S, I275V, K156E, K185E, K185M, K185N, K242N, K269M, K281A, K281D, K281E, K281H, K281N, K281S, K37F, K37L, K37M, K37Y, K395C, L279A, L357F, N224F, N260H, N Q, N277D, N277E, N277T, N314N 33L 33E, N83E 95A, N37P 434P, P434E, N37E, N434P, N37E, N434E, N37P 434E, Q169S, Q174G, Q174H, Q174N, Q311W, Q98A, Q98V, R118A, R118D, R118E, R176S, R176T, R359W, R82L, S280D, S280F, S280G, S365Y, S94E, T193D, T193E, T193G, T193V, T251T, T282V, T356C, T356L, T356V, T358M, T461L, T463A, T463V, V120E, V120G, V120M, V120S, V120W, V120Y, V131Q, V165S, V165T, V191M, V238A, V238F, V317M, V75H, W116I, W116M, W187A, W187D, W187M, W187V, W189I, W268F, Y100W, Y135D, Y135E, Y135G, Y135N, Y135P, Y135S, Y135T, Y135W, Y160D, Y160E, Y178F, Y243D, Y298V, Y363N, Y368F, Y372S, Y372T, Y396C, R393Q, P434R, V318L, T459N, H321Y, N460G, D387S, S365L, V253C, P388K, A352C, S365Q, Y396S, V379R, L405M, T81L, and R82C.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: G4Q, G7H, G7W, N25H, N Y, K5483H, Y70 100 79118D, Y135T, Y160W, R35176K, T193E, H C, T251E, K281N, G258N, G323N, G361N, G363N, G363N, G363N, G368N, G405N, G434N, G441N, G459N, G460N, G451L and Y482W, and wherein the variant has alpha-amylase activity.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: N25H, Y100W, Y T, R176K, T E and N460G, and wherein the variant has alpha-amylase activity.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: G4Q, N25H, Y100 3839E, Y T, Y135W, Y135D, Y160 38324 176K, T193E, T251E, G Q, E276R, N S, T323G, Y363E, Y363H, M382Q, L M, N460G and Y482W, and wherein the variant has alpha-amylase activity.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: G4Q, N H, R176K, G186E, T251E, L405M, W439K and Y482W, and wherein the variant has an alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at any one of SEQ ID NO:1, 3, 4, or SEQ ID NO 15-41, preferably at any one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:4, more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably at one or more amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably at any one of amino acid deletions corresponding to positions 181 and 182, 182 and 183 and 184, preferably 183 and preferably D and 184, wherein amino acid sequence numbers shown in SEQ ID NO:3 are according to the amino acid sequence numbers shown in SEQ ID NO.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: G4Q, N H, R176K, T251E, L405M, W439K and Y482W, and wherein the variant has alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at any one of SEQ ID NO:1, 3, 4, or SEQ ID NO 15-41, preferably SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, more preferably SEQ ID NO:1 or 3, most preferably the amino acid sequence shown in SEQ ID NO:1, having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably comprising a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 and 184, preferably D183 and G.times.184, wherein the amino acid sequence shown in SEQ ID NO 3 is numbered according to.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: G4Q, N H, R176K, G186E, T251E, L M and Y482W, and wherein the variant has alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at any one of SEQ ID NO:1, 3, 4, or SEQ ID NO 15-41, preferably SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, more preferably SEQ ID NO:1 or 3, most preferably the amino acid sequence shown in SEQ ID NO:1, having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably comprising a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 and 184, preferably D183 and G.times.184, wherein the amino acid sequence according to SEQ ID NO 3 is numbered.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: G4Q, N H, R176K, T E, L M and Y482W, and wherein the variant has alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion with any one of SEQ ID NOs 1, 3, 4, or 15-41, preferably any one of SEQ ID NOs 1, 3, or 4, more preferably the amino acid sequences shown in SEQ ID NOs 1 or 3, most preferably SEQ ID NO 1, having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably at least 91% comprising a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183, and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183 and G184, wherein the amino acid sequences shown in accordance with SEQ ID NO 3 are numbered.

Particularly preferably, the alpha-amylase variant having alpha-amylase activity comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions according to the numbering of the amino acid sequence shown in SEQ ID NO:3, as compared to the parent alpha-amylase: G4Q, N H, R176K, G186E, T251E, L M and Y482W, and wherein the variant has alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at any one of SEQ ID NO:1, 3, 4, or SEQ ID NO 15-41, preferably SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, more preferably SEQ ID NO:1 or 3, most preferably the amino acid sequence shown in SEQ ID NO:1, having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably comprising a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 and 184, preferably D183 and G.times.184, wherein the amino acid sequence according to SEQ ID NO 3 is numbered.

Preferably, the one or more amino acid changes, preferably substitutions, are at amino acid positions located on the surface of the alpha-amylase. Preferably, the one or more amino acid changes, preferably substitutions, located at amino acid positions of the alpha-amylase surface are selected from the following positions: numbering according to SEQ ID NO. 3, 5,7,16,18,20,22,26,28,29,32,35,36,54,70,73,75,83,86,87,90,93,94,95,96,98,113,116,125,126,128,129,131,133,135,136,146,147,149,150,151,154,158,160,169,170,172,174,175,184,191,192,208,210,224,242,249,253,256,257,268,279,297,302,303,304,306,308,309,311,312,313,318,320,321,336,337,338,345,358,370,375,376,378,379,382,383,397,398,401,405,406,416,418,421,434,442,443,446,448,455,457,458,459,461,463,465,467,471, and 474. Preferably, the alpha-amylase of the invention comprises one or more amino acid substitutions at amino acid positions located on the alpha-amylase surface, wherein the substitution at an amino acid position on the alpha-amylase surface is selected from the group consisting of: according to SEQ ID NO:3, X5F, X5E, X5M, X5Y, X5R, X5Q, X5K, X5L, X5A, X5V, X5C, X5N, X5H, X5D, X5P, X7Y, X7S, X7H, X7C, X7Q, X7F, X7P, X7W, X7R, X7K, X7E, X7V, X7N, X16F, X16R, X18M, X18F, X18K, X18T, X18I, X20C, X20S, X20L, X20M, X20T, X20D, X20Q, X20F, X20P, X20G, X20V, X20W, X20A, X20Y, X20H, X20K, X20E, X22F, X22A, X22D, X22T, X22G, X22T, X22G, X22G, X22L, X26S, X26V, X26M, X26A, X26D, X26E, X26F, X26P, X26Y, X26L, X28N, X28S, X28F, X28C, X28E, X28T, X28G, X28K, X28D, X28V, X28I, X28Q, X29Y, X29L, X29P, X29W, X29K, X29F, X29N, X29H, X29E, X29D, X29V, X29G, X29Q, X29I, X32T, X32H, X32W, X32F, X32Q, X32I, X32A, X32P, X32E, X32M, X32N, X32D, X32L, X35C, X35Y, X35P, X35T, X35M, X35H, X35A, X35V 35I, X35G 35X 35G, X35M, X35G, X35R, X22L, X26S, X26V, X26M, X26A, X26D, X26E, X26F, X26P, X26Y, X26L, X28N, X28S, X28F, X28C, X28E, X28T, X28G, X28K, X28D, X28V, X28I, X28Q, X29Y, X29L, X29P, X29W, X29K, X29F, X29N, X29H, X29E, X29D, X29V, X29G, X29Q, X29I, X32T, X32H, X32W, X32F, X32Q, X32I, X32A, X32P, X32E, X32M, X32N, X32D, X32L, X35C, X35Y, X35P, X35T, X35M, X35H, X35A, X35V, X35I, X35G, X35Q, X35D, X35N, X35R, X133A, X133N, X133K, X135M, X135D, X135E, X135T, X135W, X136D, X136E, X136F, X136M, X136P, X136H, X136C, X136A, X136W, X136L, X136N, X146C, X146H, X146K, X146T, X146E, X146S, X146D, X146W, X146F, X146L, X146G, X147M, X149E, X150Q, X150E, X150C, X151C, X151E, X154Y, X154A, X158Y, X158N, X160W, X160D, X169E, X169A, X169V, X170F, X170C, X172D, X172N, X172A, X174P, X174M, X174E, X174D, X174H, X174G, X175H, X175, X191K, X191H, X191F, X191W, X191Y, X192A, X192T, X208I, X208Y, X208F, X210D, X210C, X210E, X210N, X210F, X210Y, X210M, X210Q, X210S, X210A, X224V, X224F, X242M, X249D, X249I, X253Y, X253G, X256C, X257Y, X257L, X257V, X268G, X279P, X297M, X297K, X297S, X297H, X297V, X297E, X297F, X302H, X302I, X302Q, X302Y, X302V, X303P, X303M, X303E, X303I, X303R, X303K, X303N, X303L, X303H, X303T, X304H, X304A, X304R, X304Y, X304D, X304N, X304M, X304E, X304P, X304W, X304K, X304T, X306M, X306E, X306A, X306G, X306W, X306V, X306S, X306I, X306H, X306T, X306D, X306Q, X306Y, X306R, X308S, X309H, X309L, X309Q, X311G, X311E, X311H, X311A, X311K, X311T, X311N, X311R, X311Y, X312M, X312L, X313V, X318T, X318S, X318I, X320G, X320Y, X320L, X320N, X320A, X320D, X320K, X320Q, X320E, X320H, X320C, X320S, X321E, X321W, X321T, X321A, X321N, X321V, X321K, X336K, X337N, X337K, X337M, X337T, X337V, X337Q, X337S, X337C, X313V, X318T, X318S, X318I, X320G, X320Y, X320L, X320N, X320A, X320D, X320K, X320Q, X320E, X320H, X320C, X320S, X321E, X321W, X321T, X321A, X321N, X321V, X321K, X336K, X337N, X337K, X337M, X337T, X337V, X337Q, X337S, X337C, X383S, X383Q, X383Y, X383C, X383H, X397P, X397D, X397S, X397H, X398M, X401M, X401K, X401T, X401I, X405H, X405V, X405T, X405M, X405C, X406P, X416S, X418C, X418P, X418N, X421P, X421N, X434S, X442Q, X443T, X443H, X446P, X446I, X446V, X446F, X446L, X446R, X446Q, X446W, X448E, X448H, X448N, X448D, X455L, X457K, X457C, X457L, X457S, X457R, X457F, X457T, X457A, X457V, X457W, X457E, X457M, X457G, X458V, X458W, X458I, X458K, X459Q, X459D, X459I, X459K, X459C, X459V, X459E, X459R, X459N, X459Y, X461A, X461R, X461F, X461E, X461S, X461L, X461V, X461M, X461K, X461N, X461Q, X463L, X465P, X465V, X465H, X465R, X467A, X467E, X467H, X471E, X474P, X474H, and X474F.

Preferably, the one or more amino acid changes, preferably substitutions, are at amino acid positions located near the catalytic center of the alpha-amylase, preferably at less than 20 angstroms from the catalytic center of the alpha-amylase. Preferably, the one or more amino acid changes (preferably substitutions) located at an amino acid position near the catalytic center of the alpha-amylase, preferably less than 20 angstroms from the catalytic center of the alpha-amylase, are selected from the following positions: according to SEQ ID NO. 3, 14,15,16,17,18,19,20,51,54,59,60,71,72,73,76,109,123,125,126,172,173,174,175,176,192,193,203,234,236,237,238,240,268,270,333,334,337,338,341, and 342. Preferably, the alpha-amylase of the invention comprises one or more amino acid substitutions at an amino acid position located near the catalytic center of the alpha-amylase, preferably at a distance of less than 20 angstroms from the catalytic center of the alpha-amylase, wherein the substitution at an amino acid position located near the catalytic center of the alpha-amylase, preferably at a distance of less than 20 angstroms from the catalytic center of the alpha-amylase, is selected from the group consisting of: according to SEQ ID NO:3, X14N, X15R, X16F, X16R, X17K, X17V, X18R, X18M, X18F, X18K, X18T, X18I, X19P, X19L, X19F, X19Y, X19T, X19I, X19Q, X19C, X19H, X19S, X19M, X19K, X19G, X19D, X19A, X20C, X20S, X20L, X20M, X20T, X20D, X20Q, X20F, X20P, X20G, X20V, X20W, X20A, X20Y, X20H, X20K, X20E, X51V, X51Q, X51T, X54D, X54G, X54Q, X59T, X60T, X71D, X72T, X72E, X72D, X72N, X72C, X73Q, X73L, X73N, X76G, X76T, X76C, X76V, X76E, X76L, X109A, X123D, X123S, X125A, X125K, X125L, X125T, X20V, X20W, X20A, X20Y, X20H, X20K, X20E, X51V, X51Q, X51T, X54D, X54G, X54Q, X59T, X60T, X71D, X72T, X72E, X72D, X72N, X72C, X73Q, X73L, X73N, X76G, X76T, X76C, X76V, X76E, X76L, X109A, X123D, X123S, X125A, X125K, X125L, X125T.

Preferably, the one or more amino acid changes, preferably substitutions, are at amino acid positions located near one of the ca2+ binding sites of the alpha-amylase, preferably at less than 10 angstroms from one of the ca2+ binding sites of the alpha-amylase. Preferably, the one or more amino acid changes, preferably substitutions, located at an amino acid position near one of the ca2+ binding sites of the alpha-amylase, preferably less than 10 angstroms from one of the ca2+ binding sites of the alpha-amylase, are selected from the following positions: numbering according to SEQ ID NO. 3, 106,108,160,161,162,164,184,188,203,208,211,212,215,234,235,236,237,238,240,297,299,301,302,303,304,306,307,308,309,310,345,346,405,406,407,408,429,431, and 474. Preferably, the alpha-amylase of the invention comprises one or more amino acid substitutions at an amino acid position located near one of the ca2+ binding sites of the alpha-amylase, preferably at a distance of less than 10 angstroms from one of the ca2+ binding sites of the alpha-amylase, wherein the substitution at an amino acid position located near one of the ca2+ binding sites of the alpha-amylase, preferably at a distance of less than 10 angstroms from one of the ca2+ binding sites of the alpha-amylase, is selected from the group consisting of: according to SEQ ID NO:3, X106D, X108A, X160W, X160D, X161T, X162V, X164V, X188H, X203E, X203R, X208I, X208Y, X208F, X211D, X211N, X211C, X211T, X211E, X211G, X211V, X211Q, X211A, X211H, X211L, X211S, X212W, X212L, X212I, X212D, X212C, X215E, X215D, X215H, X215W, X215A, X234C, X235M, X235V, X235L, X236Y, X237C, X237I, X238T, X240M, X297M, X297K, X297S, X297H, X297V, X297E, X297F, X299L, X299G, X299I, X299K, X299S, X299Y, X301F, X302H, X302I, X302Q, X302Y, X302V, X303P, X303M, X303E, X303I, X303R, X303K, X303N, X303L, X303H, X303T, X304H, X304A, X235V, X235L, X236Y, X237C, X237I, X238T, X240M, X297M, X297K, X297S, X297H, X297V, X297E, X297F, X299L, X299G, X299I, X299K, X299S, X299Y, X301F, X302H, X302I, X302Q, X302Y, X302V, X303P, X303M, X303E, X303I, X303R, X303K, X303N, X303L, X303H, X303T, X304H, X304A.

Preferably, the alpha-amylase variant comprises from 1 to 50, preferably from 1 to 30 or from 1 to 25 of any of the above amino acid changes, preferably amino acid substitutions, compared to the parent alpha-amylase. Preferably, the alpha-amylase variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25 but less than 50, preferably less than 30, preferably less than 20 of the above substitutions as compared to the parent alpha-amylase.

More preferred are alpha-amylase variants wherein the number of the above substitutions is 1-30, preferably 1-25, 1-20, 1-15, 1-10 or 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 substitutions. Further preferred are alpha-amylase variants wherein the number of substitutions above is 2-30, preferably 2-25, 2-20, 2-15, 2-10, 2-8 or 2-5. Further preferred are alpha-amylase variants wherein the number of substitutions as described above is 3-30, preferably 3-25, 3-20, 3-15, 3-10, 3-8 or 3-5. Further preferred are alpha-amylase variants wherein the number of substitutions as described above is 4-30, preferably 4-25, 4-20, 4-15, 4-10 or 4-8. Preferably, the alpha-amylase variant consists of the amino acid sequence shown in SEQ ID NO. 1, except for the number of amino acid changes, preferably substitutions, listed above.

Preferably, the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 4, or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 4, preferably the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1.

In a preferred embodiment, the alpha-amylase variant of the invention comprises a combination of substitutions selected from the group consisting of:

X25H+X176K+X186E+X206Y+X251E+X482W,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X482W,

X25H+X176K+X186E+X206Y+X482W,X25H+X186E+X206Y+X405M+X482W,

X25H+X186E+X206Y+X482W,X25H+X176K+X186E+X193E+X206Y+X251E+X482W,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X482W,X25H+X176K+X186E+X482W,

X25H+X176K+X186E+X193E+X206Y+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,X25H+X176K+X186E+X206Y,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,X4Q+X206Y+X460G+X482W,

X4Q+X25H+X176K+X206Y+X251E+X460G+X482W,

X4Q+X25H+X176K+X193E+X251E+X186E+X482W,

X4Q+X193E+X206Y+X276R+X186E+X482W,X186E+X25H+X482W,

X25H+X193E+X206Y+X251E+X276R+X405M+X186E+X482W,X25H+X251E+X186E+X482W,

X25H+X160W+X176K+X186E+X251E+X405M+X482W,

X193E+X206Y+X405M+X186E+X482W,X3I+X356V,X3I+X356I,X83D+X94E,X94D+X125E,

X131I+X377Q+X410H,X48F+X94D,X48Y+X116K+X218K,X5L+X218K+X225S,

X83E+X116R+X158Y+X181E,X51V+X218K,X83E+X181E,

X4Q+X7W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X37M+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X460G,

X4Q+X25H+X176K+X186E+X206Y+X251E+X460G,

X4Q+X25Y+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X118D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X182D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X460G,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363N+X405M,

X4Q+X176K+X181D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181F+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181N+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X136E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160E+X176K+X186E+X193E+X206Y+X251E+X405M,

X7H+X25H+X176K+X186E+X206Y,X7W+X25H+X176K+X186E+X206Y,

X25D+X176K+X186E+X206Y,X25H+X186E+X206Y+X405M+X460G,

X25H+X186E+X206Y+X460G,X25H+X37M+X176K+X186E+X206Y,

X25H+X118D+X176K+X186E+X206Y,X25H+X176K+X182D+X186E+X206Y,

X25H+X176K+X186E+X206Y,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X281N,X25H+X176K+X186E+X206Y+X460G,

X25H+X176K+X186E+X206Y+X251E+X460G,X25H+X176K+X186E+X206Y+X363E,

X25H+X176K+X186E+X206Y+X363H,X25H+X176K+X186E+X206Y+X363N,

X25H+X176K+X186E+X460G,X25H+X176K+X186E+X193E+X206Y,

X25H+X176K+X186E+X193E+X206Y+X460G,

X25H+X176K+X186E+X193E+X206Y+X251E+X460G,X25H+X176K+X181D+X186E+X206Y,

X25H+X176K+X181N+X186E+X206Y,X25H+X176K+X181Q+X186E+X206Y,

X25H+X136E+X176K+X186E+X206Y,X25H+X100W+X176K+X186E+X206Y,

X25H+X135E+X176K+X186E+X206Y,X25H+X135T+X176K+X186E+X206Y,

X25H+X135W+X176K+X186E+X206Y,X25H+X160D+X176K+X186E+X206Y,

X25H+X160E+X176K+X186E+X206Y,X25Y+X176K+X186E+X206Y,

X87D+X186E+X315K+X420K,X87D+X186E+X226D+X314E+X420E+X461E,

X87D+X186E+X226D+X420E,X87D+X186E+X226D+X420E+X461E,

X87D+X186E+X314E+X471E,X87D+X186E+X311K+X314E+X420K,

X87D+X186E+X160D+X461E,X87R+X186E+X315K+X461R,X87R+X186E+X226D+X471E,

X87R+X186E+X226R+X314K+X420K+X461R,X87R+X186E+X226R+X311K+X420K,

X87R+X186E+X314K+X315K,X87R+X186E+X311K+X314E,X87R+X186E+X420E+X450R,

X87R+X186E+X450R+X452R,X186E+X315K+X420E+X452R,

X186E+X226D+X314E+X461E+X471E,X186E+X226D+X314E+X452R,

X186E+X226D+X314K+X460D+X471E+X485R,X186E+X226D+X311K+X460D,

X186E+X226D+X461E+X471E,X186E+X314E+X460D+X461E,

X186E+X314E+X420E+X452R+X461E,X186E+X314K+X450R+X460D,

X186E+X460D+X471E+X485R,X186E+X311K+X314K+X452R,

X186E+X311K+X461E+X485R,X186E+X311K+X420E+X471E,

X186E+X420K+X450R+X471E+X485R,X186E+X420K+X450R+X485R,

X186E+X452R+X461E+X471E,X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X25H+X176K+X186E+X206Y,X83E+X186E+X219D+X226R,

X83E+X186E+X219R+X226D+X314E+X452R,X83E+X186E+X219R+X226R,

X83E+X186E+X160D+X219D+X226R+X452R,X83R+X186E+X219R+X226D,

X160D+X186E+X315K+X450R,X160D+X186E+X226D+X314K+X460D+X461E,

X160D+X186E+X226D+X314K+X420E,X160D+X186E+X314K+X485R,

X160D+X186E+X420E+X452R,X160D+X186E+X420K+X460D,X186E+X276R,

X186E+X206Y,X186E+X206Y+X276R,X186E+X206Y+X276R+X405M,

X186E+X206Y+X405M,X186E+X206Y+X251E,X186E+X206Y+X251E+X276R,

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X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X473R,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X363E,

X25H+X176K+X186E+X206Y+X100W+X363E,X25H+X176K+X186E+X206Y+X135T,

X25H+X176K+X186E+X206Y+X135T+X258Q,

X25H+X176K+X186E+X206Y+X135T+X258Q+X281N,

X25H+X176K+X186E+X206Y+X135T+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X135T+X258Q+X363E,

X116K+X181T,

X116K+X181T+X225A,

x181t+x225a+x320K, and

X116K+X181T+X225A+X320K,

and wherein the variant has alpha-amylase activity. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

In a particularly preferred embodiment, the alpha-amylase variant of the invention comprises a combination of substitutions selected from the group consisting of:

X4Q+X25H,

X4Q+X25H+X176K,

X4Q+X25H+X176K+X186E,

X4Q+X25H+X176K+X186E+X251E,

X4Q+X25H+X176K+X186E+X251E+X405M,

X4Q+X25H+X176K+X186E+X251E+X405M+X439K,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X439K+X482W,

X25H+X176K,

X25H+X176K+X186E,

X176K+X186E,

X25H+X176K+X186E+X251E,

X25H+X176K+X186E+X251E+X405M,

X25H+X176K+X186E+X251E+X405M+X482W,

X25H+X176K+X186E+X251E+X405M+X439K+X482W,

X25H+X176K+X251E+X405M,

X25H+X176K+X251E+X405M+X482W,

X25H+X176K+X251E+X405M+X439K,

X25H+X176K+X251E+X405M+X439K+X482W,

X251E+X405M,

X251E+X405M+X439K,

X251E+X405M+X482W,

X251E+X405M+X439K+X482W,

X405M+X439K,

X405M+X482W,

X405M+X439K+X482W,

x4q+x25h+x176k+x251e+x405m+x439K, and

X4Q+X25H+X176K+X251E+X405M+X439K+X482W,

preferably, X25H+X176K+X186E, X25H+X176K+X186E+X251E+X405M+X482W, or

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,X25H+X176K,

X25H + X176K + X251E + X405M + X482W, or X4Q + X25H + X176K + X251E + X405M + X482W,

most preferably, the variant has an alpha-amylase activity, and wherein the alpha-amylase variant has an alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at one or more amino acids corresponding to positions 181, 182, 183 and 184, preferably at any of SEQ ID NOs 15-41, preferably at positions 181 and 182, 183 and 184, more preferably at SEQ ID NOs 4, more preferably at SEQ ID NOs 1 or 3, most preferably at amino acid sequences shown in SEQ ID NO 1 has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably at one or more amino acids corresponding to positions 181, 182, 183 and 184, preferably at positions 181 and 182, 183 and 183, preferably at positions 184, wherein amino acid sequences shown in SEQ ID NOs 1 and 184 are deleted according to the amino acid sequence numbering shown in SEQ ID NOs 3. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

In another particularly preferred embodiment, the alpha-amylase variant of the invention comprises a combination of substitutions selected from the group consisting of: the amino acid sequence depicted in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably SEQ ID No. 1, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably at any one of SEQ ID No. 1, 3, 4 or SEQ ID No. 15-41, preferably at any one of SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably at the amino acid sequence depicted in SEQ ID No. 1, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably at least 91% or at least 95% but less than 100% sequence identity, preferably at any amino acid sequence identity corresponding to one or more positions 183, preferably at positions 183, 182, 183, and amino acid sequence numbers 183, 182 and 183, preferably at positions 183, and 183, according to any one or more of SEQ ID nos. 182 and 183. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

In another particularly preferred embodiment, the alpha-amylase variant of the invention comprises a combination of substitutions selected from the group consisting of:

N25H+R176K+G186E+I206Y+R181Q,N25H+R176K+G186E+I206Y+Y135T,

N25H+R176K+G186E+I206Y+Y100W+Y363E,N25H+R176K+G186E+I206Y+Y100W+T193E,

N25H+R176K+G186E+I206Y+T251E,G4Q+N25H+R176K+G186E+I206Y+T251E+L405M,

N25H+R176K+G186E+I206Y,N25H+G186E+I206Y+L405M,N25H+G186E+I206Y,

N25H+R176K+G186E+T193E+I206Y+T251E,G4Q+R176K+G186E+T193E+I206Y+T251E+L405M,

N25H+R176K+G186E,N25H+R176K+G186E+T193E+I206Y,G4Q+N25H+R176K+G186E+T251E+L405M,

N25H+R176K+G186E+I206Y,G4Q+R176K+G186E+T193E+I206Y+T251E+L405M,

G4Q+I206Y+N460G,G4Q+N25H+R176K+I206Y+T251E+N460G,

G4Q+N25H+R176K+T193E+T251E+G186E,G4Q+T193E+I206Y+E276R+G186E,

G186E+N25H,N25H+T193E+I206Y+T251E+E276R+L405M+G186E,N25H+T251E+G186E,

N25H+Y160W+R176K+G186E+T251E+L405M,T193E+I206Y+L405M+G186E,

N25H+R176K+G186E+I206Y+G258Q+Y363E,N25H+R176K+G186E+I206Y+R181Q,

N25H+R176K+G186E+I206Y+Y100W,N25H+R176K+G186E+I206Y+Y100W+Q359R,

N25H+R176K+G186E+I206Y+Y100W+G258Q,

N25H+R176K+G186E+I206Y+Y100W+G258Q+Y363E,

N25H+R176K+G186E+I206Y+Y100W+Y135T,

N25H+R176K+G186E+I206Y+Y100W+Y135T+Y363E,N25H+R176K+G186E+I206Y+Y135T,

N25H+R176K+G186E+I206Y+Y135T+G258Q,

N25H+R176K+G186E+I206Y+Y135T+G258Q+Y363E,

N25H+R176K+G186E+I206Y+Y135T+Y363E,N25H+R176K+G186E+I206Y+Y363E,

N25H+R176K+G186E+I206Y+T251E+Y482W,N25H+R176K+G186E+I206Y+Y482W,

N25H+R176K+G186E+T193E+I206Y+T251E+Y482W,

G4Q+R176K+G186E+T193E+I206Y+T251E+L405M+Y482W,

G4Q+N25H+R176K+G186E+T251E+L405M+Y482W,

N25H+R176K+G186E+T193E+I206Y+Y482W,

G4Q+N25H+R176K+G186E+I206Y+T251E+L405M+Y482W,

N25H+G186E+I206Y+L405M+Y482W,N25H+R176K+G186E+Y482W,

N25H+R176K+G186E+T193E+I206Y+T251E+N460G,

G4Q+N25H+R176K+G186E+I206Y+T251E+N460G,

N25H+R176K+G186E+T193E+I206Y+T251E+N460G+Y482W,

G4Q+N25H+R176K+G186E+I206Y+T251E+N460G+Y482W,

N25H+R176K+G186E+N195F+T251E+Y482W,N25H+R176K+G186E+N195F+Y482W,

N25H+R176K+G186E+T193E+N195F+T251E+Y482W,

G4Q+R176K+G186E+T193E+N195F+T251E+L405M+Y482W,

N25H+R176K+G186E+T193E+N195F+Y482W,

G4Q+N25H+R176K+G186E+N195F+T251E+L405M+Y482W,

N25H+G186E+N195F+L405M+Y482W,N25H+R176K+G186E+T193E+N195F+T251E+N460G,

G4Q+N25H+R176K+G186E+N195F+T251E+N460G,

n25h+r176k+g186e+t193e+n195f+t251 e+n450g+y482W, and

G4Q+N25H+R176K+G186E+N195F+T251E+N460G+Y482W，

wherein the numbering is according to the amino acid sequence shown in SEQ ID NO. 3, and wherein the variant has alpha-amylase activity, preferably the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO. 1 or 3, preferably at the corresponding position in SEQ ID NO. 1 (numbering according to SEQ ID NO. 3).

In another particularly preferred embodiment, the alpha-amylase variant of the invention comprises a combination of substitutions selected from the group consisting of:

G4Q+N25H,

G4Q+N25H+R176K,

G4Q+N25H+R176K+G186E,

G4Q+N25H+R176K+G186E+T251E,

G4Q+N25H+R176K+G186E+T251E+L405M+W439K,

G4Q+N25H+R176K+G186E+T251E+L405M+Y482W,

G4Q+N25H+R176K+G186E+T251E+L405M+W439K+Y482W,

G4Q+N25H+R176K+T251E,

G4Q+N25H+R176K+T251E+L405M+W439K,

G4Q+N25H+R176K+T251E+L405M+Y482W,

G4Q+N25H+R176K+T251E+L405M+W439K+Y482W,

N25H+R176K,

N25H+R176K+G186E,

R176K+G186E,

N25H+R176K+G186E+T251E,

N25H+R176K+G186E+T251E+L405M,

N25H+R176K+G186E+T251E+L405M+Y482W,

N25H+R176K+G186E+T251E+L405M+W439K+Y482W,

N25H+R176K+T251E,

N25H+R176K+T251E+L405M,

n25h+r176k+t251e+l405m+y482W, and

N25H+R176K+T251E+L405M+W439K+Y482W,

preferably, N25H+R176K+G186E, N25H+R176K+G186E+T251E+L405M+Y482W,

G4Q+N25H+R176K+G186E+T251E+L405M+Y482W,N25H+R176K,

n25H+R176K+T251E+L405M+Y482W, or G4Q+N25H+R176K+T251E+L405M+Y482W,

most preferably N25H+R176K+G186E or G4Q+N25H+R176K+G186E+T251E+L405M+Y482W,

wherein the numbering is according to the amino acid sequence shown in SEQ ID No. 3, and wherein the variant has an alpha-amylase activity, preferably wherein the alpha-amylase variant comprises a deletion at one or more amino acids corresponding to any of SEQ ID nos. 1, 3, 4, or 15-41, preferably SEQ ID No. 1, SEQ ID No. 3 or 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1 has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100%, preferably at least 91% or at least 95% but less than 100%, preferably an amino acid deletion corresponding to positions 181 and 182, 183 and 184, preferably corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183 and G184, wherein the numbering is according to the amino acid sequence shown in SEQ ID No. 3. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

In another embodiment, the alpha-amylase variants of the invention having alpha-amylase activity comprise or consist of the amino acid sequence shown in SEQ ID NO. 1, wherein there are 1-50 amino acid changes described herein, preferably 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5, 2-30, 2-25, 2-20, 2-15, 2-10, 2-8 or 2-5, preferably 3-30, 3-25, 3-20, 3-15, 3-10, 3-8 or 3-5, preferably 4-30, 4-25, 4-20, 4-15, 4-10 or 4-8 amino acid changes, preferably substitutions. Preferably, the alpha-amylase variant of the invention, wherein the alpha-amylase activity is present, comprises or consists of the amino acid sequence shown in SEQ ID NO 1, wherein there are 1-50 amino acid substitutions, preferably 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5, 2-30, 2-25, 2-20, 2-15, 2-10, 2-8 or 2-5, preferably 3-30, 3-25, 3-20, 3-15, 3-10, 3-8 or 3-5, preferably 4-30, 4-25, 4-20, 4-15, 4-10 or 4-8 amino acid substitutions as described herein, and further comprises 1-50, preferably 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5, 2-30, preferably 2-30, 3-25, 3-10, 3-5, 3-25, 2-25, 3-8 or 3-5, preferably 4-25, 4-10, 3-8 or 3-5 amino acids. In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO 1, wherein there are 1-50 amino acid changes, preferably 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5, 2-30, 2-25, 2-20, 2-15, 2-10, 2-8 or 2-5, preferably 3-30, 3-25, 3-20, 3-15, 3-10, 3-8 or 3-5, preferably 4-30, 4-25, 4-20, 4-15, 4-10 or 4-8 amino acid substitutions, wherein the amino acid substitutions are selected from the group consisting of: X100D, X100F, X100K, X100L, X103A, X106D, X108A, X109A, X10A, X10C, X10E, X10F, X10L, X10W, X10Y, X113F, X113H, X113M, X113R, X113V, X113W, X113Y, X114A, X114C, X114D, X114E, X114F, X114G, X114H, X114I, X114K, X114L, X114M, X114N, X114Q, X114R, X114S, X114V, X114W, X114Y, X115C, X115D, X117K, X115M, X115N, X115Q, X115R, X115S, X115T, X115V, X116I, X116K, X116M, X116R, X116T, X117A, X117C, X116D, X117N, X117X 116D, X117W, X117X 116R, X117Y, X118A, X118D, X118E, X119H, X119P, X119R, X119S, X119Y, X120E, X120G, X120M, X120S, X120W, X120Y, X123D, X123S, X125A, X125D, X125E, X125F, X125G, X125H, X125K, X125L, X125M, X125N, X125Q, X125R, X125T, X125V, X125W, X125Y, X126D, X128C, X128E, X128L, X128M, X128W, X128Y, X129E, X12N, X12S, X131C, X131I, X131L, X131Q, X131W, X132T, X133A, X133C, X133D, X133E, X133H, X133K, X133N, X134L, X134E, X134L, X134M, X134P, X134T, X134V, X134W, X134Y, X135D, X135E, X135G, X135M, X135N, X135P, X135S, X135T, X135W, X136A, X136C, X136D, X136E, X136F, X136H, X136L, X136M, X136N, X136P, X136W, X139C, X139S, X13A, X13Y, X141V, X142C, X142E, X142F, X142L, X142M, X142Q, X142R, X142W, X142Y, X143F, X144C, X144E, X144G, X144K, X144N, X144Q, X144R, X144S, X144T, X144V, X144Y, X145A, X146C, X146D, X146E, X146F, X146G, X146H, X146K, X146L, X146S, X146T, X146W, X147M, X149E, X14N, X150C, X150E, X150Q, X151C, X151E, X154A, X154Y, X155Y, X156E, X156V, X158H, X158N, X158Y, X15R, X160D, X160E, X160W, X161T, X162V, X163A, X163Q, X163T, X164V, X165S, X165T, X165W, X169A, X169D, X169E, X169S, X169V, X16F, X16R, X170C, X170F, X172A, X172C, X172D, X172K, X172N, X173I, X173Y, X174D, X174E, X174G, X174N, X174P, X174S, X174T, X175A, X175G, X175H, X175K 176, X176K 176A, X177G, X176K 176G, X177G, X177N, X177P, X177R, X177S, X177W, X178F, X17K, X17V, X180M, X180N, X180T, X180W, X182D, X182E, X182N, X182Q, X182S, X185E, X185M, X185N, X187A, X187D, X187M, X187V, X188H, X188T, X188V, X189I, X18F, X18I, X18K, X18M, X18R, X18T, X191F, X191H, X191K, X191M, X191W, X191Y, X192A, X192T, X193D, X193E, X193G, X193V, X19A, X19C, X19D, X19F, X19G, X19H, X19I, X19K, X19L, X19M, X19P, X19Q, X19S, X19T, X19Y, X1R, X1V, X203E, X203R, X208F, X208I, X208Y, X20A, X20C, X20D, X20E, X20F, X20G, X20H, X20K, X20L, X20M, X20N, X20P, X20Q, X20S, X20T, X20V, X20W, X20Y, X210A, X210C, X210D, X210E, X210F, X210M, X210N, X210Q, X210S, X210Y, X211A, X211C, X211D, X211E, X211G, X211H, X211L, X211N, X211Q, X211S, X211T, X211V, X212C, X212D, X212I, X212L, X212W, X213A, X213C, X213M, X213R, X213S, X214E, X214P, X215A, X215D, X215E, X215H, X215G, X216W 218G, X218G, X218E, X218F, X218G, X218H, X218I, X218K, X218L, X218N, X218Q, X218S, X218T, X218V, X218W, X218Y, X219A, X219C, X219D, X219E, X219F, X219G, X219H, X219K, X219M, X219Q, X219R, X219S, X219T, X219W, X219Y, X21E, X21S, X221F, X221N, X222D, X222K, X222S, X222T, X223C, X223L, X223V, X223Y, X224F, X224V, X225A, X225C, X225E, X225F, X225H, X225I, X225N, X225R, X225S, X225Y, X226A, X226C, X226D, X226E, X226G, X226H, X226I, X226L, X226M, X226Q, X226R, X226S, X226T, X226V, X226W, X226Y, X227C, X227F, X227G, X227H, X227I, X227K, X227L, X227M, X227R, X227T, X227V, X227W, X227Y, X22A, X22D, X22E, X22F, X22G, X22K, X22L, X22M, X22Q, X22R, X22T, X22W, X22Y, X230A, X230F, X231C, X231D, X231N, X233C, X233H, X233I, X233M, X233T, X233W, X234C, X235L, X235M, X235V, X236Y, X237C, X237I, X238A, X238F, X238T, X23N, X23Q, X23W, X240M, X243D, X245H, X245M, X249D, X249I, X24G, X250V, X251A, X251E, X251F, X251L, X251M, X251S, X251T, X252C, X252I, X252S, X253C, X253G, X253Y, X255D, X255F, X255I, X255T, X255V, X256C, X257L, X257V, X257Y, X258F, X258Q, X258R, X259L, X25A, X25C, X25D, X25F, X25G, X25H, X25K, X25L, X25M, X25Q, X25S, X25W, X25Y, X260H, X261R, X262C, X262D, X262P, X262Y, X263I, X H, X264T, X264W, X268S, X268F, X26G, X26M 26F, X26M, X26P, X26S, X26V, X26Y, X270A, X270Q, X270Y, X271S, X272F, X272G, X272L, X272S, X273A, X273C, X273D, X273E, X273F, X273H, X273I, X273L, X273M, X273P, X273Q, X273R, X273V, X273W, X273Y, X274F, X274S, X275V, X276D, X276K, X276L, X276N, X276R, X276Y, X277D, X277E, X277T, X272A, X279P, X27A, X27D, X27F, X27G, X27H, X27I, X27Q, X27V, X280D, X280F, X280G, X280H, X280I, X280K, X280N, X280D, X280V, X280Y, X281E, X281H, X284A, X284F, X284H, X284L, X284M, X284N, X284Y, X285G, X285L, X285N, X285P, X286Q, X287A, X287D, X287E, X287H, X287T, X288A, X288K, X288P, X288Y, X289F, X289G, X289R, X289T, X28C, X28D, X28E, X28F, X28G, X28I, X28K, X28N, X28Q, X28S, X28T, X28V, X290D, X290M, X290N, X290Q, X290W, X291D, X291K, X291T, X292D, X292F, X292I, X292L, X292T, X292W, X293Y, X293D, X293E, X293F, X294T, X294F, X296A, X296C, X296L, X296Y, X297E, X297F, X297H, X297K, X297M, X297S, X297V, X299G, X299I, X299K, X299L, X299S, X299Y, X29D, X29E, X29F, X29G, X29H, X29I, X29K, X29L, X29N, X29P, X29Q, X29V, X29W, X29Y, X2I, X2S, X301F, X302H, X302I, X302Q, X302V, X302Y, X303E, X303H, X303I, X303K, X303L, X303M, X303N, X303P, X303R, X303T, X304A, X304D, X304E, X304H, X304K, X304M, X304N, X304P, X304R, X304T, X304W, X304Y, X306A, X306D, X306E, X306G, X306H, X306I, X306M, X306Q, X306R, X306S, X306T, X306V, X306W, X306Y, X307F, X307M, X308S, X309H, X309L, X309Q, X30A, X30E, X30F, X30G, X30H, X30I, X30K, X30L, X30M, X30Q, X30T, X30W, X30Y, X310A, X310Q, X311A, X311E, X311G, X311H, X311K, X311N, X311R, X311T, X311Y, X312L, X312M, X313V, X314C, X314E, X314K, X314Q, X315A, X315C, X315E, X315H, X315K, X315T, X318I, X318S, X318T, X319A, X319D, X319H, X319I, X319K, X319M, X319N, X319P, X319S, X319T, X319W, X31N, X31Q, X31S, X31T, X31V, X31W, X320A, X320C, X320D, X320E, X320G, X320H, X320K, X320L, X320N, X320Q, X320S, X320Y, X321A, X321E, X321K, X321N, X321T, X321V, X321W, X323A, X323G, X323K, X323L, X323V, X324K, X324L, X324M, X324W, X324Y, X326G, X326N, X326S, X326Y, X327C, X327L, X327M, X32A, X32D, X32E, X32F, X32I, X32L, X32M, X32N, X32P, X32Q, X32K, X337K, X32T, X337W, X32K, X337W, X337C, X32X 337C, X337X 32X 337X 32X 37X 32X, X337G, X337I, X337K, X337L, X337M, X337N, X337Q, X337R, X337S, X337T, X337V, X337Y, X338G, X338S, X338T, X33D, X33E, X33H, X33K, X33M, X33Q, X33R, X33Y, X341V, X342P, X343L, X343T, X343W, X343Y, X344I, X344Q, X344V, X345D, X345G, X345M, X345N, X345Q, X345S, X345T, X346A, X346C, X346D, X346G, X346H, X346N, X346Q, X348T, X34H, X34I, X34V, X350H, X350K, X350P, X351A, X351M, X354S, X354I, X354T, X355M, X355I, X356V, X357A, X358I, X358L, X358N, X358P, X358V, X359E, X35A, X35C, X35D, X35G, X35H, X35I, X35L, X35M, X35N, X35P, X35Q, X35R, X35S, X35T, X35V, X35Y, X360A, X360F, X360G, X360I, X360L, X360N, X360Q, X360R, X360S, X360T, X360V, X360Y, X362F, X362K, X362M, X362N, X362T, X362V, X362Y, X363A, X363C, X363D, X363E, X363G, X363H, X363K, X363L, X363M, X363P, X363Q, X363R, X363S, X363T, X363V, X363W, X363Y, X364A, X364C, X364G, X364K, X364L, X364N, X364S, X364T, X364V, X366I, X366L, X366T, X367E, X367S, X368A, X368F, X368L, X368N, X36A, X36E, X36G, X36I, X36K, X36M, X36N, X36P, X36Q, X36R, X36S, X36T, X36V, X370E, X370I, X372A, X372C, X372E, X372F, X372H, X372M, X372N, X372Q, X375A, X375D, X375E, X375I, X375K, X375Q, X375R, X376T, X375W, X375Y, X376G, X376I, X376M, X376Q, X376R, X376S, X376V, X377, X37C, X379A, X379L, X37L and X37L. X37G, X37M, X37P, X37T, X37V, X37W, X381E, X381V, X382A, X382H, X382K, X382L, X382N, X382Q, X382S, X383C, X383D, X383E, X383H, X383I, X383M, X383N, X383Q, X383R, X383S, X383V, X383Y, X384A, X384C, X384D, X384E, X384F, X384I, X384L, X384M, X384N, X384Q, X384R, X384T, X385V, X385W, X385Y, X385A, X385C, X385D, X385E, X385F, X385G, X385H, X385I, X385L, X385M, X385N, X385P, X385Q, X385R, X385S, X385V, X385W, X385C, X385 387E, X387N, X388E, X388F, X388H, X388I, X388M, X388R, X388V, X389G, X389H, X389K, X38N, X390D, X390F, X390M, X390N, X390P, X390R, X391A, X391F, X391G, X391K, X391M, X391N, X391Q, X391S, X391T, X391Y, X392C, X392V, X393E, X393H, X393P, X393S, X393V, X394A, X394C, X394E, X394H, X394I, X394L, X394M, X394N, X394R, X394S, X395A, X395H, X395V, X396H, X396P, X397D, X397H, X397P, X397S, X39M, X39K, X39G, X3G, X39G 3G, X3Q, X3V, X400A, X400D, X400E, X400G, X400H, X400I, X400K, X400L, X400M, X400N, X400P, X400Q, X400R, X400S, X400V, X400W, X401I, X401K, X401M, X401T, X403N, X405C, X405H, X405M, X405T, X405V, X406P, X407D, X407R, X407S, X408E, X408I, X408Q, X40I, X40S, X410H, X410I, X410K, X410L, X410P, X410R, X410Y, X413S, X414C, X414E, X414S, X415I, X416S, X418C, X418N, X418P, X41C, X41D, X41E, X41G, X41G 41S, X41S 41T 41S, X41S 41T 41A, X41S, X426D, X426W, X427C, X427F, X427G, X427K, X427Q, X427R, X427S, X427T, X427V, X429A, X429D, X429E, X429F, X429G, X429I, X429M, X429N, X429P, X429Q, X429S, X429T, X429V, X429W, X42C, X42I, X42Q, X42V, X431I, X434S, X436E, X438F, X438P, X438Y, X439K, X439C, X439P, X440V, X442Q, X443H, X445T, X445A, X445C, X445D, X445T, X445V, X446F, X446I, X446L, X446R, X446S, X446W, X447V, X448D, X448E, X448H, X448N, X449A, X449F, X449G, X449K, X449L, X449M, X449N, X449P, X449Q, X449R, X449S, X449V, X449W, X449Y, X451L, X453G, X453I, X453N, X453P, X453Y, X455L, X456L, X456M, X456R, X456S, X456V, X456W, X456Y, X457A, X457C, X457E, X457F, X457G, X457K, X457L, X457M, X457R, X457S, X457T, X457V, X457W, X458I, X458K, X458V, X458W, X459C, X459D, X459E, X459I, X459K, X459N, X459Q, X459R, X459V, X459Y, X45G, X45N, X460A, X460D, X460E, X460G, X460Q, X460R, X460S, X460T, X460V, X461A, X461E, X461F, X461K, X461L, X461M, X461N, X461Q, X461R, X461S, X461V, X462K, X463L, X465H, X465P, X465R, X465V, X467A, X467E, X467H, X468D, X468H, X470A, X470Q, X470T, X471E, X474F, X474H, X474P, X478A, X478E, X47S, X480D, X480E, X480M, X480R, X480Y, X482C, X482T, X482W, X48F, X48I, X48M, X48Y, X4A, X4C, X4K, X4M, X4Q, X4R, X4S, X51Q, X51T, X51V, X54D, X54G, X54Q, X59T, X5A, X5C, X5D, X5E, X5F, X5H, X5I, X5K, X5L, X5M, X5N, X5P, X5Q, X5R, X5V, X5Y, X60T, X63C, X63V, X6A, X6C, X6E, X6F, X6G, X6H, X6K, X6L, X6M, X6P, X6Q, X6S, X6T, X6V, X6W, X6Y, X70F, X70H, X70L, X70M, X70N, X70Y, X71D, X72C, X72D, X72E, X72N, X72T, X73L, X73N, X73Q, X75A, X75G 75L, X75G, X75T 76C, X75G, X75T 76T, X76G, X75T, X75M 76T, X76G, X75M and X76T, X7F, X7H, X7K, X7N, X7P, X7Q, X7R, X7S, X7V, X7W, X7Y, X81H, X81L, X82K, X82M, X83A, X83D, X83E, X83G, X83R, X83S, X86K, X87D, X87R, X89A, X89C, X89F, X89G, X89L, X89M, X89R, X89S, X8A, X8C, X8F, X8I, X8M, X8P, X8S, X8V, X8W, X8Y, X90A, X90D, X90E, X90F, X90G, X90I, X90M, X90N, X90Q, X90R, X90S, X90V, X90Y, X91C, X91D, X91E, X91F, X91G, X91H, X91I, X91K, X91L, X91M, X91N, X91Q, X91S, X91T, X91V, X91W, X91Y, X92D, X92M, X92V, X93K, X93Q, X94A, X94D, X94E, X94K, X94M, X94V, X94Y, X95E, X95I, X95L, X95V, X96K, X96N, X96Q, X97V, X98E, X98G, X98N, X99H, X99K, X99N, X100W, and X1G are preferably selected from: X4Q, X25H, X100W, X135E, X135T, X135W, X135D, X160W, X176K, X193E, X251E, X258Q, X276R, X299S, X323G, X363E, X363H, X382Q, X405M, X460G, and X482W, wherein optionally the alpha-amylase variant further comprises 1-50, preferably 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5, 2-30, 2-25, 2-20, 2-15, 2-10, 2-8 or 2-5, preferably 3-30, 3-25, 3-20, 3-15, 3-10, 3-8 or 3-5, preferably 4-30, 4-25, 4-20, 4-15, 4-10 or 4-8 amino acids that are conserved. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of the amino acid sequence set forth in SEQ ID NO:1, wherein there is one of the amino acid substitution combinations selected from the group consisting of:

X25H+X176K+X186E+X206Y+X251E+X482W,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X482W,

X25H+X176K+X186E+X206Y+X482W,X25H+X186E+X206Y+X405M+X482W,

X25H+X186E+X206Y+X482W,X25H+X176K+X186E+X193E+X206Y+X251E+X482W,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X482W,X25H+X176K+X186E+X482W,

X25H+X176K+X186E+X193E+X206Y+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,X25H+X176K+X186E+X206Y,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,X4Q+X206Y+X460G+X482W,

X4Q+X25H+X176K+X206Y+X251E+X460G+X482W,

X4Q+X25H+X176K+X193E+X251E+X186E+X482W,

X4Q+X193E+X206Y+X276R+X186E+X482W,X186E+X25H+X482W,

X25H+X193E+X206Y+X251E+X276R+X405M+X186E+X482W,X25H+X251E+X186E+X482W,

X25H+X160W+X176K+X186E+X251E+X405M+X482W,

X193E+X206Y+X405M+X186E+X482W,X3I+X356V,X3I+X356I,X83D+X94E,X94D+X125E,

X131I+X377Q+X410H,X48F+X94D,X48Y+X116K+X218K,X5L+X218K+X225S,

X83E+X116R+X158Y+X181E,X51V+X218K,X83E+X181E,

X4Q+X7W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X37M+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X460G,

X4Q+X25H+X176K+X186E+X206Y+X251E+X460G,

X4Q+X25Y+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X118D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X182D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X460G,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363N+X405M,

X4Q+X176K+X181D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181F+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181N+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X136E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160E+X176K+X186E+X193E+X206Y+X251E+X405M,

X7H+X25H+X176K+X186E+X206Y,X7W+X25H+X176K+X186E+X206Y,

X25D+X176K+X186E+X206Y,X25H+X186E+X206Y+X405M+X460G,

X25H+X186E+X206Y+X460G,X25H+X37M+X176K+X186E+X206Y,

X25H+X118D+X176K+X186E+X206Y,X25H+X176K+X182D+X186E+X206Y,

X25H+X176K+X186E+X206Y,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X281N,X25H+X176K+X186E+X206Y+X460G,

X25H+X176K+X186E+X206Y+X251E+X460G,X25H+X176K+X186E+X206Y+X363E,

X25H+X176K+X186E+X206Y+X363H,X25H+X176K+X186E+X206Y+X363N,

X25H+X176K+X186E+X460G,X25H+X176K+X186E+X193E+X206Y,

X25H+X176K+X186E+X193E+X206Y+X460G,

X25H+X176K+X186E+X193E+X206Y+X251E+X460G,X25H+X176K+X181D+X186E+X206Y,

X25H+X176K+X181N+X186E+X206Y,X25H+X176K+X181Q+X186E+X206Y,

X25H+X136E+X176K+X186E+X206Y,X25H+X100W+X176K+X186E+X206Y,

X25H+X135E+X176K+X186E+X206Y,X25H+X135T+X176K+X186E+X206Y,

X25H+X135W+X176K+X186E+X206Y,X25H+X160D+X176K+X186E+X206Y,

X25H+X160E+X176K+X186E+X206Y,X25Y+X176K+X186E+X206Y,

X87D+X186E+X315K+X420K,X87D+X186E+X226D+X314E+X420E+X461E,

X87D+X186E+X226D+X420E,X87D+X186E+X226D+X420E+X461E,

X87D+X186E+X314E+X471E,X87D+X186E+X311K+X314E+X420K,

X87D+X186E+X160D+X461E,X87R+X186E+X315K+X461R,X87R+X186E+X226D+X471E,

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X25H+X176K+X186E+X206Y,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X258Q+X281N,

X25H+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E+X123D,X25H+X176K+X186E+X206Y+X281N,

X25H+X176K+X186E+X206Y+X181Q,X25H+X176K+X186E+X206Y+X181Q+X193E,

X25H+X176K+X186E+X206Y+X181Q+X363E,

X25H+X176K+X186E+X206Y+X193E,

X25H+X176K+X186E+X206Y+X193E+X258Q,

X25H+X176K+X186E+X206Y+X193E+X281N+X363E,

X25H+X176K+X186E+X206Y+X193E+X363E,X25H+X176K+X186E+X206Y+X100W,

X25H+X176K+X186E+X206Y+X100W+X258Q,

X25H+X176K+X186E+X206Y+X100W+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X258Q+X363E+X135C,

X25H+X176K+X186E+X206Y+X100W+X281N,

X25H+X176K+X186E+X206Y+X100W+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X361R,

X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E+X175D,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X473R,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X363E,

X25H+X176K+X186E+X206Y+X100W+X363E,X25H+X176K+X186E+X206Y+X135T,

X25H+X176K+X186E+X206Y+X135T+X258Q,

X25H+X176K+X186E+X206Y+X135T+X258Q+X281N,

X25H+X176K+X186E+X206Y+X135T+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X135T+X258Q+X363E,

X4Q+X25H,

X4Q+X25H+X176K,

X4Q+X25H+X176K+X186E+X251E+X405M,

X4Q+X25H+X176K+X186E+X251E+X405M+X439K,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X439K+X482W,

X4Q+X25H+X176K+X251E+X405M,

X4Q+X25H+X176K+X251E+X405M+X439K,

X4Q+X25H+X176K+X251E+X405M+X482W,

X4Q+X25H+X176K+X251E+X405M+X439K+X482W,

X25H+X176K,

X25H+X176K+X186E,

X176K+X186E,

X25H+X176K+X186E+X251E,

X25H+X176K+X186E+X251E+X405M,

X25H+X176K+X186E+X251E+X405M+X482W,

X25H+X176K+X186E+X251E+X405M+X439K+X482W,

X25H+X176K+X251E,

X25H+X176K+X251E+X405M,

X25H+X176K+X251E+X405M+X482W,

X25H+X176K+X251E+X405M+X439K+X482W,

X116K+X181T,

X116K+X181T+X225A,

x181t+x225a+x320K, and

x116k+x181t+x225a+x320K, preferably selected from:

N25H+R176K+G186E+I206Y+R181Q,N25H+R176K+G186E+I206Y+Y135T,

N25H+R176K+G186E+I206Y+Y100W+Y363E,N25H+R176K+G186E+I206Y+Y100W+T193E,

N25H+R176K+G186E+I206Y+T251E,G4Q+N25H+R176K+G186E+I206Y+T251E+L405M,

N25H+R176K+G186E+I206Y,N25H+G186E+I206Y+L405M,N25H+G186E+I206Y,

N25H+R176K+G186E+T193E+I206Y+T251E,

G4Q+R176K+G186E+T193E+I206Y+T251E+L405M,N25H+R176K+G186E,

N25H+R176K+G186E+T193E+I206Y,G4Q+N25H+R176K+G186E+T251E+L405M,

N25H+R176K+G186E+I206Y,G4Q+R176K+G186E+T193E+I206Y+T251E+L405M,

G4Q+I206Y+N460G,G4Q+N25H+R176K+I206Y+T251E+N460G,

G4Q+N25H+R176K+T193E+T251E+G186E,G4Q+T193E+I206Y+E276R+G186E,

G186E+N25H,N25H+T193E+I206Y+T251E+E276R+L405M+G186E,N25H+T251E+G186E,

N25H+Y160W+R176K+G186E+T251E+L405M,T193E+I206Y+L405M+G186E,

N25H+R176K+G186E+I206Y+G258Q+Y363E,N25H+R176K+G186E+I206Y+R181Q,

N25H+R176K+G186E+I206Y+Y100W,N25H+R176K+G186E+I206Y+Y100W+Q359R,

N25H+R176K+G186E+I206Y+Y100W+G258Q,

N25H+R176K+G186E+I206Y+Y100W+G258Q+Y363E,

N25H+R176K+G186E+I206Y+Y100W+Y135T,

N25H+R176K+G186E+I206Y+Y100W+Y135T+Y363E,N25H+R176K+G186E+I206Y+Y135T,

N25H+R176K+G186E+I206Y+Y135T+G258Q,

N25H+R176K+G186E+I206Y+Y135T+G258Q+Y363E,

N25H+R176K+G186E+I206Y+Y135T+Y363E,N25H+R176K+G186E+I206Y+Y363E,

N25H+R176K+G186E+I206Y+T251E+Y482W,N25H+R176K+G186E+I206Y+Y482W,

N25H+R176K+G186E+T193E+I206Y+T251E+Y482W,

G4Q+R176K+G186E+T193E+I206Y+T251E+L405M+Y482W,

G4Q+N25H+R176K+G186E+T251E+L405M+Y482W,

N25H+R176K+G186E+T193E+I206Y+Y482W,

G4Q+N25H+R176K+G186E+I206Y+T251E+L405M+Y482W,

N25H+G186E+I206Y+L405M+Y482W,N25H+R176K+G186E+Y482W,

N25H+R176K+G186E+T193E+I206Y+T251E+N460G,

G4Q+N25H+R176K+G186E+I206Y+T251E+N460G,

N25H+R176K+G186E+T193E+I206Y+T251E+N460G+Y482W,

G4Q+N25H+R176K+G186E+I206Y+T251E+N460G+Y482W,

N25H+R176K+G186E+N195F+T251E+Y482W,N25H+R176K+G186E+N195F+Y482W,

N25H+R176K+G186E+T193E+N195F+T251E+Y482W,

G4Q+R176K+G186E+T193E+N195F+T251E+L405M+Y482W,

N25H+R176K+G186E+T193E+N195F+Y482W,

G4Q+N25H+R176K+G186E+N195F+T251E+L405M+Y482W,

N25H+G186E+N195F+L405M+Y482W,N25H+R176K+G186E+T193E+N195F+T251E+N460G,

G4Q+N25H+R176K+G186E+N195F+T251E+N460G,

N25H+R176K+G186E+T193E+N195F+T251E+N460G+Y482W,

G4Q+N25H+R176K+G186E+N195F+T251E+N460G+Y482W,

G4Q+N25H,

G4Q+N25H+R176K,

G4Q+N25H+R176K+G186E,

G4Q+N25H+R176K+G186E+T251E,

G4Q+N25H+R176K+G186E+T251E+L405M+W439K,

G4Q+N25H+R176K+G186E+T251E+L405M+Y482W,

G4Q+N25H+R176K+G186E+T251E+L405M+W439K+Y482W,

G4Q+N25H+R176K+T251E,

G4Q+N25H+R176K+T251E+L405M+W439K,

G4Q+N25H+R176K+T251E+L405M+Y482W,

G4Q+N25H+R176K+T251E+L405M+W439K+Y482W,

N25H+R176K,

N25H+R176K+G186E,

R176K+G186E,

N25H+R176K+G186E+T251E,

N25H+R176K+G186E+T251E+L405M,

N25H+R176K+G186E+T251E+L405M+Y482W,

N25H+R176K+G186E+T251E+L405M+W439K+Y482W,

N25H+R176K+T251E,

N25H+R176K+T251E+L405M,

N25H+R176K+T251E+L405M+Y482W,

N25H+R176K+T251E+L405M+W439K+Y482W,

X116K+X181T,

X116K+X181T+X225A,

x1k+x181 t+x225a+x320K, and

x181t+x225a+x320K, more preferably X116k+x181t+x225A, X181t+x225a+x320K, X116k+x181t+x225a+x320K, n25h+r17kj+g186E, n25h+r17kj+g186 e+t251 e+l404m+y482W or g4q+n25h+r17k+g180e+t251 e+l404m+y482W, most preferably n25h+r17k+g186E, g4q+n25h+r17kj+g1200e+l406m+y404w or x1k+g200a+x320K.

Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

The alpha-amylase variants of the invention having one or more amino acid changes (preferably substitutions) described herein and having alpha-amylase activity preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence of the parent amylase.

The alpha-amylase variants of the invention having one or more amino acid substitutions described herein and having alpha-amylase activity preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence of the parent amylase.

Preferably, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions as described herein, preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence shown in any of SEQ ID NO:1, 3, 4, more preferably SEQ ID NO:1 or 3, most preferably SEQ ID NO: 1.

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions as described herein, preferably has at least 91% or at least 95% but less than 100% sequence identity to the amino acid sequence shown in any of SEQ ID NO:1, 3, 4, or SEQ ID NO 15-41, preferably SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, more preferably SEQ ID NO:1 or 3, most preferably SEQ ID NO: 1.

In one embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1.

In one embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 3.

In one embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4.

In one embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 15.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 17.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 19.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 20.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 21.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 22.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 23.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 24.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 25.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 26.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 27.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 28.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 29.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 30.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 31.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 32.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 33.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO 34.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 35.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 36.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO 37.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 38.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO 39.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 40.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 41.

Preferably, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4.

Preferably, an alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO 3.

More preferably, the alpha-amylase variant of the invention having alpha-amylase activity and having one or more amino acid substitutions described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1.

The alpha-amylase variant of the invention having alpha-amylase activity preferably has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity or sequence similarity to the amino acid sequence set forth in SEQ ID NO. 1.

The alpha-amylase variant of the invention having alpha-amylase activity preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1.

The alpha-amylase variant of the invention having alpha-amylase activity preferably has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises:

(a) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% sequence identity to SEQ ID No. 1;

(b) An amino acid sequence encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to SEQ ID No. 2;

(c) An amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions to the complement of:

(i) The coding sequence of SEQ ID NO. 1; or (b)

(ii) A polynucleotide shown in SEQ ID NO. 2;

(d) An amino acid sequence encoded by a polynucleotide having at least 95% but less than 100% sequence identity to SEQ ID NO. 2, wherein the polynucleotide differs further from SEQ ID NO. 2 only by the degeneracy of the genetic code, or

(e) Fragments of (a), (b) or (c) having amylase activity.

Particularly preferred alpha-amylase variants of the invention having alpha-amylase activity have at least 91.0%, at least 91.5%, at least 92.0%, at least 92.5%, at least 93.0%, at least 93.5%, 94.0%, at least 94.5%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% but less than 100% sequence identity or sequence similarity to the amino acid sequence set forth in SEQ ID NO. 1. The alpha-amylase variant of the invention having alpha-amylase activity preferably has at least 91.0%, at least 91.5%, at least 92.0%, at least 92.5%, at least 93.0%, at least 93.5%, 94.0%, at least 94.5%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1.

The alpha-amylase variant of the invention having alpha-amylase activity preferably has at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% but less than 100% sequence identity or sequence similarity to the amino acid sequence set forth in SEQ ID NO. 1.

The alpha-amylase variant of the invention having alpha-amylase activity preferably has at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1.

The alpha-amylase variants of the invention having alpha-amylase activity preferably have at least 95.0% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1.

The alpha-amylase variant of the invention having alpha-amylase activity preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 1 and comprises an A and B domain and a C domain, wherein the amino acid sequence of the A and B domains is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identical to the amino acid sequence of SEQ ID NO. 6 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identical to the amino acid sequence of SEQ ID NO. 8.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of any of SEQ ID NO:1, 3, 4, or SEQ ID NO:15-41, preferably the amino acid sequence shown in SEQ ID NO:1, and wherein there are one or more, preferably 1-50, preferably 1-30, preferably 1-25, preferably 1-20, preferably 1-15, preferably 1-10, or preferably 1-5, of the above amino acid substitutions. In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of any of SEQ ID NO:1, 3, 4, or SEQ ID NO:15-41, preferably the amino acid sequence shown in SEQ ID NO:1, and wherein there are one or more, preferably 1-50, preferably 1-30, preferably 1-25, preferably 1-20, preferably 1-15, preferably 1-10, or preferably 1-5 amino acid substitutions as described above and 1-50, preferably 1-30, 1-20, 1-10, or 1-5 additional amino acid changes, preferably amino acid substitutions. In one embodiment, the 1-50 additional amino acid substitutions are conservative amino acid substitutions, preferably outside any functional domain, preferably outside the catalytically active domain, of the parent alpha-amylase. Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of any of SEQ ID NO. 1, 3, 4, or SEQ ID NO. 15-41, preferably the amino acid sequence shown in SEQ ID NO. 1, and wherein there are one or more amino acid substitutions as described above and 1-30, 1-20, 1-10 or 1-5 additional amino acid changes, preferably amino acid substitutions. In one embodiment, the 1-30, 1-20, 1-10 or 1-5 additional amino acid substitutions are conservative amino acid substitutions, preferably outside any functional domain, preferably outside the catalytically active domain, of the parent alpha-amylase.

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of any of SEQ ID NO:1, 3, 4, or SEQ ID NO:15-41, preferably the amino acid sequence shown in SEQ ID NO:1, and wherein there are one or more of the above amino acid substitutions and there are 1-30, 1-20, 1-10 or 1-5 additional amino acid substitutions outside the catalytically active structure. Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of any of SEQ ID NO:1, 3, 4, or SEQ ID NO:15-41, preferably the amino acid sequence shown in SEQ ID NO:1, and wherein there are one or more of the above amino acid substitutions and 1-30, 1-20, 1-10 or 1-5 additional conservative amino acid substitutions, preferably outside the catalytically active structure.

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO. 1 and wherein there are one or more, preferably 1-50, preferably 1-30, preferably 1-25, preferably 1-20, preferably 1-15, preferably 1-10 or preferably 1-5 amino acid substitutions as described above. In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO. 1 and wherein there are one or more, preferably 1-50, preferably 1-30, preferably 1-25, preferably 1-20, preferably 1-15, preferably 1-10 or preferably 1-5 amino acid substitutions as described above and 1-50, preferably 1-30, 1-20, 1-10 or 1-5 additional amino acid changes, preferably amino acid substitutions. In one embodiment, the 1-50 additional amino acid substitutions are conservative amino acid substitutions, preferably outside any functional domain, preferably outside the catalytically active domain, of the parent alpha-amylase. Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO. 1 and wherein there are one or more amino acid substitutions as described above and 1-30, 1-20, 1-10 or 1-5 additional amino acid changes, preferably amino acid substitutions. In one embodiment, the 1-30, 1-20, 1-10 or 1-5 additional amino acid substitutions are conservative amino acid substitutions, preferably outside any functional domain, preferably outside the catalytically active domain, of the parent alpha-amylase.

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO. 1, wherein there are one or more of the above amino acid substitutions and there are 1-30, 1-20, 1-10 or 1-5 additional amino acid substitutions outside the catalytically active domain. Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO. 1, wherein there are one or more of the above amino acid substitutions and there are 1-30, 1-20, 1-10 or 1-5 additional conservative amino acid substitutions outside the catalytically active domain.

The structure of alpha-amylase comprises three distinct domains A, B and C, see, e.g., machius et al, 1995, J.mol. Bio/.246:545-559. The alpha-amylase variants described herein may further comprise one or more non-catalytic CBMs (sugar binding modules, also referred to as sugar binding domains or, in particular for amylases, starch binding domains). CBM can improve binding of enzymes to substrates. The CBM is attached to the C domain. Preferably, the amylase of the invention does not comprise a sugar binding domain. Preferably, the alpha-amylase variants of the invention consist of only three domains, A, B and C domains.

As used herein, the "A and B domains" or "AB domain" of an alpha-amylase corresponds to amino acids aligned with amino acids 1-399 of SEQ ID NO. 3. As used herein, the "C domain" of an alpha-amylase corresponds to amino acids aligned with amino acids 400-485 of SEQ ID NO. 3.

Preferably, the α -amylase variant comprising one or more of the above amino acid changes, preferably insertions, deletions, substitutions or combinations thereof, preferably a substituted, is a hybrid amylase comprising domains from different parent amylases, in particular its AB domain and its C domain from different parent amylases. The alpha-amylase variants may be produced by replacing the C domain of one alpha-amylase or a portion thereof with the C domain of another alpha-amylase or a portion thereof. Preferably, the A and B domains of the alpha-amylase variants described herein have at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity, to the A and B domains of the alpha-amylase of SEQ ID NO 3.

Preferably, the A and B domains of the alpha-amylase variants described herein have at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity to SEQ ID NO. 6, meaning that the amino acid sequences forming the A and B domains have at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to SEQ ID NO. 6.

Preferably, the C domain of the alpha-amylase variant described herein comprises a C domain having at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity to the C domain of the alpha-amylase of SEQ ID No. 5.

Preferably, the C domain of the alpha-amylase variants described herein has at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 or at least 99% but less than 100% identity to SEQ ID NO. 8.

In one embodiment of the invention, the amino acid sequences forming the A and B domains have at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity with the amino acid sequence of SEQ ID NO. 6, and the amino acid sequences forming the C domains have at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity with SEQ ID NO. 8.

In one embodiment of the invention, the amino acid sequences forming the A and B domains have 100% identity to the amino acid sequence of SEQ ID NO. 6, and the amino acid sequence forming the C domain has at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity to SEQ ID NO. 8.

In one embodiment of the invention, the amino acid sequences forming the A and B domains have at least 75% identity, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity to the amino acid sequence of SEQ ID NO. 6, and the amino acid sequence forming the C domain has 100% identity to SEQ ID NO. 8.

In one embodiment, the invention relates to a method of preparing an alpha-amylase variant, comprising the steps of: preparing a hybrid from at least two different amylases, wherein the hybrid comprises an a and B domain and a C domain, and wherein the amino acid sequence of the a and B domains has at least 75%, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO:6, and the amino acid sequence of the C domain has at least 75%, preferably at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 8; and introducing one or more amino acid changes described herein into the hybrid.

In one embodiment, the alpha-amylase variant of the invention exhibits one or more improved properties compared to the parent alpha-amylase, preferably compared to the alpha-amylase shown in SEQ ID NO. 1 and/or SEQ ID NO. 3, preferably compared to the alpha-amylase shown in SEQ ID NO. 1.

Preferably, the improved property is expressed as an Improvement Factor (IF) of > 1.0. Preferably, the improvement is expressed as an improvement factor for improved heat stability and improved wash performance. Preferably, the improvement factor is equal to or greater than 1.1, equal to or greater than 1.2, equal to or greater than 1.3, equal to or greater than 1.4, equal to or greater than 1.5, equal to or greater than 1.6, equal to or greater than 1.7, equal to or greater than 1.8, equal to or greater than 1.9, or equal to or greater than 2.0. Preferably, the IF of the wash performance is equal to or greater than 1.1, equal to or greater than 1.2, or equal to or greater than 1.3. Preferably, the IF for thermal stability is equal to or greater than 1.1, equal to or greater than 1.2, equal to or greater than 1.3, equal to or greater than 1.5, or equal to or greater than 2.0.

Alternatively, the improvement in amylase properties is expressed as a percentage improvement over the parent alpha-amylase. Preferably, the alpha-amylase variant of the invention exhibits at least 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200% improved properties compared to the parent alpha-amylase, preferably compared to the alpha-amylase shown in SEQ ID NO 1 or SEQ ID NO 3, preferably compared to SEQ ID NO 1.

Alternatively, especially for stability improvement, the improvement in amylase properties is expressed as residual activity after stability challenge. Preferably, storage stability is expressed as residual activity after storage under the respective storage conditions, preferably after storage in a detergent composition (preferably in a laundry detergent or a dishwashing detergent, preferably a laundry detergent). Preferably, the residual activity of the alpha-amylase variant is increased compared to the parent amylase. Preferably, the residual activity of the alpha-amylase variant is improved by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200% compared to the parent amylase, preferably compared to the amylase shown in SEQ ID No. 1 and/or 3, preferably compared to SEQ ID No. 1. Residual activity compared to the parent amylase may also be converted into an improvement factor by forming a ratio of residual activity of the variant to residual activity of the parent amylase.

Preferably, the improved property is an improvement in one or more properties selected from the group consisting of: expression, activity, stability, thermal stability, specific activity, substrate specificity, pH dependent activity, pH stability, oxidation stability, catalytic efficiency, catalytic rate, chemical stability, pH activity, stability under storage conditions, substrate binding, substrate cleavage, substrate stability, surface properties, thermal activity, ca2+ dependence, performance in detergents, performance in laundry detergents, and performance in ADW detergents. Preferably, the improved activity is improved specific activity, substrate specificity, pH dependent activity, catalytic efficiency, catalytic rate, pH activity, substrate binding, substrate cleavage, thermal activity, ca2+ dependency, wash performance of laundry detergents and/or wash performance of ADW detergents. Preferably, the improved stability is improved thermal stability, thermal stability in a detergent composition (preferably in a laundry detergent or a dishwashing detergent composition, preferably in a laundry detergent composition), pH stability, oxidative stability, chemical stability, stability under storage conditions, substrate stability and/or thermal activity. Preferably, the improved properties are improved expression, improved solubility, improved thermal stability in the detergent composition, improved storage stability in the detergent composition and/or improved wash performance. Preferably, the improved property is improved heat stability, preferably improved heat stability in the detergent composition, improved stability in storage in the detergent composition, and/or altered wash performance. Preferably, the improved properties are improved heat stability, improved heat stability in the detergent composition, improved stability under storage conditions, preferably improved stability in storage in the detergent composition, preferably improved stability in storage in a laundry detergent and/or improved stability in storage in an ADW detergent, improved wash performance, preferably improved wash performance of a laundry detergent and/or improved wash performance of an ADW detergent.

The alpha-amylase variants described herein may be active over a wide temperature range, wherein the temperature is anywhere in the range of about 10 ℃ to about 95 ℃. The alpha-amylase variant may be active at a temperature in the range of 10 ℃ to 55 ℃, 10 ℃ to 50 ℃, 10 ℃ to 45 ℃, 10 ℃ to 40 ℃, 10 ℃ to 35 ℃, 10 ℃ to 30 ℃, or 10 ℃ to 25 ℃. The variant polypeptide may be active at a temperature range of 20 ℃ to 55 ℃, 20 ℃ to 50 ℃, 20 ℃ to 45 ℃, 20 ℃ to 40 ℃, 20 ℃ to 35 ℃, 20 ℃ to 30 ℃, or 20 ℃ to 25 ℃.

In one embodiment, the alpha-amylase variant described herein has improved thermostability, preferably improved thermostability in a detergent composition (preferably in a laundry detergent or dishwashing detergent composition, preferably in a laundry detergent composition), compared to the parent alpha-amylase. In another embodiment, the thermostability of the variant polypeptide is improved by 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃ or more than the parent alpha-amylase. In another embodiment, the increase in thermal stability is measured at a temperature between 40 ℃ and 100 ℃. In one embodiment, the thermal stability is improved at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ or 90 ℃, preferably at 60 ℃ or 70 ℃. In another embodiment, the thermal stability is improved in a temperature range between 40 ℃ and 90 ℃, preferably between 60 ℃ and 85 ℃, preferably between 60 ℃ and 80 ℃.

Thus, preferably, the improved property is improved stability, preferably thermal stability. Preferably, the alpha-amylase variant of the invention exhibiting improved stability, preferably thermostability, compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, preferably a substitution, at one or more positions corresponding to positions (numbering according to SEQ ID NO: 3): 1,2,3,4,5,6,7,8,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,45,47,48,59,60,63,70,71,72,73,75,76,81,82,83,86,89,90,91,92,93,94,95,96,97,98,99,100,103,106,108,109,113,114,115,117,119,125,126,128,129,131,132,133,134,136,139,141,142,143,144,145,146,147,149,150,151,154,155,156,160,161,162,164,165,170,172,173,174,175,176,184,188,191,203,210,211,212,213,214,215,216,218,219,221,222,223,224,225,226,227,230,231,233,234,235,236,237,238,240,242,243,245,249,250,251,252,253,255,256,257,259,261,262,263,264,265,268,270,272,273,274,276,279,280,284,285,286,287,288,289,290,291,292,293,294,295,296,297,299,299,301,302,303,304,306,307,308,309,310,311,312,313,314,315,318,318,319,320,321,323,324,326,327,336,337,338,341,342,343,344,345,346,348,350,351,352,353,354,355,357,359,360,362,363,364,366,367,368,370,372,375,376,378,379,381,382,383,384,385,387,388,389,390,391,392,393,394,396,397,398,399,400,401,403,405,406,407,408,410,413,414,415,416,418,421,424,426,427,429,431,436,438,439,440,442,443,445,446,447,448,449,451,453,455,456,457,458,459,460,461,462,463,465,467,468,470,474,478,480, and 482.

Preferably, the alpha-amylase variant of the invention exhibiting improved stability, preferably thermostability, compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising one or more amino acid substitutions selected from the group consisting of the following amino acid substitutions (numbering according to SEQ ID NO: 3): X1G, X1V, X1R, X2S, X2I, X3Q, X3K, X3F, X3G, X3L, X4Q, X4R, X4S, X4K, X4M, X4A, X4C, X5F, X5E, X5M, X5Y, X5R, X5Q, X5K, X5L, X5A, X5V, X5C, X5N, X5H, X5D, X5P, X5I, X6T, X6E, X6A, X6K, X6Q, X6V, X6M, X6G, X6L, X6H, X6W, X6F, X6P, X6S, X6C, X6Y, X7Y, X7S, X7H, X7C, X7Q, X7F, X7P, X7W, X7R, X7K, X7E, X7V, X7N, X8C, X8V, X8F, X8Y, X8P, X8W, X8A, X8S, X8M, X6K, X6Q, X6V, X6M, X6G, X6L, X6H, X6W, X6F, X6P, X6S, X6C, X6Y, X7Y, X7S, X7H, X7C, X7Q, X7F, X7P, X7W, X7R, X7K, X7E, X7V, X7N, X8C, X8V, X8F, X8Y, X8P, X8W, X8A, X8S, X8M, X22M, X22T, X22K, X22Y, X22G, X22R, X22Q, X22W, X22L, X23N, X23Q, X23W, X24G, X25H, X25Y, X25K, X25F, X25S, X25C, X25D, X25A, X25G, X25W, X25Q, X25M, X25L, X26S, X26V, X26M, X26A, X26D, X26E, X26F, X26P, X26Y, X26L, X27D, X27I, X27F, X27R, X27G, X27Q, X27V, X27T, X27A, X27H, X28N, X28S, X28F, X28C, X28E, X28T, X28G, X28K, X28D, X28V, X28I, X28Q, X28Y 29X 29Q, X29L, X29W, X29K, X29F, X29N, X29H, X29D, X29V, X29G, X29Q, X29I, X30F, X30I, X30A, X30K, X30W, X30Y, X30M, X30L, X30H, X30Q, X30G, X30E, X30T, X31S, X31V, X31Q, X31T, X31N, X31W, X32T, X32H, X32W, X32F, X32Q, X32I, X32A, X32P, X32E, X32M, X32N, X32D, X32L, X33Y, X33D, X33M, X33Q, X33K, X33R, X34H, X34V, X34I, X35C, X35Y, X35P, X35T, X35M, X35H, X35A, X35V, X35I, X35G 35M, X35G, X35N, X35R, X35S, X35L, X36V, X36I, X36T, X36K, X36A, X36S, X36R, X36M, X36E, X36P, X36G, X36N, X36Q, X37P, X37V, X37W, X37A, X37G, X38N, X39E, X39K, X40I, X40S, X41G, X41Q, X41C, X41D, X41E, X41S, X41T, X42Q, X42V, X42I, X42C, X45N, X45G, X47S, X48F, X48I, X48M, X51T, X59T, X60T, X63V, X63C, X70N, X70L, X70M, X70F, X70Y, X71D, X72T, X72E, X72D, X72N, X72C, X73Q, X73L, X73N, X75A, X75I, X75W, X75P, X75G, X75T, X76G, X76T, X76C, X76V, X76E, X76L, X81H, X82M, X82K, X83G, X83R, X83S, X86K, X89F, X89A, X89C, X89L, X89G, X89M, X89S, X89R, X90Q, X90F, X90G, X90E, X90A, X90N, X90I, X90R, X90Y, X90V, X90S, X90M, X90D, X91S, X91Y, X91K, X91Q, X91F, X91H, X91T, X91I, X91W, X91N, X91V, X91L, X91C, X91G, X91D, X91E, X91M, X92V, X92M, X92D, X93K, X93Q, X94M, X94D, X94V 95V, X95L, X96N, X96K, X97V, X98G, X98E, X98N, X99K, X99H, X99N, X100D, X100K, X100L, X100F, X103A, X106D, X108A, X109A, X113Y, X113H, X113M, X113V, X113F, X113W, X113R, X114D, X114H, X114N, X114W, X114E, X114C, X114G, X114L, X114M, X114Q, X114F, X114A, X114V, X114K, X114S, X114Y, X114R, X114I, X115C, X115K, X115N, X115T, X115M, X115S, X115Q, X115R, X115D, X115V, X116L, X117D, X117Y, X117N, X117C, X117R, X117W, X117A, X117W, X117F, X119Y, X119R, X119P, X119H, X125A, X125K, X125L, X125T, X125R, X125F, X125W, X125Q, X125V, X125Y, X125H, X125N, X125E, X125G, X125M, X126D, X128C, X128L, X128Y, X128E, X128W, X128M, X129E, X131I, X131C, X132T, X133E, X133P, X133C, X133D, X133Q, X133S, X133H, X133A, X133N, X133K, X134Y, X134W, X134F, X134P, X134E, X134V, X134L, X134M, X134I, X135M, X D, X136E, X136F, X136M, X136P, X136H 136C, X136A, X136W, X136L, X136N, X139C, X141V, X142C, X142W, X142Y, X142L, X142M, X142Q, X142F, X142E, X142R, X143F, X144Q, X144C, X144V, X144K, X144T, X144Y, X144E, X144G, X144N, X144S, X144R, X145A, X146C, X146H, X146K, X146T, X146E, X146S, X146D, X146W, X146F, X146L, X146G, X147M, X149E, X150Q, X150E, X150C, X151C, X151E, X154Y, X154A, X155Y, X156V, X160W, X161T, X164V, X165W, X144E, X169E, X170V, X170F, X172C 172W, X172C 173W, X172A, X172C, X174M, X174E, X174S, X174H, X175G, X175Q, X175H, X176K, X188V, X188H, X191K, X191H, X191F, X191W, X191Y, X203E, X203R, X210D, X210C, X210E, X210N, X210F, X210Y, X210M, X210Q, X210S, X210A, X211D, X211N, X211C, X211T, X211E, X211G, X211V, X211Q, X211A, X211H, X211L, X211S, X212W, X212L, X212I, X212D, X212C, X213A, X213S, X213M, X213R, X213C, X214P, X214E, X215D, X215H, X215W 215A, X216H, X216G, X218G 218E, X218C 218F, X218S, X218G, X218K, X218I, X218T, X218D, X218Y, X218H, X218L, X218V, X218A, X218W, X219E, X219Q, X219K, X219T, X219M, X219D, X219F, X219S, X219R, X219A, X219Y, X219H, X219W, X219G, X219C, X221N, X221F, X222S, X222T, X222D, X223V, X223Y, X223L, X223C, X224V, X224F, X225A, X225R, X225N, X225E, X225H, X225F, X225C, X225Y, X225I, X226C, X226W, X226I, X226R, X226Y, X226D, X226L, X226M, X226G, X226T, X226Q, X226S 226A, X226C, X226W, X226E, X226C, X226W, X226E, X226C, X226E, X227T, X227R, X227M, X227K, X227Y, X227V, X227F, X227I, X227H, X227L, X227G, X230A, X230F, X231N, X231C, X233T, X233W, X233H, X233C, X233I, X233Y, X233M, X234C, X235M, X235V, X235L, X236Y, X237C, X237I, X238T, X240M, X242M, X243F, X245E, X245M, X245H, X249D, X249I, X250V, X251E, X251A, X251L, X251S, X251M, X251F, X252C, X252I, X252S, X253G, X255T, X255V, X255D, X255F, X255I, X256C, X257Y, X257L, X262D, X262P, X262E, X262H, X263I, X264T, X264W, X264H, X265S, X268G, X270A, X270Y, X272L, X272G, X273H, X273V, X273L, X273M, X273C, X273D, X273W, X273F, X273A, X273E, X273P, X273Y, X273R, X273I, X274S, X276Y, X276K, X276L, X276D, X279P, X280I, X280V, X280N, X280H, X280Y, X280K, X280R, X284Y, X284A, X284L, X284F, X284H, X284N, X284M, X285N, X285L, X285G, X286Q, X287E, X H, X287A, X287D, X287T, X289K, X288P, X288A, X288F, X289G, X290Q, X290N, X290M, X290D, X290W, X291D, X291Y, X291F, X299L, X292W, X292Y, X292F, X292D, X292L, X292I, X292T, X292C, X293D, X293R, X293K, X293E, X293F, X294T, X294G, X295F, X296Y, X296L, X296C, X296A, X297M, X297K, X297S, X297H, X297V, X297E, X297F, X299L, X299G, X299I, X299K, X299S, X299Y, X301F, X302H, X302I, X302Q, X302Y, X302V, X303P, X303M, X303E, X303I, X303R, X303K, X303N, X303L, X303H, X304R 304, X304M, X304E, X304P, X304W, X304K, X304T, X306M, X306E, X306A, X306G, X306W, X306V, X306S, X306I, X306H, X306T, X306D, X306Q, X306Y, X306R, X307F, X307M, X308S, X309H, X309L, X309Q, X310Q, X310A, X311G, X311E, X311H, X311A, X311K, X311T, X311N, X311R, X311Y, X312M, X312L, X313V, X314C, X314Q, X315C, X315A, X315H, X315T, X315E, X315K, X318T, X318S, X318I, X319K, X319P, X319A, X319I, X319D 319M, X311M, X320T 320G, X320M, X320L, X320N, X320A, X320D, X320K, X320Q, X320E, X320H, X320C, X320S, X321E, X321W, X321T, X321A, X321N, X321V, X321K, X323K, X323G, X324W, X324K, X324Y, X326Y, X326N, X326G, X326S, X327M, X327C, X327L, X333I, X336K, X337N, X337K, X337M, X337T, X337V, X337Q, X337S, X337R, X337I, X337Y, X337G, X337F, X337A, X337L, X338G, X338T, X338S, X343W, X343Y, X344I, X344Q, X344V, X N, X345T, X345G, X345M 345G 345D 345, X345D 345, X346H, X346A, X346G, X346Q, X346N, X348T, X350P, X350H, X350K, X351M, X351A, X352S, X353H, X354Y, X354N, X354T, X354I, X355I, X355M, X357A, X358P, X359E, X360N, X360R, X360S, X360Y, X360V, X360L, X360A, X360I, X360T, X360G, X360F, X362T, X362M, X362V, X362N, X362Y, X362F, X362K, X363M, X363G, X363S, X363V, X363C, X363T, X363H, X363P, X363D, X363L, X363R, X363Q, X363E, X363Y, X363K, X363W, X363A, X364G, X364T, X364S, X364A, X364V, X364C, X364K, X364L, X362T, X362M, X362V, X362N, X362Y, X362F, X362K, X363M, X363G, X363S, X363V, X363C, X363T, X363H, X363P, X363D, X363L, X363R, X363Q, X363E, X363Y, X363K, X363W, X363A, X364G, X364T, X364S, X364A, X364V, X364C, X364K, X364L, X384Y, X384V, X384R, X384N, X384E, X384A, X384D, X384T, X384C, X384F, X384L, X384I, X384M, X384Q, X385G, X385V, X385T, X385Y, X385I, X385D, X385C, X385Q, X385A, X385W, X385L, X385H, X385M, X385N, X385S, X385F, X385E, X385R, X385P, X387N, X387C, X387E, X388R, X388F, X388H, X388V, X388E, X388M, X388I, X389G, X389K, X389H, X390P, X390N, X390R, X390F, X390M, X390D, X391T, X391F, X391S, X391A, X391G, X391M, X391N, X391Y, X391Q, X391K, X392C, X392V, X393V, X393E, X393H, X393S, X393P, X394C, X394L, X394R, X394S, X394A, X394E, X394N, X394M, X394H, X394I, X395V, X395M, X395A, X395H, X396P, X396H, X397P, X397D, X397S, X397H, X398M, X399P, X400L, X400P, X400I, X400S, X400A, X400R, X400Q, X400D, X400K, X400N, X400V, X400W, X400H, X400E, X400G, X400M, X401K, X401T, X401I, X403N, X405H, X405V, X405T, X405C, X406P, X407S, X407R, X407D, X408H, X410L, X413S, X414E, X414C, X414S, X415I, X415E, X416S, X418C, X418P, X418N, X421P, X421N, X424A, X426D, X426W, X427Q, X427C, X427F, X427T, X427V, X427G, X427S, X427R, X427K, X429M, X429D, X429E, X429N, X429T, X429A, X429P, X429W, X429Q, X429S, X429F, X429G, X429I, X429V, X431I, X436E, X438P, X438F, X438Y, X439K, X439C, X439P, X440V, X442Q, X443T, X443H, X445M, X445T, X445A, X445G, X445S, X445F, X445R, X445Q, X445C, X445D, X445K, X445H, X445V, X446P, X446I, X446V, X446F, X446L, X446R, X446Q, X446W, X447V, X448E, X448H, X448N, X448D, X449V, X449Y, X449F, X449L, X449P, X449G, X449W, X449M, X449N, X449K, X449Q, X449S, X449A, X451L, X453G, X453I, X453P, X453Y, X453N, X455L, X456M, X456S, X456Y, X456V, X456L, X456W, X456R, X457K, X457C, X457L, X457S, X457R, X457F, X457T, X457A, X457V, X457W, X457E, X457M, X457G, X458V, X458W, X458I, X458K, X459Q, X459D, X459I, X459K, X459E, X459Y, X460E, X460Q, X460S, X460R, X460A, X460T, X460D, X460V, X461A, X461R, X461F, X461E, X461S, X461V, X461M, X461K, X461N, X461Q, X462K, X463L, X465P, X465V, X465H, X465R, X467A, X467E, X467H, X468H, X468D, X470Q, X470T, X470A, X474P, X474H, X474F, X478A, X478E, X480Y, X480M, X480E, X480D, X480R, X482T, and X482C.

Preferably, the improved property is improved stability in a detergent composition, preferably in a laundry detergent composition, preferably improved thermal stability in a detergent composition. Preferably, the alpha-amylase variant of the invention exhibiting improved stability, preferably thermostability, in detergent compositions compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising an amino acid substitution at one or more positions (numbering according to SEQ ID NO: 3) corresponding to positions selected from the group consisting of: 4,6,20,25,27,28,33,38,41,70,72,73,75,81,93,94,96,115,125,126,128,134,139,160,174,175,176,210,213,250,251,253,276,291,292,299,314,318,319,323,326,338,343,352,356,359,363,364,368,372,378,379,382,385,387,388,390,393,396,398,400,405,418,434,447,449,459,460,461, and 465.

Preferably, the alpha-amylase variant of the invention exhibiting improved stability, preferably thermostability, in detergent compositions compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising one or more amino acid substitutions selected from the group consisting of the following amino acid substitutions (numbering according to SEQ ID NO: 3): X4Q, X6E, X6H, X20K, X25H, X25Y, X27I, X28V, X33M, X38V, X41C, X70H, X72C, X73C, X75L, X81L, X93Q, X94M, X96Q, X115T, X125K, X125R, X126D, X128C, X134F, X134Y, X139C, X160W, X174H, X175A, X176K, X210C, X210D, X213C, X250I, X251E, X253C, X276R, X291T, X292C, X299S, X314Q, X318L, X319K, X323G, X326Y, X338S, X338T, X343W, X352C, X356V, X359W, X363M, X363S, X364T, X368F, X372C, X378E, X379A, X379R, X382Q, X385W, X387E, X387N, X387S, X388E, X388K, X388Y, X390F, X393Q, X396S, X398G, X400A, X400S, X405M, X418F, X434S, X434R, X447L, X449G, X459N, X460G, X461L, X461A, and X465H.

Preferably, the alpha-amylase variant of the invention exhibiting improved stability, preferably thermostability, in detergent compositions compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising a combination of substitutions (numbering according to SEQ ID NO: 3) selected from the group consisting of:

X25H+X176K+X186E+X206Y+X251E+X482W,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X482W,

X25H+X176K+X186E+X206Y+X482W,X25H+X186E+X206Y+X405M+X482W,

X25H+X186E+X206Y+X482W,X25H+X176K+X186E+X193E+X206Y+X251E+X482W,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X482W,X25H+X176K+X186E+X482W,

X25H+X176K+X186E+X193E+X206Y+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,X25H+X176K+X186E+X206Y,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,X4Q+X206Y+X460G+X482W,

X4Q+X25H+X176K+X206Y+X251E+X460G+X482W,

X4Q+X25H+X176K+X193E+X251E+X186E+X482W,

X4Q+X193E+X206Y+X276R+X186E+X482W,X186E+X25H+X482W,

X25H+X193E+X206Y+X251E+X276R+X405M+X186E+X482W,X25H+X251E+X186E+X482W,

X25H+X160W+X176K+X186E+X251E+X405M+X482W,

X193E+X206Y+X405M+X186E+X482W,X3I+X356V,X3I+X356I,X83D+X94E,X94D+X125E,

X131I+X377Q+X410H,X48F+X94D,X48Y+X116K+X218K,X5L+X218K+X225S,

X83E+X116R+X158Y+X181E,X51V+X218K,X83E+X181E,

X4Q+X7W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X37M+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X460G,

X4Q+X25H+X176K+X186E+X206Y+X251E+X460G,

X4Q+X25Y+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X118D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X182D+X186E+X193E+X206Y+X251E+X405M,

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X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X363H+X254Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X8A,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X281N+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X363H,X25H+X176K+X186E+X206Y,

X25H+X176K+X186E+X206Y+X258Q,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X258Q+X281N,

X25H+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E+X123D,X25H+X176K+X186E+X206Y+X281N,

X25H+X176K+X186E+X206Y+X181Q,X25H+X176K+X186E+X206Y+X181Q+X193E,

X25H+X176K+X186E+X206Y+X181Q+X363E,X25H+X176K+X186E+X206Y+X193E,

X25H+X176K+X186E+X206Y+X193E+X258Q,

X25H+X176K+X186E+X206Y+X193E+X281N+X363E,

X25H+X176K+X186E+X206Y+X193E+X363E,X25H+X176K+X186E+X206Y+X100W,

X25H+X176K+X186E+X206Y+X100W+X258Q,

X25H+X176K+X186E+X206Y+X100W+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X258Q+X363E+X135C,

X25H+X176K+X186E+X206Y+X100W+X281N,

X25H+X176K+X186E+X206Y+X100W+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X361R,

X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E+X175D,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X473R,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X363E,

X25H+X176K+X186E+X206Y+X100W+X363E,X25H+X176K+X186E+X206Y+X135T,

X25H+X176K+X186E+X206Y+X135T+X258Q,

X25H+X176K+X186E+X206Y+X135T+X258Q+X281N,

x25h+x176k+x186e+x206y+x135t+x258q+x281n+x363E, and

X25H+X176K+X186E+X206Y+X135T+X258Q+X363E。

preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Preferably, the improved property is improved wash performance. Preferably, the alpha-amylase variant of the invention, which exhibits improved wash performance compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising an amino acid substitution at one or more positions corresponding to positions selected from the group consisting of SEQ ID NO: 3: 3,4,5,6,7,20,25,33,36,37,41,51,54,72,73,75,81,82,83,94,95,96,98,100,116,118,119,120,123,126,131,135,136,139,156,158,160,163,165,169,172,174,176,177,178,180,182,185,187,188,189,191,193,208,210,218,219,222,224,225,231,238,242,243,251,252,253,258,260,268,269,270,271,272,273,274,275,276,277,279,280,281,282,286,297,298,299,311,314,317,318,320,321,322,323,324,326,337,343,344,350,352,356,357,358,359,360,362,363,364,365,368,370,372,378,379,382,385,387,388,390,391,393,395,396,397,400,405,410,429,434,439,456,459,460,461,463, and 465.

Preferably, the alpha-amylase variant of the invention exhibiting improved wash performance compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising one or more amino acid substitutions (numbering according to SEQ ID NO: 3) selected from the group consisting of: X100W, X116I, X116M, X118A, X118D, X118E, X119S, X120E, X120G, X120M, X120S, X120W, X120Y, X126D, X131Q, X135D, X135E, X135G, X135N, X135N, X135P, X135S, X135T, X135T, X135W, X136E, X139S, X156E, X160D, X160E, X163A, X163Q, X163T, X165S, X165T, X169D, X169S, X174E, X174G, X174H, X174H, X174M, X174N, X176S, X176T, X177A, X177G, X177K, X177N, X177P, X177R, X177S, X177W, X178F, X180M, X180N, X180T, X180W, X182D, X182E, X182N, X182Q, X182S, X185E, X163T, X165S, X165T, X169D, X169S, X174E, X174G, X174H, X174H, X174M, X174N, X176S, X176T, X177A, X177G, X177K, X177N, X177P, X177R, X177S, X177W, X178F, X180M, X180N, X180T, X180W, X182D, X182E, X182N, X182Q, X182S, X185E, X281E, X281H, X281H, X281N, X281S, X282V, X282V, X286Q, X297M, X298V, X299S, X299S, X311G, X311W, X314Q, X314Q, X314S, X317M, X318L, X318L, X320A, X321Y, X322K, X323G, X324K, X326Y, X33L, X343W, X344V, X350P, X352C, X356C, X356L, X356V, X356V, X357C, X357F, X358M, X359W, X359W, X359W, X359W, X360T, X362C, X362M, X363E, X363H, X363M, X363N, X363S, X363Y, X363Y, X364T, X365L, X365L, X365Q, X365Q, X365Y, X368F, X368F, X36H, X36K, X370S, X372C, X372M, X372S, X372T, X372T, X378E, X379R, X37F, X37L, X37M, X37W, X37Y, X382Q, X382Q, X385W, X387E, X387N, X387S, X387S, X388E, X388H, X388K, X390F, X391K, X393Q, X395C, X395M, X396C, X396S, X397S, X400A, X400L, X400S, X405H, X405M, X41C, X41G, X429P, X434E, X434H, X434R, X434S, X439K, X456S, X459N, X459N, X460G, X460G, X461L, X461R, X463A, X463V, X465H, X5Q, X5D, X6E, X6D, X75C, X73N, X75H, X73C, X75H, X75C, X75H, X75L, X75L, X75T, X7F, X7H, X7W, X81L, X82C, X82L, X83E, X94E, X95A, X96Q, X98A, X98N, and X98V.

Preferably, the alpha-amylase variant of the invention, which exhibits improved wash performance compared to the parent alpha-amylase, preferably compared to SEQ ID No. 1, is an amylase comprising a combination of substitutions (numbering according to SEQ ID No. 3) selected from the group consisting of:

X4Q+X7W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X37M+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X460G,

X4Q+X25H+X176K+X186E+X206Y+X251E+X460G,

X4Q+X25Y+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X182D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X460G,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363N+X405M,

X4Q+X176K+X181D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181F+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181N+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X136E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160E+X176K+X186E+X193E+X206Y+X251E+X405M,

X7H+X25H+X176K+X186E+X206Y,X7W+X25H+X176K+X186E+X206Y,

X25D+X176K+X186E+X206Y,X25H+X186E+X206Y+X405M+X460G,

X25H+X186E+X206Y+X460G,X25H+X37M+X176K+X186E+X206Y,

X25H+X118D+X176K+X186E+X206Y,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X281N,X25H+X176K+X186E+X206Y+X460G,

X25H+X176K+X186E+X206Y+X251E+X460G,X25H+X176K+X186E+X206Y+X363E,

X25H+X176K+X186E+X206Y+X363H,X25H+X176K+X186E+X206Y+X363N,

X25H+X176K+X186E+X460G,X25H+X176K+X186E+X193E+X206Y,

X25H+X176K+X186E+X193E+X206Y+X460G,

X25H+X176K+X186E+X193E+X206Y+X251E+X460G,X25H+X176K+X181D+X186E+X206Y,

X25H+X176K+X181N+X186E+X206Y,X25H+X176K+X181Q+X186E+X206Y,

X25H+X136E+X176K+X186E+X206Y,X25H+X100W+X176K+X186E+X206Y,

X25H+X135D+X176K+X186E+X206Y,X25H+X135E+X176K+X186E+X206Y,

X25H+X135T+X176K+X186E+X206Y,X25H+X135W+X176K+X186E+X206Y,

X25H+X160D+X176K+X186E+X206Y,X25H+X160E+X176K+X186E+X206Y,

X25Y+X176K+X186E+X206Y,X51Q+X116K+X410I,X51V+X356I,

X33E+X54G+X83D+X116T+X337L,X3A+X116K+X172N+X323A+X356V,

X3I+X33H+X54D+X208Y+X218E+X320A,X3I+X54D+X116K+X125E+X225A,

X3I+X125D+X174D+X222D+X376L+X420D,X3I+X356I,X3I+X356V,X3I+X48Y+X116T,

X54D+X116K+X225S+X410Y,X83A+X94E+X158N+X172D+X323A,X83D+X125E,

X83E+X181E,X83E+X116R+X158Y+X181E,X169E+X377Q,X158N+X172D,

X125D+X225A+X337A+X410H,X29D+X33H+X83D+X125D+X337C+X343W,

X29E+X33K+X123D+X208I+X222D+X356V,X94D+X125E,X225A+X376G+X410Y,

X5L+X33K+X222D+X320K+X324M+X420E,X5L+X131L+X172K+X225S+X410I+X420E,

X5L+X116T+X123D+X208F,X131I+X377Q+X410H,X131L+X320K,

X116K+X181T+X225A+X320K,X116K+X125N+X158H+X225S,X116K+X131I+X158H+X174E,

X116R+X169E+X420E,X116T+X181Q+X343T,X116T+X125D+X172K+X337A+X410I,

X116T+X125E+X131I,X186E+X206Y,X186E+X206Y+X405M,X186E+X206Y+X251E,

X186E+X206Y+X251E+X276R+X405M,X186E+X206Y+X251E+X405M,

X186E+X206Y+X251E+X323G+X405M,X186E+X206Y+X323G+X405M,X186E+X405M,

X186E+X193E+X206Y+X251E,X186E+X251E+X405M,X4Q+X186E+X206Y,

X4Q+X186E+X206Y+X405M,X4Q+X186E+X206Y+X251E+X276R,

X4Q+X186E+X206Y+X251E+X405M,X4Q+X186E+X206Y+X251E+X323G+X405M,

X4Q+X186E+X193E+X206Y+X405M,X4Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X186E+X206Y,X4Q+X25H+X186E+X206Y+X405M,

X4Q+X25H+X186E+X206Y+X251E+X323A+X405M,

X4Q+X25H+X186E+X206Y+X323G+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M,X4Q+X25H+X176K+X186E+X251E,

X4Q+X25H+X176K+X186E+X251E+X405M,X4Q+X25H+X176K+X206Y+X405M+X460G,

X4Q+X25H+X176K+X206Y+X460G,X4Q+X25H+X176K+X206Y+X251E,

X4Q+X25H+X176K+X206Y+X251E+X460G,X4Q+X25H+X176K+X206Y+X323G,

X4Q+X25H+X160W+X186E+X206Y+X405M,X4Q+X25H+X160W+X186E+X206Y+X251E,

X4Q+X25H+X160W+X186E+X206Y+X323G+X405M,X4Q+X25H+X160W+X186E+X405M,

X4Q+X25H+X160W+X186E+X193E+X206Y+X276R,X4Q+X25H+X160W+X186E+X323G,

X4Q+X25H+X160W+X176K+X186E+X206Y+X251E,

X4Q+X25H+X160W+X176K+X186E+X206Y+X251E+X405M,

X4Q+X25H+X160W+X176K+X186E+X405M,

X4Q+X25H+X160W+X176K+X186E+X251E+X405M,X4Q+X25H+X160W+X176K+X206Y,

X4Q+X25H+X160W+X176K+X206Y+X251E+X460G,X4Q+X176K+X186E+X206Y+X405M,

X4Q+X176K+X186E+X206Y+X251E+X405M,X4Q+X176K+X186E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X276R+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,X4Q+X176K+X186E+X251E+X405M,

X4Q+X176K+X186E+X323G+X405M,X4Q+X176K+X206Y,X4Q+X176K+X206Y+X405M,

X4Q+X176K+X206Y+X405M+X460G,X4Q+X176K+X206Y+X460G,

X4Q+X176K+X206Y+X251E+X405M,X4Q+X176K+X206Y+X251E+X405M+X460G,

X4Q+X176K+X206Y+X251E+X323A+X460G,X4Q+X160W+X176K+X186E+X206Y+X251E,

X4Q+X160W+X176K+X186E+X251E+X405M,

X4Q+X160W+X176K+X206Y+X251E+X323G+X405M+X460G,X25H+X186E+X206Y,

X25H+X186E+X206Y+X405M,X25H+X186E+X206Y+X251E+X405M,X25H+X186E+X405M,

X25H+X186E+X193E+X206Y+X405M,X25H+X176K+X186E,

X25H+X176K+X186E+X276R+X323G+X405M,X25H+X176K+X186E+X206Y,

X25H+X176K+X186E+X206Y+X276R,X25H+X176K+X186E+X206Y+X251E,

X25H+X176K+X186E+X206Y+X251E+X405M,X25H+X176K+X186E+X405M,

X25H+X176K+X186E+X193E+X206Y,X25H+X176K+X186E+X193E+X206Y+X251E,

X25H+X176K+X186E+X193E+X206Y+X251E+X276R+X405M,

X25H+X176K+X186E+X251E+X276R,X25H+X176K+X206Y,X25H+X176K+X206Y+X405M,

X25H+X176K+X206Y+X460G,X25H+X176K+X206Y+X251E,

X25H+X176K+X206Y+X251E+X405M,X25H+X160W+X186E+X206Y,

X25H+X160W+X186E+X206Y+X251E,X25H+X160W+X186E+X206Y+X251E+X405M,

X25H+X160W+X186E+X206Y+X323G+X405M,

X25H+X160W+X176K+X186E+X206Y+X251E+X405M,

X25H+X160W+X176K+X186E+X405M,X25H+X160W+X176K+X186E+X193E+X206Y,

X25H+X160W+X176K+X186E+X193E+X206Y+X405M,

X25H+X160W+X176K+X186E+X193E+X206Y+X251E+X405M,

X25H+X160W+X176K+X186E+X251E,X25H+X160W+X176K+X186E+X251E+X405M,

X25H+X160W+X176K+X206Y+X251E+X323G+X405M,X176K+X186E+X206Y,

X176K+X186E+X206Y+X405M,X176K+X186E+X206Y+X251E+X405M,

X176K+X186E+X405M+X405M,X176K+X186E+X193E+X206Y+X405M,

X176K+X186E+X206Y+X251E+X405M,X176K+X186E+X251E+X405M,X176K+X206Y,

X176K+X206Y+X405M,X176K+X206Y+X405M+X460G,X176K+X206Y+X460G,

X176K+X206Y+X251E,X160W+X186E+X193E+X206Y+X405M,

X160W+X176K+X186E+X206Y,X160W+X176K+X186E+X405M,

X160W+X176K+X186E+X193E+X206Y,X160W+X176K+X186E+X193E+X206Y+X405M,

X160W+X176K+X186E+X193E+X206Y+X251E,X160W+X176K+X206Y+X405M,

X160W+X176K+X206Y+X323G+X405M+X460G,X186E+X193E,X4Q+X460G,

X208C+X323G,X25H+X186E,X176K+X400A,X457N+X460G+X461L,X160W+X186E,

X160W+X186Q,X160W+X434S,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X281N+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X8A+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X363E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X363H+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X363E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X363H+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X281N+X363H+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X100W+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X281N+X363E+X405M,

X4Q+X100W+X176K+X181Q+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X363H+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X363E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X363H+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X281N+X363H+X405M,

X25H+X176K+X186E+X206Y,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E,X25H+X176K+X186E+X206Y+X258Q+X363E+XN123D,

X25H+X176K+X186E+X206Y+X281N,X25H+X176K+X186E+X206Y+X363E,

X25H+X176K+X186E+X193E+X206Y,X25H+X176K+X186E+X193E+X206Y+X258Q,

X25H+X176K+X186E+X193E+X206Y+X281N+X363E,

X25H+X176K+X186E+X193E+X206Y+X363E,X25H+X176K+X181Q+X186E+X206Y,

X25H+X176K+X181Q+X186E+X206Y+X363E,X25H+X176K+X181Q+X186E+X193E+X206Y,

X25H+X100W+X176K+X186E+X206Y,X25H+X100W+X176K+X186E+X206Y+X258Q,

X25H+X100W+X176K+X186E+X206Y+X258Q+X281N,

X25H+X100W+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X100W+X176K+X186E+X206Y+X258Q+X363E,

X25H+X100W+X176K+X186E+X206Y+X281N,

X25H+X100W+X176K+X186E+X206Y+X281N+X363E,

X25H+X100W+X176K+X186E+X206Y+X361R,X25H+X100W+X176K+X186E+X206Y+X363E,

X25H+X100W+X176K+X186E+X193E+X206Y,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X473R,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X281N,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X281N+X363E,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X363E,

X25H+X100W+X176K+X186E+X193E+X206Y+X363E,

X25H+X100W+X176K+X181Q+X186E+X206Y+X363E,

X25H+X100W+X176K+X181Q+X186E+X193E+X206Y,

X25H+X100W+X176K+X181Q+X186E+X193E+X206Y+X258Q+X281N,

X25H+X100W+X176K+X181Q+X186E+X193E+X206Y+X258Q+X363E,

X25H+X100W+X135C+X176K+X186E+X206Y+X258Q+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q+X281N,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y+X281N,

X25H+X100W+X135T+X176K+X186E+X206Y+X281N+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y+X363E,

X25H+X100W+X135T+X176K+X186E+X193E+X206Y,

X25H+X100W+X135T+X176K+X186E+X193E+X206Y+X258Q+X363E,

X25H+X100W+X135T+X176K+X186E+X193E+X206Y+X363E,

X25H+X100W+X135T+X176K+X181Q+X186E+X206Y,

X25H+X100W+X135T+X176K+X181Q+X186E+X206Y+X258Q+X281N,

X25H+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y,

X25H+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X258Q,

X25H+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X363E,

X25H+X135T+X176K+X186E+X206Y,X25H+X135T+X176K+X186E+X206Y+X258Q,

X25H+X135T+X176K+X186E+X206Y+X258Q+X281N,

X25H+X135T+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X135T+X176K+X186E+X206Y+X258Q+X363E,

X25H+X135T+X176K+X186E+X206Y+X281N,

X25H+X135T+X176K+X186E+X206Y+X281N+X363E,

X25H+X135T+X176K+X186E+X206Y+X281N+X363E+X415S,

X25H+X135T+X176K+X186E+X206Y+X363E,

X25H+X135T+X176K+X186E+X193E+X206Y+X258Q,

X25H+X135T+X176K+X181Q+X186E+X206Y+X258Q,

x25h+x135t+x176k+x181q+x186e+x206y+x258q+x363E, and

X25H+X135T+X176K+X181Q+X186E+X193E+X206Y+X258Q+X281N。

preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Preferably, the alpha-amylase variant of the invention, which exhibits improved wash performance and/or improved stability compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising a combination of substitutions (numbering according to SEQ ID NO: 3) selected from the group consisting of:

X4Q+X7W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X37M+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X460G,

X4Q+X25H+X176K+X186E+X206Y+X251E+X460G,

X4Q+X25Y+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X182D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X460G,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363N+X405M,

X4Q+X176K+X181D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181F+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181N+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X136E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160E+X176K+X186E+X193E+X206Y+X251E+X405M,

X7H+X25H+X176K+X186E+X206Y,X7W+X25H+X176K+X186E+X206Y,

X25D+X176K+X186E+X206Y,X25H+X186E+X206Y+X405M+X460G,

X25H+X186E+X206Y+X460G,X25H+X37M+X176K+X186E+X206Y,

X25H+X118D+X176K+X186E+X206Y,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X281N,X25H+X176K+X186E+X206Y+X460G,

X25H+X176K+X186E+X206Y+X251E+X460G,X25H+X176K+X186E+X206Y+X363E,

X25H+X176K+X186E+X206Y+X363H,X25H+X176K+X186E+X206Y+X363N,

X25H+X176K+X186E+X460G,X25H+X176K+X186E+X193E+X206Y,

X25H+X176K+X186E+X193E+X206Y+X460G,

X25H+X176K+X186E+X193E+X206Y+X251E+X460G,X25H+X176K+X181D+X186E+X206Y,

X25H+X176K+X181N+X186E+X206Y,X25H+X176K+X181Q+X186E+X206Y,

X25H+X136E+X176K+X186E+X206Y,X25H+X100W+X176K+X186E+X206Y,

X25H+X135D+X176K+X186E+X206Y,X25H+X135E+X176K+X186E+X206Y,

X25H+X135T+X176K+X186E+X206Y,X25H+X135W+X176K+X186E+X206Y,

X25H+X160D+X176K+X186E+X206Y,X25H+X160E+X176K+X186E+X206Y,

X25Y+X176K+X186E+X206Y,X51Q+X116K+X410I,X51V+X356I,

X33E+X54G+X83D+X116T+X337L,X3A+X116K+X172N+X323A+X356V,

X3I+X33H+X54D+X208Y+X218E+X320A,X3I+X54D+X116K+X125E+X225A,

X3I+X125D+X174D+X222D+X376L+X420D,X3I+X356I,X3I+X356V,X3I+X48Y+X116T,

X54D+X116K+X225S+X410Y,X83A+X94E+X158N+X172D+X323A,X83D+X125E,

X83E+X181E,X83E+X116R+X158Y+X181E,X169E+X377Q,X158N+X172D,

X125D+X225A+X337A+X410H,X29D+X33H+X83D+X125D+X337C+X343W,

X29E+X33K+X123D+X208I+X222D+X356V,X94D+X125E,X225A+X376G+X410Y,

X5L+X33K+X222D+X320K+X324M+X420E,X5L+X131L+X172K+X225S+X410I+X420E,

X5L+X116T+X123D+X208F,X131I+X377Q+X410H,X131L+X320K,

X116K+X181T+X225A+X320K,X116K+X125N+X158H+X225S,X116K+X131I+X158H+X174E,

X116R+X169E+X420E,X116T+X181Q+X343T,X116T+X125D+X172K+X337A+X410I,

X116T+X125E+X131I,X186E,X186E+X206Y,X186E+X206Y+X405M,X186E+X206Y+X251E,

X186E+X206Y+X251E+X276R+X405M,X186E+X206Y+X251E+X405M,

X186E+X206Y+X251E+X323G+X405M,X186E+X206Y+X323G+X405M,X186E+X405M,

X186E+X193E+X206Y+X251E,X186E+X251E+X405M,X4Q+X186E+X206Y,

X4Q+X186E+X206Y+X405M,X4Q+X186E+X206Y+X251E+X276R,

X4Q+X186E+X206Y+X251E+X405M,X4Q+X186E+X206Y+X251E+X323G+X405M,

X4Q+X186E+X193E+X206Y+X405M,X4Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X186E+X206Y,X4Q+X25H+X186E+X206Y+X405M,

X4Q+X25H+X186E+X206Y+X251E+X323A+X405M,

X4Q+X25H+X186E+X206Y+X323G+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M,X4Q+X25H+X176K+X186E+X251E,

X4Q+X25H+X176K+X186E+X251E+X405M,X4Q+X25H+X176K+X206Y+X405M+X460G,

X4Q+X25H+X176K+X206Y+X460G,X4Q+X25H+X176K+X206Y+X251E,

X4Q+X25H+X176K+X206Y+X251E+X460G,X4Q+X25H+X176K+X206Y+X323G,

X4Q+X25H+X160W+X186E+X206Y+X405M,X4Q+X25H+X160W+X186E+X206Y+X251E,

X4Q+X25H+X160W+X186E+X206Y+X323G+X405M,X4Q+X25H+X160W+X186E+X405M,

X4Q+X25H+X160W+X186E+X193E+X206Y+X276R,X4Q+X25H+X160W+X186E+X323G,

X4Q+X25H+X160W+X176K+X186E+X206Y+X251E,

X4Q+X25H+X160W+X176K+X186E+X206Y+X251E+X405M,

X4Q+X25H+X160W+X176K+X186E+X405M,

X4Q+X25H+X160W+X176K+X186E+X251E+X405M,X4Q+X25H+X160W+X176K+X206Y,

X4Q+X25H+X160W+X176K+X206Y+X251E+X460G,X4Q+X176K+X186E+X206Y+X405M,

X4Q+X176K+X186E+X206Y+X251E+X405M,X4Q+X176K+X186E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X276R+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,X4Q+X176K+X186E+X251E+X405M,

X4Q+X176K+X186E+X323G+X405M,X4Q+X176K+X206Y,X4Q+X176K+X206Y+X405M,

X4Q+X176K+X206Y+X405M+X460G,X4Q+X176K+X206Y+X460G,

X4Q+X176K+X206Y+X251E+X405M,X4Q+X176K+X206Y+X251E+X405M+X460G,

X4Q+X176K+X206Y+X251E+X323A+X460G,X4Q+X160W+X176K+X186E+X206Y+X251E,

X4Q+X160W+X176K+X186E+X251E+X405M,

X4Q+X160W+X176K+X206Y+X251E+X323G+X405M+X460G,X25H+X186E+X206Y,

X25H+X186E+X206Y+X405M,X25H+X186E+X206Y+X251E+X405M,X25H+X186E+X405M,

X25H+X186E+X193E+X206Y+X405M,X25H+X176K+X186E,

X25H+X176K+X186E+X276R+X323G+X405M,X25H+X176K+X186E+X206Y,

X25H+X176K+X186E+X206Y+X276R,X25H+X176K+X186E+X206Y+X251E,

X25H+X176K+X186E+X206Y+X251E+X405M,X25H+X176K+X186E+X405M,

X25H+X176K+X186E+X193E+X206Y+X251E,

X25H+X176K+X186E+X193E+X206Y+X251E+X276R+X405M,

X25H+X176K+X186E+X251E+X276R,X25H+X176K+X206Y,X25H+X176K+X206Y+X405M,

X25H+X176K+X206Y+X460G,X25H+X176K+X206Y+X251E,

X25H+X176K+X206Y+X251E+X405M,X25H+X160W+X186E+X206Y,

X25H+X160W+X186E+X206Y+X251E,X25H+X160W+X186E+X206Y+X251E+X405M,

X25H+X160W+X186E+X206Y+X323G+X405M,

X25H+X160W+X176K+X186E+X206Y+X251E+X405M,

X25H+X160W+X176K+X186E+X405M,X25H+X160W+X176K+X186E+X193E+X206Y,

X25H+X160W+X176K+X186E+X193E+X206Y+X405M,

X25H+X160W+X176K+X186E+X193E+X206Y+X251E+X405M,

X25H+X160W+X176K+X186E+X251E,X25H+X160W+X176K+X186E+X251E+X405M,

X25H+X160W+X176K+X206Y+X251E+X323G+X405M,X176K+X186E+X206Y,

X176K+X186E+X206Y+X405M,X176K+X186E+X206Y+X251E+X405M,

X176K+X186E+X405M+X405M,X176K+X186E+X193E+X206Y+X405M,

X176K+X186E+X251E+X405M,X176K+X206Y,X176K+X206Y+X405M,

X176K+X206Y+X405M+X460G,X176K+X206Y+X460G,X176K+X206Y+X251E,

X160W+X186E+X193E+X206Y+X405M,X160W+X176K+X186E+X206Y,

X160W+X176K+X186E+X405M,X160W+X176K+X186E+X193E+X206Y,

X160W+X176K+X186E+X193E+X206Y+X405M,

X160W+X176K+X186E+X193E+X206Y+X251E,X160W+X176K+X206Y+X405M,

X160W+X176K+X206Y+X323G+X405M+X460G,X186E+X193E,X4Q+X460G,

X208C+X323G,X25H+X186E,X176K+X400A,X457N+X460G+X461L,X160W+X186E,

X160W+X186Q,X160W+X434S,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X281N+X363E+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X8A+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X363E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X363H+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X363E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X258Q+X363H+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X281N+X363H+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X100W+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X281N+X363E+X405M,

X4Q+X100W+X176K+X181Q+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X363H+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X135T+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X281N+X363E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X258Q+X363H+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X281N+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X258Q+X281N+X405M,

X4Q+X135T+X176K+X181Q+X186E+X193E+X206Y+X251E+X281N+X363H+X405M,

X25H+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E+XN123D,

X25H+X176K+X186E+X193E+X206Y+X258Q,

X25H+X176K+X186E+X193E+X206Y+X281N+X363E,

X25H+X176K+X186E+X193E+X206Y+X363E,X25H+X176K+X181Q+X186E+X206Y+X363E,

X25H+X176K+X181Q+X186E+X193E+X206Y,X25H+X100W+X176K+X186E+X206Y+X258Q,

X25H+X100W+X176K+X186E+X206Y+X258Q+X281N,

X25H+X100W+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X100W+X176K+X186E+X206Y+X258Q+X363E,

X25H+X100W+X176K+X186E+X206Y+X281N,

X25H+X100W+X176K+X186E+X206Y+X281N+X363E,

X25H+X100W+X176K+X186E+X206Y+X361R,X25H+X100W+X176K+X186E+X206Y+X363E,

X25H+X100W+X176K+X186E+X193E+X206Y,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X473R,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X281N,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X281N+X363E,

X25H+X100W+X176K+X186E+X193E+X206Y+X258Q+X363E,

X25H+X100W+X176K+X186E+X193E+X206Y+X363E,

X25H+X100W+X176K+X181Q+X186E+X206Y+X363E,

X25H+X100W+X176K+X181Q+X186E+X193E+X206Y,

X25H+X100W+X176K+X181Q+X186E+X193E+X206Y+X258Q+X281N,

X25H+X100W+X176K+X181Q+X186E+X193E+X206Y+X258Q+X363E,

X25H+X100W+X135C+X176K+X186E+X206Y+X258Q+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q+X281N,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y+X258Q+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y+X281N,

X25H+X100W+X135T+X176K+X186E+X206Y+X281N+X363E,

X25H+X100W+X135T+X176K+X186E+X206Y+X363E,

X25H+X100W+X135T+X176K+X186E+X193E+X206Y,

X25H+X100W+X135T+X176K+X186E+X193E+X206Y+X258Q+X363E,

X25H+X100W+X135T+X176K+X186E+X193E+X206Y+X363E,

X25H+X100W+X135T+X176K+X181Q+X186E+X206Y,

X25H+X100W+X135T+X176K+X181Q+X186E+X206Y+X258Q+X281N,

X25H+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y,

X25H+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X258Q,

X25H+X100W+X135T+X176K+X181Q+X186E+X193E+X206Y+X363E,

X25H+X135T+X176K+X186E+X206Y+X258Q,

X25H+X135T+X176K+X186E+X206Y+X258Q+X281N,

X25H+X135T+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X135T+X176K+X186E+X206Y+X258Q+X363E,

X25H+X135T+X176K+X186E+X206Y+X281N,

X25H+X135T+X176K+X186E+X206Y+X281N+X363E,

X25H+X135T+X176K+X186E+X206Y+X281N+X363E+X415S,

X25H+X135T+X176K+X186E+X206Y+X363E,

X25H+X135T+X176K+X186E+X193E+X206Y+X258Q,

X25H+X135T+X176K+X181Q+X186E+X206Y+X258Q,

X25H+X135T+X176K+X181Q+X186E+X206Y+X258Q+X363E,

X25H+X135T+X176K+X181Q+X186E+X193E+X206Y+X258Q+X281N,

X25H+X176K+X186E+X206Y+X251E+X482W,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X482W,

X25H+X176K+X186E+X206Y+X482W,X25H+X186E+X206Y+X405M+X482W,

X25H+X186E+X206Y+X482W,X25H+X176K+X186E+X193E+X206Y+X251E+X482W,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X482W,X25H+X176K+X186E+X482W,

X25H+X176K+X186E+X193E+X206Y+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,X4Q+X206Y+X460G+X482W,

X4Q+X25H+X176K+X206Y+X251E+X460G+X482W,

X4Q+X25H+X176K+X193E+X251E+X186E+X482W,

X4Q+X193E+X206Y+X276R+X186E+X482W,X186E+X25H+X482W,

X25H+X193E+X206Y+X251E+X276R+X405M+X186E+X482W,X25H+X251E+X186E+X482W,

x25h+x160w+x176k+x186e+x251e+x405m+x482W, and

X193E+X206Y+X405M+X186E+X482W。

preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Preferably, the improved property is improved expression. Preferably, the alpha-amylase variant of the invention exhibiting improved expression compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising an amino acid substitution at one or more positions corresponding to positions selected from the group consisting of SEQ ID NO: 3: 4. 6, 20, 25, 27, 28, 37, 70, 72, 75, 81, 94, 96, 160, 175, 176, 193, 276, 277, 299, 323, 368, 372, 382, 400, 405, 434, 459, and 461.

Preferably, the alpha-amylase variant of the invention exhibiting improved expression compared to the parent alpha-amylase, preferably compared to SEQ ID No. 1, is an amylase comprising one or more amino acid substitutions selected from the group consisting of amino acid substitutions (numbering according to SEQ ID No. 3): X4Q, X6E, X K, X25H, X Y, X5483V, X37 7970H, X72C, X3575L, X81L, X94M, X96Q, X160W, X175A, X176A, X193A, X276 277A, X299A, X323A, X368A, X372A, X382A, X52400A, X434A, X459N and X461L. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Preferably, the improved property is improved solubility. Preferably, the alpha-amylase variant of the invention, which exhibits improved solubility compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising an amino acid substitution at one or more positions corresponding to positions selected from the group consisting of SEQ ID NO: 3: 4. 25, 83, 87, 160, 176, 193, 219, 226, 251, 311, 314, 315, 378, 405, 439, 459, 460, 461, and 471.

Preferably, the alpha-amylase variant of the invention exhibiting improved solubility compared to the parent alpha-amylase, preferably compared to SEQ ID NO:1, is an amylase comprising one or more amino acid substitutions (numbering according to SEQ ID NO: 3) selected from the group consisting of: X4K, X H, X83E, X87D, X87R, X160D, X4815 193 5235 193E, X219D, X219R, X226R, X D, X E, X311K, X314E, X314K, X K, X378K, X405M, X439 459R, X460G, X D, X461E and X471E. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

In another embodiment, an alpha-amylase variant of the invention having alpha-amylase activity comprises one or more of the above amino acid substitutions and one or more additional amino acids of a specific type, preferably one or more additional amino acid changes, preferably amino acid substitutions, as compared to the parent alpha-amylase.

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises 1-50 additional amino acid changes, preferably 1-35, 1-31, 1-30, 1-20, 1-15, 1-10 or 1-5 amino acid changes, preferably substitutions, compared to the parent alpha-amylase.

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises a deletion of 1-4, preferably 1-2 amino acids compared to the parent alpha-amylase.

In one embodiment, these additional amino acid changes are conservative substitutions and/or substitutions described below.

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises one or more, preferably at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 25 or all amino acids of the additional change, preferably additional substitution, at positions selected from 9, 130, 179, 181, 186, 190, 195, 202, 206, 244, 402, 419, 420, 422, 423, 428, 430, 435, 441, 444, 450, 452, 454, 466, 469, 473, 475, 476, 479, 483 and 485, wherein the alpha-amylase variant has alpha-amylase activity, preferably one or more amino acid substitutions selected from the following amino acid substitutions: X430C, X430D, X430E, X430F, X430G, X430I, X430L, X430P, X430Q, X430S, X430T, X430V, X435E, X435K, X435P, X435R, X435S, X435A, X435D, X437A, X437L, X437T, X437W, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X444E, X444H, X444K, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450I, X450L, X450M, X450P, X450Q, X450R, X450T, X430C, X430D, X430E, X430F, X430G, X430I, X430L, X430P, X430Q, X430S, X430T, X430V, X435E, X435K, X435P, X435R, X435S, X435A, X435D, X437A, X437L, X437T, X437W, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X444E, X444H, X444K, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450I, X450L, X450M, X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X452F, X452I, X452K, X452M, X452N, X452R, X452T, X454A, X454E, X454K, X454L, X454M, X454P, X454S, X454T, X466E, X466W, X469F, X469L, X469Y, X473H, X473Q, X473R, X475A, X475K, X475N, X475E, X475L, X476G, X476E, X479A, X479I, X479K, X479M, X483F, X483I, X483L, X483Q, X483R, X485K, and X485R, wherein according to SEQ ID NO:3, and the amino acid sequence shown in 3. Preferably, the amino acid residue at the above position (i.e.X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Preferably, the alpha-amylase variant of the invention having alpha-amylase activity comprises one or more, preferably at most 1, at most 2, at most 3, at most 4, at most 5 or all amino acids of an additional change, preferably an additional substitution, at a position selected from 181, 186, 195, 206, 441 and 473, preferably at position 186, 195 or 206, more preferably at position 206 or 195, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO:3, preferably wherein the alpha-amylase variant further comprises one or more additional substitutions selected from the group consisting of: X181D, X181E, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, X181Y, X186A, X186C, X186D, X186E, X186F, X186H, X186I, X186K, X186L, X186M, X186N, X186Q, X186R, X186S, X186V, X186W, X186Y, X190H, X195F, X195H, X195K, X195L, X195W, X195Y, X206C, X206H, X206L, X206M, X206Y, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X473H, X473Q and X473R (numbering according to SEQ ID NO: 3).

Most preferably, as additional changes, (preferably in combination with one or more additional changes described above), an alpha-amylase variant of the invention having one or more amino acid changes (preferably substitutions) described herein and having alpha-amylase activity comprises one or more amino acid deletions at positions corresponding to positions selected from (numbering according to SEQ ID NO: 3) 181, 182, 183 and 184, preferably two or more amino acid deletions at positions selected from 181, 182, 183 and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 or 183 and 184 (numbering according to SEQ ID NO: 3). Preferably, the alpha-amylase variants of the invention having one or more amino acid changes (preferably substitutions) described herein and having alpha-amylase activity comprise a deletion of one or more amino acids corresponding to positions 183 and 184, preferably two amino acid deletions corresponding to positions 183 and 184 (numbering according to SEQ ID NO: 3). Preferably, the alpha-amylase variants of the invention having one or more amino acid changes (preferably substitutions) described herein and having alpha-amylase activity comprise deletions corresponding to one or more, preferably two or more, most preferably two amino acids, preferably D183 and G184, positions selected from the group consisting of R181, G182, D183 and G184, wherein numbering is according to the amino acid sequence set forth in SEQ ID No. 3.

Preferably, the present invention relates to an alpha-amylase variant of a parent alpha-amylase, wherein the variant comprises:

(i) Amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of: 1,2,3,4,5,6,7,8,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,45,47,48,51,54,59,60,63,70,71,72,73,75,76,81,82,83,86,87,89,90,91,92,93,94,95,96,97,98,99,100,103,106,108,109,113,114,115,116,117,119,120,123,125,126,128,129,131,132,133,134,135,136,139,141,142,143,144,145,146,147,149,150,151,154,155,156,158,160,161,162,163,164,165,169,170,172,173,174,175,176,177,178,180,182,184,185,187,188,189,191,192,193,203,208,210,211,212,213,214,215,216,218,219,221,222,223,224,225,226,227,230,231,233,234,235,236,237,238,240,242,243,245,249,250,251,252,253,255,256,257,258,259,260,261,262,263,264,265,268,269,270,271,272,273,274,275,276,277,279,280,281,282,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,301,302,303,304,306,307,308,309,310,311,312,313,314,315,317,318,319,320,321,322,323,324,326,327,333,334,336,337,338,341,342,343,344,345,346,348,350,351,352,353,354,355,356,357,358,359,360,362,363,364,365,366,367,368,370,372,375,376,378,379,381,382,383,384,385,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,403,405,406,407,408,410,413,414,415,416,418,421,424,426,427,429,431,432,434,436,438,439,440,442,443,445,446,447,448,449,451,453,455,456,457,458,459,460,461,462,463,465,467,468,470,471,474,478,480 and 482 according to the amino acid sequence set forth in SEQ ID NO. 3;

(ii) The variant has at least 60%, preferably at least 80%, but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO. 1,

(iii) The variant comprises a deletion of one or more, preferably two or more, most preferably two amino acids, preferably D183 and G184 corresponding to positions selected from R181, G182, D183 and G184, wherein according to the amino acid sequence numbering shown in SEQ ID No. 3,

(iv) Optionally, the variant comprises one or more additional amino acid substitutions as described herein,

(v) The variant comprises the improved properties described herein, and

(vi) The variant has alpha-amylase activity, preferably wherein the parent amylase is SEQ ID NO. 1.

In an alternative embodiment, the invention relates to an alpha-amylase variant of a parent alpha-amylase, wherein the variant comprises:

(i) One or more amino acid substitutions selected from the group consisting of: X9D, X9F, X9K, X9N, X9P, X9Q, X9S, X9T, X9Y, X179G, X181D, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, X181Y, X186A, X186C, X186D, X186F, X186H, X186I, X186K, X186L, X186M, X186R, X186V, X186W, X186Y, X190H, X195H, X195K, X195L, X195W, X195Y, X206C, X206H, X206M, X206Y, X244A, X244C, X244D, X244E, X244F, X244G, X244H, X244M, X244N, X244V, X402T X419C, X420D, X420E, X420G, X420H, X420K, X420L, X420Q, X422C, X422N, X422H, X423F, X423M, X423Q, X423S, X428C, X428D, X428E, X428G, X428I, X428K, X428L, X428M, X428N, X428R, X428S, X428V, X428W, X428Y, X430A, X430C, X430D, X430E, X430F, X430G, X430L, X430P, X430Q, X430S, X430T, X430V, X435K, X435P, X435S, X435A, X435D, X437L, X437T, X437W, X441C, X441K, X441L, X441M, X441S, X444H, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450L, X450M, X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X452F, X452I, X452K, X452M, X452N, X452T, X454A, X454E, X454K, X454L, X454P, X454S, X454T, X466E, X466W, X469F, X469L, X469Y, X475A, X475K, X475E, X475L, X479I, X479K, X479M, X483F, X483L, X483Q, and X483R, preferably selected from one or more amino acid substitutions: X181D, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, X181Y, X186A, X186C, X186D, X186F, X186H, X186I, X186K, X186L, X186M, X186R, X186V, X186W, X186Y, X195H, X195K, X195L, X195W, X195Y, X206C, X206H, X206M, X206Y, X441C, X441K, X441L, X441M and X441S,

Wherein numbering is according to the amino acid sequence shown in SEQ ID NO. 3, and wherein the variant has an alpha-amylase activity, preferably wherein the parent alpha-amylase of the alpha-amylase variant of the invention is an amylase according to SEQ ID NO. 1 or SEQ ID NO. 3 or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of the alpha-amylase variant is an amylase according to SEQ ID NO. 1,

(ii) The variant has at least 60%, preferably at least 80% but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 1,

(iii) The variant comprises one or more, preferably two or more, most preferably two amino acid deletions corresponding to positions selected from R181, G182, D183 and G184, preferably D183 and G184, wherein the amino acid sequence numbers as set forth in SEQ ID No. 3 are followed by

(iv) The variant comprises the improved properties described herein, and

(v) The variant has alpha-amylase activity.

The invention also relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the alpha-amylase variant has an alpha-amylase activity according to SEQ ID NO:3, which variant comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at one or more positions corresponding to positions selected from 13, 17, 22, 23, 27, 28, 32, 36, 42, 51, 70, 75, 83, 89, 92, 95, 96, 154, 192, 215, 222, 226, 233, 245, 277, 282, 285, 288, 297, 312, 338, 341, 343, 353, 355, 356, 376, 379, 381, 389, 429, 432, 442, 451, 459 and 463, compared to the parent alpha-amylase, wherein preferably the alpha-amylase variant has at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% identity with the amino acid sequence set forth in any one of SEQ ID NOs 1, 3, 4 or 15-41, preferably SEQ ID NO1, SEQ ID NO 3 or SEQ ID NO 4, more preferably SEQ ID NO1 or 3, most preferably SEQ ID NO 1. The invention also relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at one or more positions corresponding to positions selected from 13, 17, 23, 27, 36, 42, 51, 89, 92, 192, 215, 222, 233, 245, 277, 282, 288, 297, 312, 338, 341, 343, 353, 355, 356, 376, 379, 381, 389, 429, 432, 442 and 451 compared to the parent alpha-amylase according to the numbering of the amino acid sequence shown in SEQ ID NO:3, wherein preferably the alpha-amylase variant is identical to SEQ ID NO:1, 3, 4 or SEQ ID NO:15-41, preferably SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1 has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably the alpha-amylase variant further comprises a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183 and G184, wherein numbering is according to the amino acid sequence shown in SEQ ID No. 3. The invention also relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at one or more positions corresponding to positions selected from 13, 18, 146, 163, 170, 188, 224, 238, 242, 245, 353, 385, 388, 405, 432, 442, 474 and 478, compared to the parent alpha-amylase according to the numbering of the amino acid sequence shown in SEQ ID NO:3, wherein preferably the alpha-amylase variant hybridizes to SEQ ID NO:1, 3, 4 or SEQ ID NO:15-41, preferably SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4, more preferably SEQ ID No. 1 or 3, most preferably the amino acid sequence shown in SEQ ID No. 1 has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, preferably the alpha-amylase variant further comprises a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183 and G184, wherein numbering is according to the amino acid sequence shown in SEQ ID No. 3.

The present invention also relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the variant comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at one or more positions corresponding to positions selected from the group consisting of: 1,2,3,5,7,8,9,11,16,18,19,25,26,29,30,31,35,37,40,41,43,44,46,48,49,50,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,71,72,73,74,77,78,79,80,81,84,85,86,87,88,90,91,93,94,97,98,101,102,103,104,105,106,107,109,110,111,112,113,114,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,151,152,153,155,157,158,159,160,161,162,163,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,185,186,187,188,189,190,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,216,217,218,219,220,224,225,227,228,229,230,231,232,235,238,239,241,242,243,244,246,247,248,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,269,270,272,273,274,275,276,278,279,280,281,283,284,286,287,291,292,293,294,295,296,298,299,300,302,303,304,305,306,307,310,311,313,314,315,316,317,319,320,321,322,323,324,325,328,329,330,331,332,334,335,337,339,340,342,344,345,346,347,349,350,354,357,359,360,361,362,363,364,365,368,369,371,372,373,374,375,377,380,382,383,384,385,386,387,388,390,391,393,394,395,396,397,398,400,401,402,403,404,405,406,407,408,409,410,411,412,414,415,416,417,418,419,420,421,422,423,425,427,428,430,431,433,434,435,437,438,439,441,444,445,446,447,449,450,452,454,455,457,458,460,461,462,464,465,466,467,469,470,471,472,473,474,475,476,477,478,479,481,483,484 and 485, wherein preferably the alpha-amylase variant has at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1, preferably the alpha-amylase variant comprises one or more, preferably at most 1, at most 2, at most 3, at most 4, at most 5 or all amino acids of an additional change, preferably an additional substitution, at a position selected from 181, 186, 195, 206, 441 and 473, preferably at position 186, 195 or 206, more preferably at position 206 and/or 195, wherein according to the amino acid sequence numbering set forth in SEQ ID NO. 3, preferably wherein the alpha-amylase variant further comprises one or more additional substitutions (according to the numbering of SEQ ID NO. 3) selected from the group consisting of: X181D, X181E, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, X181Y, X186A, X186C, X186D, X186E, X186F, X186H, X186I, X186K, X186L, X186M, X186N, X186Q, X186R, X186S, X186V, X186W, X186Y, X190H, X195F, X195H, X195K, X195L, X195W, X195Y, X206C, X206H, X206L, X206M, X206Y, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X473H, X473Q and X473R, preferably the alpha-amylase variant further comprises one or more amino acids corresponding to positions selected from the group consisting of 181,182, and 183D, preferably the amino acid sequence of amino acids 184, and 183D, and 183 ID, preferably in sequence numbers X182, and 183G, according to the preferred amino acid sequences.

The invention also relates to an alpha-amylase variant of a parent alpha-amylase having alpha-amylase activity, wherein the alpha-amylase variant has an alpha-amylase activity according to SEQ ID NO:3, most preferably a substitution, wherein the alpha-amylase variant has at least 95% identity, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, preferably the alpha-amylase variant comprises one or more amino acid changes, preferably insertions, deletions, substitutions or combinations thereof, at positions corresponding to positions selected from the group consisting of 25, 116, 176, 181, 183, 186, 206, 405, 408, 409, 410, 418, 419, 420, 422, 423, 437, 441, 444, 446, 449, 452, 458, 460, 466, 471, 473, 475, 484 and 485, most preferably the alpha-amylase variant comprises one or more, preferably at positions 206 and/or 195, preferably at most 1, at most 2, at most 3, at most 4, at most 5, preferably at most more amino acids, preferably at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, preferably the alpha-amylase variant comprises one or more amino acid changes at positions selected from the group consisting of 181, 186, 195, 441, or 473, and 473, preferably at positions 206 and/or 206, 3, more preferably the amino acid changes set forth in SEQ ID NO:3, more amino acid sequence set forth below: X181D, X181E, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, X181Y, X186A, X186C, X186D, X186E, X186F, X186H, X186I, X186K, X186L, X186M, X186N, X186Q, X186R, X186S, X186V, X186W, X186Y, X190H, X195F, X195H, X195K, X195L, X195W, X195Y, X206C, X206H, X206L, X206M, X206Y, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X473H, X473Q and X473R (numbering according to SEQ ID NO: 3), preferably the α -amylase further comprises one or more deletions of amino acids corresponding to positions 182, 184, 183D, and 183D, preferably amino acid residues corresponding to positions 182 and 183G, and 183, in the preferred amino acid sequences shown in the amino acid sequences.

Nucleic acid constructs

The invention also relates to polynucleotides encoding the alpha-amylase variants of the invention. Preferably, the polynucleotide is a codon optimized polynucleotide for improving expression in a specific host cell, preferably a bacillus cell.

In another embodiment, the invention also relates to a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions, preferably very high stringency conditions, with (i) the mature polypeptide coding sequence described herein or (ii) the full length complement of (i).

Thus, the invention also relates to a nucleic acid, preferably an isolated, synthetic and/or recombinant nucleic acid, comprising:

(a) A nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% identity to SEQ ID No. 2, wherein the nucleic acid encodes a polypeptide having amylase activity;

(b) A nucleic acid sequence encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% identity to SEQ ID No. 1 (wherein the polypeptide has amylase activity) or encoding any alpha-amylase variant described herein having amylase activity;

(c) Polynucleotides which hybridize under high stringency conditions, preferably very high stringency conditions, to the complement of the following sequences:

(i) The coding sequence of SEQ ID NO. 1 or the coding sequence of any of the alpha-amylase variants described herein having alpha-amylase activity; or (b)

(ii) A polynucleotide shown in SEQ ID NO. 2;

(d) A fragment of (a), (b) or (c), wherein the fragment encodes a polypeptide having amylase activity; or (b)

(e) A nucleic acid sequence that is fully complementary to any one of (a) to (d);

(f) A polynucleotide differing from any of the nucleic acid sequences recited in (a) through (e) only by the degeneracy of the genetic code.

The invention also relates to nucleic acid constructs, preferably expression cassettes, comprising the polynucleotides described herein.

Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region, which in eukaryotes typically comprises a polyadenylation site. Other regulatory elements may include transcriptional enhancers and translational enhancers. Intronic sequences may also be added to the 5' untranslated region (UTR) or coding sequence to increase the amount of mature message that accumulates in the cytosol. The expression cassette may be part of a vector or may be integrated into the genome of a host cell and replicated together with the genome of its host cell. Expression cassettes are generally capable of increasing or decreasing expression.

The invention also relates to expression vectors comprising the polynucleotides or nucleic acid constructs described herein. The expression vector may be a low copy number vector or a high copy number vector.

Vectors used herein can provide segments for transcription and translation of foreign polynucleotides upon transformation into a host cell or an organelle of a host cell. Such other segments may include regulatory nucleotide sequences, one or more origins of replication required to maintain and/or replicate the vector in a particular cell type, one or more selectable markers, polyadenylation signals, suitable sites for inserting foreign coding sequences, such as multiple cloning sites, and the like. One example is when the vector needs to be maintained in a bacterial cell as an episomal genetic element (e.g., plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include f1-ori and colE1.

The vector may replicate without integrating into the host cell genome, such as a plasmid in a bacterial host cell, or it may integrate a portion or all of its DNA into the host cell genome, resulting in replication and expression of its DNA.

Polynucleotides encoding alpha-amylase variants may be introduced into the vector by standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide comprising the coding sequence may be suitably introduced (transformed, transduced, transfected, etc.) into a host cell or an organelle of a host cell. Cloning vectors suitable for expression of the polynucleotide sequences in a host cell or in an organelle of a host cell may be selected.

Host cells

The invention also relates to a host cell comprising a polynucleotide encoding an alpha-amylase variant described herein, a nucleic acid construct described herein or an expression vector described herein. In one embodiment of the invention, the vector is used to transform a host cell.

Polynucleotides encoding the alpha-amylase variants described herein may be introduced transiently or stably into a host cell, and may be maintained in the host cell in non-integrated form, e.g., as a plasmid. In general, stable transformation is due to integration of a nucleic acid comprising an exogenous coding sequence into a chromosome or as an episome (separate nuclear DNA fragments). In general, transient transformation is caused by the fact that a nucleic acid comprising a foreign nucleic acid sequence is not integrated into the chromosome or is present as an episome. Alternatively, a polynucleotide encoding an alpha-amylase variant described herein may be integrated into the host genome.

Nucleic acids may be introduced into host cells, for example, but not limited to, by protoplast transformation (see, e.g., chang and Cohen,1979,Molecular General Genetics 168:111-115), by using competent cells (see, e.g., young and Spizezen, 1961,Journal of Bacteriology 81:823-829 or Dubinau and Davidoff-Abelson,1971,Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., shigekawa and Dower,1988,Biotechniques 6:742-751), or by conjugation (see, e.g., koehler and Thorne,1987,Journal of Bacteriology 169:5271-5278). Specific transformation protocols for various types of host cells are known in the art (for E.coli protoplast transformation see, e.g., hanahan,1983, J.mol. Biol. 166:557-580).

A variety of host cells can be used to express the nucleic acid constructs described herein. Host cells comprising the genetic constructs described herein may be obtained by one of the methods described herein for introducing a polynucleotide into such host cells. The host cells of the invention do not naturally express the alpha-amylase variant. Thus, the host cell is a recombinant host cell; the nucleic acid constructs described herein are heterologous to the host cell.

In one embodiment, the host cell is a prokaryote or eukaryote. In another embodiment, the host cell is a bacterium, archaebacteria, fungal cell, yeast cell, or eukaryotic cell. In another embodiment, the host cell is a non-human host cell.

In one embodiment, the host cell is a bacterial cell. The bacterial host cell may be any gram positive or gram negative bacterium. Gram positive bacteria include, but are not limited to, bacillus (Bacillus), brevibacterium (Brevibacterium), corynebacterium (Corynebacterium), streptococcus (Streptococcus), streptomyces (Streptomyces), staphylococcus (Staphylococcus), enterococcus (Enterococcus), lactobacillus (Lactobacillus), lactococcus (Lactobacillus), clostridium (Clostridium), geobacillus (Geobacillus) and Bacillus (oceanobacter). Gram negative bacteria include, but are not limited to, escherichia, pseudomonas, salmonella, campylobacter, helicobacter, acetobacter, flavobacterium, fusobacterium, and Gluconobacter. In a specific embodiment, the bacterial host cell is an E.coli cell. In one embodiment, the host cell is a bacterial cell. In a specific embodiment, the host cell belongs to the genus Escherichia or Bacillus.

In the methods of the invention, the bacterial host cell may be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, bacillus alcalophilus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus stearothermophilus), bacillus circulans (Bacillus circulans), bacillus clausii (Bacillus coagulans), bacillus coagulans (Bacillus coagulans), bacillus firmus, bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus lentus), bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus pumilus), bacillus stearothermophilus (Bacillus stearothermophilus), bacillus methylotrophicus (Bacillus methylotrophicus), bacillus cereus (Bacillus cereus), bacillus paralicheniformis (Bacillus paralicheniformis), bacillus subtilis (Bacillus subtilis), and Bacillus thuringiensis (Bacillus thuringiensis) cells. In one embodiment, the bacterial host cell is a Bacillus amyloliquefaciens, bacillus pumilus, bacillus lentus, bacillus licheniformis, bacillus stearothermophilus, or Bacillus subtilis cell. In preferred embodiments, the bacterial host cell is a Bacillus licheniformis cell, a Bacillus pumilus cell, or a Bacillus subtilis cell. Preferably, the bacterial host cell is a bacillus licheniformis cell.

In the method of the present invention, the process, the bacterial host cell may be Lactobacillus acidophilus (), lactobacillus plantarum (), lactobacillus gossypii (), lactobacillus bulgaricus (), lactobacillus reuteri (), escherichia coli, staphylococcus aureus (), corynebacterium glutamicum (), corynebacterium aceti (), corynebacterium acetoacidophilus (), corynebacterium meyeri (), corynebacterium ammoniagenes (), corynebacterium thermoaminogenes (), corynebacterium febrile (), streptococcus suis (), brevibacterium flavum (), brevibacterium lactofermentum (), pseudomonas pseudomonad (), pseudomonas syringae (), streptomyces coelicolor (), streptomyces lividi (), streptomyces lividans (), streptomyces albus (), streptomyces avermitis (), gluconobacter oxydan (), gluconobacter aceti (), fusobutyrate (), streptococcus equi (), streptococcus pyogenes (), streptococcus mammitis (), streptococcus (E.suis () Streptococcus equi subsp zooepidemicus (Streptococcus equi subsp. Zooepidemicus) or Basfia succiniciproducens.

In another embodiment, the bacterial host cell may additionally comprise modifications, such as deletions or disruptions, to other genes that may be detrimental to the production, recovery, or use of the polypeptide of interest. In one embodiment, the bacterial host cell is a protease deficient cell. In another embodiment, a bacterial host cell, such as a bacillus cell, comprises a disruption or deletion of an extracellular protease gene, including but not limited to aprE, mpr, vpr, bpr and/or epr. In one embodiment, the bacterial host cell does not produce spores. In another embodiment, a bacterial host cell, such as a bacillus cell, comprises a disruption or deletion of spoIIAC, sigE, and/or sigG. In one embodiment, a bacterial host cell, such as a bacillus cell, comprises a deletion of one of the genes involved in the biosynthesis of the surface active peptide, such as srfA, srfB, srfC and/or srfD. See, for example, U.S. patent 5958728. In another embodiment, the bacterial host cell comprises a disruption or deletion of one of the genes involved in polyglutamic acid biosynthesis. Other genes detrimental to the production, recovery or use of the polypeptide of interest may also be disrupted or deleted, including but not limited to the amyE gene.

In another embodiment, the bacterial host cell is a standard E.coli cloning host cell, including but not limited to DH 5. Alpha (Invitrogen), DH10B (Invitrogen), omnimax (Invitrogen), INV110 (Invitrogen), TOP10 (Invitrogen), HB101 (Promega), SURE (Stratagene), XL1-Blue (Stratagen), TG1 (Lucigen), and JM109 (NEB). In another embodiment, the bacterial host cell is a standard bacillus subtilis cloning host cell, including, but not limited to, bacillus subtilis harboring a defective hsd (RI) R-M-locus, such as bacillus subtilis IG-20 (BGSC 1a 436), or a defective hsdRM1 mutation, such as bacillus subtilis 1012WT (Mobitec).

Alternative other host cells include, but are not limited to: aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), hansenula polymorpha (Hansenula polymorpha), thermomyces lanuginosus (Thermomyces lanuginosus), fusarium oxysporum (Fusarium oxysporum), fusarium heterosporum (Fusarium heterosporum), pichia pastoris (also known as Komagataella phaffii), myceliophthora thermophila (Myceliopthora thermophile) (C1), themothelomyces thermophila, schizosaccharomyces pombe (Schizosaccharomyces pombe), trichoderma (Trichoderma), preferably Trichoderma reesei (Trichoderma reesei), and Saccharomyces (Saccharomyces), preferably Saccharomyces cerevisiae (Saccharomyces cerevisiae), or Rhizomucor (Rhizomucor).

Preparation method

Another embodiment of the invention is a method of obtaining an alpha-amylase variant of a parent alpha-amylase comprising the steps of:

a) According to SEQ ID NO:3, the number of the amino acid sequence shown in 3, corresponding to the selection from 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 47, 48, 51, 54, 59, 60, 63, 70, 71, 72, 73, 75, 76, 81, 82, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 103, 106, 108, 109, 113, 114, 115, 116, 117, 119, 120, 123, 125, 126, 128, 129, 131, 132, 133, 134, 135, 136, 139, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 154, 155, 156, 158, 160, 161, 162, 163, 170, 172, 180, 176, 182, 176. 184, 185, 187, 188, 189, 191, 192, 193, 203, 208, 210, 211, 212, 213, 214, 215, 216, 218, 219, 221, 222, 223, 224, 225, 226, 227, 230, 231, 233, 234, 235, 236, 237, 238, 240, 242, 243, 245, 249, 250, 251, 252, 253, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 284, 285, 286, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 301, 302, 303, 304, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 317, 319, 320, 321, 322, 323, 324, 326, 327, 333, 334, 336, 337, 338, 342 344. 345, 346, 348, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 362, 363, 364, 365, 366, 367, 368, 370, 372, 375, 376, 378, 379, 381, 382, 383, 384, 385, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 405, 406, 407, 408, 410, 413, 414, 415, 416, 418, 421, 424, 426, 427, 429, 431, 432, 434, 436, 438, 439, 440, 442, 443, 445, 446, 447, 448, 449, 451, 453, 455, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 470, 471, 474, 480 and 482, preferably a combination thereof, is introduced into the parent enzyme, preferably a starch ID of SEQ ID 1;

b) Alternatively, deletions are introduced at two or more positions of the parent alpha-amylase, said positions being two or more of R181, G182, D183, and G184 numbered according to the amino acid sequence shown in SEQ ID NO 3;

the method thus provides an alpha-amylase variant of the parent alpha-amylase,

wherein the variant has at least 60% sequence identity to the amino acid sequence of the polypeptide of SEQ ID No. 1, and wherein the alpha-amylase variant has alpha-amylase activity, and preferably wherein the alpha-amylase variant has improved properties relative to the parent.

Methods for introducing amino acid changes, such as substitutions or deletions, in protein sequences are well known in the art. Variants may be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semisynthetic gene construction, random mutagenesis, shuffling, and the like. Site-directed mutagenesis is a technique whereby one or more (several) mutations are made at one or more defined sites in a polynucleotide encoding a parent. Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis may also be performed in vitro by cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid comprising the polynucleotide encoding the parent, followed by ligation of the oligonucleotide comprising the mutation in the polynucleotide. Typically, the restriction enzymes used to digest the plasmid and oligonucleotide are identical, allowing the cohesive ends of the plasmid and insert to be ligated to one another. See, e.g., scherer and Davis,1979,Proc.Natl.Acad.Sci.USA 76:4949-4955; and Barton et al, 1990,Nucleic Acids Res.18:7349-4966. Site-directed mutagenesis may also be accomplished in vivo by methods known in the art. See, for example, U.S. patent application publication No. 2004/0171154; storici et al 2001,Nature Biotechnol.19:773-776; kren et al, 1998, nat. Med.4:285-290; and Calissano and Macino,1996,Fungal Genet.Newslett.43:15-16. Any site-directed mutagenesis procedure can be used in the present invention. There are a number of commercially available kits that can be used to prepare variants.

Synthetic gene construction requires in vitro synthesis of the designed polynucleotide molecule to encode the polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as the multiplex microchip-based techniques described by Tian et al (2004,Nature 432:1050-1054) and the like, in which oligonucleotides are synthesized and assembled on a photo-programmable microfluidic chip.

Known mutagenesis, recombination and/or shuffling methods can be used followed by relevant screening procedures to prepare and test single or multiple amino acid substitutions, deletions and/or insertions, such as Reidhaar-Olson and Sauer,1988,Science 241:53-57; bowie and Sauer,1989,Proc.Natl.Acad.Sci.USA 86:2152-2156; WO 95/17413; or those disclosed in WO 95/22625. Other methods that may be used include error-prone PCR, phage display (e.g., lowman et al, 1991,Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al, 1986,Gene 46:145;Ner et al, 1988, DNA 7:127).

The mutagenesis/shuffling method can be combined with high throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al, 1999,Nature Biotechnology 17:893-896). The mutagenized DNA molecules encoding the active polypeptides may be recovered from the host cells and rapidly sequenced using methods standard in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.

Semisynthetic gene construction is accomplished by combining aspects of synthetic gene construction and/or site-directed mutagenesis and/or random mutagenesis and/or shuffling. A typical feature of semi-synthetic construction is the process of using synthetic polynucleotide fragments in conjunction with PCR techniques. Thus, defined regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, while still other regions can be subject to error-prone PCR or non-error-prone PRC amplification. The polynucleotide subsequences may then be shuffled.

The alpha-amylase variants obtained can be produced on an industrial scale and subsequently purified. Industrial production of an enzyme is typically accomplished by culturing a host cell (also known as fermentation) that expresses the enzyme. Suitable host cells are described herein. The nucleic acid sequences encoding the alpha-amylase variants described herein may be transformed into a host cell, which is then cultured under conditions suitable for the host cell to produce the alpha-amylase variants. In a preferred embodiment, the alpha-amylase variant is purified from the host cell.

Accordingly, in yet another embodiment, the present invention relates to a method of producing an alpha-amylase variant comprising the steps of:

(a) Providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding an alpha-amylase variant described herein by introducing the nucleic acid construct comprising a polynucleotide encoding an alpha-amylase variant described herein into a host cell;

(b) Culturing the recombinant host cell of step (a) under conditions conducive for expression of the polynucleotide; and

(c) Optionally, recovering the alpha-amylase variant encoded by the polynucleotide.

The host cells are typically cultured in a suitable nutrient medium to grow the recombinant cells and express the desired protein. At the end of the fermentation, the fermentation broth is collected and may be further processed, wherein the fermentation broth comprises a liquid fraction and a solid fraction. The enzyme of interest may be further purified from the fermentation broth.

The alpha-amylase variants described herein may be secreted from the microbial cells (into the liquid fraction of the fermentation broth) or not (and thus comprised in the cells of the fermentation broth). Depending on this, the alpha-amylase variant may be recovered from the liquid fraction of the fermentation broth or from the cell lysate. Preferably, the alpha-amylase variant is secreted from the cell into the fermentation broth, preferably by a secretion signal peptide added to the amino acid sequence end of the alpha-amylase variant. Recovery of the alpha-amylase variant may be achieved by methods known to those skilled in the art. Suitable methods for recovering proteins from fermentation broths include, but are not limited to, collection, centrifugation, filtration, extraction, and precipitation. If the product of interest precipitates or crystallizes in the fermentation broth, or at least partially binds to the particulate matter of the fermentation broth, additional processing steps may be required to liberate the protein of interest from the biomass or to solubilize the protein crystals of interest and precipitate. US 6316240B1 and WO 2008/110498 A1 describe a method for recovering a protein of interest precipitated and/or crystallized during fermentation from a fermentation broth. In the case where the desired protein is contained in cells of the fermentation broth, it may be desirable to release the product of interest from the cells. Release from the cells may be achieved, for example, but not limited to, by performing cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, sonication, french press, or a combination thereof.

The alpha-amylase variants may be purified from the fermentation broth by methods known in the art. For example, the alpha-amylase variant may be isolated from the fermentation broth by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing and size exclusion), electrophoresis procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., protein Purification, j. -c. janson and Lars Ryden, editors, VCH Publishers, new York, 1989).

Composition and method for producing the same

The purified solution of the alpha-amylase variants described herein may be further processed to form a composition comprising an alpha-amylase, i.e., an "alpha-amylase variant preparation". Thus, also claimed herein are compositions comprising an alpha-amylase variant described herein and at least one other ingredient.

The alpha-amylase variant formulation may be solid or liquid. Protein formulations may be obtained by using techniques known in the art. For example, but not limited thereto, the solid enzyme preparation may be obtained by extrusion or granulation. Suitable extrusion and pelletisation techniques are known in the art and are described, for example, in WO 94/1944A1 and WO 97/43482A 1. The liquid enzyme preparation may comprise an amount of enzyme of 0.1wt% to 40wt%, 0.5wt% to 30wt%, 1wt% to 25wt%, 1wt% to 10wt%, or preferably 1 to 6wt%, each relative to the total weight of the enzyme preparation.

Preferably, the enzyme preparation comprises an alpha-amylase variant of the invention in an amount of 1-100g of active alpha-amylase variant per kg of enzyme preparation, preferably 5-80g/kg, 5-60g/kg or 10-40g/kg.

In one embodiment, the enzyme preparation, in particular the liquid enzyme preparation, further comprises one or more further compounds selected from the group consisting of solvents, salts, pH regulators, preservatives, enzyme stabilizers and thickeners. The solvent may be water and/or an organic solvent. The aqueous enzyme preparations of the invention may comprise more than about 50wt%, more than about 60wt%, more than about 70wt% or more than about 80wt% of water, all relative to the total weight of the enzyme preparation. The organic solvent may be a water-miscible solvent. The organic solvent may be one or more selected from glycerol, propylene glycol, polypropylene glycol and polyethylene glycol.

In one embodiment, the amylase formulation comprises at least one preservative. Preferably, a preservative means a substance added to the liquid composition for preservation, which means that more preferably, compounds known to have preservation characteristics contained in the liquid composition formed during production are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1, 3-diol, and formic acid in acid form or its salt form, and 4,4' -dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the invention comprises at least one preservative in an amount of less than 10ppm, such as from 2ppm to 5wt%, relative to the total weight of the liquid composition. Preferably, the amylase preparation is preservative-free, meaning that the preservative content is less than 1ppm.

In one embodiment, the enzyme preparation described herein may comprise at least one enzyme stabilizer. Preferred enzyme stabilizers are selected from the group consisting of polyols (e.g., 1, 3-propanediol, ethylene glycol, glycerol, and 1, 2-propanediol), salts (e.g., caCl2, mgCl2, naCl), formic acid, formate salts (e.g., sodium formate), acetate salts, and any combination thereof. If protease is present, protease inhibitors may be added, preferably selected from borates (borates), boric acid (bonic acid), bonic acid (e.g., phenylboronic acid (phenylboronic acid) such as 4-FPBA), peptide aldehydes, peptide acetals, and peptide aldehyde bisulfite adducts (peptide aldehyde hydrosulfite adduct), preferably peptide aldehydes such as Z-VAL-H or Z-GAY-H.

The liquid enzyme preparation of the invention may comprise residual components such as salts derived from the fermentation medium, cell debris derived from the production host cells, metabolites produced by the production host cells during fermentation. In one embodiment, the residual ingredients may be included in the liquid enzyme preparation in an amount of less than 30wt%, less than 20wt%, less than 10wt% or less than 5wt%, relative to the total weight of the aqueous enzyme preparation.

Second enzyme

In another embodiment, a composition comprising an alpha-amylase variant described herein further comprises one or more second enzymes different from the alpha-amylase variant. Preferably, the second enzyme is selected from: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, aminopeptidases, amylases, asparaginase, carbohydrases, carboxypeptidases, catalases, cellulases, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, esterases, alpha-galactosidases, beta-galactosidases, glucoamylases, alpha-glucosidase, beta-glucosidase, hyaluronan synthases, invertases, laccases, lipases, mannosidases, mutanases, oxidases, pectolyses, peroxidases, phytases, polyphenol oxidases, proteases, ribonucleases, transglutaminases, and xylanases. In particular, the second enzyme is selected from the group consisting of a protease, a second amylase, a lipase, a cellulase, a laccase, a mannanase, a pectinase, a xylanase, a nuclease, and any combination thereof. Preferably, the second enzyme is selected from the group consisting of amylases, proteases, cellulases, mannanases and lipases which are different from the amylases of the invention. Preferably, the second enzyme is selected from the group consisting of proteases, mannanases and lipases, especially preferably proteases or mannanases, most preferably proteases, preferably subtilisins.

Protease enzyme

Proteases are active proteins that exert "protease activity" or "proteolytic activity". Proteolytic activity is related to the protease or the rate at which the proteolytic enzyme degrades the protein over a defined time period.

Preferably, the second enzyme different from the alpha-amylase variant is a protease having at least 40% to 100% identity to the full length polypeptide sequence of any of SEQ ID NOs 10-14. In one embodiment, the protease comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the full-length polypeptide sequence set forth in SEQ ID NO. 10, 11, 12, 13 or 14, preferably SEQ ID NO. 10.

Preferably, the protease used in combination with the alpha-amylase variants described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to SEQ ID No. 10, and further comprises amino acid substitutions in one or more of the following positions: 3. 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (numbered according to BPN'), and which has proteolytic activity. In one embodiment, the protease is not mutated at positions Asp32, his64 and Ser221 (numbered according to BPN'). Preferably, the protease used in combination with the alpha-amylase variants described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to SEQ ID No. 10, and which is further characterized as follows: the amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (a), or glycine (G), or serine (S), preferably glutamic acid (E), is present at position 101 (numbered according to BPN'), and has proteolytic activity. Most preferred is a protease having at least 80% but less than 100% sequence identity to SEQ ID NO 10 and having the amino acid glutamic acid (E) at position 101 (numbered according to BPN') and having proteolytic activity. The protease may comprise amino acid substitutions at position 101, such as R101E alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and/or 274 (numbered according to BPN'), and have proteolytic activity. In one embodiment, the protease comprises one or more additional substitutions: (a) threonine (3T) at position 3, (b) isoleucine (4I) at position 4, (c) alanine, threonine or arginine (63A, 63T or 63R) at position 63, (D) aspartic acid or glutamic acid (156D or 156E) at position 156, (E) proline (194P) at position 194, (f) methionine (199M) at position 199, (G) isoleucine (205I) at position 205, (h) aspartic acid, glutamic acid or glycine (217D, 217E or 217G) at position 217, (I) a combination of two or more amino acids according to (a) to (h). Suitable proteases may be at least 80% identical to SEQ ID NO. 10 and have the following characteristics: comprising one amino acid (according to (a) - (h)) or a combination according to (i), together with amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (numbered according to BPN'), and having proteolytic activity. In one embodiment, the protease is at least 80% identical to SEQ ID NO. 10 and comprises a mutation (numbered according to BPN') R101E or S3T+V4I+V205I or S3T+V4I+R101E+V205I or S3T+V4I+V199M+V205I+L217D and has proteolytic activity. In another embodiment, the protease comprises an amino acid sequence having at least 80% identity to SEQ ID No. 10 and is further characterized by: comprises S3T+V4I+S9R+A15T+V68A+D99S+R101S+A103S+I200V+N218D (numbered according to BPN'), and has proteolytic activity. In another embodiment, the protease may have an amino acid sequence at least 80% identical to SEQ ID No. 10 and is further characterized by: comprises R101E, and one or more mutations selected from S156D, L262E, Q137H, S3T, R45E, D, Q, P55N, T W, Y, L, Q D, M, N, T, G D, R, S87E, G97S, A D, E, R, S106A, W, N117W, N120V, D, K, W, N125W, N129W, N136W, N161W, N163A, W, N171W, N172 185W, N199W, N209W, N222W, N238W, N244W, N261T, D and L262N, Q, D (numbered according to BPN'), and has proteolytic activity.

Lipase enzyme

"Lipase", "lipolytic enzyme" and "lipase" all refer to class EC 3.1.1 enzymes ("carboxylesterase hydrolase"). Lipase refers to an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; enzymes having cutinase activity may be referred to herein as cutinases), sterol esterase activity (EC 3.1.1.13) and/or wax ester hydrolase activity (EC 3.1.1.50). Lipases include lipases of bacterial or fungal origin.

In one aspect of the invention, suitable lipases (component (b)) are selected from the following: lipases from Humicola (synonymous with Thermomyces), for example from Humicola lanuginosa (H.lanuginosa) (Thermomyces), described in EP 258068, EP 305116, WO 92/05249 and WO 2009/109500, or from Humicola insolens (H.insolens), described in WO 96/13580; lipase derived from Rhizomucor miehei (Rhizomucor miehei) as described in WO 92/05249; lipases from strains of the genus Pseudomonas, some of which are now more known as Burkholderia (Burkholderia), for example from Pseudomonas alcaligenes (P.alcaligenes) or Pseudomonas pseudoalcaligenes (P.pseudoalcaligenes) (EP 218272, WO 94/25578, WO 95/30744, WO 95/35381, WO 96/00292), pseudomonas cepacia (P.cepacia) (EP 331376), pseudomonas stonecrop (P.stutzeri) (GB 1372034), pseudomonas fluorescens (P.fluoscens), pseudomonas strain SD705 (WO 95/06720 and WO 96/27002), pseudomonas Kang Xinjia (P.wisconsiensis) (WO 96/12012), pseudomonas mendocina (Pseudomonas mendocina) (WO 95/14783), pseudomonas pod (P.glae) (WO 95/35381, WO 96/00292); lipase from streptomyces griseus (Streptomyces griseus) (WO 2011/150157) and streptomyces pristinaespiralis (WO 2012/137147), streptomyces GDSL lipase (WO 2010/064555); lipase from Thermobifida fusca (Thermobifida fusca) disclosed in WO 2011/084412; lipase from geobacillus stearothermophilus (Geobacillus stearothermophilus) disclosed in WO 2011/084417; bacillus lipases, for example, as disclosed in WO 00/60063, lipases from Bacillus subtilis (as disclosed in Dartois et al (1992), biochemica et Biophysica Acta,1131,253-360 or WO 2011/084599), bacillus stearothermophilus (JP S64-074992) or Bacillus pumilus (WO 91/16422); lipase from candida antarctica (Candida antarctica) as disclosed in WO 94/01541; cutinases from Pseudomonas mendocina (US 5389536, WO 88/09367); cutinase from Magnaporthe grisea (WO 2010/107560); cutinases from Fusarum solani pisi as disclosed in WO 90/09446, WO 00/34450 and WO 01/92502; and cutinases from Humicola lanuginosa as disclosed in WO 00/34450 and WO 01/92502.

Such suitable lipase variants are, for example, those developed by the methods disclosed in WO 95/22615, WO 97/04079, WO 97/77202, WO 00/60063, WO 2007/087508, EP 407225 and EP 260105.

Commercially available lipases include, but are not limited to, lipolase under the trade name Lipolase ^TM 、Lipex ^TM 、Lipolex ^TM And lipoclear ^TM (Novozymes A/S), lumafast (originally from Genencor), preferenz L (DuPont) and Lipomax (Gist-Brocades/now DSM).

In one embodiment, the lipase is selected from fungal triacylglycerol lipases (EC category 3.1.1.3). The fungal triacylglycerol lipase may be selected from the group consisting of a lipase of Thermomyces lanuginosus. In one embodiment, the thermomyces lanuginosus lipase is selected from the group consisting of triacylglycerol lipases of amino acids 1-269 of SEQ ID NO. 2 according to US5869438 and variants thereof having lipolytic activity.

The thermomyces lanuginosus lipase may be selected from variants with lipolytic activity which are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the full length polypeptide sequence of amino acids 1-269 of SEQ ID No. 2 of US 5869438.

The thermomyces lanuginosus lipase may be selected from variants with lipolytic activity comprising only conservative mutations which do not involve the functional domain of amino acids 1-269 of SEQ ID No. 2 of US 5869438. The lipolytic active lipase variant of this embodiment may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the full length polypeptide sequence of amino acids 1-269 of SEQ ID NO. 2 of US 5869438.

The thermomyces lanuginosus lipase may be selected from variants with lipolytic activity comprising the following amino acid substitutions compared to amino acids 1-269 of SEQ ID No. 2 of US 5869438: T231R and N233R. The lipase variant may further comprise one or more of the following amino acid exchanges compared to amino acids 1-269 of SEQ ID NO. 2 of US 5869438: Q4V, V60S, A150G, L227G, P256K.

The thermomyces lanuginosus lipase may be selected from variants with lipolytic activity comprising amino acid substitutions T231R, N233R, Q4V, V60S, A150G, L227G, P K within the polypeptide sequence of amino acids 1-269 of SEQ ID No. 2 of US5869438 and being at least 95%, at least 96% or at least 97% similar to the full length polypeptide sequence of amino acids 1-269 of SEQ ID No. 2 of US 5869438.

The thermomyces lanuginosus lipase may be selected from variants with lipolytic activity comprising amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID No. 2 of US5869438 and being at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar as compared to the full length polypeptide sequence of amino acids 1-269 of SEQ ID No. 2 of US 586948.

The thermomyces lanuginosus lipase may be a lipolytic active variant of amino acids 1-269 of SEQ ID No. 2 of US5869438, wherein the variant of amino acids 1-269 of SEQ ID No. 2 of US586948 is characterized by comprising the amino acid substitutions T231R and N233R.

The thermomyces lanuginosus lipase may be selected from variants with lipolytic activity comprising amino acid substitutions N11K/A18K/G23K/K24A/V77I/D130A/V154I/V187T/T189Q or N11K/A18K/G23K/K24A/L75R/V77I/D130A/V154I/V187T/T189Q within the polypeptide sequence of amino acids 1-269 of SEQ ID NO:1 of WO2015/01009 and being at least 95%, at least 96% or at least 97% similar to the full length polypeptide sequence of amino acids 1-269 of SEQ ID NO:1 of WO 2015/01009.

Amylase enzyme

Amylases other than the alpha-amylase variants described herein may be selected from alpha-or beta-amylases (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase other than the alpha-amylase variant is also an alpha-amylase (EC 3.2.1.1).

The amylase of the invention has "amylolytic activity" or "amylase activity" involved in the (endo) hydrolysis of glycosidic bonds in polysaccharides. Alpha-amylase activity may be determined by assays known to those of skill in the art for measuring alpha-amylase activity.

In one aspect of the invention, the amylase other than the alpha-amylase variant may be selected from the group consisting of:

an amylase from Bacillus licheniformis with SEQ ID NO. 2 as described in WO 95/10603. Suitable variants are described in WO 95/10603, which comprise one or more substitutions in the following positions: 15,23,105,106,124,128,133,154,156,178,179,181,188,190,197,201,202,207,208,209,211,243,264,304,305,391,408 and 444, which have amylolytic activity. Variants are described in SEQ ID NO. 4 of WO 94/02597, WO 94/018134, WO 97/043424 and WO 99/019467.

An amylase from Bacillus stearothermophilus having SEQ ID NO. 6 or an amylase having an optional C-terminal truncation relative to the wild-type sequence as disclosed in WO 02/10355. Suitable variants of SEQ ID NO. 6 include variants comprising a deletion at position 181 and/or 182 and/or a substitution at position 193.

An amylase from Bacillus species (Bacillus sp.) 707 having SEQ ID NO. 6 as disclosed in WO 99/19467. Preferred variants of SEQ NO. 6 are those having substitutions, deletions or insertions in one or more of the following positions: r181, G182, H183, G184, N195, I206, E212, E216 and K269.

An amylase from Bacillus halmapalus having SEQ ID NO. 2 or SEQ ID NO. 7 as described in WO 96/23872, also described herein as SP-722. Preferred variants are described in WO 97/3296, WO 99/194671 and WO 2013/001078.

An amylase from Bacillus species DSM 12649 with SEQ ID NO. 4 as disclosed in WO 00/22103.

An amylase from bacillus strain TS-23 having SEQ ID No. 2 as disclosed in WO 2009/061380. Other amylases which may be used are the amylase of SEQ ID NO. 2 of WO 2009/061380 or variants thereof having 90% sequence identity to SEQ ID NO. 2. Preferred variants of SEQ ID NO. 2 are variants having substitutions, deletions or insertions in one or more of the following positions: q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. A more preferred variant of SEQ ID NO. 2 is one having a substitution in one or more of the following positions: Q87E, R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M L, F202Y, N225E, R, N272E, R, 243Q, a, E, d, Y305R, R309A, Q R, Q359E, K444E and G475K and/or variants with deletions in positions R180 and/or S181. The most preferred amylase variant of SEQ ID NO. 2 is one having the following substitutions: N128C + k178l + t182G + y305R + G475K, N128C + k178l + T182G + f202Y + y305R + D319T + G475K, S a + n168c + k178l + t182G + y305R + G475K or s125a + n168c + T131I + T165I + k178l + t182G + Y305R + G475K wherein the variant optionally further comprises a substitution at position 243 and/or a deletion at positions 180 and/or 181.

Amylase from a Cellularomyces species (Cytophaga sp.) having SEQ ID NO:1 as disclosed in WO 2013/184577.

An amylase from Bacillus megaterium DSM 90 having SEQ ID NO. 1 as disclosed in WO 2010/104675.

An amylase from a Bacillus species comprising amino acids 1 to 485 of SEQ ID NO. 2 as described in WO 00/60060.

An amylase from bacillus amyloliquefaciens, or a variant thereof, as described in WO 2016/092009, preferably selected from the group of amylases according to SEQ ID NO: 3.

An amylase having SEQ ID NO:12 as described in WO 2006/002643 or an amylase variant comprising substitutions Y295F and M202LITV within the SEQ ID NO: 12.

The amylase described in WO 2011/098531 having SEQ ID NO:6 or comprising substituted amylase variants at one or more positions selected from 193[ G, A, S, T or M ],195[ F, W, Y, L, I or V ],197[ F, W, Y, L or V ],198[ Q or N ],200[ F, W, Y, L, I or V ],203[ F, W, Y, L, I or V ],206[ F, W, Y, N, L, I, V, H, Q, D or E ],210[ F, W, Y, L, I or V ],212[ F, W, Y, L, I or V ],213[ G, A, S, T or M ] and 243[ F, W, Y, L, I or V ] within the SEQ ID NO: 6.

An amylase having SEQ ID NO. 1 as described in WO 2013/001078, or comprising altered amylase variants at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476 and G477 within the SEQ ID NO. 1.

An amylase having a SEQ ID NO:2 as described in WO 2013/00087 or an amylase variant comprising a deletion of positions 181+182, or 182+183, or 183+184 within the SEQ ID NO:2, optionally comprising one or two or more modifications in any position corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 within the SEQ ID NO: 2.

An amylase consisting of a hybrid alpha-amylase from the above-mentioned amylase, for example as described in WO 2006/066594; other examples of amylases are hybrid alpha-amylases comprising residues 1-33 of the Bacillus amyloliquefaciens-derived alpha-amylase shown in SEQ ID NO. 6 of WO 2006/066594 and residues 36-483 of the Bacillus licheniformis alpha-amylase shown in SEQ ID NO. 4 of WO 2006/066594, or variants thereof having 90% sequence identity. Preferred variants of the hybrid alpha-amylase are variants having substitutions, deletions or insertions in one or more of the following positions: g48, T49, G107, H156, a181, N190, M197, I201, a209, and Q264. The most preferred variants of hybrid alpha-amylases comprising residues 1-33 of SEQ ID NO:6 of WO 2006/066594 and residues 36-483 of SEQ ID NO:4 of WO 2006/066594 are variants having the following substitutions: M197T, H156Y+A181T+N190F+A209V+Q264S or G48+T49+G107+H156+A181+N190+I201+A209+Q264.

A hybrid amylase of WO 2014/183920 having a and B domain at least 90% identical to SEQ ID No. 2 of WO 2014/183920 and a C domain at least 90% identical to SEQ ID No. 6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO. 23 of WO 2014/183920 and has amylolytic activity;

a hybrid amylase of WO 2014/183921 having an a and B domain with at least 75% identity to SEQ ID No. 2, SEQ ID No. 15, SEQ ID No. 20, SEQ ID N0:23, SEQ ID No. 29, SEQ ID No. 26, SEQ ID No. 32 and SEQ ID No. 3 disclosed in WO 2014/183921 and a C domain with at least 90% identity to SEQ ID No. 6 of WO 2014/183921, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid alpha-amylase is at least 95% identical to SEQ ID NO. 30 disclosed in WO 2014/183921 and has amylolytic activity.

Other amylases are variants of SEQ ID NO. 1 of WO 2016/203064 having at least 75% sequence identity to SEQ ID NO. 1. Preferred variants are variants comprising modifications in one or more positions corresponding to positions 1, 54, 56, 72, 109, 113, 116, 134, 140, 159, 167, 169, 172, 173, 174, 181, 182, 183, 184, 189, 194, 195, 206, 255, 260, 262, 265, 284, 289, 304, 305, 347, 391, 395, 439, 469, 444, 473, 476 and 477 of SEQ ID No. 1, wherein the alpha-amylase variant has at least 75% but less than 100% sequence identity to SEQ ID No. 1.

Further examples of amylases are the alpha-amylase of WO 2001/066712 having SEQ ID NO. 12 or variants having at least 90% sequence identity to SEQ ID NO. 12. Preferred amylase variants are those having substitutions, deletions or insertions in one or more of the following positions of SEQ ID NO:12 in WO 2001/066712: r28, R118, N174; r181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; r320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions R118K, N195F, R K and R458K, as well as variants additionally having substitutions in one or more positions selected from M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and a339, most preferably variants additionally having substitutions in all of these positions.

In one embodiment, the at least one amylase is selected from commercially available amylases including, but not limited to, those under the trade name Duramyl ^TM 、Termamyl ^TM 、Fungamyl ^TM 、Stainzyme ^TM 、Stainzyme Plus ^TM 、Natalase ^TM Liquozyme X and BAN ^TM 、Amplify ^TM 、Amplify Prime ^TM (from Novozymes A/S) and Rapid ^TM 、Purastar ^TM 、Powerase ^TM 、Effectenz ^TM (M100 from DuPont), preference z ^TM (S1000, S110, S210 and F1000; from DuPont), excellenz ^TM (S3300)、PrimaGreen ^TM (ALL；DuPont)、Optisize ^TM (DuPont) product sold.

Mannanase

As used herein, a "mannanase" is an enzyme selected from the group consisting of mannanase degrading enzymes. The mannanase may be selected from the group consisting of beta-mannosidase (EC 3.2.1.25), endo-1, 4-beta-mannosidase (EC 3.2.1) 78) and 1, 4-beta-mannobiosidase (EC 8.1.100). Preferably, the mannanase is selected from the group of endo-1, 4-beta-mannosidase (EC3.2.1.78), enzyme group which may be referred to herein as endo-beta-1, 4-D-mannanase, beta-mannanase or mannanase.

Mannanases may be selected from mannanases derived from bacillus organisms, such as the mannanases described in the following documents: JP-0304706[ beta-mannanase from Bacillus species ], JP-63056289[ alkaline, thermostable beta-mannanase ], JP-63036774[ Bacillus microorganism FERM P-8856 producing beta-mannanase and beta-mannosidase at alkaline pH ], JP-08051975[ alkaline beta-mannanase from Alkalophila species AM-001 ], WO97/11164[ mannanase from Bacillus amyloliquefaciens ], WO91/18974[ mannanase active at extreme pH and temperature ], WO97/11164[ mannanase from Bacillus amyloliquefaciens ], WO2014/100018[ endo- (3-mannanase) cloned from Bacillus circulans or Bacillus lentus strain CMG1240 (Bleman 1; see US 5476775) ]. Suitable mannanases are described in WO 99/064619.

Mannanases may be selected from mannanases derived from a Trichoderma organism, as disclosed in WO 93/24622.

Mannanases may be selected from commercially available mannanases, e.g. Mannaway ^TM (Novozymes A/S) or Preferenz ^TM (M100)(DuPont)。

Detergent composition

In one embodiment, the invention relates to the use of an alpha-amylase variant in a detergent composition. Accordingly, the present invention also relates to detergent compositions comprising the alpha-amylase variants described herein and one or more detergent ingredients. The one or more detergent ingredients may be selected from other enzymes other than the alpha-amylase of the invention, surfactants, defoamers, builders, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, desiccants, brighteners, buffers, preservatives, anti-corrosion additives, dyes, and perfumes.

Preferably, at least one component of the detergent is a surfactant. Preferably, at least one other ingredient of the detergent is a second enzyme as described herein which is different from the alpha-amylase of the invention.

The detergent ingredients may have more than one function in the end use of the detergent formulation, so any detergent ingredient mentioned in the context of the specific functions herein may have other functions in the end use of the detergent formulation as well. The function of a particular detergent ingredient in the end use of a detergent formulation generally depends on its amount in the detergent formulation, i.e. the effective amount of the detergent ingredient. The type and/or amount of detergent ingredients in the detergent formulation will vary depending on the desired application, such as washing white textiles, colored textiles and wool. The ingredient(s) selected also depend on the physical form of the detergent formulation (liquid, solid, gel, pouch or tablet presentation, etc.). The ingredient(s) selected (e.g. for laundry washing formulations) also depend on regional habits, which relate themselves to the washing temperature used, the machine of the washing machine (vertical axis vs. horizontal axis washing machine), the water consumption per washing cycle, etc., as well as geographical features such as the average hardness of the water.

In one embodiment, the detergent formulation is a formulation of two or more detergent ingredients, wherein at least one ingredient is effective in stain removal, at least one ingredient is effective in providing optimal cleaning conditions, and at least one ingredient is effective in maintaining the physical characteristics of the detergent.

The detergent composition may be a liquid or solid detergent composition or a combination of liquid and solid detergent compositions. The liquid detergent composition is preferably a gel detergent composition. The solid detergent composition may be a soap bar or a powder detergent composition, preferably a powder detergent composition, wherein the powder detergent composition may be compressed into tablets.

The detergent composition may be a unit dose or multi-dose composition. The detergent composition may be in the form of a pouch, including a multi-compartment pouch. The detergent composition may be a laundry detergent or a dishwashing detergent composition suitable for home care and/or industrial and institutional (I & I) cleaning. Both laundry and dishwashing compositions can be in the form of hand wash or automatic wash compositions. Preferably, the dishwashing composition is an automatic dishwashing detergent (ADW).

The detergent pouch may be of any form, shape and material suitable for containing the composition, for example not allowing the composition to be released from the pouch prior to contact with water. The bag is made of a water-soluble film that encloses an interior volume. The interior volume may be divided into compartments of the bag. Preferred films are polymeric materials, preferably polymers that form a film or sheet. Preferred polymers, copolymers or derivatives thereof are selected from the group consisting of polyacrylate and water soluble acrylate copolymers, methyl cellulose, carboxymethyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC). Preferably, the level of polymer in the film (e.g., PVA) is at least about 60%. The preferred average molecular weight is typically from about 20000 to about 150000. The film may also be a blend composition comprising a blend of hydrolytically degradable and water soluble polymers such as polylactide and polyvinyl alcohol (known under the trade designation M8630, sold by Chris Craft in. Prod. Of Gary, ind., US) plus a plasticizer such as glycerol, ethylene glycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch may contain a solid laundry cleaning composition or a portion of ingredients and/or a liquid cleaning composition or a portion of ingredients separated by a water-soluble film. The compartments of the liquid component may be compositionally different from the compartments containing the solid (see e.g. US 2009/0011970).

In one embodiment, the alpha-amylase variant of the invention may be added to the detergent composition in an amount corresponding to 1-1000mg of active alpha-amylase variant per kg of detergent composition, preferably 5-500mg/kg, 5-300mg/kg, 10-200mg/kg or 50-200 mg/kg.

In one embodiment, the detergent formulation has a pH in the range of 5-12, preferably in the range of 6-11, more preferably in the range of 6-10, 7-9 and 7.5-8.5. In one embodiment, the formulation is a detergent formulation, preferably a liquid detergent formulation.

In one embodiment, the detergent formulations of the present invention comprise one or more surfactants. "surfactant" (synonymous herein with "surfactant") refers to an organic chemical that changes the properties of a liquid at an interface when added to the liquid. Surfactants are referred to as nonionic, anionic, cationic or amphoteric surfactants, depending on their ionic charge.

The detergent compositions of the present invention may comprise one or more surfactants, which may be anionic and/or cationic and/or nonionic and/or semi-polar and/or zwitterionic surfactants or mixtures thereof. In a preferred embodiment, the detergent composition of the present invention comprises at least one surfactant. In a particular embodiment, the detergent composition of the present invention comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) are typically present at a level of about 0.1-60wt%, such as 1-40wt%, 3-20wt%, or 3-10wt%. The surfactant(s) are selected based on the desired cleaning application and include any conventional surfactant known in the art. Any surfactant known in the art for use in detergents may be used. Non-limiting examples of surfactants are disclosed in McCutcheon's 2016Detergents and Emulsifiers and McCutcheon's 2016Functional Materials, north american and international versions, MC Publishing Co, 2016. Other useful examples are disclosed in earlier versions of the same publication known to those skilled in the art.

When included therein, the detergent typically comprises from about 1 to 40wt%, such as from 5 to 30wt%, from 5 to 15wt%, or from 20 to 25wt% of the anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, especially Linear Alkylbenzenesulfonates (LAS), isomers of LAS, branched Alkylbenzenesulfonates (BABS), phenylalkansulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2, 3-diylbis (sulfates), hydroxyalkanesulfonates and disulfonates, alkyl Sulfates (AS) such AS Sodium Dodecyl Sulfate (SDS), fatty Alcohol Sulfates (FAS), primary Alcohol Sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known AS alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary Alkane Sulfonates (SAS), paraffin Sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES), including Methyl Ester Sulfonates (MES), alkyl or alkenyl succinic acids, dodecenyl/tetradecyl succinic acid (DTSA), fatty acid derivatives of amino acids, di-and mono-esters or soaps of sulfo succinic acid, and combinations thereof.

When included therein, the detergent typically contains from about 0 to about 10wt% cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanolamine quaternary ammonium salts (quats) (admeq), cetyl Trimethyl Ammonium Bromide (CTAB), dimethyl distearyl ammonium chloride (DSDMAC) and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated Quaternary Ammonium (AQA) compounds, and combinations thereof.

When included therein, the detergent typically contains from about 0.2 to 40wt% nonionic surfactant, for example from 0.5 to 30wt%, especially from 1 to 20wt%, from 3 to 10wt%, from 3 to 5wt% or from 8 to 12wt%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated Fatty Alcohols (PFA), alkyl esters of alkoxyfatty acids such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty Acid Monoethanolamides (FAM), fatty Acid Diethanolamides (FADA), ethoxylated Fatty Acid Monoethanolamides (EFAM), propoxylated Fatty Acid Monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide GA or fatty acid glucamide FAGA), and products available under the trade names SPAN and TWEEN, and combinations thereof.

When included therein, the detergent typically contains from about 0 to about 10wt% of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include Amine Oxides (AO) such as alkyl dimethyl amine oxides, N- (cocoalkyl) -N, N-dimethyl amine oxides and N- (tallow alkyl) -N, N-bis- (2-hydroxyethyl) amine oxides, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

When included therein, the detergent typically contains about 0-10wt% of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines, alkyl dimethyl betaines, sulfobetaines, and combinations thereof.

The detergent formulations of the present invention may comprise one or more compounds selected from complexing agents (chelating agents), precipitating agents and ion exchange compounds, which may form water soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building agents," but are not meant to limit such compounds to this function in the end use of the detergent formulation.

In one embodiment, the detergent formulation of the present invention comprises at least one builder selected from non-phosphate based builders such as sodium gluconate, citrate, silicate, carbonate, phosphonate, aminocarboxylate, polycarboxylate, polysulfonate and polyphosphonate. In one embodiment, the detergent formulation of the present invention comprises a strong chelating builder. Preferably, the detergent compositions of the present invention are phosphate-free, meaning substantially free of phosphate-based builders. Herein, "substantially free of phosphate" is understood to mean that the sum of the phosphate and polyphosphate content is in the range of 10ppm to 1wt%, as determined by gravimetric method, based on the corresponding detergent composition of the present invention. In another preferred embodiment, the detergent formulation comprises a phosphonate, wherein the phosphonate is preferably DTPMP and/or HEDP.

In one embodiment, the detergent formulation of the present invention comprises at least one "citrate salt" selected from the group consisting of mono-and di-alkali metal salts of citric acid, especially the mono-sodium salt, preferably the trisodium salt, the ammonium or substituted ammonium salt of citric acid, and citric acid itself. Citrate can be used as an anhydrous compound or hydrate, for example as sodium citrate dihydrate. The total amount of citrate may be in the range of 0wt% to about 20wt%, in the range of about 0.5wt% to about 10wt%, or in the range of 1-5wt%, all relative to the total weight of the detergent formulation. In one embodiment, the detergent formulation of the present invention comprises a total amount of citrate in the range of about 1-3% relative to the total weight of the detergent formulation.

The detergent compositions of the present invention may comprise one or more silicates. In the context of the present invention, "silicate" includes in particular sodium disilicate and sodium metasilicate, aluminosilicates, such as sodium aluminosilicate, such as zeolite a (i.e. Na ₁₂ (AlO ₂ ) ₁₂ (SiO ₂ ) ₁₂ *27H ₂ O) and sheet silicates, in particular of the formula alpha-Na ₂ Si ₂ O ₅ 、β-Na ₂ Si ₂ O ₅ And delta-Na ₂ Si ₂ O ₅ Those of (3).

The detergent compositions of the present invention may comprise one or more carbonates. The term "carbonate" includes alkali metal carbonates and alkali metal bicarbonates, preferably sodium salts. Especially suitable is sodium carbonate (Na ₂ CO ₃ )。

The detergent compositions of the present invention may comprise one or more phosphonates. "phosphonate" includes, but is not limited to, 2-phosphonate butane-1, 2, 4-tricarboxylic acid (PBTC); ethylenediamine tetra (methylenephosphonic acid) (EDTMPA); 1-hydroxyethane-1, 1-diphosphonic acid (HEDP), CH ₂ C(OH)[PO(OH) ₂ ] ₂ The method comprises the steps of carrying out a first treatment on the surface of the Aminotri (methylenephosphonic Acid) (ATMP), N [ CH ₂ PO(OH) ₂ ] ₃ The method comprises the steps of carrying out a first treatment on the surface of the Aminotri (methylenephosphonic acid), sodium salt (ATMP), N [ CH ] ₂ PO(ONa) ₂ ] ₃ The method comprises the steps of carrying out a first treatment on the surface of the 2-hydroxyethyl iminobis (methylenephosphonic acid), HOCH ₂ CH ₂ N[CH ₂ PO(OH) ₂ ] ₂ The method comprises the steps of carrying out a first treatment on the surface of the Diethylenetriamine penta (methylenephosphonic acid) (DTPMP), (HO) ₂ POCH ₂ N[CH ₂ CH ₂ N[CH ₂ PO(OH) ₂ ] ₂ ] ₂ The method comprises the steps of carrying out a first treatment on the surface of the Diethylene triamine penta (methylene phosphonic acid), sodium salt, C ₉ H _(28-x) N ₃ Na _x O ₁₅ P ₅ (x=7); hexamethylenediamine (tetramethylene phosphonic acid), potassium salt, C ₁₀ H _(28-x) N ₂ K _x O ₁₂ P ₄ (x=6); and bis (hexamethylene) triamine (pentamethylene phosphonic acid), (HO) ₂ )POCH ₂ N[(CH ₂ ) ₂ N[CH ₂ PO(OH) ₂ ] ₂ ] ₂ . Salts thereof may also be suitable.

The detergent compositions of the present invention may comprise one or more aminocarboxylate salts. Non-limiting examples of suitable "aminocarboxylates" include, but are not limited to: diethanolglycine (DEG), dimethylglycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethylamino diacetic acid, ethylenediamine tetraacetic acid (EDTA), N- (2-hydroxyethyl) iminodiacetic acid (HEIDA), hydroxyethylethylenediamine triacetic acid, N-hydroxyethylethylenediamine triacetic acid (HEDTA), hydroxyethylethylenediamine tetraacetic acid, diethylenetriamine pentaacetic acid (DTPA) and methylglycine diacetic acid (MGDA), glutamic diacetic acid (GLDA), iminodisuccinic acid (IDS), hydroxyiminodisuccinic acid, ethylenediamine disuccinic acid (EDDS), aspartic acid diacetic acid, and alkali metal or ammonium salts thereof. Other suitable are aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N, N-diacetic acid (ASDA), aspartic acid-N-monopropionic Acid (ASMP), N- (2-sulfomethyl) aspartic acid (SMAS), N- (2-sulfoethyl) aspartic acid (SEAS), N- (2-sulfomethyl) glutamic acid (SMGL, N- (2-sulfoethyl) glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), alpha-alanine-N, N-diacetic acid (alpha-ALDA), serine-N, N-diacetic acid (SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, examples of cations having at least one permanently quaternized nitrogen atom include tetramethyl ammonium, tetraethyl ammonium, dimethyl diethyl ammonium and N-C ₁₀ -C ₂₀ -alkyl trimethylammonium. With at least one temporaryExamples of cations of quaternized nitrogen atoms include protonated amines and ammonia, such as monomethyl ammonium, dimethyl ammonium, trimethyl ammonium, monoethyl ammonium, diethyl ammonium, triethyl ammonium, n-C ₁₀ -C ₂₀ -alkyldimethylammonium, 2-hydroxyethylammonium, bis (2-hydroxyethyl) ammonium, tris (2-hydroxyethyl) ammonium, N-methyl-2-hydroxyethylammonium, N-dimethyl-2-hydroxyethylamine, in particular NH4 ⁺ 。

In one embodiment, the detergent composition of the present invention comprises more than one builder. Preferably, the detergent composition of the present invention comprises less than 0.2wt% nitrilotriacetic acid (NTA), or from 0.01 to 0.1wt% NTA, relative to the total weight of the detergent composition.

In one embodiment, the detergent composition of the present invention comprises at least one amino carboxylate selected from methylglycine diacetic acid (MGDA), glutamic diacetic acid (GLDA) and their respective salts, e.g. their base (e.g. sodium) salts, in an amount ranging from 0.1wt% to 25.0wt%, from 1.0wt% to 18.0wt%, from 3.0wt% to 15.0wt%, from 3.0wt% to 10.0wt% or from 5.0wt% to 8.0wt% relative to the total weight of the detergent composition.

The detergent formulations of the present invention may comprise one or more hydrotropes. The one or more hydrotropes may be selected from organic solvents such as ethanol, isopropanol, ethylene glycol, 1, 2-propanediol, and other organic solvents known in the art to be miscible with water under normal conditions without limitation. In one embodiment, the detergent formulation of the present invention comprises 1, 2-propanediol in a total amount in the range of 5-10wt%, preferably about 6wt%, all relative to the total weight of the detergent formulation. Other non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium Xylenesulfonate (SXS), sodium Cumene Sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

In one embodiment, the detergent composition comprises at least one preservative. Preferably, a preservative means a substance added to the liquid composition for preservation, which means that more preferably, compounds known to have preservation characteristics contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1, 3-diol, and formic acid in acid form or its salt form, and 4,4' -dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the invention comprises at least one preservative in an amount of less than 10ppm, such as from 2ppm to 5wt%, relative to the total weight of the liquid composition. Preferably, the liquid composition is preservative-free, meaning that the preservative content is less than 1ppm.

The detergent formulation may comprise one or more other enzymes other than the alpha-amylase variant of the invention selected from the group consisting of proteases, amylases other than the alpha-amylase variant of the invention, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, dnases, pectinases, pectate lyases, pectolyases, carbohydrases, arabinanases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases. Preferably, the detergent formulation comprises at least the protease disclosed herein, preferably a protease comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS 10-14, preferably SEQ ID NO 10. Particularly preferred additional enzymes are disclosed elsewhere herein, and the relevant description is also hereby incorporated by reference in this section of the specification.

The detergent formulation may comprise a water soluble source of calcium and/or magnesium ions. In one embodiment, the detergent formulation comprises at least one enzyme stabilizer selected from the group consisting of the polyols and water-soluble salts described above.

The detergent formulation may comprise at least one enzyme stabilizer selected from the boron-containing compounds and peptide stabilizers described above.

In one embodiment, the present invention relates to a method of providing a liquid detergent formulation, more preferably a liquid laundry detergent formulation, comprising the steps of mixing in one or more steps:

(a) At least one alpha-amylase variant of the invention, preferably wherein the amylase is provided within an enzyme preparation of the invention; and

(b) At least one detergent ingredient selected from the group consisting of surfactants, builders and hydrotropes, present in an amount effective for cleaning performance or for maintaining the physical characteristics of the detergent.

Preferably, the detergent composition is according to table 1a below.

An alternative detergent formulation is a detergent composition according to table 1b below.

One or more enzymes described herein may be added to the detergent formulations shown in table 1a and table 1 b.

Application method

The invention also relates to the use of the alpha-amylase variants described herein in cleaning processes, such as laundry or hard surface cleaning, including home care and I & I cleaning. The invention therefore also relates to the use of a composition comprising an alpha-amylase variant as described herein for providing improved alpha-amylase stability in a detergent and/or improved wash performance of a detergent composition.

Accordingly, the present invention also relates to a method for providing a detergent composition having improved stability and/or wash performance of an alpha-amylase comprising using a detergent composition comprising:

a) An alpha-amylase variant described herein; and

b) One or more detergent ingredients.

Description of the preferred embodiments

Particularly preferred herein are:

preferred embodiment 1.) an alpha-amylase variant of a parent alpha-amylase, wherein the variant comprises:

(i) a) amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of: 1,2,3,4,5,6,7,8,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,45,47,48,51,54,59,60,63,70,71,72,73,75,76,81,82,83,86,87,89,90,91,92,93,94,95,96,97,98,99,100,103,106,108,109,113,114,115,116,117,119,120,123,125,126,128,129,131,132,133,134,135,136,139,141,142,143,144,145,146,147,149,150,151,154,155,156,158,160,161,162,163,164,165,169,170,172,173,174,175,176,177,178,180,182,184,185,187,188,189,191,192,193,203,208,210,211,212,213,214,215,216,218,219,221,222,223,224,225,226,227,230,231,233,234,235,236,237,238,240,242,243,245,249,250,251,252,253,255,256,257,258,259,260,261,262,263,264,265,268,269,270,271,272,273,274,275,276,277,279,280,281,282,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,301,302,303,304,306,307,308,309,310,311,312,313,314,315,317,318,319,320,321,322,323,324,326,327,333,334,336,337,338,341,342,343,344,345,346,348,350,351,352,353,354,355,356,357,358,359,360,362,363,364,365,366,367,368,370,372,375,376,378,379,381,382,383,384,385,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,403,405,406,407,408,410,413,414,415,416,418,421,424,426,427,429,431,432,434,436,438,439,440,442,443,445,446,447,448,449,451,453,455,456,457,458,459,460,461,462,463,465,467,468,470,471,474,478,480 and 482 are provided with a plurality of openings,

Or (b)

b) According to the numbering of the amino acid sequence shown in SEQ ID NO. 3, one or more amino acid substitutions, compared to the parent sequence, selected from the group consisting of: X9D, X9F, X9K, X9N, X9P, X9Q, X9S, X9T, X9Y, X179G, X181D, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, X181Y, X186A, X186C, X186D, X186F, X186H, X186I, X186K, X186L, X186M, X186R, X186V, X186W, X186Y, X190H, X195H, X195K, X195L, X195W, X195Y, X206C, X206H, X206M, X206Y, X244A, X244C, X244D, X244E, X244F, X244G, X244H, X244M, X244N, X244V, X402T X419C, X420D, X420E, X420G, X420H, X420K, X420L, X420Q, X422C, X422N, X422H, X423F, X423M, X423Q, X423S, X428C, X428D, X428E, X428G, X428I, X428K, X428L, X428M, X428N, X428R, X428S, X428V, X428W, X428Y, X430A, X430C, X430D, X430E, X430F, X430G, X430L, X430P, X430Q, X430S, X430T, X430V, X435K, X435P, X435S, X435A, X435D, X437L, X437T, X437W, X441C, X441K, X441L, X441M, X441S, X444H, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450L, X450M, X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X452F, X452I, X452K, X452M, X452N, X452T, X454A, X454E, X454K, X454L, X454P, X454S, X454T, X466E, X466W, X469F, X469L, X469Y, X475A, X475K, X475E, X475L, X479I, X479K, X479M, X483F, X483L, X483Q, and X483R, preferably selected from one or more amino acid substitutions: X181D, X181F, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, X181Y, X186A, X186C, X186D, X186F, X186H, X186I, X186K, X186L, X186M, X186R, X186V, X186W, X186Y, X195H, X195K, X195L, X195W, X195Y, X206C, X206H, X206M, X206Y, X441C, X441K, X441L, X441M and X441S,

Or (b)

c) According to the numbering of the amino acid sequence shown in SEQ ID NO. 3, one or more amino acid substitutions, compared to the parent sequence, are selected from the following positions: 1,2,3,5,7,8,9,11,16,18,19,25,26,29,30,31,35,37,40,41,43,44,46,48,49,50,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,71,72,73,74,77,78,79,80,81,84,85,86,87,88,90,91,93,94,97,98,101,102,103,104,105,106,107,109,110,111,112,113,114,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,151,152,153,155,157,158,159,160,161,162,163,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,185,186,187,188,189,190,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,216,217,218,219,220,224,225,227,228,229,230,231,232,235,238,239,241,242,243,244,246,247,248,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,269,270,272,273,274,275,276,278,279,280,281,283,284,286,287,291,292,293,294,295,296,298,299,300,302,303,304,305,306,307,310,311,313,314,315,316,317,319,320,321,322,323,324,325,328,329,330,331,332,334,335,337,339,340,342,344,345,346,347,349,350,354,357,359,360,361,362,363,364,365,368,369,371,372,373,374,375,377,380,382,383,384,385,386,387,388,390,391,393,394,395,396,397,398,400,401,402,403,404,405,406,407,408,409,410,411,412,414,415,416,417,418,419,420,421,422,423,425,427,428,430,431,433,434,435,437,438,439,441,444,445,446,447,449,450,452,454,455,457,458,460,461,462,464,465,466,467,469,470,471,472,473,474,475,476,477,478,479,481,483,484 and 485 are provided with a series of switches,

(ii) The variant has at least 91%, at least 91.5%, at least 92.0%, at least 92.5%, at least 93.0%, at least 93.5%, 94.0%, at least 94.5%, more preferably at least 95%, preferably at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%, preferably at least 91% or at least 95% but less than 100% sequence identity to any of SEQ ID NOs 1, 3, 4, or any of SEQ ID NOs 15-41, preferably the amino acid sequence shown in SEQ ID NOs 1, and

(iii) The variant has alpha-amylase activity.

Preferred embodiment 2.): the alpha-amylase variant of the foregoing preferred embodiment, wherein the parent alpha-amylase of the alpha-amylase variant is an amylase of SEQ ID NO. 1 or SEQ ID NO. 3 or any alpha-amylase having at least 60% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 3, most preferably the parent alpha-amylase of the alpha-amylase variant is an amylase of SEQ ID NO. 1, preferably the resulting amino acid residue of said amino acid substitution is different from the amino acid residue in the corresponding position in SEQ ID NO. 3 or 5.

Preferred embodiment 3.): the alpha-amylase of any of the preceding preferred embodiments, wherein the alpha-amylase variant comprises 1-30, preferably 1-25, 1-20, 1-15, 1-10 or 1-5, 2-30, 2-25, 2-20, 2-15, 2-10, 2-8, or 2-5, preferably 3-30, 3-25, 3-20, 3-15, 3-10, 3-8, or 3-5, preferably 4-30, 4-25, 4-20, 4-15, 4-10, or 4-8 amino acid substitutions at the positions described above.

Preferred embodiment 4.): the alpha-amylase of any of the preceding preferred embodiments, wherein the alpha-amylase variant optionally further comprises 1-30, 1-20, 1-10 or 1-5 additional conservative amino acid substitutions, preferably outside any functional domain, preferably outside the catalytically active domain, of the parent alpha-amylase.

Preferred embodiment 5): the alpha-amylase of any of the preceding preferred embodiments, wherein the variant comprises an a and B domain and a C domain, wherein the amino acid sequence of the a and B domains is at least 75% but less than 100% identical to the amino acid sequence of SEQ ID No. 6 and the amino acid sequence of the C domain is at least 75% but less than 100% identical to the amino acid sequence of SEQ ID No. 8.

Preferred embodiment 6): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the one or more amino acid substitutions are selected from the group consisting of: positions 4, 25, 100, 135, 276, 160, 176, 193, 251, 258, 276, 299, 323, 363, 382, 405, 460 and 482 or selected from positions 4, 7, 25, 37, 70, 100, 118, 135, 160, 176, 193, 210, 251, 281, 258, 323, 361, 363, 368, 405, 434, 441, 459, 460, 451 and 482, preferably selected from positions 25, 100, 135, 176, 193 and 460.

Preferred embodiment 7): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the one or more amino acid substitutions, according to the numbering of the amino acid sequence set forth in SEQ ID NO:3, are selected from the group consisting of: X100D, X100F, X100K, X100L, X103A, X106D, X108A, X109A, X10A, X10C, X10E, X10F, X10L, X10W, X10Y, X113F, X113H, X113M, X113R, X113V, X113W, X113Y, X114A, X114C, X114D, X114E, X114F, X114G, X114H, X114I, X114K, X114L, X114M, X114N, X114Q, X114R, X114S, X114V, X114W, X114Y, X115C, X115D, X117K, X115M, X115N, X115Q, X115R, X115S, X115T, X115V, X116I, X116K, X116M, X116R, X116T, X117A, X117C, X116D, X117N, X117X 116D, X117W, X117X 116R, X117Y, X118A, X118D, X118E, X119H, X119P, X119R, X119S, X119Y, X120E, X120G, X120M, X120S, X120W, X120Y, X123D, X123S, X125A, X125D, X125E, X125F, X125G, X125H, X125K, X125L, X125M, X125N, X125Q, X125R, X125T, X125V, X125W, X125Y, X126D, X128C, X128E, X128L, X128M, X128W, X128Y, X129E, X12N, X12S, X131C, X131I, X131L, X131Q, X131W, X132T, X133A, X133C, X133D, X133E, X133H, X133K, X133N, X134L, X134E, X134L, X134M, X134P, X134T, X134V, X134W, X134Y, X135D, X135E, X135G, X135M, X135N, X135P, X135S, X135T, X135W, X136A, X136C, X136D, X136E, X136F, X136H, X136L, X136M, X136N, X136P, X136W, X139C, X139S, X13A, X13Y, X141V, X142C, X142E, X142F, X142L, X142M, X142Q, X142R, X142W, X142Y, X143F, X144C, X144E, X144G, X144K, X144N, X144Q, X144R, X144S, X144T, X144V, X144Y, X145A, X146C, X146D, X146E, X146F, X146G, X146H, X146K, X146L, X146S, X146T, X146W, X147M, X149E, X14N, X150C, X150E, X150Q, X151C, X151E, X154A, X154Y, X155Y, X156E, X156V, X158H, X158N, X158Y, X15R, X160D, X160E, X160W, X161T, X162V, X163A, X163Q, X163T, X164V, X165S, X165T, X165W, X169A, X169D, X169E, X169S, X169V, X16F, X16R, X170C, X170F, X172A, X172C, X172D, X172K, X172N, X173I, X173Y, X174D, X174E, X174G, X174N, X174P, X174S, X174T, X175A, X175G, X175H, X175K 176, X176K 176A, X177G, X176K 176G, X177G, X177N, X177P, X177R, X177S, X177W, X178F, X17K, X17V, X180M, X180N, X180T, X180W, X182D, X182E, X182N, X182Q, X182S, X185E, X185M, X185N, X187A, X187D, X187M, X187V, X188H, X188T, X188V, X189I, X18F, X18I, X18K, X18M, X18R, X18T, X191F, X191H, X191K, X191M, X191W, X191Y, X192A, X192T, X193D, X193E, X193G, X193V, X19A, X19C, X19D, X19F, X19G, X19H, X19I, X19K, X19L, X19M, X19P, X19Q, X19S, X19T, X19Y, X1R, X1V, X203E, X203R, X208F, X208I, X208Y, X20A, X20C, X20D, X20E, X20F, X20G, X20H, X20K, X20L, X20M, X20N, X20P, X20Q, X20S, X20T, X20V, X20W, X20Y, X210A, X210C, X210D, X210E, X210F, X210M, X210N, X210Q, X210S, X210Y, X211A, X211C, X211D, X211E, X211G, X211H, X211L, X211N, X211Q, X211S, X211T, X211V, X212C, X212D, X212I, X212L, X212W, X213A, X213C, X213M, X213R, X213S, X214E, X214P, X215A, X215D, X215E, X215H, X215G, X216W 218G, X218G, X218E, X218F, X218G, X218H, X218I, X218K, X218L, X218N, X218Q, X218S, X218T, X218V, X218W, X218Y, X219A, X219C, X219D, X219E, X219F, X219G, X219H, X219K, X219M, X219Q, X219R, X219S, X219T, X219W, X219Y, X21E, X21S, X221F, X221N, X222D, X222K, X222S, X222T, X223C, X223L, X223V, X223Y, X224F, X224V, X225A, X225C, X225E, X225F, X225H, X225I, X225N, X225R, X225S, X225Y, X226A, X226C, X226D, X226E, X226G, X226H, X226I, X226L, X226M, X226Q, X226R, X226S, X226T, X226V, X226W, X226Y, X227C, X227F, X227G, X227H, X227I, X227K, X227L, X227M, X227R, X227T, X227V, X227W, X227Y, X22A, X22D, X22E, X22F, X22G, X22K, X22L, X22M, X22Q, X22R, X22T, X22W, X22Y, X230A, X230F, X231C, X231D, X231N, X233C, X233H, X233I, X233M, X233T, X233W, X234C, X235L, X235M, X235V, X236Y, X237C, X237I, X238A, X238F, X238T, X23N, X23Q, X23W, X240M, X243D, X245H, X245M, X249D, X249I, X24G, X250V, X251A, X251E, X251F, X251L, X251M, X251S, X251T, X252C, X252I, X252S, X253C, X253G, X253Y, X255D, X255F, X255I, X255T, X255V, X256C, X257L, X257V, X257Y, X258F, X258Q, X258R, X259L, X25A, X25C, X25D, X25F, X25G, X25H, X25K, X25L, X25M, X25Q, X25S, X25W, X25Y, X260H, X261R, X262C, X262D, X262P, X262Y, X263I, X H, X264T, X264W, X268S, X268F, X26G, X26M 26F, X26M, X26P, X26S, X26V, X26Y, X270A, X270Q, X270Y, X271S, X272F, X272G, X272L, X272S, X273A, X273C, X273D, X273E, X273F, X273H, X273I, X273L, X273M, X273P, X273Q, X273R, X273V, X273W, X273Y, X274F, X274S, X275V, X276D, X276K, X276L, X276N, X276R, X276Y, X277D, X277E, X277T, X272A, X279P, X27A, X27D, X27F, X27G, X27H, X27I, X27Q, X27V, X280D, X280F, X280G, X280H, X280I, X280K, X280N, X280D, X280V, X280Y, X281E, X281H, X284A, X284F, X284H, X284L, X284M, X284N, X284Y, X285G, X285L, X285N, X285P, X286Q, X287A, X287D, X287E, X287H, X287T, X288A, X288K, X288P, X288Y, X289F, X289G, X289R, X289T, X28C, X28D, X28E, X28F, X28G, X28I, X28K, X28N, X28Q, X28S, X28T, X28V, X290D, X290M, X290N, X290Q, X290W, X291D, X291K, X291T, X292D, X292F, X292I, X292L, X292T, X292W, X293Y, X293D, X293E, X293F, X294T, X294F, X296A, X296C, X296L, X296Y, X297E, X297F, X297H, X297K, X297M, X297S, X297V, X299G, X299I, X299K, X299L, X299S, X299Y, X29D, X29E, X29F, X29G, X29H, X29I, X29K, X29L, X29N, X29P, X29Q, X29V, X29W, X29Y, X2I, X2S, X301F, X302H, X302I, X302Q, X302V, X302Y, X303E, X303H, X303I, X303K, X303L, X303M, X303N, X303P, X303R, X303T, X304A, X304D, X304E, X304H, X304K, X304M, X304N, X304P, X304R, X304T, X304W, X304Y, X306A, X306D, X306E, X306G, X306H, X306I, X306M, X306Q, X306R, X306S, X306T, X306V, X306W, X306Y, X307F, X307M, X308S, X309H, X309L, X309Q, X30A, X30E, X30F, X30G, X30H, X30I, X30K, X30L, X30M, X30Q, X30T, X30W, X30Y, X310A, X310Q, X311A, X311E, X311G, X311H, X311K, X311N, X311R, X311T, X311Y, X312L, X312M, X313V, X314C, X314E, X314K, X314Q, X315A, X315C, X315E, X315H, X315K, X315T, X318I, X318S, X318T, X319A, X319D, X319H, X319I, X319K, X319M, X319N, X319P, X319S, X319T, X319W, X31N, X31Q, X31S, X31T, X31V, X31W, X320A, X320C, X320D, X320E, X320G, X320H, X320K, X320L, X320N, X320Q, X320S, X320Y, X321A, X321E, X321K, X321N, X321T, X321V, X321W, X323A, X323G, X323K, X323L, X323V, X324K, X324L, X324M, X324W, X324Y, X326G, X326N, X326S, X326Y, X327C, X327L, X327M, X32A, X32D, X32E, X32F, X32I, X32L, X32M, X32N, X32P, X32Q, X32K, X337K, X32T, X337W, X32K, X337W, X337C, X32X 337C, X337X 32X 337X 32X 37X 32X, X337G, X337I, X337K, X337L, X337M, X337N, X337Q, X337R, X337S, X337T, X337V, X337Y, X338G, X338S, X338T, X33D, X33E, X33H, X33K, X33M, X33Q, X33R, X33Y, X341V, X342P, X343L, X343T, X343W, X343Y, X344I, X344Q, X344V, X345D, X345G, X345M, X345N, X345Q, X345S, X345T, X346A, X346C, X346D, X346G, X346H, X346N, X346Q, X348T, X34H, X34I, X34V, X350H, X350K, X350P, X351A, X351M, X354S, X354I, X354T, X355M, X355I, X356V, X357A, X358I, X358L, X358N, X358P, X358V, X359E, X35A, X35C, X35D, X35G, X35H, X35I, X35L, X35M, X35N, X35P, X35Q, X35R, X35S, X35T, X35V, X35Y, X360A, X360F, X360G, X360I, X360L, X360N, X360Q, X360R, X360S, X360T, X360V, X360Y, X362F, X362K, X362M, X362N, X362T, X362V, X362Y, X363A, X363C, X363D, X363E, X363G, X363H, X363K, X363L, X363M, X363P, X363Q, X363R, X363S, X363T, X363V, X363W, X363Y, X364A, X364C, X364G, X364K, X364L, X364N, X364S, X364T, X364V, X366I, X366L, X366T, X367E, X367S, X368A, X368F, X368L, X368N, X36A, X36E, X36G, X36I, X36K, X36M, X36N, X36P, X36Q, X36R, X36S, X36T, X36V, X370E, X370I, X372A, X372C, X372E, X372F, X372H, X372M, X372N, X372Q, X375A, X375D, X375E, X375I, X375K, X375Q, X375R, X376T, X375W, X375Y, X376G, X376I, X376M, X376Q, X376R, X376S, X376V, X377, X37C, X379A, X379L, X37L and X37L. X37G, X37M, X37P, X37T, X37V, X37W, X381E, X381V, X382A, X382H, X382K, X382L, X382N, X382Q, X382S, X383C, X383D, X383E, X383H, X383I, X383M, X383N, X383Q, X383R, X383S, X383V, X383Y, X384A, X384C, X384D, X384E, X384F, X384I, X384L, X384M, X384N, X384Q, X384R, X384T, X385V, X385W, X385Y, X385A, X385C, X385D, X385E, X385F, X385G, X385H, X385I, X385L, X385M, X385N, X385P, X385Q, X385R, X385S, X385V, X385W, X385C, X385 387E, X387N, X388E, X388F, X388H, X388I, X388M, X388R, X388V, X389G, X389H, X389K, X38N, X390D, X390F, X390M, X390N, X390P, X390R, X391A, X391F, X391G, X391K, X391M, X391N, X391Q, X391S, X391T, X391Y, X392C, X392V, X393E, X393H, X393P, X393S, X393V, X394A, X394C, X394E, X394H, X394I, X394L, X394M, X394N, X394R, X394S, X395A, X395H, X395V, X396H, X396P, X397D, X397H, X397P, X397S, X39M, X39K, X39G, X3G, X39G 3G, X3Q, X3V, X400A, X400D, X400E, X400G, X400H, X400I, X400K, X400L, X400M, X400N, X400P, X400Q, X400R, X400S, X400V, X400W, X401I, X401K, X401M, X401T, X403N, X405C, X405H, X405M, X405T, X405V, X406P, X407D, X407R, X407S, X408E, X408I, X408Q, X40I, X40S, X410H, X410I, X410K, X410L, X410P, X410R, X410Y, X413S, X414C, X414E, X414S, X415I, X416S, X418C, X418N, X418P, X41C, X41D, X41E, X41G, X41G 41S, X41S 41T 41S, X41S 41T 41A, X41S, X426D, X426W, X427C, X427F, X427G, X427K, X427Q, X427R, X427S, X427T, X427V, X429A, X429D, X429E, X429F, X429G, X429I, X429M, X429N, X429P, X429Q, X429S, X429T, X429V, X429W, X42C, X42I, X42Q, X42V, X431I, X434S, X436E, X438F, X438P, X438Y, X439K, X439C, X439P, X440V, X442Q, X443H, X445T, X445A, X445C, X445D, X445T, X445V, X446F, X446I, X446L, X446R, X446S, X446W, X447V, X448D, X448E, X448H, X448N, X449A, X449F, X449G, X449K, X449L, X449M, X449N, X449P, X449Q, X449R, X449S, X449V, X449W, X449Y, X451L, X453G, X453I, X453N, X453P, X453Y, X455L, X456L, X456M, X456R, X456S, X456V, X456W, X456Y, X457A, X457C, X457E, X457F, X457G, X457K, X457L, X457M, X457R, X457S, X457T, X457V, X457W, X458I, X458K, X458V, X458W, X459C, X459D, X459E, X459I, X459K, X459N, X459Q, X459R, X459V, X459Y, X45G, X45N, X460A, X460D, X460E, X460G, X460Q, X460R, X460S, X460T, X460V, X461A, X461E, X461F, X461K, X461L, X461M, X461N, X461Q, X461R, X461S, X461V, X462K, X463L, X465H, X465P, X465R, X465V, X467A, X467E, X467H, X468D, X468H, X470A, X470Q, X470T, X471E, X474F, X474H, X474P, X478A, X478E, X47S, X480D, X480E, X480M, X480R, X480Y, X482C, X482T, X482W, X48F, X48I, X48M, X48Y, X4A, X4C, X4K, X4M, X4Q, X4R, X4S, X51Q, X51T, X51V, X54D, X54G, X54Q, X59T, X5A, X5C, X5D, X5E, X5F, X5H, X5I, X5K, X5L, X5M, X5N, X5P, X5Q, X5R, X5V, X5Y, X60T, X63C, X63V, X6A, X6C, X6E, X6F, X6G, X6H, X6K, X6L, X6M, X6P, X6Q, X6S, X6T, X6V, X6W, X6Y, X70F, X70H, X70L, X70M, X70N, X70Y, X71D, X72C, X72D, X72E, X72N, X72T, X73L, X73N, X73Q, X75A, X75G 75L, X75G, X75T 76C, X75G, X75T 76T, X76G, X75T, X75M 76T, X76G, X75M and X76T, X7F, X7H, X7K, X7N, X7P, X7Q, X7R, X7S, X7V, X7W, X7Y, X81H, X81L, X82K, X82M, X83A, X83D, X83E, X83G, X83R, X83S, X86K, X87D, X87R, X89A, X89C, X89F, X89G, X89L, X89M, X89R, X89S, X8A, X8C, X8F, X8I, X8M, X8P, X8S, X8V, X8W, X8Y, X90A, X90D, X90E, X90F, X90G, X90I, X90M, X90N, X90Q, X90R, X90S, X90V, X90Y, X91C, X91D, X91E, X91F, X91G, X91H, X91I, X91K, X91L, X91M, X91N, X91Q, X91S, X91T, X91V, X91W, X91Y, X92D, X92M, X92V, X93K, X93Q, X94A, X94D, X94E, X94K, X94M, X94V, X94Y, X95E, X95I, X95L, X95V, X96K, X96N, X96Q, X97V, X98E, X98G, X98N, X99H, X99K, X99N, X100W, and X1G.

Preferred embodiment 8): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the alpha-amylase variant further comprises a deletion at one or more amino acids corresponding to positions selected from 181, 182, 183 and 184, preferably two or more amino acid deletions corresponding to positions selected from 181, 182, 183 and 184, preferably amino acid deletions corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletions d183 and g184, wherein numbering is according to the amino acid sequence set forth in SEQ ID No. 3.

Preferred embodiment 9.): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the variant exhibits one or more improved properties relative to said parent alpha-amylase, preferably wherein the improved properties are measured as an Improvement Factor (IF) of > 1.0, and wherein preferably the improvement factor is equal to or greater than 1.2, preferably equal to or greater than 1.3.

Preferred embodiment 10): an alpha-amylase variant of any of the preceding preferred embodiments, wherein, according to the coding for the amino acid sequence shown in SEQ ID NO:3, the one or more amino acid substitutions are selected from the group consisting of amino acid substitutions X4 100 135 135 135 135 160 176 193, 276, 363 363 382, 460G and X482W, or wherein the one or more amino acid substitutions are selected from the group consisting of amino acid substitutions X4 7 25 25 37, 100, 118, 160, 193, 251, 281, 323, 361, 363, 434, 441, 459, 451L and X482W, preferably wherein the one or more amino acid substitutions are selected from the group consisting of amino acid substitutions X25, 100, 176, 193E and X460G.

Preferred embodiment 11.): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the variant comprises a combination of substitutions selected from the group consisting of:

X25H+X176K+X186E+X206Y+X251E+X482W,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X482W,

X25H+X176K+X186E+X206Y+X482W,X25H+X186E+X206Y+X405M+X482W,

X25H+X186E+X206Y+X482W,X25H+X176K+X186E+X193E+X206Y+X251E+X482W,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X482W,X25H+X176K+X186E+X482W,

X25H+X176K+X186E+X193E+X206Y+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,X25H+X176K+X186E+X206Y,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,X4Q+X206Y+X460G+X482W,

X4Q+X25H+X176K+X206Y+X251E+X460G+X482W,

X4Q+X25H+X176K+X193E+X251E+X186E+X482W,

X4Q+X193E+X206Y+X276R+X186E+X482W,X186E+X25H+X482W,

X25H+X193E+X206Y+X251E+X276R+X405M+X186E+X482W,X25H+X251E+X186E+X482W,

X25H+X160W+X176K+X186E+X251E+X405M+X482W,

X193E+X206Y+X405M+X186E+X482W,X3I+X356V,X3I+X356I,X83D+X94E,X94D+X125E,

X131I+X377Q+X410H,X48F+X94D,X48Y+X116K+X218K,X5L+X218K+X225S,

X83E+X116R+X158Y+X181E,X51V+X218K,X83E+X181E,

X4Q+X7W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X37M+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X460G,

X4Q+X25H+X176K+X186E+X206Y+X251E+X460G,

X4Q+X25Y+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X118D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X182D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X460G,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363H+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X363N+X405M,

X4Q+X176K+X181D+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181F+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181N+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X136E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135E+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X135W+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160D+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X160E+X176K+X186E+X193E+X206Y+X251E+X405M,

X7H+X25H+X176K+X186E+X206Y,X7W+X25H+X176K+X186E+X206Y,

X25D+X176K+X186E+X206Y,X25H+X186E+X206Y+X405M+X460G,

X25H+X186E+X206Y+X460G,X25H+X37M+X176K+X186E+X206Y,

X25H+X118D+X176K+X186E+X206Y,X25H+X176K+X182D+X186E+X206Y,

X25H+X176K+X186E+X206Y,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X281N,X25H+X176K+X186E+X206Y+X460G,

X25H+X176K+X186E+X206Y+X251E+X460G,X25H+X176K+X186E+X206Y+X363E,

X25H+X176K+X186E+X206Y+X363H,X25H+X176K+X186E+X206Y+X363N,

X25H+X176K+X186E+X460G,X25H+X176K+X186E+X193E+X206Y,

X25H+X176K+X186E+X193E+X206Y+X460G,

X25H+X176K+X186E+X193E+X206Y+X251E+X460G,X25H+X176K+X181D+X186E+X206Y,

X25H+X176K+X181N+X186E+X206Y,X25H+X176K+X181Q+X186E+X206Y,

X25H+X136E+X176K+X186E+X206Y,X25H+X100W+X176K+X186E+X206Y,

X25H+X135E+X176K+X186E+X206Y,X25H+X135T+X176K+X186E+X206Y,

X25H+X135W+X176K+X186E+X206Y,X25H+X160D+X176K+X186E+X206Y,

X25H+X160E+X176K+X186E+X206Y,X25Y+X176K+X186E+X206Y,

X87D+X186E+X315K+X420K,X87D+X186E+X226D+X314E+X420E+X461E,

X87D+X186E+X226D+X420E,X87D+X186E+X226D+X420E+X461E,

X87D+X186E+X314E+X471E,X87D+X186E+X311K+X314E+X420K,

X87D+X186E+X160D+X461E,X87R+X186E+X315K+X461R,X87R+X186E+X226D+X471E,

X87R+X186E+X226R+X314K+X420K+X461R,X87R+X186E+X226R+X311K+X420K,

X87R+X186E+X314K+X315K,X87R+X186E+X311K+X314E,X87R+X186E+X420E+X450R,

X87R+X186E+X450R+X452R,X186E+X315K+X420E+X452R,

X186E+X226D+X314E+X461E+X471E,X186E+X226D+X314E+X452R,

X186E+X226D+X314K+X460D+X471E+X485R,X186E+X226D+X311K+X460D,

X186E+X226D+X461E+X471E,X186E+X314E+X460D+X461E,

X186E+X314E+X420E+X452R+X461E,X186E+X314K+X450R+X460D,

X186E+X460D+X471E+X485R,X186E+X311K+X314K+X452R,

X186E+X311K+X461E+X485R,X186E+X311K+X420E+X471E,

X186E+X420K+X450R+X471E+X485R,X186E+X420K+X450R+X485R,

X186E+X452R+X461E+X471E,X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X25H+X176K+X186E+X206Y,X83E+X186E+X219D+X226R,

X83E+X186E+X219R+X226D+X314E+X452R,X83E+X186E+X219R+X226R,

X83E+X186E+X160D+X219D+X226R+X452R,X83R+X186E+X219R+X226D,

X160D+X186E+X315K+X450R,X160D+X186E+X226D+X314K+X460D+X461E,

X160D+X186E+X226D+X314K+X420E,X160D+X186E+X314K+X485R,

X160D+X186E+X420E+X452R,X160D+X186E+X420K+X460D,X186E+X276R,

X186E+X206Y,X186E+X206Y+X276R,X186E+X206Y+X276R+X405M,

X186E+X206Y+X405M,X186E+X206Y+X251E,X186E+X206Y+X251E+X276R,

X186E+X206Y+X251E+X276R+X405M,X186E+X206Y+X251E+X405M,

X186E+X206Y+X251E+X323G+X405M,X186E+X206Y+X323G+X405M,X186E+X405M,

X186E+X193E+X206Y+X405M,X186E+X193E+X206Y+X251E,X186E+X251E,

X186E+X251E+X405M,X4Q+X186E,X4Q+X186E+X276R,X4Q+X186E+X206Y,

X4Q+X186E+X206Y+X276R+X405M,X4Q+X186E+X206Y+X405M,

X4Q+X186E+X206Y+X251E+X276R,X4Q+X186E+X206Y+X251E+X276R+X405M,

X4Q+X186E+X206Y+X251E+X405M,X4Q+X186E+X206Y+X251E+X323G+X405M,

X4Q+X186E+X405M,X4Q+X186E+X193E+X206Y+X276R,

X4Q+X186E+X193E+X206Y+X405M,X4Q+X186E+X193E+X206Y+X251E+X405M,

X4Q+X186E+X251E+X276R+X405M,X4Q+X25H+X186E+X206Y,

X4Q+X25H+X186E+X206Y+X276R,X4Q+X25H+X186E+X206Y+X276R+X405M,

X4Q+X25H+X186E+X206Y+X405M,X4Q+X25H+X186E+X206Y+X251E+X323A+X405M,

X4Q+X25H+X186E+X206Y+X323G+X405M,X4Q+X25H+X186E+X405M,

X4Q+X25H+X176K+X186E+X206Y+X276R,

X4Q+X25H+X176K+X186E+X206Y+X276R+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X276R+X405M,

X4Q+X25H+X176K+X186E+X206Y+X251E+X405M,X4Q+X25H+X176K+X186E+X251E,

X4Q+X25H+X176K+X186E+X251E+X405M,X4Q+X25H+X176K+X206Y,

X4Q+X25H+X176K+X206Y+X405M+X460G,X4Q+X25H+X176K+X206Y+X460G,

X4Q+X25H+X176K+X206Y+X251E,X4Q+X25H+X176K+X206Y+X251E+X276R+X405M,

X4Q+X25H+X176K+X206Y+X251E+X460G,X4Q+X25H+X176K+X206Y+X323G,

X4Q+X25H+X160W+X186E+X206Y+X405M,X4Q+X25H+X160W+X186E+X206Y+X251E,

X4Q+X25H+X160W+X186E+X206Y+X251E+X276R+X405M,

X4Q+X25H+X160W+X186E+X206Y+X323G+X405M,X4Q+X25H+X160W+X186E+X405M,

X4Q+X25H+X160W+X186E+X193E+X206Y+X276R,X4Q+X25H+X160W+X186E+X323G,

X4Q+X25H+X160W+X169R+X186E+X206Y+X276R,

X4Q+X25H+X160W+X176K+X186E+X276R+X405M,

X4Q+X25H+X160W+X176K+X186E+X206Y+X276R,

X4Q+X25H+X160W+X176K+X186E+X206Y+X251E,

X4Q+X25H+X160W+X176K+X186E+X206Y+X251E+X405M,

X4Q+X25H+X160W+X176K+X186E+X405M,

X4Q+X25H+X160W+X176K+X186E+X251E+X276R+X405M,

X4Q+X25H+X160W+X176K+X186E+X251E+X405M,X4Q+X25H+X160W+X176K+X206Y,

X4Q+X25H+X160W+X176K+X206Y+X405M,X4Q+X25H+X160W+X176K+X206Y+X460G,

X4Q+X25H+X160W+X176K+X206Y+X251E+X460G,X4Q+X176K+X186E+X276R+X405M,

X4Q+X176K+X186E+X206Y+X276R,X4Q+X176K+X186E+X206Y+X276R+X405M,

X4Q+X176K+X186E+X206Y+X405M,X4Q+X176K+X186E+X206Y+X251E+X276R+X405M,

X4Q+X176K+X186E+X206Y+X251E+X405M,X4Q+X176K+X186E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X276R+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X251E+X276R+X405M,X4Q+X176K+X186E+X251E+X405M,

X4Q+X176K+X186E+X323G+X405M,X4Q+X176K+X206Y,X4Q+X176K+X206Y+X276R,

X4Q+X176K+X206Y+X405M,X4Q+X176K+X206Y+X405M+X460G,

X4Q+X176K+X206Y+X460G,X4Q+X176K+X206Y+X251E+X276R+X405M,

X4Q+X176K+X206Y+X251E+X276R+X405M+X460G,X4Q+X176K+X206Y+X251E+X405M,

X4Q+X176K+X206Y+X251E+X405M+X460G,X4Q+X176K+X206Y+X251E+X323A+X460G,

X4Q+X160W+X186E+X276R+X405M,X4Q+X160W+X186E+X206Y,

X4Q+X160W+X186E+X206Y+X276R+X323G,X4Q+X160W+X186E+X206Y+X405M,

X4Q+X160W+X186E+X206Y+X251E,X4Q+X160W+X186E+X206Y+X251E+X276R+X405M,

X4Q+X160W+X186E+X405M,X4Q+X160W+X186E+X251E+X405M,

X4Q+X160W+X176K+X186E,X4Q+X160W+X176K+X186E+X276R+X405M,

X4Q+X160W+X176K+X186E+X206Y+X251E,

X4Q+X160W+X176K+X186E+X251E+X276R+X405M,

X4Q+X160W+X176K+X186E+X251E+X405M,X4Q+X160W+X176K+X206Y,

X4Q+X160W+X176K+X206Y+X251E+X276R+X460G,

X4Q+X160W+X176K+X206Y+X251E+X323G+X405M+X460G,X25H+X186E,

X25H+X186E+X206Y,X25H+X186E+X206Y+X276R+X405M,

X25H+X186E+X206Y+X276R+X323G,X25H+X186E+X206Y+X405M,

X25H+X186E+X206Y+X251E+X276R+X323G+X405M,X25H+X186E+X206Y+X251E+X405M,

X25H+X186E+X405M,X25H+X186E+X202I+X206Y+X405M,

X25H+X186E+X193E+X206Y+X276R+X405M,X25H+X186E+X193E+X206Y+X405M,

X25H+X186E+X193E+X206Y+X251E+X276R+X405M,X25H+X186E+X251E,

X25H+X186E+X251E+X276R,X25H+X176K+X186E,

X25H+X176K+X186E+X276R+X323G+X405M,X25H+X176K+X186E+X206Y,

X25H+X176K+X186E+X206Y+X276R,X25H+X176K+X186E+X206Y+X251E,

X25H+X176K+X186E+X206Y+X251E+X276R+X405M,

X25H+X176K+X186E+X206Y+X251E+X405M,X25H+X176K+X186E+X405M,

X25H+X176K+X186E+X193E+X206Y,X25H+X176K+X186E+X193E+X206Y+X251E,

X25H+X176K+X186E+X193E+X206Y+X251E+X276R+X405M,

X25H+X176K+X186E+X251E+X276R,X25H+X176K+X206Y,X25H+X176K+X206Y+X276R,

X25H+X176K+X206Y+X276R+X405M,X25H+X176K+X206Y+X276R+X460G,

X25H+X176K+X206Y+X405M,X25H+X176K+X206Y+X460G,X25H+X176K+X206Y+X251E,

X25H+X176K+X206Y+X251E+X276R+X405M+X460G,

X25H+X176K+X206Y+X251E+X276R+X460G,X25H+X176K+X206Y+X251E+X405M,

X25H+X160W+X186E+X206Y,X25H+X160W+X186E+X206Y+X251E,

X25H+X160W+X186E+X206Y+X251E+X276R,

X25H+X160W+X186E+X206Y+X251E+X276R+X323G+X405M,

X25H+X160W+X186E+X206Y+X251E+X405M,

X25H+X160W+X186E+X206Y+X323G+X405M,

X25H+X160W+X186E+X251E+X276R+X405M,

X25H+X160W+X176K+X186E+X206Y+X276R,

X25H+X160W+X176K+X186E+X206Y+X276R+X405M,

X25H+X160W+X176K+X186E+X206Y+X251E+X276R+X323G,

X25H+X160W+X176K+X186E+X206Y+X251E+X405M,

X25H+X160W+X176K+X186E+X405M,X25H+X160W+X176K+X186E+X193E+X206Y,

X25H+X160W+X176K+X186E+X193E+X206Y+X405M,

X25H+X160W+X176K+X186E+X193E+X206Y+X251E+X405M,

X25H+X160W+X176K+X186E+X251E,X25H+X160W+X176K+X186E+X251E+X276R,

X25H+X160W+X176K+X186E+X251E+X405M,

X25H+X160W+X176K+X186E+X323G+X405M,

X25H+X160W+X176K+X206Y+X276R+X323G+X405M,

X25H+X160W+X176K+X206Y+X460G,X25H+X160W+X176K+X206Y+X251E,

X25H+X160W+X176K+X206Y+X251E+X323G+X405M,X176K+X186E+X206Y,

X176K+X186E+X206Y+X276R,X176K+X186E+X206Y+X276R+X405M,

X176K+X186E+X206Y+X405M,X176K+X186E+X206Y+X251E+X405M,

X176K+X186E+X405M,X176K+X186E+X193E+X206Y+X405M,

X176K+X186E+X193E+X206Y+X251E+X405M,X176K+X186E+X193E+X405M,

X176K+X186E+X251E+X276R+X405M,X176K+X186E+X251E+X405M,X176K+X206Y,

X176K+X206Y+X276R,X176K+X206Y+X276R+X460G,X176K+X206Y+X405M,

X176K+X206Y+X405M+X460G,X176K+X206Y+X460G,X176K+X206Y+X251E,

X176K+X206Y+X251E+X405M,X160W+X186E,X160W+X186E+X276R+X405M,

X160W+X186E+X206Y+X276R,X160W+X186E+X206Y+X276R+X405M,

X160W+X186E+X206Y+X405M,X160W+X186E+X206Y+X251E,

X160W+X186E+X206Y+X251E+X405M,X160W+X186E+X405M,

X160W+X186E+X193E+X206Y,X160W+X186E+X193E+X206Y+X405M,

X160W+X186E+X251E+X276R,X160W+X186E+X251E+X276R+X405M,

X160W+X186E+X323G+X405M,X160W+X176K+X186E+X276R+X405M,

X160W+X176K+X186E+X206Y,X160W+X176K+X186E+X206Y+X251E+X405M,

X160W+X176K+X186E+X405M,X160W+X176K+X186E+X193E+X206Y,

X160W+X176K+X186E+X193E+X206Y+X405M,

X160W+X176K+X186E+X193E+X206Y+X251E,

X160W+X176K+X206Y+X276R+X405M+X460G,

X160W+X176K+X206Y+X276R+X323G+X460G,X160W+X176K+X206Y+X405M,

X160W+X176K+X206Y+X405M+X460G,X160W+X176K+X206Y+X460G,

X160W+X176K+X206Y+X251E+X276R,X160W+X176K+X206Y+X251E+X405M+X460G,

X160W+X176K+X206Y+X323G+X405M+X460G,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X281N+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X258Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X281N+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N+X363H+X272V,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X281N+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X281N+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X363H+X254Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X281N+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X8A,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X281N+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q+X281N,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X363H,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X363E,

X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X363H,X25H+X176K+X186E+X206Y,

X25H+X176K+X186E+X206Y+X258Q,X25H+X176K+X186E+X206Y+X258Q,

X25H+X176K+X186E+X206Y+X258Q+X281N,

X25H+X176K+X186E+X206Y+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E,

X25H+X176K+X186E+X206Y+X258Q+X363E+X123D,X25H+X176K+X186E+X206Y+X281N,

X25H+X176K+X186E+X206Y+X181Q,X25H+X176K+X186E+X206Y+X181Q+X193E,

X25H+X176K+X186E+X206Y+X181Q+X363E,X25H+X176K+X186E+X206Y+X193E,

X25H+X176K+X186E+X206Y+X193E+X258Q,

X25H+X176K+X186E+X206Y+X193E+X281N+X363E,

X25H+X176K+X186E+X206Y+X193E+X363E,X25H+X176K+X186E+X206Y+X100W,

X25H+X176K+X186E+X206Y+X100W+X258Q,

X25H+X176K+X186E+X206Y+X100W+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X258Q+X363E+X135C,

X25H+X176K+X186E+X206Y+X100W+X281N,

X25H+X176K+X186E+X206Y+X100W+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X361R,

X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E+X175D,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X181Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X473R,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N,

X25H+X176K+X186E+X206Y+X100W+X135T+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X258Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X281N+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X258Q+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X363E,

X25H+X176K+X186E+X206Y+X100W+X135T+X363E,

X25H+X176K+X186E+X206Y+X100W+X363E,X25H+X176K+X186E+X206Y+X135T,

X25H+X176K+X186E+X206Y+X135T+X258Q,

X25H+X176K+X186E+X206Y+X135T+X258Q+X281N,

x25h+x176k+x186e+x206y+x135t+x258q+x281n+x363E, and

X25H+X176K+X186E+X206Y+X135T+X258Q+X363E,

X25H+X176K+X251E+X405M,

X25H+X176K+X251E+X405M+X482W,

X25H+X176K+X251E+X405M+X439K,

X25H+X176K+X251E+X405M+X439K+X482W,

X251E+X405M,

X251E+X405M+X439K,

X251E+X405M+X482W,

X251E+X405M+X439K+X482W,

X405M+X439K,

X405M+X482W,

X405M+X439K+X482W,

X4Q+X25H+X176K+X251E+X405M+X439K,

X4Q+X25H+X176K+X251E+X405M+X439K+X482W,

X116K+X181T,

X116K+X181T+X225A,

x181t+x225a+x320K, and

x116k+x181t+x225a+x320K, preferably selected from:

X4Q+X25H,

X4Q+X25H+X176K,

X4Q+X25H+X176K+X186E,

X4Q+X25H+X176K+X186E+X251E,

X4Q+X25H+X176K+X186E+X251E+X405M,

X4Q+X25H+X176K+X186E+X251E+X405M+X439K,

X4Q+X25H+X176K+X186E+X251E+X405M+X482W,

X4Q+X25H+X176K+X186E+X251E+X405M+X439K+X482W,

X25H+X176K,

X25H+X176K+X186E,

X176K+X186E,

X25H+X176K+X186E+X251E,

X25H+X176K+X186E+X251E+X405M,

x25h+x176k+x186e+x251e+x405m+x482W, and

X25H+X176K+X186E+X251E+X405M+X439K+X482W,

X25H+X176K+X251E+X405M,

X25H+X176K+X251E+X405M+X482W,

X25H+X176K+X251E+X405M+X439K,

X25H+X176K+X251E+X405M+X439K+X482W,

X251E+X405M,

X251E+X405M+X439K,

X251E+X405M+X482W,

X251E+X405M+X439K+X482W,

X405M+X439K,

X405M+X482W,

X405M+X439K+X482W,

X4Q+X25H+X176K+X251E+X405M+X439K,

X4Q+X25H+X176K+X251E+X405M+X439K+X482W,

X116K+X181T,

X116K+X181T+X225A,

X116K+X181T+X225A+X320K,

x181t+x225a+x320K, and

X181T+X225A+X320K,

most preferably, x1k+x181 t+x225A, x16k+x181 t+x225 a+x25h+x176K, x25h+x176 k+x176 k+x186E, x25h+x176k+x186e+x251e+x405m+x482W, or x4q+x25h+x176k+x186e+x251e+x405m+x482W, most preferably, x25h+x176k+x186E, x4q+x25h+x176k+x186e+x251e+x405m+x482W, or x16k+x181 t+x225a+x320K.

Preferred embodiment 12): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the amino acid residue at an unspecified position (i.e., X) corresponds to the amino acid residue shown in SEQ ID NO:1 or 3, preferably at the corresponding position in SEQ ID NO:1 (numbering according to SEQ ID NO: 3).

Preferred embodiment 13): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the variant comprises deletions D183 and G184 according to the numbering of SEQ ID No. 3.

Preferred embodiment 14.): an alpha-amylase variant of any of the preceding preferred embodiments, further comprising additional amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of: 9,130,179,181,186,190,195,202,206,244,402,419,420,422,423,428,430,435,441,444,450,452,454,466,469,473,475,476,479,483 and 485, preferably one or more additional amino acid substitutions selected from the following amino acid substitutions: X430C, X430D, X430E, X430F, X430G, X430I, X430L, X430P, X430Q, X430S, X430T, X430V, X435E, X435K, X435P, X435R, X435S, X435A, X435D, X437A, X437L, X437T, X437W, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X444E, X444H, X444K, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450I, X450L, X450M, X450P, X450Q, X450R, X450T, X430C, X430D, X430E, X430F, X430G, X430I, X430L, X430P, X430Q, X430S, X430T, X430V, X435E, X435K, X435P, X435R, X435S, X435A, X435D, X437A, X437L, X437T, X437W, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X444E, X444H, X444K, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450I, X450L, X450M, X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X452F, X452I, X452K, X452M, X452N, X452R, X452T, X454A, X454E, X454K, X454L, X454M, X454P, X454S, X454T, X466E, X466W, X469F, X469L, X469Y, X473H, X473Q, X473R, X475A, X475K, X475N, X475E, X475L, X476G, X476E, X479A, X479I, X479K, X479M, X483F, X483I, X483L, X483Q, X483R, X485K, and X485R, wherein according to SEQ ID NO:3, and numbering the amino acid sequences shown in 3.

Preferred embodiment 15): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the number of substitutions compared to SEQ ID NO:1 is 1-30, preferably 1-25, 1-20, 1-15, 1-10, or 1-5, 2-30, 2-25, 2-20, 2-15, 2-10, 2-8, or 2-5, preferably 3-30, 3-25, 3-20, 3-15, 3-10, 3-8, or 3-5, preferably 4-30, 4-25, 4-20, 4-15, 4-10, or 4-8.

Preferred embodiment 16): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the variant comprises or consists of the amino acid sequence shown in SEQ ID No. 1, and wherein there are one or more, preferably 1-30, amino acid substitutions selected from the group consisting of: X100D, X100F, X100K, X100L, X103A, X106D, X108A, X109A, X10A, X10C, X10E, X10F, X10L, X10W, X10Y, X113F, X113H, X113M, X113R, X113V, X113W, X113Y, X114A, X114C, X114D, X114E, X114F, X114G, X114H, X114I, X114K, X114L, X114M, X114N, X114Q, X114R, X114S, X114V, X114W, X114Y, X115C, X115D, X117K, X115M, X115N, X115Q, X115R, X115S, X115T, X115V, X116I, X116K, X116M, X116R, X116T, X117A, X117C, X116D, X117N, X117X 116D, X117W, X117X 116R, X117Y, X118A, X118D, X118E, X119H, X119P, X119R, X119S, X119Y, X120E, X120G, X120M, X120S, X120W, X120Y, X123D, X123S, X125A, X125D, X125E, X125F, X125G, X125H, X125K, X125L, X125M, X125N, X125Q, X125R, X125T, X125V, X125W, X125Y, X126D, X128C, X128E, X128L, X128M, X128W, X128Y, X129E, X12N, X12S, X131C, X131I, X131L, X131Q, X131W, X132T, X133A, X133C, X133D, X133E, X133H, X133K, X133N, X134L, X134E, X134L, X134M, X134P, X134T, X134V, X134W, X134Y, X135D, X135E, X135G, X135M, X135N, X135P, X135S, X135T, X135W, X136A, X136C, X136D, X136E, X136F, X136H, X136L, X136M, X136N, X136P, X136W, X139C, X139S, X13A, X13Y, X141V, X142C, X142E, X142F, X142L, X142M, X142Q, X142R, X142W, X142Y, X143F, X144C, X144E, X144G, X144K, X144N, X144Q, X144R, X144S, X144T, X144V, X144Y, X145A, X146C, X146D, X146E, X146F, X146G, X146H, X146K, X146L, X146S, X146T, X146W, X147M, X149E, X14N, X150C, X150E, X150Q, X151C, X151E, X154A, X154Y, X155Y, X156E, X156V, X158H, X158N, X158Y, X15R, X160D, X160E, X160W, X161T, X162V, X163A, X163Q, X163T, X164V, X165S, X165T, X165W, X169A, X169D, X169E, X169S, X169V, X16F, X16R, X170C, X170F, X172A, X172C, X172D, X172K, X172N, X173I, X173Y, X174D, X174E, X174G, X174N, X174P, X174S, X174T, X175A, X175G, X175H, X175K 176, X176K 176A, X177G, X176K 176G, X177G, X177N, X177P, X177R, X177S, X177W, X178F, X17K, X17V, X180M, X180N, X180T, X180W, X182D, X182E, X182N, X182Q, X182S, X185E, X185M, X185N, X187A, X187D, X187M, X187V, X188H, X188T, X188V, X189I, X18F, X18I, X18K, X18M, X18R, X18T, X191F, X191H, X191K, X191M, X191W, X191Y, X192A, X192T, X193D, X193E, X193G, X193V, X19A, X19C, X19D, X19F, X19G, X19H, X19I, X19K, X19L, X19M, X19P, X19Q, X19S, X19T, X19Y, X1R, X1V, X203E, X203R, X208F, X208I, X208Y, X20A, X20C, X20D, X20E, X20F, X20G, X20H, X20K, X20L, X20M, X20N, X20P, X20Q, X20S, X20T, X20V, X20W, X20Y, X210A, X210C, X210D, X210E, X210F, X210M, X210N, X210Q, X210S, X210Y, X211A, X211C, X211D, X211E, X211G, X211H, X211L, X211N, X211Q, X211S, X211T, X211V, X212C, X212D, X212I, X212L, X212W, X213A, X213C, X213M, X213R, X213S, X214E, X214P, X215A, X215D, X215E, X215H, X215G, X216W 218G, X218G, X218E, X218F, X218G, X218H, X218I, X218K, X218L, X218N, X218Q, X218S, X218T, X218V, X218W, X218Y, X219A, X219C, X219D, X219E, X219F, X219G, X219H, X219K, X219M, X219Q, X219R, X219S, X219T, X219W, X219Y, X21E, X21S, X221F, X221N, X222D, X222K, X222S, X222T, X223C, X223L, X223V, X223Y, X224F, X224V, X225A, X225C, X225E, X225F, X225H, X225I, X225N, X225R, X225S, X225Y, X226A, X226C, X226D, X226E, X226G, X226H, X226I, X226L, X226M, X226Q, X226R, X226S, X226T, X226V, X226W, X226Y, X227C, X227F, X227G, X227H, X227I, X227K, X227L, X227M, X227R, X227T, X227V, X227W, X227Y, X22A, X22D, X22E, X22F, X22G, X22K, X22L, X22M, X22Q, X22R, X22T, X22W, X22Y, X230A, X230F, X231C, X231D, X231N, X233C, X233H, X233I, X233M, X233T, X233W, X234C, X235L, X235M, X235V, X236Y, X237C, X237I, X238A, X238F, X238T, X23N, X23Q, X23W, X240M, X243D, X245H, X245M, X249D, X249I, X24G, X250V, X251A, X251E, X251F, X251L, X251M, X251S, X251T, X252C, X252I, X252S, X253C, X253G, X253Y, X255D, X255F, X255I, X255T, X255V, X256C, X257L, X257V, X257Y, X258F, X258Q, X258R, X259L, X25A, X25C, X25D, X25F, X25G, X25H, X25K, X25L, X25M, X25Q, X25S, X25W, X25Y, X260H, X261R, X262C, X262D, X262P, X262Y, X263I, X H, X264T, X264W, X268S, X268F, X26G, X26M 26F, X26M, X26P, X26S, X26V, X26Y, X270A, X270Q, X270Y, X271S, X272F, X272G, X272L, X272S, X273A, X273C, X273D, X273E, X273F, X273H, X273I, X273L, X273M, X273P, X273Q, X273R, X273V, X273W, X273Y, X274F, X274S, X275V, X276D, X276K, X276L, X276N, X276R, X276Y, X277D, X277E, X277T, X272A, X279P, X27A, X27D, X27F, X27G, X27H, X27I, X27Q, X27V, X280D, X280F, X280G, X280H, X280I, X280K, X280N, X280D, X280V, X280Y, X281E, X281H, X284A, X284F, X284H, X284L, X284M, X284N, X284Y, X285G, X285L, X285N, X285P, X286Q, X287A, X287D, X287E, X287H, X287T, X288A, X288K, X288P, X288Y, X289F, X289G, X289R, X289T, X28C, X28D, X28E, X28F, X28G, X28I, X28K, X28N, X28Q, X28S, X28T, X28V, X290D, X290M, X290N, X290Q, X290W, X291D, X291K, X291T, X292D, X292F, X292I, X292L, X292T, X292W, X293Y, X293D, X293E, X293F, X294T, X294F, X296A, X296C, X296L, X296Y, X297E, X297F, X297H, X297K, X297M, X297S, X297V, X299G, X299I, X299K, X299L, X299S, X299Y, X29D, X29E, X29F, X29G, X29H, X29I, X29K, X29L, X29N, X29P, X29Q, X29V, X29W, X29Y, X2I, X2S, X301F, X302H, X302I, X302Q, X302V, X302Y, X303E, X303H, X303I, X303K, X303L, X303M, X303N, X303P, X303R, X303T, X304A, X304D, X304E, X304H, X304K, X304M, X304N, X304P, X304R, X304T, X304W, X304Y, X306A, X306D, X306E, X306G, X306H, X306I, X306M, X306Q, X306R, X306S, X306T, X306V, X306W, X306Y, X307F, X307M, X308S, X309H, X309L, X309Q, X30A, X30E, X30F, X30G, X30H, X30I, X30K, X30L, X30M, X30Q, X30T, X30W, X30Y, X310A, X310Q, X311A, X311E, X311G, X311H, X311K, X311N, X311R, X311T, X311Y, X312L, X312M, X313V, X314C, X314E, X314K, X314Q, X315A, X315C, X315E, X315H, X315K, X315T, X318I, X318S, X318T, X319A, X319D, X319H, X319I, X319K, X319M, X319N, X319P, X319S, X319T, X319W, X31N, X31Q, X31S, X31T, X31V, X31W, X320A, X320C, X320D, X320E, X320G, X320H, X320K, X320L, X320N, X320Q, X320S, X320Y, X321A, X321E, X321K, X321N, X321T, X321V, X321W, X323A, X323G, X323K, X323L, X323V, X324K, X324L, X324M, X324W, X324Y, X326G, X326N, X326S, X326Y, X327C, X327L, X327M, X32A, X32D, X32E, X32F, X32I, X32L, X32M, X32N, X32P, X32Q, X32K, X337K, X32T, X337W, X32K, X337W, X337C, X32X 337C, X337X 32X 337X 32X 37X 32X, X337G, X337I, X337K, X337L, X337M, X337N, X337Q, X337R, X337S, X337T, X337V, X337Y, X338G, X338S, X338T, X33D, X33E, X33H, X33K, X33M, X33Q, X33R, X33Y, X341V, X342P, X343L, X343T, X343W, X343Y, X344I, X344Q, X344V, X345D, X345G, X345M, X345N, X345Q, X345S, X345T, X346A, X346C, X346D, X346G, X346H, X346N, X346Q, X348T, X34H, X34I, X34V, X350H, X350K, X350P, X351A, X351M, X354S, X354I, X354T, X355M, X355I, X356V, X357A, X358I, X358L, X358N, X358P, X358V, X359E, X35A, X35C, X35D, X35G, X35H, X35I, X35L, X35M, X35N, X35P, X35Q, X35R, X35S, X35T, X35V, X35Y, X360A, X360F, X360G, X360I, X360L, X360N, X360Q, X360R, X360S, X360T, X360V, X360Y, X362F, X362K, X362M, X362N, X362T, X362V, X362Y, X363A, X363C, X363D, X363E, X363G, X363H, X363K, X363L, X363M, X363P, X363Q, X363R, X363S, X363T, X363V, X363W, X363Y, X364A, X364C, X364G, X364K, X364L, X364N, X364S, X364T, X364V, X366I, X366L, X366T, X367E, X367S, X368A, X368F, X368L, X368N, X36A, X36E, X36G, X36I, X36K, X36M, X36N, X36P, X36Q, X36R, X36S, X36T, X36V, X370E, X370I, X372A, X372C, X372E, X372F, X372H, X372M, X372N, X372Q, X375A, X375D, X375E, X375I, X375K, X375Q, X375R, X376T, X375W, X375Y, X376G, X376I, X376M, X376Q, X376R, X376S, X376V, X377, X37C, X379A, X379L, X37L and X37L. X37G, X37M, X37P, X37T, X37V, X37W, X381E, X381V, X382A, X382H, X382K, X382L, X382N, X382Q, X382S, X383C, X383D, X383E, X383H, X383I, X383M, X383N, X383Q, X383R, X383S, X383V, X383Y, X384A, X384C, X384D, X384E, X384F, X384I, X384L, X384M, X384N, X384Q, X384R, X384T, X385V, X385W, X385Y, X385A, X385C, X385D, X385E, X385F, X385G, X385H, X385I, X385L, X385M, X385N, X385P, X385Q, X385R, X385S, X385V, X385W, X385C, X385 387E, X387N, X388E, X388F, X388H, X388I, X388M, X388R, X388V, X389G, X389H, X389K, X38N, X390D, X390F, X390M, X390N, X390P, X390R, X391A, X391F, X391G, X391K, X391M, X391N, X391Q, X391S, X391T, X391Y, X392C, X392V, X393E, X393H, X393P, X393S, X393V, X394A, X394C, X394E, X394H, X394I, X394L, X394M, X394N, X394R, X394S, X395A, X395H, X395V, X396H, X396P, X397D, X397H, X397P, X397S, X39M, X39K, X39G, X3G, X39G 3G, X3Q, X3V, X400A, X400D, X400E, X400G, X400H, X400I, X400K, X400L, X400M, X400N, X400P, X400Q, X400R, X400S, X400V, X400W, X401I, X401K, X401M, X401T, X403N, X405C, X405H, X405M, X405T, X405V, X406P, X407D, X407R, X407S, X408E, X408I, X408Q, X40I, X40S, X410H, X410I, X410K, X410L, X410P, X410R, X410Y, X413S, X414C, X414E, X414S, X415I, X416S, X418C, X418N, X418P, X41C, X41D, X41E, X41G, X41G 41S, X41S 41T 41S, X41S 41T 41A, X41S, X426D, X426W, X427C, X427F, X427G, X427K, X427Q, X427R, X427S, X427T, X427V, X429A, X429D, X429E, X429F, X429G, X429I, X429M, X429N, X429P, X429Q, X429S, X429T, X429V, X429W, X42C, X42I, X42Q, X42V, X431I, X434S, X436E, X438F, X438P, X438Y, X439K, X439C, X439P, X440V, X442Q, X443H, X445T, X445A, X445C, X445D, X445T, X445V, X446F, X446I, X446L, X446R, X446S, X446W, X447V, X448D, X448E, X448H, X448N, X449A, X449F, X449G, X449K, X449L, X449M, X449N, X449P, X449Q, X449R, X449S, X449V, X449W, X449Y, X451L, X453G, X453I, X453N, X453P, X453Y, X455L, X456L, X456M, X456R, X456S, X456V, X456W, X456Y, X457A, X457C, X457E, X457F, X457G, X457K, X457L, X457M, X457R, X457S, X457T, X457V, X457W, X458I, X458K, X458V, X458W, X459C, X459D, X459E, X459I, X459K, X459N, X459Q, X459R, X459V, X459Y, X45G, X45N, X460A, X460D, X460E, X460G, X460Q, X460R, X460S, X460T, X460V, X461A, X461E, X461F, X461K, X461L, X461M, X461N, X461Q, X461R, X461S, X461V, X462K, X463L, X465H, X465P, X465R, X465V, X467A, X467E, X467H, X468D, X468H, X470A, X470Q, X470T, X471E, X474F, X474H, X474P, X478A, X478E, X47S, X480D, X480E, X480M, X480R, X480Y, X482C, X482T, X482W, X48F, X48I, X48M, X48Y, X4A, X4C, X4K, X4M, X4Q, X4R, X4S, X51Q, X51T, X51V, X54D, X54G, X54Q, X59T, X5A, X5C, X5D, X5E, X5F, X5H, X5I, X5K, X5L, X5M, X5N, X5P, X5Q, X5R, X5V, X5Y, X60T, X63C, X63V, X6A, X6C, X6E, X6F, X6G, X6H, X6K, X6L, X6M, X6P, X6Q, X6S, X6T, X6V, X6W, X6Y, X70F, X70H, X70L, X70M, X70N, X70Y, X71D, X72C, X72D, X72E, X72N, X72T, X73L, X73N, X73Q, X75A, X75G 75L, X75G, X75T 76C, X75G, X75T 76T, X76G, X75T, X75M 76T, X76G, X75M and X76T, X7F, X7H, X7K, X7N, X7P, X7Q, X7R, X7S, X7V, X7W, X7Y, X81H, X81L, X82K, X82M, X83A, X83D, X83E, X83G, X83R, X83S, X86K, X87D, X87R, X89A, X89C, X89F, X89G, X89L, X89M, X89R, X89S, X8A, X8C, X8F, X8I, X8M, X8P, X8S, X8V, X8W, X8Y, X90A, X90D, X90E, X90F, X90G, X90I, X90M, X90N, X90Q, X90R, X90S, X90V, X90Y, X91C, X91D, X91E, X91F, X91G, X91H, X91I, X91K, X91L, X91M, X91N, X91Q, X91S, X91T, X91V, X91W, X91Y, X92D, X92M, X92V, X93K, X93Q, X94A, X94D, X94E, X94K, X94M, X94V, X94Y, X95E, X95I, X95L, X95V, X96K, X96N, X96Q, X97V, X98E, X98G, X98N, X99H, X99K, X99N, X100W and X1G, wherein according to SEQ ID NO:3, preferably further comprising 1-10 conservative amino acid substitutions and/or further comprising additional amino acid substitutions at 1-10 positions corresponding to positions selected from the group consisting of: 9,130,179,181,186,190,195,202,206,244,402,419,420,422,423,428,430,435,441,444,450,452,454,466,469,473,475,476,479,483 and 485, preferably 1-10 additional amino acid substitutions selected from the following amino acid substitutions: X430C, X430D, X430E, X430F, X430G, X430I, X430L, X430P, X430Q, X430S, X430T, X430V, X435E, X435K, X435P, X435R, X435S, X435A, X435D, X437A, X437L, X437T, X437W, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X444E, X444H, X444K, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450I, X450L, X450M, X450P, X450Q, X450R, X450T, X430C, X430D, X430E, X430F, X430G, X430I, X430L, X430P, X430Q, X430S, X430T, X430V, X435E, X435K, X435P, X435R, X435S, X435A, X435D, X437A, X437L, X437T, X437W, X441C, X441D, X441K, X441L, X441M, X441N, X441S, X444E, X444H, X444K, X444M, X444N, X444R, X444T, X450C, X450D, X450E, X450H, X450I, X450L, X450M, X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X452F, X452I, X452K, X452M, X452N, X452R, X452T, X454A, X454E, X454K, X454L, X454M, X454P, X454S, X454T, X466E, X466W, X469F, X469L, X469Y, X473H, X473Q, X473R, X475A, X475K, X475N, X475E, X475L, X476G, X476E, X479A, X479I, X479K, X479M, X483F, X483I, X483L, X483Q, X483R, X485K, and X485R, wherein according to SEQ ID NO:3, and numbering the amino acid sequences shown in 3.

Preferred embodiment 17): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the variant comprises a combination of substitutions selected from any of tables 8-12, preferably selected from: according to the numbering of SEQ ID NO. 3,

N25H+R176K+G186E+I206Y+R181Q,N25H+R176K+G186E+I206Y+Y135T,

N25H+R176K+G186E+I206Y+Y100W+Y363E,N25H+R176K+G186E+I206Y+Y100W+T193E,

N25H+R176K+G186E+I206Y+T251E,G4Q+N25H+R176K+G186E+I206Y+T251E+L405M,

N25H+R176K+G186E+I206Y,N25H+G186E+I206Y+L405M,N25H+G186E+I206Y,

N25H+R176K+G186E+T193E+I206Y+T251E,

G4Q+R176K+G186E+T193E+I206Y+T251E+L405M,N25H+R176K+G186E,

N25H+R176K+G186E+T193E+I206Y,G4Q+N25H+R176K+G186E+T251E+L405M,

N25H+R176K+G186E+I206Y,G4Q+R176K+G186E+T193E+I206Y+T251E+L405M,

G4Q+I206Y+N460G,G4Q+N25H+R176K+I206Y+T251E+N460G,

G4Q+N25H+R176K+T193E+T251E+G186E,G4Q+T193E+I206Y+E276R+G186E,

G186E+N25H,N25H+T193E+I206Y+T251E+E276R+L405M+G186E,N25H+T251E+G186E,

N25H+Y160W+R176K+G186E+T251E+L405M,T193E+I206Y+L405M+G186E,

N25H+R176K+G186E+I206Y+G258Q+Y363E,N25H+R176K+G186E+I206Y+R181Q,

N25H+R176K+G186E+I206Y+Y100W,N25H+R176K+G186E+I206Y+Y100W+Q359R,

N25H+R176K+G186E+I206Y+Y100W+G258Q,

N25H+R176K+G186E+I206Y+Y100W+G258Q+Y363E,

N25H+R176K+G186E+I206Y+Y100W+Y135T,

N25H+R176K+G186E+I206Y+Y100W+Y135T+Y363E,N25H+R176K+G186E+I206Y+Y135T,

N25H+R176K+G186E+I206Y+Y135T+G258Q,

N25H+R176K+G186E+I206Y+Y135T+G258Q+Y363E,

N25H+R176K+G186E+I206Y+Y135T+Y363E,N25H+R176K+G186E+I206Y+Y363E,

N25H+R176K+G186E+I206Y+T251E+Y482W,N25H+R176K+G186E+I206Y+Y482W,

N25H+R176K+G186E+T193E+I206Y+T251E+Y482W,

G4Q+R176K+G186E+T193E+I206Y+T251E+L405M+Y482W,

G4Q+N25H+R176K+G186E+T251E+L405M+Y482W,

N25H+R176K+G186E+T193E+I206Y+Y482W,

G4Q+N25H+R176K+G186E+I206Y+T251E+L405M+Y482W,

N25H+G186E+I206Y+L405M+Y482W,N25H+R176K+G186E+Y482W,

N25H+R176K+G186E+T193E+I206Y+T251E+N460G,

G4Q+N25H+R176K+G186E+I206Y+T251E+N460G,

N25H+R176K+G186E+T193E+I206Y+T251E+N460G+Y482W,

G4Q+N25H+R176K+G186E+I206Y+T251E+N460G+Y482W,

N25H+R176K+G186E+N195F+T251E+Y482W,N25H+R176K+G186E+N195F+Y482W,

N25H+R176K+G186E+T193E+N195F+T251E+Y482W,

G4Q+R176K+G186E+T193E+N195F+T251E+L405M+Y482W,

N25H+R176K+G186E+T193E+N195F+Y482W,

G4Q+N25H+R176K+G186E+N195F+T251E+L405M+Y482W,

N25H+G186E+N195F+L405M+Y482W,N25H+R176K+G186E+T193E+N195F+T251E+N460G,

G4Q+N25H+R176K+G186E+N195F+T251E+N460G,

n25h+r176k+g186e+t193e+n195f+t251 e+n450g+y482W, and

G4Q+N25H+R176K+G186E+N195F+T251E+N460G+Y482W，

wherein numbering is according to SEQ ID NO. 3.

Preferred embodiment 18): a polynucleotide encoding an alpha-amylase variant of any of the preceding embodiments.

Preferred embodiment 19): a nucleic acid construct or expression vector comprising the polynucleotide of embodiment 18).

Preferred embodiment 20): a host cell comprising the polynucleotide of embodiment 18.), the nucleic acid construct of embodiment 19), or the expression vector of embodiment 19).

Preferred embodiment 21): a composition comprising an alpha-amylase variant as described in the preceding embodiment, wherein the composition comprises one or more second enzymes different from the alpha-amylase variant, preferably wherein the second enzyme is a protease, a lipase or a mannanase, preferably a mannanase or a protease, particularly preferably a protease, preferably a subtilisin having at least 60% sequence identity to any of SEQ ID NOs 10-14, preferably to SEQ ID NO 1, preferably wherein the protease comprises glutamic acid at position 101 according to the BPN' numbering.

Preferred embodiment 22): a composition comprising an alpha-amylase variant as described in the preceding preferred embodiment, wherein the composition is a detergent composition, preferably comprising an alpha-amylase variant in the preceding preferred embodiment in an amount of 1-1000mg active alpha-amylase variant/kg detergent composition, preferably 5-500mg/kg, 5-300mg/kg, 10-200mg/kg or 50-200mg/kg, preferably comprising at least one other ingredient selected from the group consisting of other enzymes than alpha-amylase of the invention, surfactants, defoamers, builders, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, desiccants, brighteners, buffers, preservatives, anti-corrosion additives, dyes and perfumes, preferably at least one ingredient of the detergent is a surfactant and/or a builder, preferably a strongly chelating builder.

Preferred embodiment 23.): embodiments 21.) and 22.), wherein the detergent composition is a liquid or a solid, preferably a laundry detergent composition or an Automatic Dishwashing (ADW) detergent composition, preferably in a pouch form.

Preferred embodiment 24.): a method of producing an alpha-amylase variant comprising:

a. Culturing the host cell of embodiment 20) under conditions suitable for expression of the alpha-amylase variant; and

b. recovering the alpha-amylase variant.

Preferred embodiment 25.): use of the alpha-amylase variant described in the preceding embodiments in a cleaning process such as laundry or hard surface cleaning.

Preferred embodiment 26): an alpha-amylase variant of any of the preceding preferred embodiments, wherein the one or more amino acid substitutions are according to the numbering of the amino acid sequence set forth in SEQ ID NO: 3:

a) Selected from positions 4,429 and 459 or

b) Selected from positions 4,25,116,176,225,251,320,400,405,408,410,418,429,446,449,458,459,460,471 and 482, or

c) Selected from positions 4,25,176,251,405 and 482, or

d) Selected from positions 4,6,10,12,13,14,15,17,20,21,22,23,24,27,28,32,34,36,38,39,42,45,47,51,70,75,76,83,89,92,95,96,99,100,108,115,154,156,164,191,192,213,215,221,222,223,226,233,234,236,237,240,245,249,268,271,277,282,285,288,289,290,297,301,308,309,312,326,327,333,336,338,341,343,348,351,352,353,355,356,358,366,367,370,376,379,381,389,392,399,413,424,426,429,432,436,440,442,443,448,451,453,456,459,463,468 and 480, or

e) Selected from positions 4,429 and 459, or

f) Selected from positions 4,10,12,13,14,15,17,20,21,23,24,27,34,36,38,39,42,45,47,51,89,92,99,100,108,115,164,191,192,213,221,222,223,233,234,236,237,240,245,249,268,271,282,288,289,290,297,301,308,309,312,326,327,333,336,338,341,343,348,351,352,353,355,356,358,366,367,370,376,379,381,389,392,413,424,426,429,432,440,442,443,448,451,453,456,468 and 480, or

g) Selected from positions 4 and 429, or

h) Selected from positions 13,17,22,23,27,28,32,36,42,51,70,75,83,89,92,95,96,154,192,215,222,226,233,245,277,282,285,288,297,312,338,341,343,353,355,356,376,379,381,389,429,432,442,451,459 and 463, or

i) Selected from positions 13,17,23,27,36,42,51,89,92,192,215,222,233,245,277,282,288,297,312,338,341,343,353,355,356,376,379,381,389,429,432,442 and 451, or

j) Selected from positions 13,18,146,163,170,188,224,238,242,245,353,385,388,405,432,442,474 and 478, or

Selected from positions 1,2,3,5,7,8,9,11,16,18,19,25,26,29,30,31,35,37,40,41,43,44,46,48,49,50,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,71,72,73,74,77,78,79,80,81,84,85,86,87,88,90,91,93,94,97,98,101,102,103,104,105,106,107,109,110,111,112,113,114,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,151,152,153,155,157,158,159,160,161,162,163,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,185,186,187,188,189,190,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,216,217,218,219,220,224,225,227,228,229,230,231,232,235,238,239,241,242,243,244,246,247,248,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,269,270,272,273,274,275,276,278,279,280,281,283,284,286,287,291,292,293,294,295,296,298,299,300,302,303,304,305,306,307,310,311,313,314,315,316,317,319,320,321,322,323,324,325,328,329,330,331,332,334,335,337,339,340,342,344,345,346,347,349,350,354,357,359,360,361,362,363,364,365,368,369,371,372,373,374,375,377,380,382,383,384,385,386,387,388,390,391,393,394,395,396,397,398,400,401,402,403,404,405,406,407,408,409,410,411,412,414,415,416,417,418,419,420,421,422,423,425,427,428,430,431,433,434,435,437,438,439,441,444,445,446,447,449,450,452,454,455,457,458,460,461,462,464,465,466,467,469,470,471,472,473,474,475,476,477,478,479,481,483,484 and 485, or

k) Selected from positions 25,116,176,181,183,186,206,225,251,320,400,402,405,408,409,410,418,419,420,422,423,437,441,444,446,449,452,458,460,466,471,473,475,484 and 485, or

l) is selected from positions 25 and 176, or

m) is selected from positions 4,25,176,251,405,439 and 482, or

n) is selected from positions 25,176 and 186, or

o) is selected from positions 4,25,176,186,251,405,439 and 482, or

p) is selected from the group consisting of positions 116,181,225 and 320,

preferably selected from 25 and 176, or selected from 4,25,176,251,405,439 and 482, or selected from 116,181,225 and 320.

Examples

Example 1

Improved parent alpha-amylases

The ALBA amylase (SEQ ID NO: 3) was modified by deletion of amino acids G182 and D183, resulting in an increased stability of the amylase. The resulting amylase variant is amylase A shown in SEQ ID NO. 4. The wash performance of amylase A was compared to amylase B (shown in SEQ ID NO: 1) using a mini wash test (microscale wash trials) and a wash tester (Launderometer), which was used as the parent amylase for the alpha-amylase variants described herein.

For this mini-cleaning performance test, the soil was removed from the stale corn starch stain (CS-126,aged corn starch stains) in 3.3g/L ES1-C (as described in Table 1B above) using amylase A and B, ES1-C diluted with water (15 DEG dH) at room temperature, amylase added to the wash at 0.02ppm, 0.05ppm and 0.1ppm for 60 minutes, then rinsed under tap water for 5 minutes, and dried.

The wash performance is measured as the color brightness of the washed textile. Brightness may also be expressed as the intensity of light reflected from a sample when illuminated with white light. When the sample is stained, the intensity of the reflected light is lower than that of a clean sample. In other words, a cleaner sample will reflect more light and have a higher intensity. Thus, the intensity of the reflected light can be used to measure the wash performance. Color measurements were made using a digital color metric, with the average of the RGB values on each stain as the result. Subtracting the value of the detergent base gives the ratio of the performance of amylase B to the performance of amylase a, and values above 100% represent higher performance than that of amylase a.

In table 2 below, the average of at least 2 experiments is given.

Amylase enzyme	0ppm	0.02ppm	0.05ppm	0.1ppm
					A	0	100％	100％	100％
B	0	151％	191％	188％

The same wash test was performed at room temperature using different detergents according to formulation a of table 1a above, wherein amylase was added to the wash liquor at 0.02ppm, 0.05ppm or 0.1ppm for 60 minutes, then rinsed under tap water for 5 minutes, dried and the reflected light intensity was measured as an average of RGB values. Subtracting the value of the detergent base gives the ratio of the performance of amylase B to the performance of amylase a, and values above 100% represent higher performance than that of amylase a.

In table 3 below, the average of at least 2 experiments is given.

Amylase enzyme	0ppm	0.02ppm	0.05ppm	0.1ppm
					A	0％	100％	100％	100％
B	0％	153％	158％	213％

For the wash test with the wash tester, the different soil stains (CS-129 old tapioca starch, EMPA161 cotton starch, CS-26 corn starch, CS-28 rice starch, CS-28 tapioca starch) were washed with amylase A and B in 3.5g/L commercial Persil Non-Bio diluted with water (15 DEG dH), wherein amylase was added to the wash liquor at 0.02ppm, 0.05ppm or 0.1ppm for 40 minutes, then rinsed under running water for 5 minutes, dried and the reflected light intensity was measured as an average of RGB values. Subtracting the value of the detergent base gives the ratio of the performance of amylase B to the performance of amylase a, and values above 100% represent higher performance than that of amylase a.

In table 4 below, the average of at least 2 experiments is given.

	0ppm	0.01ppm	0.02ppm	0.04ppm
					EMPA161	-	182％	159％	165％
CS26	-	290％	245％	186％
					CS28	-	289％	206％	147％
CS129	-	217％	193％	174％
					CS29	-	284％	277％	257％

As is evident from the above table, amylase B is superior to amylase A in all detergents tested.

Example 2

Improved temperature stability of alpha-amylase variants of a parent amylase

More stable variants were identified using a heat treatment assay. Thus, the supernatant of each single mutant Bacillus licheniformis expressing amylase B (test in example 1; SEQ ID NO: 1) was diluted separately in 384 well plates using 100mM HEPES (pH 8), amylase B being a positive control. The dilutions were split into two plates, one plate stored at 8 ℃ and representing baseline activity, and the other plate incubated at 92 ℃ for 10 minutes and representing treatment values. For activity measurement, 3 μl of sample was added to 27 μl of 1.1% red starch solution (in 100mm HEPES pH 8). The mixture was incubated at room temperature for 10 minutes. The reaction was stopped by adding 60 μl ice-cold ethanol (95%), after centrifugation 40 μl of supernatant was removed and added to a new 384 well plate. The absorbance at 510nm was recorded. Residual activity was calculated by dividing the absorbance value of the treated sample by the absorbance value of the baseline sample. The improvement factor is the ratio of the residual activity of the variant to the residual activity of amylase B (SEQ ID NO: 1). The amylase variants (numbering according to SEQ ID NO: 3) with improved temperature stability relative to the parent are given in the table.

The results are given in table 5 below:

example 3

Single point mutations of SEQ ID NO. 1 storage stability at 40℃relative to amylase B (SEQ ID NO: 1) in detergents according to formulation A of Table 1a above.

To further assess whether improved thermostability is also associated with an increase in stability in detergents, variants of amylase B (SEQ ID NO: 1) were formulated in detergents and tested for residual activity after storage for 3 days at 40 ℃.

Storage in liquid laundry detergents was performed by incubating amylase in the detergent according to formulation a of table 1a above. Thus, 5. Mu.L of diluted supernatant from amylase expressing Bacillus licheniformis was added to 45. Mu.L of detergent. The mixture was incubated at 40℃and after the indicated time points, 5. Mu.L of the detergent mixture was removed and 45. Mu.L of MOPS buffer (50 mM, supplemented with 1mM CaCl ₂ pH 7.0). To 25. Mu.L of this mixture was added 25. Mu.L of the affinity amylase reagent. The activity is the slope at 405nm, calculated as 5point MaxV over 5 minutes. Residual activity was calculated by dividing the activity after the storage time by the activity of the sample at time point 0. The residual activity was compared with that of reference amylase B (SEQ ID NO: 1).

In Table 6 below, mutations (numbered according to SEQ ID NO: 3) with improved residual activity relative to amylase B (SEQ ID NO: 1) are given.

Table 6: storage stability at 40℃in detergent formulation A relative to amylase B (SEQ ID NO: 1). Residual activity was given after 3 days of challenge.

Example 4

Storage stability in ES1-C detergents at 40℃for 7 days.

To further assess whether improved thermostability is also associated with an increase in stability in detergents, variants of amylase B (SEQ ID NO: 1) were formulated in detergents and tested for residual activity after storage for 7 days at 40 ℃.

Liquid coating by incubation of amylase in ES1-C (as described in Table 1b above) detergentStorage in laundry detergents. Thus, 5. Mu.L of diluted supernatant from amylase expressing Bacillus licheniformis was added to 45. Mu.L of detergent. The mixture was incubated at 40℃and after the indicated time points, 5. Mu.L of the detergent mixture was removed and 45. Mu.L of MOPS buffer (50 mM, supplemented with 1mM CaCl ₂ pH 7.0). To 25. Mu.L of this mixture was added 25. Mu.L of the affinity amylase reagent. The activity is the slope at 405nm, calculated as 5 points MaxV within 5 minutes. Residual activity was calculated by dividing the activity after the storage time by the activity of the sample at time point 0. The residual activity was compared with that of reference amylase B (SEQ ID NO: 1). In Table 7 below, mutations with improved residual activity relative to SEQ ID NO. 1 (numbering according to SEQ ID NO. 3) are given.

Table 7: storage stability at 40℃in detergents ES1-C relative to amylase B (SEQ ID NO: 1). Challenge residual activity after 3 days.

Example 5

Stabilization of mutant combinatorial libraries

The mutations in amylase B (SEQ ID NO: 1) are combined to produce an amylase having multiple mutations relative to amylase B (SEQ ID NO: 1). To further evaluate whether improved thermostability is also associated with improved stability in detergents, variants of amylase B (SEQ ID NO: 1) were formulated in detergents according to formulation A of Table 1a above and tested for residual activity after the indicated time points. Storage at 40℃or 50℃with or without protease (if 0.3g/L of subtilisin (BLAP) from Bacillus lentus containing the R101E mutation (BPN' numbering) is added).

Storage in liquid laundry detergents is performed by incubating amylase in the detergent. Thus, 25. Mu.L of diluted supernatant from amylase expressing Bacillus licheniformis was added to 475. Mu.L of detergent. The mixture was incubated at 40℃and after the indicated time point, 10. Mu.L of the detergent mixture was removed and diluted with 90. Mu.L of MOPS buffer (50 mM, supplemented with 1mM CaCl2 pH 7.0). To 50. Mu.L of this mixture was added 50. Mu.LInfinity amylase reagent. The activity is the slope at 405nm, calculated as 5 points MaxV within 5 minutes. Residual activity was calculated by dividing the activity after the storage time by the activity of the sample at time point 0. The residual activity was compared with that of reference amylase B (SEQ ID NO: 1). The numbering of the following table is carried out according to SEQ ID NO. 3.

In table 8 below, the residual activity after 28 days of storage is shown, where storage is at 40 ℃ in the presence of protease.

In table 9 below, the residual activity after 7 days of storage in detergent is shown, where storage is at 40 ℃ in the absence of protease.

Variation on the parent Amylase (SEQ ID NO: 1)	Residual Activity [%]
		Parent strain	21％
N3I+T356V	23％
		N3I+T356I	24％
N83D+S94E	24％
		S94D+S125E	25％
V131I+H377Q+V410H	25％
		W48F+S94D	27％
W48Y+W116K+R218K	32％
		T5L+R218K+T225S	33％
N83E+W116R+R158Y+R181E	34％
		A51V+R218K	35％
N83E+R181E	43％

In Table 10 below, the residual activity after 7 days of storage in the detergent is shown, where the storage is at 40℃in the presence of protease (0.3 g/L protease in the detergent).

In table 11 below, the residual activity after 8 days of storage in the detergent is shown, where storage is at 50 ℃ in the absence of protease.

In table 12 below, the residual activity after 7 days of storage in the detergent is shown, where storage at 40 ℃ is in the absence of protease.

Example 6

Washing test

For the mini-wash test, CS-126 was used as a fabric, and the wash performance of variants of amylase B (SEQ ID NO: 1) containing several amino acid substitutions at the positions of interest was evaluated using the method described in example 1.

Table 13 below shows the results (numbered according to SEQ ID NO: 3) for amylase B variants containing single amino acid substitutions.

Table 14 below shows the results for amylase B variants containing multiple amino acid substitutions (numbered according to SEQ ID NO: 3).

Sequence listing

<110> Basf European company (BASF SE)

Hangao stock limited and two-party company (Henkel AG & Co. KGAA)

<120> amylase variants

<130> 210083

<150> EP21158484.2

<151> 2021-02-22

<150> EP21213738.4

<151> 2021-12-10

<160> 41

<170> BiSSAP 1.3.6 and according to WIPO Std.25

<210> 1

<211> 483

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 1

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Gly Trp Asp Trp Glu Val Asp Thr Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn

210 215 220

Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr Gly

245 250 255

Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile Gly Ala

260 265 270

Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly Gly Asn

290 295 300

Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg His Pro

305 310 315 320

Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu

325 330 335

Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Thr Gln Arg

385 390 395 400

Asp Tyr Leu Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp

405 410 415

Gly Val His Ala Asp Ser Gly Leu Ala Thr Leu Met Ser Asp Gly Pro

420 425 430

Gly Gly Ser Lys Trp Met Glu Val Gly Lys Asn Asn Ala Gly Glu Val

435 440 445

Trp Tyr Asp Ile Thr Gly Asn Gln Thr Asn Thr Val Thr Ile Asn Lys

450 455 460

Asp Gly Trp Gly Gln Phe His Val Ser Gly Gly Ser Val Ser Ile Tyr

465 470 475 480

Val Gln Gln

<210> 2

<211> 1449

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 2

catcacaatg gcacaaacgg taccatgatg caatattttg agtggtacct cccaaatgac 60

ggaaatcatt ggaacagatt gcgctctgat gcaagcaacc ttaaagataa gggcataaca 120

gctgtttgga ttccgccagc atggaaaggc gccagtcaaa acgacgtcgg atatggagcg 180

tacgatctgt atgatttagg cgagtttaat cagaaaggca cagtaagaac gaaatatgga 240

accaggaatc aacttcaggc ggctgtgacg gccctcaagt ctaatggcat tcaggtgtat 300

ggcgacgttg tgatgaacca caaaggagga gctgatgcga cagaatgggt tagagcagtc 360

gaagtaaatc ctagcaatcg aaaccaggag gtgagcggtg attacacaat cgaggcatgg 420

accaaattcg actttcctgg acgtggcaac acacatagta attttaagtg gagatggtat 480

cacttcgatg gcgtagactg ggatcaatct agacagttac agaaccggat ctacaaattt 540

cgcggaaaag gctgggattg ggaagttgat acggagaacg gcaattatga ttacttgatg 600

tacgcagata tcgacatgga tcatccggaa gtggtcaatg agttacgtaa ttggggcgtc 660

tggtacacta acacactggg ccttgacgga ttccgcattg atgcggttaa acatatcaaa 720

tattcgttta cgcgcgactg gttaactcat gtccggaata caacaggcaa gaatatgttt 780

gctgttgcag aattttggaa aaacgatatc ggcgcgattg aaaattatct ttcaaagact 840

aactggaatc attccgtatt cgacgtgcct ttgcactaca acctgtataa tgcgtcgcgc 900

tccggcggta actatgacat gcgacaaatc ttcaacggca cagtcgttca gcgtcacccc 960

acacacgctg ttacatttgt agacaaccat gacagccaac cggaggaagc tcttgaaagc 1020

ttcgtagaag aatggtttaa gcctctggcc tatgcgctga cgcttacgcg ggatcaagga 1080

tacccgagcg tgttttatgg agattactat ggcattccga cgcatggcgt gccagccatg 1140

aaatcaaaaa ttgatccgat actcgaagcc agacaaaaat atgcatatgg tactcaacgg 1200

gattatttgg accatccgga tgtcattggt tggacccgtg aaggagatgg agtacatgct 1260

gactctggct tagccacact gatgtccgat ggtcctggag gctcaaaatg gatggaagtg 1320

ggtaagaaca atgcaggtga agtttggtat gatattaccg gcaatcagac gaatacggtt 1380

actattaata aagatggttg gggacaattt catgtctcag gcggctctgt ctcaatctat 1440

gttcagcag 1449

<210> 3

<211> 485

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 3

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr

245 250 255

Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile

260 265 270

Gly Ala Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly

290 295 300

Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg

305 310 315 320

His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys

385 390 395 400

Gln Asn Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys Ala Gly

435 440 445

Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Asn Asn

485

<210> 4

<211> 483

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 4

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Gly Trp Asp Trp Glu Val Asp Thr Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn

210 215 220

Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr Gly

245 250 255

Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile Gly Ala

260 265 270

Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly Gly Asn

290 295 300

Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg His Pro

305 310 315 320

Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu

325 330 335

Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys Gln Asn

385 390 395 400

Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro

420 425 430

Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys Ala Gly Gln Val

435 440 445

Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp

465 470 475 480

Val Asn Asn

<210> 5

<211> 486

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 5

Ala Thr Ile Asn Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Ala

1 5 10 15

Pro Asn Asp Gly Asn His Trp Lys Arg Leu His Thr Asp Ala Gly Asn

20 25 30

Leu Ala Gln Lys Gly Ile Thr Ser Val Trp Ile Pro Pro Ala Tyr Lys

35 40 45

Gly Thr Thr Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp

50 55 60

Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Ile Glu Ala Leu His Lys Gln Asn Ile

85 90 95

Asn Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Tyr

100 105 110

Thr Glu Thr Val Thr Ala Val Glu Val Asp Arg Asn Asn Arg Asn Ile

115 120 125

Glu Val Ser Gly Asp Tyr Glu Ile Asn Ala Trp Thr Gly Phe Asn Phe

130 135 140

Pro Gly Arg Gly Asp Thr His Ser Asn Phe Lys Trp Lys Trp Tyr His

145 150 155 160

Phe Asp Gly Thr Asp Trp Asp Glu Gly Arg Lys Leu Asn Arg Ile Tyr

165 170 175

Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Phe Asp His

195 200 205

Pro Asp Val Ala Asn Glu Met Lys Asn Trp Gly Thr Trp Tyr Ala Asn

210 215 220

Glu Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Asp

225 230 235 240

His Glu Tyr Leu Arg Asp Trp Val Asn His Val Arg Gln Gln Thr Gly

245 250 255

Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Ile Gln Thr

260 265 270

Leu Asn Asn Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Val Phe Asp

275 280 285

Ala Pro Leu His Tyr Asn Phe His Tyr Ala Ser Lys Gly Asn Gly Asn

290 295 300

Tyr Asp Met Arg Asn Ile Leu Asn Gly Thr Val Met Gln Asn His Pro

305 310 315 320

Ala Leu Ala Val Thr Leu Val Glu Asn His Asp Ser Gln Pro Gly Gln

325 330 335

Ser Leu Glu Ser Val Val Ser Pro Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Phe Ile Leu Thr Arg Ala Glu Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Thr Ser Gly Asn Ser Ser Tyr Glu Ile Pro Ala Leu Lys

370 375 380

Asp Lys Ile Asp Pro Ile Leu Met Ala Arg Lys Asn Phe Ala Tyr Gly

385 390 395 400

Thr Gln Arg Asp Tyr Leu Asp His Pro Asp Val Ile Gly Trp Thr Arg

405 410 415

Glu Gly Asp Gly Val His Ala Asp Ser Gly Leu Ala Thr Leu Ile Ser

420 425 430

Asp Gly Pro Gly Gly Ser Lys Trp Met Glu Val Gly Lys Asn Asn Ala

435 440 445

Gly Glu Val Trp Tyr Asp Met Thr Gly Asn Gln Thr Asn Thr Val Thr

450 455 460

Ile Asn Lys Asp Gly Trp Gly Gln Phe His Val Ser Gly Gly Ser Val

465 470 475 480

Ser Ile Tyr Val Gln Gln

485

<210> 6

<211> 399

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 6

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr

245 250 255

Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile

260 265 270

Gly Ala Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly

290 295 300

Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg

305 310 315 320

His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly

385 390 395

<210> 7

<211> 86

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 7

Lys Gln Asn Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg

1 5 10 15

Glu Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser

20 25 30

Asp Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys Ala

35 40 45

Gly Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr

50 55 60

Ile Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val

65 70 75 80

Ser Ile Trp Val Asn Asn

85

<210> 8

<211> 86

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 8

Thr Gln Arg Asp Tyr Leu Asp His Pro Asp Val Ile Gly Trp Thr Arg

1 5 10 15

Glu Gly Asp Gly Val His Ala Asp Ser Gly Leu Ala Thr Leu Ile Ser

20 25 30

Asp Gly Pro Gly Gly Ser Lys Trp Met Glu Val Gly Lys Asn Asn Ala

35 40 45

Gly Glu Val Trp Tyr Asp Met Thr Gly Asn Gln Thr Asn Thr Val Thr

50 55 60

Ile Asn Lys Asp Gly Trp Gly Gln Phe His Val Ser Gly Gly Ser Val

65 70 75 80

Ser Ile Tyr Val Gln Gln

85

<210> 9

<211> 400

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 9

Ala Thr Ile Asn Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Ala

1 5 10 15

Pro Asn Asp Gly Asn His Trp Lys Arg Leu His Thr Asp Ala Gly Asn

20 25 30

Leu Ala Gln Lys Gly Ile Thr Ser Val Trp Ile Pro Pro Ala Tyr Lys

35 40 45

Gly Thr Thr Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp

50 55 60

Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Ile Glu Ala Leu His Lys Gln Asn Ile

85 90 95

Asn Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Tyr

100 105 110

Thr Glu Thr Val Thr Ala Val Glu Val Asp Arg Asn Asn Arg Asn Ile

115 120 125

Glu Val Ser Gly Asp Tyr Glu Ile Asn Ala Trp Thr Gly Phe Asn Phe

130 135 140

Pro Gly Arg Gly Asp Thr His Ser Asn Phe Lys Trp Lys Trp Tyr His

145 150 155 160

Phe Asp Gly Thr Asp Trp Asp Glu Gly Arg Lys Leu Asn Arg Ile Tyr

165 170 175

Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Phe Asp His

195 200 205

Pro Asp Val Ala Asn Glu Met Lys Asn Trp Gly Thr Trp Tyr Ala Asn

210 215 220

Glu Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Asp

225 230 235 240

His Glu Tyr Leu Arg Asp Trp Val Asn His Val Arg Gln Gln Thr Gly

245 250 255

Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Ile Gln Thr

260 265 270

Leu Asn Asn Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Val Phe Asp

275 280 285

Ala Pro Leu His Tyr Asn Phe His Tyr Ala Ser Lys Gly Asn Gly Asn

290 295 300

Tyr Asp Met Arg Asn Ile Leu Asn Gly Thr Val Met Gln Asn His Pro

305 310 315 320

Ala Leu Ala Val Thr Leu Val Glu Asn His Asp Ser Gln Pro Gly Gln

325 330 335

Ser Leu Glu Ser Val Val Ser Pro Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Phe Ile Leu Thr Arg Ala Glu Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Thr Ser Gly Asn Ser Ser Tyr Glu Ile Pro Ala Leu Lys

370 375 380

Asp Lys Ile Asp Pro Ile Leu Met Ala Arg Lys Asn Phe Ala Tyr Gly

385 390 395 400

<210> 10

<211> 269

<212> PRT

<213> Bacillus lentus (Bacillus lentus)

<400> 10

Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Asp Gly Arg Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

165 170 175

Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 11

<211> 275

<212> PRT

<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)

<400> 11

Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu

1 5 10 15

His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp

20 25 30

Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala

35 40 45

Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His

50 55 60

Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly

65 70 75 80

Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu

85 90 95

Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu

100 105 110

Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125

Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala

130 135 140

Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly

145 150 155 160

Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala

165 170 175

Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val

180 185 190

Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr

195 200 205

Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser

210 215 220

Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn

225 230 235 240

Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys

245 250 255

Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala

260 265 270

Ala Ala Gln

275

<210> 12

<211> 269

<212> PRT

<213> Bacillus gibsonii (Bacillus gibsonii)

<400> 12

Gln Gln Thr Val Pro Trp Gly Ile Thr Arg Val Gln Ala Pro Thr Val

1 5 10 15

His Asn Arg Gly Ile Thr Gly Ser Gly Val Lys Val Ala Ile Leu Asp

20 25 30

Thr Gly Ile Ala Gln His Ser Asp Leu Thr Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Ser Thr Thr Ala Asp Leu Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Ile

65 70 75 80

Gly Val Ala Pro Ser Ala Asp Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Asn Gly Arg Gly Ser Val Ser Gly Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Ala Thr Asn Asn Met His Ile Ala Asn Met Ser Leu Gly Ser Asp Ala

115 120 125

Pro Ser Thr Thr Leu Glu Arg Ala Val Asn Tyr Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Ile Ala Ala Thr Gly Asn Asn Gly Thr Gly Ser Ile Gly

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

165 170 175

Asn Asn Arg Arg Ala Ser Phe Ser Gln Tyr Gly Thr Gly Ile Asp Ile

180 185 190

Val Ala Pro Gly Val Gly Ile Gln Ser Thr Tyr Leu Asn Asn Ser Tyr

195 200 205

Ala Ser Met Pro Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Asn Ala Thr Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Asn Leu Gly Asn Ser Ser Gln

245 250 255

Phe Gly Ser Gly Leu Val Asn Ala Asp Ala Ala Thr Arg

260 265

<210> 13

<211> 275

<212> PRT

<213> Bacillus pumilus (Bacillus pumilus)

<400> 13

Ala Gln Thr Val Pro Tyr Gly Ile Pro Gln Ile Lys Ala Pro Ala Val

1 5 10 15

His Ala Gln Gly Tyr Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile His Ala Ala His Pro Asp Leu Asn Val Ala Gly Gly Ala

35 40 45

Ser Phe Val Pro Ser Glu Pro Asn Ala Thr Gln Asp Phe Gln Ser His

50 55 60

Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asp Asn Thr Ile Gly

65 70 75 80

Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu

85 90 95

Asp Arg Asn Gly Asp Gly Gln Tyr Ser Trp Ile Ile Ser Gly Ile Glu

100 105 110

Trp Ala Val Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125

Pro Asn Gly Ser Thr Ala Leu Lys Asn Ala Val Asp Thr Ala Asn Asn

130 135 140

Arg Gly Val Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Phe Gly

145 150 155 160

Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Asp Ser Thr Ile Ala

165 170 175

Val Ala Asn Val Asn Ser Asn Asn Val Arg Asn Ser Ser Ser Ser Ala

180 185 190

Gly Pro Glu Leu Asp Val Ser Ala Pro Gly Thr Ser Ile Leu Ser Thr

195 200 205

Val Pro Ser Ser Gly Tyr Thr Ser Tyr Thr Gly Thr Ser Met Ala Ser

210 215 220

Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys Tyr Pro Asn

225 230 235 240

Leu Ser Thr Ser Gln Val Arg Gln Arg Leu Glu Asn Thr Ala Thr Pro

245 250 255

Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala

260 265 270

Ala Ser Asn

275

<210> 14

<211> 311

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 14

Ala Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn

1 5 10 15

Asp Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala

20 25 30

Val Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser

35 40 45

Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly

50 55 60

Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val

65 70 75 80

Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro

85 90 95

Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly

100 105 110

Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala

115 120 125

Ser Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser

130 135 140

Ala Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys

145 150 155 160

Gly Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn

165 170 175

Thr Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala

180 185 190

Leu Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser

195 200 205

Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg

210 215 220

Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr

225 230 235 240

Thr Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His

245 250 255

Val Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser

260 265 270

His Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp

275 280 285

Ile Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly

290 295 300

Phe Gly Tyr Pro Arg Val Lys

305 310

<210> 15

<211> 483

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 15

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Ser Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn

210 215 220

Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly

245 250 255

Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala

260 265 270

Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Asn

290 295 300

Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro

305 310 315 320

Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu

325 330 335

Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His

385 390 395 400

Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro

420 425 430

Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asp Lys Ala Gly Gln Val

435 440 445

Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp

465 470 475 480

Val Lys Arg

<210> 16

<211> 485

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 16

His His Asp Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn

1 5 10 15

Val Pro Asn Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln

20 25 30

Asn Leu Lys Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Lys Ala Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Phe Thr Glu Arg Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn

130 135 140

Phe Pro Gly Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Ala Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met

195 200 205

Asp His Pro Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr

210 215 220

Ala Asn Thr Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His

225 230 235 240

Ile Lys Phe Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln

245 250 255

Thr Gly Lys Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu

260 265 270

Gly Ala Leu Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser

290 295 300

Gly Asn Tyr Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg

305 310 315 320

His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro

325 330 335

Gly Glu Ala Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Thr Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln

370 375 380

Gln Ile Asp Pro Leu Leu Lys Ala Arg Gln Gln Tyr Ala Tyr Gly Thr

385 390 395 400

Gln His Asp Tyr Leu Asp Asn Gln Asp Val Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asp Ser Ala His Ala Gly Ser Gly Leu Ala Thr Val Met Ser Asp

420 425 430

Gly Pro Gly Gly Ser Lys Thr Met Tyr Val Gly Thr Ala His Ala Gly

435 440 445

Gln Val Phe Lys Asp Ile Thr Gly Asn Arg Thr Asp Thr Val Thr Ile

450 455 460

Asn Ser Ala Gly Asn Gly Thr Phe Pro Cys Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Lys Gln

485

<210> 17

<211> 485

<212> PRT

<213> Cellulars species (Cytophaga sp.)

<400> 17

Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro

1 5 10 15

Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu

20 25 30

Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly

35 40 45

Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu

50 55 60

Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys

65 70 75 80

Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln

85 90 95

Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr

100 105 110

Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu

115 120 125

Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro

130 135 140

Gly Arg Gly Thr Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe His Phe

145 150 155 160

Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys

165 170 175

Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn

180 185 190

Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro

195 200 205

Asp Val Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu

210 215 220

Val Gly Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe

225 230 235 240

Ser Phe Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys

245 250 255

Glu Met Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu

260 265 270

Asn Asn Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala

275 280 285

Pro Leu His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr

290 295 300

Asp Met Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr

305 310 315 320

Lys Ala Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser

325 330 335

Leu Glu Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe

340 345 350

Ile Leu Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met

355 360 365

Tyr Gly Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser

370 375 380

Lys Ile Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr

385 390 395 400

Gln Arg Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asp Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp

420 425 430

Gly Pro Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly

435 440 445

Glu Ile Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile

450 455 460

Gly Ser Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser

465 470 475 480

Val Trp Val Gln Gln

485

<210> 18

<211> 484

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 18

Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp Asp

1 5 10 15

Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ala

20 25 30

Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr

35 40 45

Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly

85 90 95

Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp

100 105 110

Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn

115 120 125

Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile

165 170 175

Tyr Lys Phe Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr

180 185 190

Glu Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp

195 200 205

His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val

210 215 220

Asn Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile

225 230 235 240

Lys Tyr Thr Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr

245 250 255

Gly Lys Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn

260 265 270

Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe

275 280 285

Asp Ala Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly

290 295 300

Tyr Phe Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln

305 310 315 320

Pro Ser Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly

325 330 335

Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr

340 345 350

Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly

355 360 365

Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys

370 375 380

Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln

385 390 395 400

Arg Asp Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly

405 410 415

Ile Asp Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly

420 425 430

Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys

435 440 445

Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn

450 455 460

Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile

465 470 475 480

Trp Val Ala Lys

<210> 19

<211> 482

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 19

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp

100 105 110

Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn

115 120 125

Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile

165 170 175

Tyr Lys Phe Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn

180 185 190

Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro

195 200 205

Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn Thr

210 215 220

Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr

225 230 235 240

Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly Lys

245 250 255

Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala Leu

260 265 270

Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp Val

275 280 285

Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Asn Tyr

290 295 300

Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro Met

305 310 315 320

His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu Ser

325 330 335

Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala Leu

340 345 350

Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr

355 360 365

Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile Asp

370 375 380

Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His Asp

385 390 395 400

Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn Thr

405 410 415

Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly

420 425 430

Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln Val Trp

435 440 445

His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala Asp

450 455 460

Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp Val

465 470 475 480

Lys Arg

<210> 20

<211> 485

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 20

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr

210 215 220

Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala

245 250 255

Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly

290 295 300

Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys

305 310 315 320

His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr

385 390 395 400

Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asp Lys Ala Gly

435 440 445

Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Lys Arg

485

<210> 21

<211> 485

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 21

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser

20 25 30

Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly His Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala

245 250 255

Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly

290 295 300

Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg

305 310 315 320

His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys

385 390 395 400

Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly

435 440 445

Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Asn Lys

485

<210> 22

<211> 514

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 22

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala His His Asn

20 25 30

Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His Leu Pro Asn

35 40 45

Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser Asn Leu Arg

50 55 60

Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp Lys Gly Thr

65 70 75 80

Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly

85 90 95

Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Arg Ser

100 105 110

Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly Val Gln Val

115 120 125

Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Ala Thr Glu

130 135 140

Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn Gln Glu Ile

145 150 155 160

Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp Phe Pro Gly

165 170 175

Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr His Phe Asp

180 185 190

Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg Ile Tyr Lys

195 200 205

Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp Ser Glu Asn

210 215 220

Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His Pro

225 230 235 240

Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn Thr

245 250 255

Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr

260 265 270

Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly Lys

275 280 285

Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala Leu

290 295 300

Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp Val

305 310 315 320

Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Asn Tyr

325 330 335

Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro Met

340 345 350

His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu Ser

355 360 365

Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala Leu

370 375 380

Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr

385 390 395 400

Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile Asp

405 410 415

Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His Asp

420 425 430

Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn Thr

435 440 445

Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly

450 455 460

Gly Glu Lys Trp Met Tyr Val Gly Gln Asp Lys Ala Gly Gln Val Trp

465 470 475 480

His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala Asp

485 490 495

Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp Val

500 505 510

Lys Arg

<210> 23

<211> 582

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 23

Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp Asp

1 5 10 15

Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ala

20 25 30

Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr

35 40 45

Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly

85 90 95

Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp

100 105 110

Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn

115 120 125

Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile

165 170 175

Tyr Lys Phe Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr

180 185 190

Glu Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp

195 200 205

His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val

210 215 220

Asn Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile

225 230 235 240

Lys Tyr Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr

245 250 255

Gly Lys Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn

260 265 270

Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe

275 280 285

Asp Ala Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr

290 295 300

Phe Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro

305 310 315 320

Ser Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln

325 330 335

Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile

370 375 380

Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg

385 390 395 400

Asp Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile

405 410 415

Asp Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro

420 425 430

Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val

435 440 445

Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp

465 470 475 480

Val Ala Lys Thr Ser Asn Val Thr Phe Thr Val Asn Asn Ala Thr Thr

485 490 495

Thr Ser Gly Gln Asn Val Tyr Val Val Ala Asn Ile Pro Glu Leu Gly

500 505 510

Asn Trp Asn Thr Ala Asn Ala Ile Lys Met Asn Pro Ser Ser Tyr Pro

515 520 525

Thr Trp Lys Ala Thr Ile Ala Leu Pro Gln Gly Lys Ala Ile Glu Phe

530 535 540

Lys Phe Ile Lys Lys Asp Gln Ala Gly Asn Val Ile Trp Glu Ser Thr

545 550 555 560

Ser Asn Arg Thr Tyr Thr Val Pro Phe Ser Ser Thr Gly Ser Tyr Thr

565 570 575

Ala Ser Trp Asn Val Pro

580

<210> 24

<211> 480

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 24

Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu

1 5 10 15

Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu

20 25 30

Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly

35 40 45

Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu

50 55 60

Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys

65 70 75 80

Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn

85 90 95

Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr

100 105 110

Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp

115 120 125

Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser

130 135 140

Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe

145 150 155 160

Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Ile Phe Arg

165 170 175

Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn

180 185 190

Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val

195 200 205

Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp

210 215 220

Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr

225 230 235 240

Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu

245 250 255

Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe

260 265 270

Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu

275 280 285

Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met

290 295 300

Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala

305 310 315 320

Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu

325 330 335

Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu

340 345 350

Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly

355 360 365

Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu

370 375 380

Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe

385 390 395 400

Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg

405 410 415

Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser

420 425 430

Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp

435 440 445

Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp

450 455 460

Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln

465 470 475 480

<210> 25

<211> 483

<212> PRT

<213> Bacillus halmapalus

<400> 25

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Ser Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn

210 215 220

Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly

245 250 255

Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala

260 265 270

Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Asn

290 295 300

Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro

305 310 315 320

Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu

325 330 335

Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His

385 390 395 400

Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro

420 425 430

Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln Val

435 440 445

Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp

465 470 475 480

Val Lys Arg

<210> 26

<211> 483

<212> PRT

<213> Bacillus halmapalus

<400> 26

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Tyr Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Ser Glu

180 185 190

Phe Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn

210 215 220

Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Phe Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly

245 250 255

Lys Gly Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala

260 265 270

Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Arg Gly Asn

290 295 300

Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro

305 310 315 320

Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu

325 330 335

Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His

385 390 395 400

Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro

420 425 430

Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln Val

435 440 445

Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Ala Asn Phe Ser Val Asn Lys Gly Ser Val Ser Ile Trp

465 470 475 480

Val Lys Arg

<210> 27

<211> 514

<212> PRT

<213> Bacillus halmapalus

<400> 27

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala His His Asn

20 25 30

Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His Leu Pro Asn

35 40 45

Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser Asn Leu Arg

50 55 60

Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp Lys Gly Thr

65 70 75 80

Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly

85 90 95

Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Arg Ser

100 105 110

Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly Val Gln Val

115 120 125

Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Ala Thr Glu

130 135 140

Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn Gln Glu Ile

145 150 155 160

Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp Phe Pro Gly

165 170 175

Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr His Phe Asp

180 185 190

Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg Ile Tyr Lys

195 200 205

Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp Ser Glu Asn

210 215 220

Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His Pro

225 230 235 240

Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn Thr

245 250 255

Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr

260 265 270

Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly Lys

275 280 285

Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala Leu

290 295 300

Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp Val

305 310 315 320

Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Asn Tyr

325 330 335

Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro Met

340 345 350

His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu Ser

355 360 365

Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala Leu

370 375 380

Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr

385 390 395 400

Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile Asp

405 410 415

Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His Asp

420 425 430

Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn Thr

435 440 445

Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly

450 455 460

Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln Val Trp

465 470 475 480

His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala Asp

485 490 495

Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp Val

500 505 510

Lys Arg

<210> 28

<211> 482

<212> PRT

<213> Bacillus halmapalus

<400> 28

His Asn Gly Thr Asn Ala Thr Met Met Gln Tyr Phe Glu Trp His Leu

1 5 10 15

Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser Asn

20 25 30

Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp Lys

35 40 45

Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp

50 55 60

Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr

65 70 75 80

Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly Val

85 90 95

Gln Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Ala

100 105 110

Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn Gln

115 120 125

Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Tyr Thr Lys Phe Asp Phe

130 135 140

Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr His

145 150 155 160

Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg Ile

165 170 175

Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Ser Glu Phe

180 185 190

Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Met Asp His Pro

195 200 205

Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn Thr

210 215 220

Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys Phe

225 230 235 240

Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly Lys

245 250 255

Gly Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala Leu

260 265 270

Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val Phe Asp Val

275 280 285

Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Arg Gly Asn Tyr

290 295 300

Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro Met

305 310 315 320

His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu Ser

325 330 335

Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala Leu

340 345 350

Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr

355 360 365

Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile Asp

370 375 380

Pro Ile Leu Ala Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His Asp

385 390 395 400

Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn Thr

405 410 415

Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly

420 425 430

Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln Val Trp

435 440 445

His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala Asp

450 455 460

Gly Trp Ala Asn Phe Ser Val Asn Lys Gly Ser Val Ser Ile Trp Val

465 470 475 480

Lys Arg

<210> 29

<211> 483

<212> PRT

<213> Bacillus licheniformis (Bacillus licheniformis)

<400> 29

Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro

1 5 10 15

Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu

20 25 30

Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly

35 40 45

Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu

50 55 60

Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys

65 70 75 80

Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn

85 90 95

Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr

100 105 110

Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val

115 120 125

Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro

130 135 140

Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe

145 150 155 160

Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys

165 170 175

Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn

180 185 190

Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val

195 200 205

Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln

210 215 220

Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe

225 230 235 240

Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met

245 250 255

Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn

260 265 270

Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu

275 280 285

His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met

290 295 300

Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser

305 310 315 320

Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu

325 330 335

Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu

340 345 350

Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly

355 360 365

Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile

370 375 380

Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His

385 390 395 400

Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp

405 410 415

Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro

420 425 430

Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr

435 440 445

Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser

450 455 460

Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr

465 470 475 480

Val Gln Arg

<210> 30

<211> 485

<212> PRT

<213> Bacillus halmapalus

<400> 30

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr

210 215 220

Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala

245 250 255

Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly

290 295 300

Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys

305 310 315 320

His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr

385 390 395 400

Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly

435 440 445

Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Lys Arg

485

<210> 31

<211> 485

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 31

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr

245 250 255

Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile

260 265 270

Gly Ala Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly

290 295 300

Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg

305 310 315 320

His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala

340 345 350

Cys Ala Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys

385 390 395 400

Gln Asn Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys Ala Gly

435 440 445

Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Asn Asn

485

<210> 32

<211> 481

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 32

Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp

1 5 10 15

Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp

20 25 30

Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser

35 40 45

Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu

50 55 60

Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu

65 70 75 80

Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr

85 90 95

Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp

100 105 110

Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val Ile Ser

115 120 125

Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro Gly Arg

130 135 140

Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly

145 150 155 160

Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Gln

165 170 175

Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn Tyr Asp

180 185 190

Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val Ala Ala

195 200 205

Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln Leu Asp

210 215 220

Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg

225 230 235 240

Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr

245 250 255

Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu

260 265 270

Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr

275 280 285

Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met Arg Lys

290 295 300

Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr

305 310 315 320

Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr

325 330 335

Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg

340 345 350

Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys

355 360 365

Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile Glu Pro

370 375 380

Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His Asp Tyr

385 390 395 400

Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp Ser Ser

405 410 415

Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly

420 425 430

Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr Trp His

435 440 445

Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Glu Gly

450 455 460

Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln

465 470 475 480

Arg

<210> 33

<211> 483

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 33

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser

20 25 30

Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn

210 215 220

Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Thr Gly

245 250 255

Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala

260 265 270

Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly Asn

290 295 300

Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg His Pro

305 310 315 320

Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu

325 330 335

Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Pro Gln His

385 390 395 400

Asp Tyr Leu Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp

405 410 415

Ser Ser His Pro Lys Ser Gly Leu Ala Thr Leu Ile Thr Asp Gly Pro

420 425 430

Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr

435 440 445

Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser

450 455 460

Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr

465 470 475 480

Val Gln Lys

<210> 34

<211> 484

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 34

Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp Asp

1 5 10 15

Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ala

20 25 30

Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr

35 40 45

Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly

85 90 95

Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp

100 105 110

Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn

115 120 125

Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile

165 170 175

Tyr Lys Phe Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr

180 185 190

Glu Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp

195 200 205

His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val

210 215 220

Asn Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile

225 230 235 240

Lys Tyr Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr

245 250 255

Gly Lys Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn

260 265 270

Lys Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe

275 280 285

Asp Ala Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly

290 295 300

Tyr Phe Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln

305 310 315 320

Pro Ser Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly

325 330 335

Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr

340 345 350

Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly

355 360 365

Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys

370 375 380

Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln

385 390 395 400

Arg Asp Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly

405 410 415

Ile Asp Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly

420 425 430

Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys

435 440 445

Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn

450 455 460

Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile

465 470 475 480

Trp Val Ala Lys

<210> 35

<211> 613

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 35

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Thr

20 25 30

Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp Asp Leu Pro

35 40 45

Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ala Asn Leu

50 55 60

Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly

65 70 75 80

Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu

85 90 95

Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly Thr Lys

100 105 110

Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly Met Gln

115 120 125

Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Gly Thr

130 135 140

Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn Gln Glu

145 150 155 160

Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro

165 170 175

Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe

180 185 190

Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys

195 200 205

Phe Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn

210 215 220

Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro

225 230 235 240

Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr

245 250 255

Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr

260 265 270

Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys

275 280 285

Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu

290 295 300

His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe Asp Ala

305 310 315 320

Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe

325 330 335

Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser

340 345 350

Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser

355 360 365

Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe

370 375 380

Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr

385 390 395 400

Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp

405 410 415

Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp

420 425 430

Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp

435 440 445

Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly

450 455 460

Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe

465 470 475 480

Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp

485 490 495

Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Ile Trp Val

500 505 510

Ala Lys Thr Ser Asn Val Thr Phe Thr Val Asn Asn Ala Thr Thr Thr

515 520 525

Ser Gly Gln Asn Val Tyr Val Val Ala Asn Ile Pro Glu Leu Gly Asn

530 535 540

Trp Asn Thr Ala Asn Ala Ile Lys Met Asn Pro Ser Ser Tyr Pro Thr

545 550 555 560

Trp Lys Ala Thr Ile Ala Leu Pro Gln Gly Lys Ala Ile Glu Phe Lys

565 570 575

Phe Ile Lys Lys Asp Gln Ala Gly Asn Val Ile Trp Glu Ser Thr Ser

580 585 590

Asn Arg Thr Tyr Thr Val Pro Phe Ser Ser Thr Gly Ser Tyr Thr Ala

595 600 605

Ser Trp Asn Val Pro

610

<210> 36

<211> 483

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 36

His His Asp Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn

1 5 10 15

Val Pro Asn Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln

20 25 30

Asn Leu Lys Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Lys Ala Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Phe Thr Glu Arg Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn

130 135 140

Phe Pro Gly Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Ala Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu

180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His

195 200 205

Pro Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr Ala Asn

210 215 220

Thr Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys

225 230 235 240

Phe Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln Thr Gly

245 250 255

Lys Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu Gly Ala

260 265 270

Leu Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala Phe Asp

275 280 285

Val Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser Gly Asn

290 295 300

Tyr Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg His Pro

305 310 315 320

Ser His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu

325 330 335

Ala Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Thr Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln Gln Ile

370 375 380

Asp Pro Leu Leu Lys Ala Arg Gln Gln Tyr Ala Tyr Gly Arg Gln His

385 390 395 400

Asp Tyr Phe Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Ala Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro

420 425 430

Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Lys Ala Gly Glu Val

435 440 445

Trp His Asp Met Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn Gln

450 455 460

Asp Gly Trp Gly His Phe Phe Val Asn Gly Gly Ser Val Ser Val Trp

465 470 475 480

Val Lys Arg

<210> 37

<211> 484

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 37

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser

20 25 30

Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp

100 105 110

Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn

115 120 125

Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile

165 170 175

Tyr Lys Phe Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr

180 185 190

Glu Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp

195 200 205

His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr

210 215 220

Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile

225 230 235 240

Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr

245 250 255

Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly

260 265 270

Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe

275 280 285

Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly

290 295 300

Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His

305 310 315 320

Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly

325 330 335

Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr

340 345 350

Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly

355 360 365

Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys

370 375 380

Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln

385 390 395 400

His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly

405 410 415

Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly

420 425 430

Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln

435 440 445

Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn

450 455 460

Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile

465 470 475 480

Trp Val Lys Arg

<210> 38

<211> 483

<212> PRT

<213> Bacillus subtilis (Bacillus subtilis)

<400> 38

His His Asn Gly Thr Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Lys Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Ala Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Thr Lys Ala Trp Asp Trp Glu Val Asp Thr Glu

180 185 190

Phe Gly Asn Tyr Asp Tyr Leu Leu Tyr Ala Asp Ile Asp Met Asp His

195 200 205

Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn

210 215 220

Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys

225 230 235 240

Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Ile Gly

245 250 255

Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala

260 265 270

Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp

275 280 285

Val Pro Leu His Phe Asn Leu Tyr Tyr Ala Ser Lys Ser Gly Gly Asn

290 295 300

Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Lys His Pro

305 310 315 320

Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu

325 330 335

Ser Leu Glu Ser Phe Val Arg Glu Trp Phe Lys Pro Leu Ala Tyr Ala

340 345 350

Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp

355 360 365

Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile

370 375 380

Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg Gln Asn

385 390 395 400

Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn

405 410 415

Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Ala

420 425 430

Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly Gln Val

435 440 445

Trp Thr Asp Ile Thr Gly Asn Lys Ala Gly Thr Val Thr Ile Asn Ala

450 455 460

Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp

465 470 475 480

Val Asn Lys

<210> 39

<211> 485

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 39

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala

20 25 30

Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Gly Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn

115 120 125

Gln Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys

165 170 175

Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Ile Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr

245 250 255

Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Ala Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly

290 295 300

Gly Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys

305 310 315 320

His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Gly Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser

370 375 380

Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr

385 390 395 400

Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly

435 440 445

Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser

465 470 475 480

Val Trp Val Lys Gln

485

<210> 40

<211> 485

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 40

His His Asp Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn

1 5 10 15

Val Pro Asn Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln

20 25 30

Asn Leu Lys Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly

65 70 75 80

Thr Lys Ala Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Phe Thr Glu Arg Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn

130 135 140

Phe Pro Gly Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Ala Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met

195 200 205

Asp His Pro Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr

210 215 220

Ala Asn Thr Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His

225 230 235 240

Ile Lys Phe Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln

245 250 255

Thr Gly Lys Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu

260 265 270

Gly Ala Leu Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser

290 295 300

Gly Asn Tyr Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg

305 310 315 320

His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro

325 330 335

Gly Glu Ala Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Thr Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln

370 375 380

Gln Ile Asp Pro Leu Leu Lys Ala Arg Gln Gln Tyr Ala Tyr Gly Arg

385 390 395 400

Gln His Asp Tyr Phe Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Ala Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Lys Ala Gly

435 440 445

Glu Val Trp His Asp Met Thr Gly Asn Arg Ser Gly Thr Val Thr Ile

450 455 460

Asn Gln Asp Gly Trp Gly His Phe Phe Val Asn Gly Gly Ser Val Ser

465 470 475 480

Val Trp Val Lys Arg

485

<210> 41

<211> 485

<212> PRT

<213> Bacillus species (Bacillus sp.)

<400> 41

His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser

20 25 30

Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp

35 40 45

Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr

50 55 60

Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly

65 70 75 80

Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly

85 90 95

Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp

100 105 110

Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn

115 120 125

Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp

130 135 140

Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr

145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg

165 170 175

Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp

180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met

195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr

210 215 220

Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His

225 230 235 240

Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala

245 250 255

Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu

260 265 270

Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val

275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly

290 295 300

Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg

305 310 315 320

His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro

325 330 335

Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala

340 345 350

Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr

355 360 365

Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser

370 375 380

Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg

385 390 395 400

Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp

420 425 430

Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly

435 440 445

Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile

450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser

465 470 475 480

Ile Trp Val Asn Lys

485

Claims (20)

1. An alpha-amylase variant of a parent alpha-amylase, wherein the variant comprises: (i) According to the numbering of the amino acid sequence shown in SEQ ID NO:3, amino acid changes at one or more positions corresponding to positions selected from the following: 25, 1, 2, 3, 4, 5, 6, 7 ,8,10,12,13,14,15,16,17,18,19,20,21,22,23,24,26,27,28,29,30,31,32,33,34,35 ,36,37,38,39,40,41,42,45,47,48,51,54,59,60,63,70,71,72,73,75,76,81,82,83,86 ,87,89,90,91,92,93,94,95,96,97,98,99,100,103,106,108,109,113,114,115,116,117,119,120,123 ,125,126,128,129,131,132,133,134,135,136,139,141,142,143,144,145,146,147,149,150,151,154,155,156,158 ,160,161,162,163,164,165,169,170,172,173,174,175,176,177,178,180,182,184,185,187,188,189,191,192,193 ,203,208,210,211,212,213,214,215,216,218,219,221,222,223,224,225,226,227,230,231,233,234,235,236,237 ,238,240,242,243,245,249,250,251,252,253,255,256,257,258,259,260,261,262,263,264,265,268,269,270,271 ,272,273,274,275,276,277,279,280,281,282,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298 ,299,301,302,303,304,306,307,308,309,310,311,312,313,314,315,317,318,319,320,321,322,323,324,326,327 ,333,334,336,337,338,341,342,343,344,345,346,348,350,351,352,353,354,355,356,357,358,359,360,362,363 ,364,365,366,367,368,370,372,375,376,378,379,381,382,383,384,385,387,388,389,390,391,392,393,394,395 ,396,397,398,399,400,401,403,405,406,407,408,410,413,414,415,416,418,421,424,426,427,429,431,432,434 ,436,438,439,440,442,443,445,446,447,448,449,451,453,455,456,457,458,459,460,461,462,463,465,467,468 , 470, 471, 474, 478, 480 and 482, (ii) The variant is consistent with any one of SEQ ID NO: 1, 3, 4, or SEQ ID NO: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, more preferably SEQ ID NO: 1 The amino acid sequence shown in has a sequence identity of at least 60% but less than 100%, preferably at least 91% or at least 95% but less than 100%, and (iii) The variant has alpha-amylase activity, preferably wherein the parent alpha-amylase of the alpha-amylase variant is the amylase of SEQ ID NO: 1. 2. The alpha-amylase variant of claim 1, wherein the variant comprises one or more amino acid substitutions, wherein, according to the numbering of the amino acid sequence shown in SEQ ID NO: 3, the one or more amino acid substitutions are selected from the following Amino acid substitution group consisting of: X25H, X25A, X25C, X25D, X25F, X25G, X25K, X25L, X25M, X25Q, 0A. X10C, X10E, X10F, X10L, X10W, X10Y, 14K, X114L, X114M, X114N, X114Q, X114R, X114S, X114V, X114W, X114Y, X115C, X115D, 16R, X116T, X117A, X117C, X117D, X117F, X117I, X117N, X117P, X117R, X117W, X117Y, X118A, 20Y, X123D, X123S, X125A, X125D, X125E, X125F, X125G, X125H, X125K, X125L, X125M, X125N, 28Y, X129E, X12N, X12S, X131C, X131I, X131L, X131Q, X131W, X132T, X133A, X133C, X133D, 34M, X134P, X134T, X134V, X134W, X134Y, X135D, X135E, X135G, X135M, X135N, X135P, X135S, 36W, X139C, X139S, X13A, X13Y, X141V, X142C, X142E, X142F, X142L, 44V, X144Y, X145A, X146C, X146D, X146E, X146F, X146G, X146H, X146K, X146L, X146S, X146T, 6E, X156V, X158H, X158N, X158Y, X15R, X160D, X160E, X160W, X161T, X162V, X163A, ,X170F,X172A,X172C,X172D, X172K, X172N, X173I, X173Y, X174D, X174E, X174G, 77A, X177G, X177K, X177N, X177P, X177R, X177S, X177W, X178F, X17K, X17V, X180M, X180N, M, X187V, X188H, X188T, X188V, X189I, X18F, X18I, X18K, X18M, X18R, X18T, X191F, X191H, ,X19F,X19G,X19H, X19I, X19K, X19L, X19M, X19P, X19Q, X19S, X19T, X19Y, ,X20L, X20M, X20N, X20P, X20Q, X20S, X20T, X20V, X20W, X20Y, , X211E, X211G, X211H, X211L, X211N, X211Q, X211S, X211T, X211V, X212C, X212D, 15E, X215H, X215W, X216G, X216H, X218A, X218C, X218D, X218E, X218F, X218G, X218H, X218I, 19E, X219F, X219G, X219H, X219K, X219M, X219Q, X219R, X219S, X219T, X219W, X219Y, X21E, X21S, V, X225A, X225C, X225E, X225F, X225H, X225I, X225N, X225R, X225S, X225Y, X226A, X226C, 26W, X226Y, X227C, X227F, X227G, X227H, X227I, X227K, X227L, X227M, X227R, X227T, X227V, 2Y, X230A, X230F, X231C, X231D, X231N, X233C, X233H, X233I, X233M, X233T, 3N, X23Q, X23W, X240M, X242M, X242N, X243D, X243F, X245E, X245H, 3C, X253G, X253Y, X255D, X255F, X255I, X255T, X255V, X256C, X257L, X257V, X257Y, X258F, 64H, X264T, X264W, X265S, X268F, X268G, X269M, X26A, X26D, X26E, X26F, X26L, X26M, X273D, X273E, X273F, X273H, X273I, X273L, X273M, X273P, X273Q, X273R, X273V, 77E, X277T, X279A, X279P, X27A, X27D, X27F, X27G, X27H, X27I, X27Q, X27R, X27T, X27V, ,X281H,X284A,X284F, X284H, X284L, X284M, X284N, X284Y, X285G, X285L, X285N, 89G, X289R, X289T, X28C, X28D, X28E, X28F, X28G, X28I, X28K, X28N, X28Q, X28S, X292I, X292L, X292T, X292W, X292Y, X293D, X293E, X293F, X293K, X293R, X294G, 97V, X299G, X299I, X299K, X299L, X299S, X299Y, X29D, X29E, X29F, X29G, X29H, X29I, 02Y, X303E, X303H, X303I, X303K, X303L, X303M, X303N, X303P, X303R, 04Y, X306A, X306D, X306E, X306G, X306H, X306I, X306M, X306Q, X306R, X306S, X306T, X306V, 30I, X30K, X30L, X30M, X30Q, X30T, X30W, X30Y, X310A, X310Q, X311A, X311E, X314Q, X315A, X315C, X315E, X315H, X315K, X315T, X318I, X318S, X31V, X31W, X320A, X320C, X320D, X320E, X320G, X320H, X320K, X320L, X320N, X320Q, X320S, 23L, X323V, X324K, X324L, X324M, X324W, X324Y, X326G, X326N, X326S, X326Y, X327C, X327L, 3I, X334T, X336K, X337A, X337C, X337F, X337G, X337I, X337K, X337L, X337M, X337N, , X33K, X33M, X33Q, X33R, X33Y, X341V, X342P, X343L, X343T, X343W, X343Y, 6G, X346H, X346N, X346Q, X348T, X34H, X34I, X34V, X350H, X350K, X350P, X351A, X351M, , X358N, X358P, X358V, X359E, X35A, X35C, X35D, X35G, X35H, X35I, X35L, X35M, X35N, X35P, X360R, X360S, X360T, X360V, X360Y, X362F, X362K, X362M, X362N, X362T, 63Q, X363R, X363S, X363T, X363V, X363W, X363Y, X364A, X364C, X364G, X364K, X364L, X364N, 6A, X36E, X36G, X36I, X36K, X36M, X36N, X36P, X36Q, X36R, X36S, X36T, X36V, I, X375K, X375Q, X375R, X375T, X375W, X375Y, X376G, X376I, X376K, X376L, X376M, 7A, X37G, X37M, X37P, X37T, X37V, X37W, X381E, X381V, X382A, X382H, X382K, X382L, S, X383V, X383Y, X384A, X384C, X384D, X384E, X384F, X384I, X384L, X384M, X384N, X384Q, 85I, X385L, X385M, X385N, X385P, X385Q, X385R, X385S, X385T, X385V, X385W, X385Y, X387C, 8N, X390D, X390F, X390M, X390N, X390P, X390R, X391A, X391F, X391G, X391K, X391M, X391N, 94C, X394E, X394H, X394I, X394L, X394M, X394N, X394R, X394S, X395A, X395H, X395M, X395V, X3K, X3L, X3Q, X3V, X400A, X400D, X400E, X400G, X400H, X400I, X400K, T, X403N, X405C, X405H, X405M, X405T, X405V, X406P, X407D, X407R, X407S, X408E, E. X414S, X415E, X415I, X416S, X418C, X418N, X418P, X41C, X41D, X41E, X41G, X41Q, 7S, X427T, X427V, X429A, X429D, X429E, X429F, X429G, X429I, X429M, X429N, X429P, X438P, X438Y, X439K, X439C, X439P, X440V, X442Q, X443H, X443T, X445A, X445C, X445D, 46L, X446P, X446Q, X446R, X446V, X446W, X447V, X448D, X448E, X448H, X448N, X449A, X449F, 51L, X453G, X453I, X453N, X453P, X453Y, X455L, X456L, X456M, X456R, 57V, X457W, X458I, X458K, X458V, X458W, X459C, X459D, X459E, X459I, X459K, X459N, X459Q, T, X460V, X461A, X461E, X461F, X461K, X461L, X461M, X461N, X461Q, X461R, X461S, X461V, 70Q, X470T, X471E, X474F, X474H, X474P, X478A, X478E, X47S, X480D, Q、X51T、 X51V, X54D, X54G, X54Q, X59T, X5A, X5C, X5D, X5E, X6C, X6E, X6F, X6G, X6H, X6K, X6L, X6M, X6P, X6Q, X6S, X72N, X72T, X73L, X73N, X73Q, X75A, X75G, X75I, X7Q, X7R, X7S, X7V, X7W, X7Y, X81H, X81L, X82K, X89R, X89S, X8A, X8C, X8F, X8I, X8M, X8P, X8S, X8V, X91C, X91D, X91E, X91F, X91G, X91H, X91I, X91K, X91L, X91M, 4E. X94K, X94M, X94V, X94Y, X95E, X95I, X95L, X95V, X96K, Preferably selected from X4Q, X25H, X100W, X135E, X135T, X135W, X135D, 482W, and wherein the variant has alpha-amylase activity. 3. An alpha-amylase variant according to any one of the preceding claims, wherein the variant comprises a combination of substitutions selected from: X25H+X176K、 X25H+X176K+X186E、 X25H+X176K+X186E+X251E+X405M, X25H+X176K+X186E+X251E+X405M+X482W、 X25H+X176K+X186E+X251E+X405M+X439K, X25H+X176K+X186E+X251E+X405M+X439K+X482W、 X25H+X176K+X251E+X405M, X25H+X176K+X251E+X405M+X482W、 X25H+X176K+X251E+X405M+X439K, X25H+X176K+X251E+X405M+X439K+X482W、 X176K+X186E、 X251E+X405M、 X251E+X405M+X439K、 X251E+X405M+X482W、 X251E+X405M+X439K+X482W、 X405M+X439K、 X405M+X482W、 X405M+X439K+X482W、 X4Q+X25H+X176K+X186E+X251E+X405M+X439K, X4Q+X25H+X176K+X186E+X251E+X405M+X439K+X482W、 X4Q+X25H+X176K+X251E+X405M+X439K, X4Q+X25H+X176K+X251E+X405M+X439K+X482W、 X116K+X181T+X225A、 X181T+X225A+X320K、 X116K+X181T+X225A+X320K、 X25H+X176K+X186E+X206Y+X251E+X482W、 X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X482W、 X25H+X176K+X186E+X206Y+X482W、 X25H+X186E+X206Y+X405M+X482W、 X25H+X186E+X206Y+X482W、 X25H+X176K+X186E+X193E+X206Y+X251E+X482W、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X482W、 X25H+X176K+X186E+X482W、 X25H+X176K+X186E+X193E+X206Y+X482W、 X4Q+X25H+X176K+X186E+X251E+X405M+X482W、 X25H+X176K+X186E+X206Y、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X206Y+X460G+X482W、 X4Q+X25H+X176K+X206Y+X251E+X460G+X482W、 X4Q+X25H+X176K+X193E+X251E+X186E+X482W、 X4Q+X193E+X206Y+X276R+X186E+X482W、 X186E+X25H+X482W、 X25H+X193E+X206Y+X251E+X276R+X405M+X186E+X482W、 X25H+X251E+X186E+X482W、 X25H+X160W+X176K+X186E+X251E+X405M+X482W、 X193E+X206Y+X405M+X186E+X482W、 X3I+X356V、 X3I+X356I、 X83D+X94E、 X94D+X125E、 X131I+X377Q+X410H、 X48F+X94D、 X48Y+X116K+X218K、 X5L+X218K+X225S、 X83E+X116R+X158Y+X181E、 X51V+X218K、 X83E+X181E、 X4Q+X7W+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X37M+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X25D+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X25H+X176K+X186E+X206Y+X251E+X405M+X460G, X4Q+X25H+X176K+X186E+X206Y+X251E+X460G, X4Q+X25Y+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X118D+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X182D+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X186E+X193E+X206Y+X251E+X258Q+X405M, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X460G, X4Q+X176K+X186E+X193E+X206Y+X251E+X363E+X405M, X4Q+X176K+X186E+X193E+X206Y+X251E+X363H+X405M, X4Q+X176K+X186E+X193E+X206Y+X251E+X363N+X405M, X4Q+X176K+X181D+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X181F+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X181N+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X181Q+X186E+X193E+X206Y+X251E+X405M, X4Q+X136E+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X100W+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X135D+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X135E+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X135T+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X135W+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X160D+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X160E+X176K+X186E+X193E+X206Y+X251E+X405M, X7H+X25H+X176K+X186E+X206Y、 X7W+X25H+X176K+X186E+X206Y、 X25D+X176K+X186E+X206Y、 X25H+X186E+X206Y+X405M+X460G, X25H+X186E+X206Y+X460G、 X25H+X37M+X176K+X186E+X206Y、 X25H+X118D+X176K+X186E+X206Y、 X25H+X176K+X182D+X186E+X206Y、 X25H+X176K+X186E+X206Y、 X25H+X176K+X186E+X206Y+X258Q、 X25H+X176K+X186E+X206Y+X281N、 X25H+X176K+X186E+X206Y+X460G、 X25H+X176K+X186E+X206Y+X251E+X460G, X25H+X176K+X186E+X206Y+X363E、 X25H+X176K+X186E+X206Y+X363H、 X25H+X176K+X186E+X206Y+X363N、 X25H+X176K+X186E+X460G、 X25H+X176K+X186E+X193E+X206Y、 X25H+X176K+X186E+X193E+X206Y+X460G、 X25H+X176K+X186E+X193E+X206Y+X251E+X460G, X25H+X176K+X181D+X186E+X206Y、 X25H+X176K+X181N+X186E+X206Y、 X25H+X176K+X181Q+X186E+X206Y、 X25H+X136E+X176K+X186E+X206Y、 X25H+X100W+X176K+X186E+X206Y、 X25H+X135E+X176K+X186E+X206Y、 X25H+X135T+X176K+X186E+X206Y、 X25H+X135W+X176K+X186E+X206Y、 X25H+X160D+X176K+X186E+X206Y、 X25H+X160E+X176K+X186E+X206Y、 X25Y+X176K+X186E+X206Y、 X87D+X186E+X315K+X420K、 X87D+X186E+X226D+X314E+X420E+X461E, X87D+X186E+X226D+X420E、 X87D+X186E+X226D+X420E+X461E、 X87D+X186E+X314E+X471E、 X87D+X186E+X311K+X314E+X420K, X87D+X186E+X160D+X461E、 X87R+X186E+X315K+X461R、 X87R+X186E+X226D+X471E、 X87R+X186E+X226R+X314K+X420K+X461R、 X87R+X186E+X226R+X311K+X420K, X87R+X186E+X314K+X315K, X87R+X186E+X311K+X314E、 X87R+X186E+X420E+X450R、 X87R+X186E+X450R+X452R、 X186E+X315K+X420E+X452R、 X186E+X226D+X314E+X461E+X471E、 X186E+X226D+X314E+X452R、 X186E+X226D+X314K+X460D+X471E+X485R、 X186E+X226D+X311K+X460D, X186E+X226D+X461E+X471E、 X186E+X314E+X460D+X461E、 X186E+X314E+X420E+X452R+X461E、 X186E+X314K+X450R+X460D、 X186E+X460D+X471E+X485R、 X186E+X311K+X314K+X452R、 X186E+X311K+X461E+X485R、 X186E+X311K+X420E+X471E、 X186E+X420K+X450R+X471E+X485R、 X186E+X420K+X450R+X485R、 X186E+X452R+X461E+X471E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M, X25H+X176K+X186E+X206Y、 X83E+X186E+X219D+X226R、 X83E+X186E+X219R+X226D+X314E+X452R、 X83E+X186E+X219R+X226R、 X83E+X186E+X160D+X219D+X226R+X452R、 X83R+X186E+X219R+X226D、 X160D+X186E+X315K+X450R、 X160D+X186E+X226D+X314K+X460D+X461E、 X160D+X186E+X226D+X314K+X420E、 X160D+X186E+X314K+X485R、 X160D+X186E+X420E+X452R、 X160D+X186E+X420K+X460D、 X186E+X276R、 X186E+X206Y、 X186E+X206Y+X276R、 X186E+X206Y+X276R+X405M, X186E+X206Y+X405M、 X186E+X206Y+X251E、 X186E+X206Y+X251E+X276R、 X186E+X206Y+X251E+X276R+X405M, X186E+X206Y+X251E+X405M, X186E+X206Y+X251E+X323G+X405M, X186E+X206Y+X323G+X405M, X186E+X405M、 X186E+X193E+X206Y+X405M、 X186E+X193E+X206Y+X251E、 X186E+X251E、 X186E+X251E+X405M、 X4Q+X186E、 X4Q+X186E+X276R、 X4Q+X186E+X206Y、 X4Q+X186E+X206Y+X276R+X405M、 X4Q+X186E+X206Y+X405M、 X4Q+X186E+X206Y+X251E+X276R、 X4Q+X186E+X206Y+X251E+X276R+X405M, X4Q+X186E+X206Y+X251E+X405M, X4Q+X186E+X206Y+X251E+X323G+X405M, X4Q+X186E+X405M、 X4Q+X186E+X193E+X206Y+X276R、 X4Q+X186E+X193E+X206Y+X405M、 X4Q+X186E+X193E+X206Y+X251E+X405M, X4Q+X186E+X251E+X276R+X405M, X4Q+X25H+X186E+X206Y、 X4Q+X25H+X186E+X206Y+X276R、 X4Q+X25H+X186E+X206Y+X276R+X405M, X4Q+X25H+X186E+X206Y+X405M、 X4Q+X25H+X186E+X206Y+X251E+X323A+X405M, X4Q+X25H+X186E+X206Y+X323G+X405M, X4Q+X25H+X186E+X405M、 X4Q+X25H+X176K+X186E+X206Y+X276R、 X4Q+X25H+X176K+X186E+X206Y+X276R+X405M, X4Q+X25H+X176K+X186E+X206Y+X251E+X276R+X405M, X4Q+X25H+X176K+X186E+X206Y+X251E+X405M, X4Q+X25H+X176K+X186E+X251E、 X4Q+X25H+X176K+X186E+X251E+X405M, X4Q+X25H+X176K+X206Y、 X4Q+X25H+X176K+X206Y+X405M+X460G、 X4Q+X25H+X176K+X206Y+X460G、 X4Q+X25H+X176K+X206Y+X251E、 X4Q+X25H+X176K+X206Y+X251E+X276R+X405M, X4Q+X25H+X176K+X206Y+X251E+X460G、 X4Q+X25H+X176K+X206Y+X323G、 X4Q+X25H+X160W+X186E+X206Y+X405M, X4Q+X25H+X160W+X186E+X206Y+X251E、 X4Q+X25H+X160W+X186E+X206Y+X251E+X276R+X405M, X4Q+X25H+X160W+X186E+X206Y+X323G+X405M, X4Q+X25H+X160W+X186E+X405M, X4Q+X25H+X160W+X186E+X193E+X206Y+X276R、 X4Q+X25H+X160W+X186E+X323G, X4Q+X25H+X160W+X169R+X186E+X206Y+X276R、 X4Q+X25H+X160W+X176K+X186E+X276R+X405M, X4Q+X25H+X160W+X176K+X186E+X206Y+X276R、 X4Q+X25H+X160W+X176K+X186E+X206Y+X251E、 X4Q+X25H+X160W+X176K+X186E+X206Y+X251E+X405M, X4Q+X25H+X160W+X176K+X186E+X405M, X4Q+X25H+X160W+X176K+X186E+X251E+X276R+X405M, X4Q+X25H+X160W+X176K+X186E+X251E+X405M, X4Q+X25H+X160W+X176K+X206Y、 X4Q+X25H+X160W+X176K+X206Y+X405M, X4Q+X25H+X160W+X176K+X206Y+X460G, X4Q+X25H+X160W+X176K+X206Y+X251E+X460G, X4Q+X176K+X186E+X276R+X405M, X4Q+X176K+X186E+X206Y+X276R、 X4Q+X176K+X186E+X206Y+X276R+X405M, X4Q+X176K+X186E+X206Y+X405M, X4Q+X176K+X186E+X206Y+X251E+X276R+X405M, X4Q+X176K+X186E+X206Y+X251E+X405M, X4Q+X176K+X186E+X405M、 X4Q+X176K+X186E+X193E+X206Y+X276R+X405M, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X186E+X251E+X276R+X405M, X4Q+X176K+X186E+X251E+X405M, X4Q+X176K+X186E+X323G+X405M, X4Q+X176K+X206Y、 X4Q+X176K+X206Y+X276R、 X4Q+X176K+X206Y+X405M、 X4Q+X176K+X206Y+X405M+X460G、 X4Q+X176K+X206Y+X460G、 X4Q+X176K+X206Y+X251E+X276R+X405M, X4Q+X176K+X206Y+X251E+X276R+X405M+X460G, X4Q+X176K+X206Y+X251E+X405M, X4Q+X176K+X206Y+X251E+X405M+X460G、 X4Q+X176K+X206Y+X251E+X323A+X460G, X4Q+X160W+X186E+X276R+X405M, X4Q+X160W+X186E+X206Y、 X4Q+X160W+X186E+X206Y+X276R+X323G, X4Q+X160W+X186E+X206Y+X405M, X4Q+X160W+X186E+X206Y+X251E、 X4Q+X160W+X186E+X206Y+X251E+X276R+X405M, X4Q+X160W+X186E+X405M、 X4Q+X160W+X186E+X251E+X405M, X4Q+X160W+X176K+X186E、 X4Q+X160W+X176K+X186E+X276R+X405M, X4Q+X160W+X176K+X186E+X206Y+X251E、 X4Q+X160W+X176K+X186E+X251E+X276R+X405M, X4Q+X160W+X176K+X186E+X251E+X405M, X4Q+X160W+X176K+X206Y、 X4Q+X160W+X176K+X206Y+X251E+X276R+X460G, X4Q+X160W+X176K+X206Y+X251E+X323G+X405M+X460G, X25H+X186E、 X25H+X186E+X206Y、 X25H+X186E+X206Y+X276R+X405M, X25H+X186E+X206Y+X276R+X323G、 X25H+X186E+X206Y+X405M、 X25H+X186E+X206Y+X251E+X276R+X323G+X405M, X25H+X186E+X206Y+X251E+X405M, X25H+X186E+X405M、 X25H+X186E+X202I+X206Y+X405M, X25H+X186E+X193E+X206Y+X276R+X405M, X25H+X186E+X193E+X206Y+X405M、 X25H+X186E+X193E+X206Y+X251E+X276R+X405M, X25H+X186E+X251E、 X25H+X186E+X251E+X276R、 X25H+X176K+X186E、 X25H+X176K+X186E+X276R+X323G+X405M, X25H+X176K+X186E+X206Y、 X25H+X176K+X186E+X206Y+X276R、 X25H+X176K+X186E+X206Y+X251E、 X25H+X176K+X186E+X206Y+X251E+X276R+X405M, X25H+X176K+X186E+X206Y+X251E+X405M, X25H+X176K+X186E+X405M, X25H+X176K+X186E+X193E+X206Y、 X25H+X176K+X186E+X193E+X206Y+X251E、 X25H+X176K+X186E+X193E+X206Y+X251E+X276R+X405M, X25H+X176K+X186E+X251E+X276R、 X25H+X176K+X206Y、 X25H+X176K+X206Y+X276R、 X25H+X176K+X206Y+X276R+X405M, X25H+X176K+X206Y+X276R+X460G、 X25H+X176K+X206Y+X405M、 X25H+X176K+X206Y+X460G、 X25H+X176K+X206Y+X251E、 X25H+X176K+X206Y+X251E+X276R+X405M+X460G, X25H+X176K+X206Y+X251E+X276R+X460G、 X25H+X176K+X206Y+X251E+X405M, X25H+X160W+X186E+X206Y、 X25H+X160W+X186E+X206Y+X251E、 X25H+X160W+X186E+X206Y+X251E+X276R、 X25H+X160W+X186E+X206Y+X251E+X276R+X323G+X405M, X25H+X160W+X186E+X206Y+X251E+X405M, X25H+X160W+X186E+X206Y+X323G+X405M, X25H+X160W+X186E+X251E+X276R+X405M, X25H+X160W+X176K+X186E+X206Y+X276R、 X25H+X160W+X176K+X186E+X206Y+X276R+X405M, X25H+X160W+X176K+X186E+X206Y+X251E+X276R+X323G, X25H+X160W+X176K+X186E+X206Y+X251E+X405M, X25H+X160W+X176K+X186E+X405M, X25H+X160W+X176K+X186E+X193E+X206Y、 X25H+X160W+X176K+X186E+X193E+X206Y+X405M, X25H+X160W+X176K+X186E+X193E+X206Y+X251E+X405M, X25H+X160W+X176K+X186E+X251E、 X25H+X160W+X176K+X186E+X251E+X276R、 X25H+X160W+X176K+X186E+X251E+X405M, X25H+X160W+X176K+X186E+X323G+X405M, X25H+X160W+X176K+X206Y+X276R+X323G+X405M, X25H+X160W+X176K+X206Y+X460G, X25H+X160W+X176K+X206Y+X251E、 X25H+X160W+X176K+X206Y+X251E+X323G+X405M, X176K+X186E+X206Y、 X176K+X186E+X206Y+X276R、 X176K+X186E+X206Y+X276R+X405M、 X176K+X186E+X206Y+X405M、 X176K+X186E+X206Y+X251E+X405M, X176K+X186E+X405M、 X176K+X186E+X193E+X206Y+X405M、 X176K+X186E+X193E+X206Y+X251E+X405M, X176K+X186E+X193E+X405M, X176K+X186E+X251E+X276R+X405M, X176K+X186E+X251E+X405M, X176K+X206Y、 X176K+X206Y+X276R、 X176K+X206Y+X276R+X460G、 X176K+X206Y+X405M、 X176K+X206Y+X405M+X460G、 X176K+X206Y+X460G、 X176K+X206Y+X251E、 X176K+X206Y+X251E+X405M、 X160W+X186E、 X160W+X186E+X276R+X405M, X160W+X186E+X206Y+X276R、 X160W+X186E+X206Y+X276R+X405M, X160W+X186E+X206Y+X405M, X160W+X186E+X206Y+X251E、 X160W+X186E+X206Y+X251E+X405M, X160W+X186E+X405M、 X160W+X186E+X193E+X206Y、 X160W+X186E+X193E+X206Y+X405M, X160W+X186E+X251E+X276R、 X160W+X186E+X251E+X276R+X405M, X160W+X186E+X323G+X405M, X160W+X176K+X186E+X276R+X405M, X160W+X176K+X186E+X206Y、 X160W+X176K+X186E+X206Y+X251E+X405M, X160W+X176K+X186E+X405M, X160W+X176K+X186E+X193E+X206Y、 X160W+X176K+X186E+X193E+X206Y+X405M, X160W+X176K+X186E+X193E+X206Y+X251E、 X160W+X176K+X206Y+X276R+X405M+X460G, X160W+X176K+X206Y+X276R+X323G+X460G, X160W+X176K+X206Y+X405M, X160W+X176K+X206Y+X405M+X460G、 X160W+X176K+X206Y+X460G、 X160W+X176K+X206Y+X251E+X276R、 X160W+X176K+X206Y+X251E+X405M+X460G, X160W+X176K+X206Y+X323G+X405M+X460G, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q+X281N+X363H、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X258Q+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X281N、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X281N+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X281N+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X258Q, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X258Q+X281N、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X258Q+X281N+X363H、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X281N+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X181Q+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N+X363H、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X281N+X363H+X272V, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X258Q+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X281N+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X281N+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X281N+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X258Q+X363H、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X281N+X363H、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X181Q+X363H+X254Q, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X281N+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X258Q+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X258Q+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X281N+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X181Q+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X135T+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X100W+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X8A, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X281N+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X258Q+X363H、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q+X281N, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X181Q+X258Q+X363H、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X363E, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X135T+X363H, X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X363E、 X4Q+X176K+X186E+X193E+X206Y+X251E+X405M+X363H、 X25H+X176K+X186E+X206Y、 X25H+X176K+X186E+X206Y+X258Q、 X25H+X176K+X186E+X206Y+X258Q、 X25H+X176K+X186E+X206Y+X258Q+X281N、 X25H+X176K+X186E+X206Y+X258Q+X281N+X363E、 X25H+X176K+X186E+X206Y+X258Q+X363E、 X25H+X176K+X186E+X206Y+X258Q+X363E+X123D, X25H+X176K+X186E+X206Y+X281N、 X25H+X176K+X186E+X206Y+X181Q、 X25H+X176K+X186E+X206Y+X181Q+X193E、 X25H+X176K+X186E+X206Y+X181Q+X363E、 X25H+X176K+X186E+X206Y+X193E、 X25H+X176K+X186E+X206Y+X193E+X258Q、 X25H+X176K+X186E+X206Y+X193E+X281N+X363E、 X25H+X176K+X186E+X206Y+X193E+X363E、 X25H+X176K+X186E+X206Y+X100W、 X25H+X176K+X186E+X206Y+X100W+X258Q、 X25H+X176K+X186E+X206Y+X100W+X258Q+X281N、 X25H+X176K+X186E+X206Y+X100W+X258Q+X281N+X363E、 X25H+X176K+X186E+X206Y+X100W+X258Q+X363E、 X25H+X176K+X186E+X206Y+X100W+X258Q+X363E+X135C, X25H+X176K+X186E+X206Y+X100W+X281N、 X25H+X176K+X186E+X206Y+X100W+X281N+X363E、 X25H+X176K+X186E+X206Y+X100W+X361R、 X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E、 X25H+X176K+X186E+X206Y+X100W+X181Q+X281N+X363E+X175D, X25H+X176K+X186E+X206Y+X100W+X181Q+X193E、 X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X281N, X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X258Q+X363E, X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X281N+X363E, X25H+X176K+X186E+X206Y+X100W+X181Q+X193E+X363E、 X25H+X176K+X186E+X206Y+X100W+X181Q+X363E、 X25H+X176K+X186E+X206Y+X100W+X193E、 X25H+X176K+X186E+X206Y+X100W+X193E+X258Q, X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X473R、 X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N, X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X281N+X363E, X25H+X176K+X186E+X206Y+X100W+X193E+X258Q+X363E、 X25H+X176K+X186E+X206Y+X100W+X193E+X363E、 X25H+X176K+X186E+X206Y+X100W+X135T、 X25H+X176K+X186E+X206Y+X100W+X135T+X258Q、 X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N, X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X281N+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X258Q+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X281N、 X25H+X176K+X186E+X206Y+X100W+X135T+X281N+X363E、 X25H+X176K+X186E+X206Y+X100W+X135T+X181Q、 X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X258Q+X281N+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X281N+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E、 X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X258Q+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X181Q+X193E+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X193E、 X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X258Q+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X193E+X363E, X25H+X176K+X186E+X206Y+X100W+X135T+X363E、 X25H+X176K+X186E+X206Y+X100W+X363E、 X25H+X176K+X186E+X206Y+X135T、 X25H+X176K+X186E+X206Y+X135T+X258Q、 X25H+X176K+X186E+X206Y+X135T+X258Q+X281N、 X25H+X176K+X186E+X206Y+X135T+X258Q+X281N+X363E, and X25H+X176K+X186E+X206Y+X135T+X258Q+X363E, wherein the variant is numbered according to the amino acid sequence shown in SEQ ID NO:3, and wherein the variant has alpha-amylase activity. 4. The alpha-amylase variant of any one of the preceding claims, wherein the variant is identical to any one of SEQ ID NO: 1, 3, 4, or SEQ ID NO 15-41, preferably SEQ ID NO: 1 The amino acid sequence shown has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, Sequence identity of at least 91% or at least 95% but less than 100% is preferred. 5. The alpha-amylase variant of any one of the preceding claims, wherein the variant comprises A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are identical to the amino acids of SEQ ID NO: 6 The sequence is at least 75% but less than 100% identical, and the amino acid sequence of the C domain is at least 75% but less than 100% identical to the amino acid sequence of SEQ ID NO:8. 6. An alpha-amylase variant according to any one of the preceding claims, wherein the variant is relative to said parent alpha-amylase, preferably relative to what is shown in SEQ ID NO: 1 and/or SEQ ID NO: 3 The parent α-amylase preferably exhibits one or more improved properties relative to the parent α-amylase shown in SEQ ID NO: 1, preferably the α-amylase variant has improved expression, activity, and stability. properties, thermal stability, specific activity, substrate specificity, pH-dependent activity, pH stability, oxidative stability, catalytic efficiency, catalytic rate, chemical stability, pH activity, stability under storage conditions, substrate binding , substrate cleavage, substrate stability, surface properties, thermal activity, Ca2+ dependence, washing performance, washing performance in laundry detergents, and/or washing performance in ADW detergents, preferably, the improved The properties are improved thermal stability, improved storage stability in detergent compositions and/or improved washing performance, most preferably improved thermal stability, preferably improved in detergent compositions, preferably in laundry washing. Thermal stability in detergent or dishwashing compositions, preferably in laundry detergent compositions. 7. The alpha-amylase variant of claim 7, wherein the improved properties are expressed as an improvement factor (IF) >1.0, and wherein preferably the improvement factor is equal to or greater than 1.1, preferably equal to or greater than 1.2, more preferably equal to or greater than 1.3. 8. The alpha-amylase variant of any one of the preceding claims, further comprising a deletion at one or more amino acids corresponding to a position selected from the group consisting of 181, 182, 183 and 184, preferably at a position corresponding to a position selected from the group consisting of 181 Comprising deletions at two or more amino acids at positions 182, 183 and 184, preferably comprising deletions of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, wherein according to SEQ ID NO: 3 Amino acid sequence number. 9. The alpha-amylase variant of any one of the preceding claims, wherein compared to SEQ ID NO: 1, the number of substitutions is 1-30, preferably 1-25, 1-20, 1- 15, 1-10, 2-10, 3-10, or 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 10,11,12,13,14,15,15,16,17,18,19,20,21,22,23,24 or 25 replaced. 10. The alpha-amylase variant of any one of the preceding claims, wherein the variant comprises an amino acid substitution set forth in SEQ ID NO: 1 with one or more, preferably 1 to 30 amino acid substitutions selected from the group consisting of The amino acid sequence may consist of: X25H, X25A, X25C, X25D, X25F, X25G, X25K, X25L, X25M, X25S, X25W, X25Y, X100F, X100K, X100L, X103A, X100K, X100L, X103A, X100L, X103A,, X103A,, X103A,,, X103A,,, X103A,,, X103A, X103A,,, X103A, X103A,,, X103A,,,,,,,, are X106D, X108A, X109A, X10A, X10C, X10E, X10F, X10L, X10W, X10Y, X113F, 4I, X114K, X114L, X114M, X114N, X114Q, X114R, X114S, X114V, X114W, X114Y, 16M, X116R, X116T, X117A, X117C, X117D, X117F, X117I, X117N, X117P, X117R, X117W, X117Y, 20W, X120Y, X123D, X123S, X125A, X125D, X125E, X125F, X125G, X125H, X125K, X125L, X125M, 28W, X128Y, X129E, X12N, X12S, X131C, X131I, X131L, X131Q, X131W, X132T, X133A, X133C, 34L, X134M, X134P, X134T, X134V, X134W, X134Y, X135D, X135E, X135G, X135M, X135N, X135P, 36P, X136W, X139C, X139S, X13A, X13Y, X141V, X142C, X142E, X142F, X142L, X142M, X142Q, 4T, X144V, X144Y, X145A, X146C, X146D, X146E, X146F, X146G, X146H, X146K, X146L, X146S, 5Y, X156E, X156V, X158H, X158N, X158Y, X15R, X160D, X160E, X160W, X161T, X162V, X163A, ,X170C,X170F,X172A,X172C, X172D, X172K, X172N, X173I, X173Y, X174D, X174E, X174G, 76T, X177A, X177G, X177K, X177N, X177P, X177R, X177S, X177W, X178F, X17K, X17V, X180M, D. X187M, X187V, X188H, X188T, X188V, X189I, X18F, X18I, X18K, X18M, X18R, X18T, C, X19D, X19F, X19G, X19H, X19I, X19K, X19L, X19M, X19P, X19Q, X19S, X19T, ,X20K, X20L, X20M, X20N, X20P, X20Q, X20S, X20T, X20V, X211D, X211E, X211G, X211H, X211L, X211N, X211Q, X211S, X211T, X211V, X212C, 15D, X215E, X215H, X215W, X216G, X216H, X218A, X218C, X218D, X218E, X218F, X218G, X218H, 19D, X219E, X219F, X219G, X219H, X219K, X219M, X219Q, X219R, X219S, X219T, X219W, X219Y, F, X224V, X225A, X225C, X225E, X225F, X225H, X225I, X225N, X225R, X225S, X225Y, X226A, 26V, X226W, X226Y, X227C, X227F, X227G, X227H, X227I, X227K, X227L, X227M, X227R, X227T, 22W, X22Y, X230A, X230F, X231C, X231D, X231N, X233C, X233H, X233I, 38T, X23N, X23Q, X23W, X240M, X242M, X242N, X243D, X243F, X245E, X245H, X245M, X249D, 2S, X253C, X253G, X253Y, X255D, X255F, X255I, X255T, X255V, X256C, X257L, X257V, X257Y, 63I, X264H, X264T, X264W, X265S, X268F, X268G, X269M, X26A, X26D, X26E, X26F, X26L, X273C, X273D, X273E, X273F, X273H, X273I, X273L, X273M, X273P, X273Q, X273R, 77D, X277E, X277T, X279A, X279P, X27A, X27D, X27F, X27G, X27H, X27I, X27Q, X27R, X281E, X281H, X284A, X284F, X284H, X284L, X284M, X284N, X284Y, X285G, X285L, 89F, X289G, X289R, X289T, X28C, X28D, X28E, X28F, X28G, X28I, X28K, X28N, X28Q, X28S, 292F, X292I, X292L, X292T, X292W, X292Y, X293D, X293E, X293F, X293K, X293R, 97S, X297V, X299G, X299I, X299K, X299L, X299S, X299Y, X29D, X29E, X29F, X29G, X29H, 02V, X302Y, X303E, X303H, X303I, X303K, X303L, X303M, X303N, X303P, 04W, X304Y, X306A, X306D, X306E, X306G, X306H, X306I, X306M, X306Q, X306R, X306S, X30H, X30I, X30K, X30L, X30M, X30Q, X30T, X30W, X30Y, X310A, X310Q, X311A, 314K, X314Q, X315A, X315C, X315E, X315H, X315K, X315T, X318I, X318S, X318T, X319A, X319D, , X31T, X31V, X31W, X320A, X320C, X320D, X320E, X320G, X320H, X320K, X320L, X320N, 23K, X323L, X323V, X324K, X324L, X324M, X324W, X324Y, X326G, X326N, X326S, X326Y, X327C, 2W, X333I, X334T, X336K, X337A, X337C, X337F, X337G, X337I, X337K, X337L, E. X33H, X33K, X33M, X33Q, X33R, X33Y, X341V, X342P, X343L, X343T, X343W, X343Y, D. X346G, X346H, X346N, X346Q, X348T, X34H, X34I, X34V, X350H, X350K, X350P, X351A, , X358L, X358N, X358P, X358V, X359E, X35A, X35C, X35D, X35G, X35H, X35I, X35L, X360Q, X360R, X360S, X360T, X360V, X360Y, X362F, X362K, X362M, X362N, 63P, X363Q, X363R, X363S, X363T, X363V, X363W, X363Y, X364A, X364C, X364G, X364K, X364L, 68N, X36A, X36E, X36G, X36I, X36K, X36M, X36N, X36P, X36Q, X36R, X36S, X36T, X36V, , X375I, X375K, X375Q, X375R, X375T, X375W, X375Y, X376G, X376I, X376K, X376L, 79S, X37A, X37G, X37M, X37P, X37T, X37V, X37W, X381E, X381V, X382A, X382H, X382K, , X383S, X383V, X383Y, X384A, X384C, X384D, X384E, X384F, X384I, X384L, X384M, X384N, 85H, X385I, X385L, X385M, X385N, X385P, X385Q, X385R, X385S, X385T, X385V, X385W, X385Y, 89K, X38N, X390D, X390F, X390M, X390N, X390P, X390R, X391A, X391F, X391G, X391K, X391M, 94A, X394C, X394E, X394H, X394I, X394L, X394M, X394N, X394R, X394S, X395A, X395H, X395M, G, X3I, X3K, X3L, X3Q, X3V, X400A, X400D, X400E, X400G, X400H, X400I, X401T, X403N, X405C, X405H, X405M, X405T, X405V, X406P, X407D, X407R, X407S, X408E, C. X414E, X414S, X415E, X415I, X416S, X418C, X418N, X418P, X41C, X41D, X41E, X41G, X41Q, 7R, X427S, X427T, X427V, X429A, X429D, X429E, X429F, X429G, X429I, X429M, X429N, X438F, X438P, X438Y, X439K, X439C, X439P, X440V, X442Q, X443H, X443T, X445A, X445C, 46I, X446L, X446P, X446Q, X446R, X446V, X446W, X447V, X448D, X448E, X448H, X448N, X449A, 49Y, X451L, X453G, X453I, X453N, X453P, X453Y, X455L, X456L, X456M, X456R, X456S, X456V, 57T, X457V, X457W, X458I, X458K, X458V, X458W, X459C, X459D, X459E, X459I, X459K, X459N, S, X460T, X460V, X461A, X461E, X461F, X461K, X461L, X461M, X461N, X461Q, X461R, X461S, 70A, X470Q, X470T, X471E, X474F, X474H, X474P, X478A, X478E, X47S, 4S, X51Q, X51T, X51V, X54D, X54G, X54Q, X59T, X5A, X5C, X5D, X6A, X6C, X6E, X6F, X6G, X6H, X6K, X6L, X6M, X6P, X6Q, X72E, X72N, X72T, X73L, X73N, X73Q, X75A, X75G, X7P, X7Q, X7R, X7S, X7V, X7W, X7Y, X81H, X81L, X89M, X89R, X89S, X8A, X8C, X8F, X8I, X8M, X8P, X8S, X90Y, X91C, X91D, X91E, X91F, X91G, X91H, X91I, 4D. X94E, X94K, X94M, X94V, X94Y, X95E, X95I, X95L, X95V, X96K, X96N, 11. The alpha-amylase variant of any one of the preceding claims, further comprising an alpha-amylase variant selected from the group consisting of 9, 130, 179, 181, 186, 190, 195, 202, 206, 244, 402, 419, 420, Comprising additional amino acid changes at one or more of positions 422, 423, 428, 430, 435, 441, 444, 450, 452, 454, 466, 469, 473, 475, 476, 479, 483 and 485, preferably Other substitutions, preferably one or more other amino acid substitutions selected from the following amino acid substitutions: X9D, X9F, X9K, X9L, X9N, X9P, X9Q, X9S, X9T, X9Y, X130V, X181H, X181I, X181N, X181Q, X181S, X181T, X181V, X181W, 86Q, X186R, X186S, X186V, X186W, X186Y, X190H, X195F, X195H, X195K, X195L, X195W, 4H, X244M, X244N, X244Q, X244V, X402T , X419C, X420D, X420E, X420G, X420H, X420K, X420L, X428I, X428K, X428L, X428M, X428N , X428R, X428S, X428V, X428W, X428Y, X430A, X430C, X435P, X435R, X435S, X435A, X435D , X437A, X437L, X437T, X437W, X441C, X441D, X441K, X450E, X450H, X450I, X450L, X450M , X450P, X450Q, X450R, X450T, X452A, X452C, X452E, X454S, X454T, X466E, X466W, X469F , X469L, X469Y, X473H, X473Q, X473R, X475A, X475K, X483R, X485K and X485R, which according to SEQ The amino acid sequence number shown in ID NO:3. 12. A polynucleotide encoding an alpha-amylase variant according to any one of the preceding claims. 13. A nucleic acid construct or expression vector comprising the polynucleotide of claim 12. 14. A host cell comprising the polynucleotide of claim 12, the nucleic acid construct of claim 13, or the expression vector of claim 13. 15. A composition comprising an alpha-amylase variant according to any one of claims 1 to 11 and at least one other ingredient. 16. The composition of claim 15, which is a detergent composition, preferably a laundry detergent composition or an automatic dishwashing (ADW) detergent composition. 17. The composition of claim 16, wherein the composition contains one or more surfactants and/or one or more builders, preferably strongly chelating builders. 18. The composition of any one of claims 15-17, wherein the composition comprises one or more second enzymes different from the alpha-amylase variant of any one of the preceding claims, wherein said The one or more second enzymes are preferably selected from the group consisting of protease, second amylase, lipase, cellulase, laccase, mannanase, pectinase, xylanase and nuclease, and are particularly preferably selected from the group consisting of Proteases, mannanases and lipases, most preferably proteases. 19. A method of producing an alpha-amylase variant, comprising: a. Culturing the host cell of claim 14 under conditions suitable for expression of the alpha-amylase variant; and b. Recovery of the alpha-amylase variant. 20. Use of an alpha-amylase variant according to any one of claims 1 to 11 in cleaning processes, such as laundry or hard surface cleaning.