JP2025526738A - Amylase variants - Google Patents

Abstract

The present invention provides novel amylase enzymes, more specifically genetically engineered amylase enzymes, compositions containing the enzymes, and methods of making and using the enzymes or compositions containing the enzymes.
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Description

The present invention provides novel amylase enzymes. More specifically, it provides genetically engineered amylase enzymes, compositions containing the enzymes, and methods for making and using the enzymes or compositions containing the enzymes.

Enzymes are increasingly being used in a variety of applications as a sustainable alternative to petrochemicals. They are biodegradable and can be catalytically active already at low temperatures, resulting in reduced energy consumption. In particular, in the detergent industry, enzymes are being implemented in cleaning formulations to improve cleaning efficiency and reduce energy consumption in the washing process.

Amylases are enzymes capable of hydrolyzing starch. Amylases are therefore used in the removal of starch stains and are added to detergent compositions for this purpose. In detergent applications, amylases must be stable at high temperatures and/or under the denaturing conditions of detergents and wash solutions. Therefore, there is a need for new amylase enzymes that meet these requirements.

The present invention relates to amylase variants of a parent amylase, said variants comprising:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) amino acid substitutions at one or more, preferably two or more amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO:3; and (iii) at least 60%, preferably at least 91%, but less than 100% sequence identity to any of SEQ ID NOs:1, 3, 4, or SEQ ID NOs:15-41, preferably SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1 (preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1).

In one embodiment, the amylase variant of the invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3; and (iii) any of SEQ ID NOs: 1, 3, 4, or 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5% of SEQ ID NO: 1; At least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

In another embodiment, the present invention relates to a polynucleotide encoding an amylase variant.

Furthermore, the present invention relates to a composition containing an amylase variant. Preferably, the composition containing an amylase variant is a detergent composition.

The present invention further relates to methods for producing the amylase variants and methods for using the amylase variants, particularly for cleaning objects.

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

While the present invention will be described with reference to specific embodiments, this description should not be construed in a limiting sense.

DEFINITIONS Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the art.

Before describing exemplary embodiments of the present invention in detail, definitions important for understanding the present invention are provided. Unless otherwise stated or apparent from the nature of the definition, the definitions apply to all compounds, methods, and uses described herein.

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

In the context of the present invention, the terms "about" and "approximately" refer to a margin of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically refers to a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated numerical value.

Furthermore, the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," etc. in this specification and claims are used to distinguish between similar components and do not necessarily describe sequential or chronological order. It is to be understood that terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein may be performed in orders other than those described or illustrated herein. When the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," "i," "ii," etc. refer to steps in a method, use, or assay, unless otherwise specified in this application, as described herein above or below, there is no time gap or inconsistent time interval between the steps, i.e., the steps may be performed simultaneously, or there may be a time gap of seconds, minutes, hours, days, weeks, months, or even years between such steps.

Throughout this application, various articles are referenced. The entire disclosures of these articles and the references cited therein in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The term "comprises" should be understood to be open-ended. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprises." Hereinafter, when a group is defined as comprising at least a certain number of members, this is meant to encompass groups consisting only of these members.

A "variant" enzyme differs from a "parent" enzyme by certain amino acid modifications, preferably amino acid substitutions at one or more amino acid positions.

When describing variants of the present invention, abbreviations for single amino acids are used according to the generally accepted IUPAC one-letter or three-letter amino acid abbreviations.

"Amino acid modification," as used herein, refers to an amino acid substitution, deletion, or insertion.

A "substitution" is described by adding the original amino acid followed by the number of its position in the amino acid sequence, followed by the amino acid that replaces the original amino acid. For example, a substitution of histidine with alanine at position 120 would be designated "His120Ala" or "H120A." A substitution may also be described by simply naming the resulting amino acid in the variant without specifying the parent amino acid at this position, e.g., "X120A" or "120A" or "Xaa120Ala" or "120Ala."

A "deletion" is described by adding the number of the position in the amino acid sequence after the original amino acid, followed by an *. Thus, a deletion of glycine at position 150 would be designated "Gly150*" or "G150*." Alternatively, the deletion may be indicated by, for example, "deletion of D183 and G184."

An "insertion" is described by adding the number of the position in the amino acid sequence after the original amino acid, followed by the original amino acid and the added amino acid. For example, an insertion of a lysine at position 180 next to a glycine would be designated as "Gly180GlyLys" or "G180GK." If two or more amino acid residues are inserted, e.g., Lys and Ala after Gly180, this can be indicated as "Gly180GlyLysAla" or "G195GKA."

When a substitution and insertion occur at the same position, this may be designated "S99SD+S99A" or, for short, "S99AD." Variants containing multiple alterations are separated by "+", e.g., "Arg170Tyr+Gly195Glu," "R170Y+G195E," or "X170Y+X195E" representing the substitution of arginine and glycine with tyrosine and glutamic acid at positions 170 and 195, respectively. Alternatively, multiple alterations may be separated by spaces or commas, e.g., "R170Y G195E" or "R170Y,G195E," respectively. When different alternative modifications can be introduced at a certain position, the different modifications are separated by commas, e.g., "Arg170Tyr,Glu" and "R170T,E" represent the substitution of arginine at position 170 with tyrosine or glutamic acid, respectively. Alternative substitutions at a particular position may also be indicated as "X120A,G,H," "120A,G,H," "X120A/G/H," or "120A/G/H." Alternatively, the different modifications or optional substitutions may be indicated in square brackets, e.g., "Arg170[Tyr,Gly]" or "Arg170{Tyr,Gly}," or, in summary, "R170[Y,G]" or "R170{Y,G}."

The term "native" (or naturally occurring or wild-type or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide as found in nature (i.e., without any human intervention).

The term "heterologous" (or exogenous or foreign or recombinant or non-natural or non-naturally occurring) polypeptide is defined herein as a polypeptide that is not native to the host cell; a polypeptide native to the host cell in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recombinant DNA techniques to modify the native polypeptide; or a polypeptide native to the host cell in which expression has been quantitatively altered or expression is directed from a genomic location different from that of the native host cell as a result of manipulation of the host cell's DNA by recombinant DNA techniques, e.g., by a stronger promoter. Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-natural or non-naturally occurring) polynucleotide refers to a polynucleotide that is not native to the host cell; a polypeptide native to the host cell in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recombinant DNA techniques to alter native nucleotides; a polynucleotide native to the host cell whose expression is quantitatively altered as a result of recombinant DNA techniques, e.g., manipulation of the polynucleotide's regulatory elements by a stronger promoter; or a polynucleotide that is native to the host cell but is not integrated into its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques. In reference to the relationship between two or more polynucleotide sequences or the relationship between two or more amino acid sequences, the term "heterologous" is used to characterize the fact that the two or more polynucleotide sequences or two or more amino acid sequences do not naturally occur in that particular combination with each other.

For purposes of this invention, "recombinant" (or transgenic) with respect to a cell or organism means that the cell or organism contains a heterologous polynucleotide, which has been introduced by man using genetic techniques. With respect to a polynucleotide, "recombinant" means
Includes all constructs produced by using genetic/recombinant DNA technology in which either (a) the sequence of a polynucleotide or a portion thereof, or (b) one or more gene control sequences operably linked to a polynucleotide, including but not limited to a promoter therefor, or (c) both a) and b) are not located in their wild-type genetic environment or have been modified by man.

"Synthetic" compounds are obtained by in vitro chemical and/or enzymatic synthesis.

Variant polynucleotide and variant polypeptide sequences can be defined by their sequence identity when compared to another sequence. Sequence identity is usually expressed as "% sequence identity" or "% identity." To calculate sequence identity, the first step is to generate a sequence alignment. According to the present invention, a pairwise global alignment is generated, meaning that two sequences are aligned over their entire length, usually by using a mathematical method called an alignment algorithm.

According to the present invention, alignment means that two sequences must be aligned over their entire length using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, pp. 443-453). Preferably, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used for the present invention, using the program's default parameters (polynucleotides: gap open = 10.0, gap extension = 0.5, and matrix = EDNAFULL; polypeptides: gap open = 10.0, gap extension = 0.5, and matrix = EBLOSUM62). After aligning the two sequences, in a second step, an identity value is determined from the resulting alignment. For this purpose, % identity is calculated by dividing the number of identical residues by the length of the alignment region showing each of the sequences of the present invention over its entire length and multiplying by 100: % identity = (identical residues / length of the alignment region showing each of the sequences of the present invention over its entire length) * 100.

To calculate the percent identity of two nucleic acid sequences, the same applies as for calculating the percent identity of two amino acid sequences, with several criteria. For nucleic acid sequences encoding proteins, pairwise alignments should be performed over the entire length of the coding region of the subject sequence from the start codon to the stop codon, excluding introns. Introns present in the other sequence to which the subject sequence is compared should also be removed for pairwise alignments. After aligning the two sequences, in a second step, an identity value is determined from the resulting alignment. The percent identity is calculated as follows: %-identity = (identical residues/length of the aligned region representing the subject sequence from the start codon to the stop codon, excluding introns, over its entire length) * 100.

Furthermore, a preferred alignment program for nucleic acid sequences that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453) is "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program's default parameters (gap open = 10.0, gap extension = 0.5, and matrix = EDNAFULL).

Various polypeptides can be defined by their sequence similarity when compared to another sequence. Sequence similarity is usually provided as "% sequence similarity" or "%-similarity". % sequence similarity considers that a defined set of amino acids share similar properties, e.g., due to their size, hydrophobicity, charge, or other characteristics. Herein, the replacement of one amino acid with a similar amino acid may be referred to as a "conservative mutation". Similar amino acids according to the present invention are defined as follows, which shall also be applied for determining %-similarity according to the present invention, and are according to the BLOSUM62 matrix, as used, for example, by the program "NEEDLE", which is one of the most used amino acid similarity matrices for database searching and sequence alignment:
Amino acid A is similar to amino acid S,
Amino acid D is similar to amino acid E;N,
The amino acid E is similar to the amino acids D; K; and Q.
The amino acid F is similar to the amino acid W; Y,
The amino acid H is similar to the amino acid N;Y,
Amino acid I is similar to amino acids L; M; V;
The amino acid K is similar to the amino acids E; Q; and R;
The amino acid L is similar to the amino acids I; M; and V;
The amino acid M is similar to the amino acids I; L; and V;
The amino acid N is similar to the amino acids D; H; S,
The amino acid Q is similar to the amino acids E; K; and R;
The amino acid R is similar to the amino acids K; Q,
The amino acid S is similar to the amino acids A, N, and T,
The amino acid T is similar to the amino acid S,
Amino acid V is similar to amino acids I; L; M;
The amino acid W is similar to the amino acid F; Y,
The amino acid Y is similar to the amino acids F; H; and W.

To calculate sequence similarity, in a first step, a sequence alignment is generated as described above. After aligning the two sequences, in a second step, a similarity value is determined from the resulting alignment. For this purpose, %-similarity is calculated by dividing the number of identical residues plus similar residues by the length of the alignment region representing the sequence of the present invention over its entire length, and multiplying by 100: %-similarity = [(identical residues + similar residues) / length of the alignment region representing the sequence of the present invention over its entire length] * 100.

For nucleic acids, similar sequences can also be determined by hybridization using the respective stringency conditions. The term "high stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures, for probes at least 100 nucleotides in length, in 5x SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C. The carrier material is finally washed three times for 15 minutes each at 65°C using 2x SSC, 0.2% SDS. The term "very high stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures, for probes at least 100 nucleotides in length, in 5x SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C. The carrier material is finally washed three times for 15 minutes each using 2x SSC, 0.2% SDS at 70°C.

As used herein, a "fragment" or "subsequence" refers to 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 a full-length amino acid sequence, respectively, but still possesses the same or similar activity and/or function. Preferably, a functional fragment is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 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. A functional fragment comprises consecutive nucleotides or amino acids compared to the original nucleic acid or original amino acid sequence, respectively.

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

The term "vector," as used herein, includes any type of construct suitable for carrying an exogenous polynucleotide sequence for transfer to another cell or for stable or transient expression within a given cell. The term "vector," as used herein, encompasses any type of cloning vehicle, such as, but not limited to, plasmids, phagemids, viral vectors (e.g., phages), bacteriophages, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vector specific to a particular host of interest. The exogenous polynucleotide sequence typically includes a coding sequence, which may be referred to herein as a "gene of interest." The gene of interest may include introns and exons, depending on the type of origin or destination host cell.

The terms "introduction of a polynucleotide" or "transformation of a polynucleotide," as referred to herein, encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for transfer. That is, the term "transformation of a polynucleotide," as used herein, is independent of vectors, shuttle systems, or host cells, and it not only refers to polynucleotide transfer methods of transformation as known in the art (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but also encompasses any additional type of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

A polynucleotide that encodes a polypeptide may be "expressed." The term "expression" or "gene expression" refers to the transcription of a gene or genes or a genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA, with or without subsequent translation of the latter into a protein. This process includes transcription of DNA and processing of the resulting mRNA product.

The terms "purification" or "purifying" refer 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, which compartment or phase does not necessarily have to be separated by a physical barrier. Examples of such different compartments are two compartments, i.e., a filtrate and a retentate, separated by a filtration membrane or cloth; examples of such different phases are a pellet and a supernatant, or a cake and a filtrate, respectively. The solution obtained after purifying the protein of interest from the fermentation broth is referred to herein as a "purified enzyme solution."

"Protein formulation" (or "enzyme preparation"), e.g., "protein variant formulation," refers to any non-complex formulation containing a small number of ingredients, which serve the purpose of stabilizing the proteins contained in the protein formulation and/or the protein formulation itself. Preferably, non-complex protein formulations contain proteins at higher concentrations than complex formulations, e.g., detergent compositions. Thus, preferably, non-complex protein formulations are concentrated protein variant formulations. Preferably, non-complex protein formulations contain 2-120 mg/g of active enzyme, while complex formulations, such as detergent compositions, contain 0.002-6 mg/g of active enzyme.

"Enzyme properties" include, but are not limited to, catalytic activity, substrate/cofactor specificity, product specificity, stability over time, thermal stability, pH stability, and chemical stability. "Enzyme activity" or "catalytic activity" refers to the catalytic effect exerted by an enzyme, expressed as units per milligram of enzyme (specific activity) or molecules of substrate converted per minute per molecule of enzyme (molecular activity). Enzyme activity can be specified by the actual function of the enzyme, such as a protease that exerts proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds, a lipase that exerts lipolytic activity by the hydrolytic cleavage of ester bonds, or an amylase activity involved in the hydrolysis of glycosidic bonds in polysaccharides.

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

To determine and quantify changes in the catalytic activity of an enzyme stored or used under specified conditions over time, the "initial enzyme activity" is measured under defined conditions at time zero (100%) and at a later specified time point (x%). By comparing the measured values, the extent of potential decrease in enzyme activity can be determined. The extent of decrease in enzyme activity determines the stability or instability of the enzyme.

An "enzyme inhibitor," as used herein, is a compound that slows or stops enzyme activity. Enzyme inhibitors often also stabilize enzymes in their three-dimensional structure. Thus, enzyme inhibitors typically also act as "enzyme stabilizers."

"pH stability" refers to the ability of an enzyme to exhibit enzymatic activity after exposure to a particular pH value.

The terms "thermostable," "thermostable," or "thermally dependent activity" refer to the ability of an enzyme to exhibit catalytic activity or cleaning performance, preferably in a detergent composition (preferably in Model ES1-C detergent), after exposure to elevated temperatures, preferably 40°C, for 28 days, preferably 56 days, or 92°C for at least 10 minutes.

The terms "detergent stability" or "stability under storage in a detergent composition" refer to the ability of an enzyme to exhibit catalytic activity or cleaning performance after storage in a detergent composition, preferably in a detergent composition (preferably in Model ES1-C detergent), at a temperature of 40°C or 50°C for 28 days, preferably 56 days.

"Improvement Factor" (IF) is preferably the degree of improvement of an enzyme variant in a particular property over its respective parent enzyme. Improvement of an enzyme variant relative to its respective parent enzyme is preferably characterized by an improvement factor (IF) of >1.0. Alternatively, improvement factor can be expressed as a percentage, e.g., an IF of 1.1 is equivalent to 110%.

As used herein, the "washing 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 added to the detergent composition by the enzyme's performance. The term "washing performance" is used herein equally for laundry and hard surface cleaning. Washing performance is compared under appropriate wash conditions. The term "appropriate wash conditions" is used herein to refer to the conditions, particularly the wash temperature, time, wash dynamics, foam concentration, detergent type, and water hardness, that are actually used in homes in the detergent market segment. The term "improved wash performance" is used to indicate that a better end result is obtained in stain removal under appropriate wash conditions, or that less enzyme by weight is required to achieve the same end result relative to the corresponding control conditions.

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

"Detergent composition," "detergent formulation," or "detergent" refers to a composition designed to clean soiled materials. Detergent compositions according to the present invention include detergent compositions for various applications, such as laundry and hard surface cleaning. The term "detergent component" is defined herein to refer to a class of chemicals that may be used in a detergent composition. A typical detergent component is a surfactant. "Surfactant" (used synonymously herein with "surface active agent") refers to an organic chemical that, when added to a liquid, alters the interfacial properties of that liquid. Depending on their ionic charge, surfactants are referred to as nonionic, anionic, cationic, or amphoteric. The term "effective amount of detergent component" includes the amount of a particular component that provides effective stain removal and/or effective cleaning conditions (e.g., pH, temperature, water hardness), the amount of a particular component that effectively provides optical benefits (e.g., optical brightness, dye transfer prevention, color care), and the amount of a particular component that effectively facilitates processing (maintaining physical characteristics during processing, storage, and use; e.g., rheology modifiers, hydrotropes, drying agents).

The term "laundry" or "washing" refers to the process of treating textiles and/or fabrics with a solution containing the detergent composition of the present invention, both in domestic and industrial laundering. The laundering process can be carried out by using a technical device, such as a domestic or industrial washing machine. Alternatively, the laundering process can be done manually.

The term "textile" means any textile material, including yarns (yarns made from natural or synthetic fibers used for knitting or weaving), intermediate products of yarns, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics made from these materials, such as garments, cloth, and other articles. The terms "fabric" (fabrics made by weaving, knitting, or felting fibers) or "garment" (any article of clothing made from textiles) as used herein also include the broader term textile.

The term "fiber" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are of plant (such as flax, jute, and cotton) or animal origin and contain proteins such as collagen, keratin, and fibroin (e.g., silk, sheep's wool, angora, mohair, and cashmere). Examples of fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as Elastofine, or polyamide fibers such as nylon. A fiber may be a single fiber or part of a textile, such as knitwear, woven fabric, or nonwoven fabric.

The term "hard surface cleaning" refers to both domestic and industrial hard surface cleaning and refers to the process of treating a hard surface with a solution containing the detergent composition of the present invention. Hard surfaces can include any hard surface in the home or industry, such as floors, furniture, walls, sanitary ceramics, glass, medical equipment, cutlery, and metal surfaces, including dishware. A specific form of hard surface cleaning is dishwashing, which can be manual dishwashing (MDW) or automatic dishwashing (ADW).

The term "dishwashing" refers to all forms of washing dishes, for example, by manual or automatic dishwashing. Washing dishware includes, but is not limited to, cleaning all forms of ceramics, such as plates, cups, glasses, and bowls; all forms of cutlery, such as spoons, knives, forks, and serving utensils; and ceramics, plastics such as melamine, metals, china, glass, and acrylic.

Cleaning performance is evaluated under relevant cleaning conditions. In this specification, the term "relevant washing conditions" refers to the conditions actually used in a washing machine, automatic dishwasher or manual washing process, in particular the washing temperature, time, washing equipment, foam concentration, detergent type and water hardness.

The term "medical device cleaning" refers to the cleaning process when reusable medical devices are reprocessed. Medical device cleaning methods can be divided into two categories: manual and mechanical/automatic cleaning methods. Manual cleaning is used when a mechanical unit is not available or when the medical device to be cleaned is too fragile or too difficult to clean with a mechanical unit. Mechanical/automatic cleaning methods remove soils and microorganisms through an automatic cleaning and rinsing process, including ultrasonic cleaning and washing.

In the detergent field, the term "stain" is typically used in reference to laundry, e.g., cleaning of textiles, fabrics, or fibers, while the term "soil" is typically used in reference to hard surface cleaning, e.g., cleaning of dishes and cutlery. However, in this specification, the terms "stain" and "soil" will be used interchangeably.

"Metal ion-trapping builders," as used herein, differ from precipitating builders in that when the builder is initially used in an amount sufficient to combine with all of the calcium ions in an aqueous solution at neutral pH and 7° dH hardness (German hardness), a modest amount of precipitate is formed. "Potent builders" are classified as highly efficient chelating agents that can strongly bind divalent cations such as Ca2+, with a stability constant (Log _KCa ) of the logarithm of the cation/chelating agent complex greater than 4, particularly greater than 5, greater than 6, or greater than 7. The stability constants are determined at an ionic strength of 0.1 M and a temperature of 25°C. "Potent metal ion-trapping builders" combine both of the above properties.

The term "low temperature," as used herein, refers to a temperature range below 40°C, preferably 10 to 40°C, more preferably 20 to 40°C, and more preferably 20 to 35°C.

DETAILED DESCRIPTION The present invention provides novel amylase enzymes, more specifically amylase variants, methods of making the amylase variants, compositions comprising the amylase variants, and methods of using the amylase variants or compositions comprising the amylase variants.

Amylase Variants The present invention relates to amylase variants of a parent amylase, said variants comprising:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) amino acid substitutions at one or more, preferably two or more amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO:3; and (iii) at least 60%, preferably at least 91%, but less than 100% sequence identity to any of SEQ ID NOs:1, 3, 4, or SEQ ID NOs:15-41, preferably SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1 (preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1).

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

The amylases and amylase variants of the present invention have "amylolytic activity" or "amylase activity." "Amylolytic activity" or "amylase activity" describes the ability to hydrolyze glucosidic bonds in polysaccharides. Amylase activity can be determined by assays for measuring amylase activity known to those skilled in the art. Examples of assays for measuring amylase activity are the Phadebas assay or the EPS assay ("Infinity Reagent"). In the Phadebas assay, amylase activity is determined by utilizing Phadebas tablets as a substrate (Phadebas Amylase Test, provided by Magle Life Science). Starch is hydrolyzed by the amylase to produce soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of amylase activity. The absorbance measured is directly proportional to the specific activity (activity/mg of pure amylase protein) of the amylase of interest under a given set of conditions.

Alternatively, amylase activity can also be determined by a method utilizing ethylidene-4-nitrophenyl-alpha-D-maltoheptaside (EPS). D-maltoheptaside is a block oligosaccharide that can be cleaved by endo-amylase. After cleavage, the alpha-glucosidase included in the kit digests the substrate, liberating free PNP molecules that have a yellow color, which can be measured by visible spectrophotometry at 405 nm. Kits containing the EPS substrate and alpha-glucosidase are manufactured, for example, by Roche Costum Biotech (cat. no. 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity per mg of enzyme) of the amylase of interest under a given set of conditions.

In one embodiment, the amylase variants of the invention exhibit 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% of the amylolytic activity of the parent amylase. In one embodiment, the amylase variants of the invention exhibit the same or increased amylolytic activity compared to the parent amylase. Preferably, the amylase variants of the invention exhibit increased amylolytic activity compared to the parent amylase.

Preferably, the parent amylase for the amylase variant of the present invention is an amylase having at least 60% sequence identity with any of SEQ ID NOs: 1, 3, or 4, or any of SEQ ID NOs: 15-41, and preferably, the parent amylase for the amylase variant of the present invention is an amylase according to any of SEQ ID NOs: 1, 3, or 4, or any of SEQ ID NOs: 15-41. Most preferably, the parent amylase for the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

The present invention relates to amylase variants comprising amino acid modifications, preferably insertions, deletions, substitutions, or a combination thereof, most preferably substitutions, at two or more positions corresponding to positions selected from the group consisting of 25, 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of the amino acid sequence set forth in SEQ ID NO: 3.

In an alternative manner, amino acid positions 25, 116, 176, 181, 186, 195, 206, 225, 320, and 482 (according to the numbering of SEQ ID NO:3) can be described with respect to the numbering of SEQ ID NO:1. In this alternative manner of describing amino acid positions of the present invention, amino acid positions 25, 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO:3 correspond to amino acid positions 25, 116, 176, 181, 184, 193, 204, 223, 318, and 480 according to the numbering of SEQ ID NO:1.

Preferably, the parent amylase for the amylase variant of the present invention is an amylase having at least 60% sequence identity with any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NOs:15-41, and more preferably, the parent amylase for the amylase variant of the present invention is an amylase according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NOs:15-41. Most preferably, the parent amylase for the amylase variant is an amylase according to SEQ ID NO:1.

Preferably, the present invention relates to an amylase variant comprising two or more amino acid substitutions selected from the group consisting of X25H, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3.

Preferably, the present invention relates to an amylase variant comprising, compared to the parent sequence, two or more amino acid substitutions selected from the group consisting of: X25H, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, X482W and amino acid substitutions at these positions for amino acids similar to the designated amino acid substitutions at these positions according to the numbering of SEQ ID NO: 3, i.e. amino acid substitutions at these positions for amino acids similar to the designated target amino acids, wherein said variant has amylase activity; preferably, the parent amylase for the amylase variant of the present invention is an amylase according to SEQ ID NO: 1 or any amylase having at least 60% sequence identity with SEQ ID NO: 1; most preferably, the parent amylase for the amylase variant is an amylase according to SEQ ID NO: 1;
Amino acid A is similar to amino acid S,
Amino acid D is similar to amino acid E;N,
The amino acid E is similar to the amino acids D; K; and Q;
The amino acid F is similar to the amino acid W; Y,
The amino acid H is similar to the amino acid N;Y,
Amino acid I is similar to amino acids L; M; V;
The amino acid K is similar to the amino acids E; Q; and R;
The amino acid L is similar to the amino acids I; M; and V;
The amino acid M is similar to the amino acids I; L; and V;
The amino acid N is similar to the amino acids D; H; S,
The amino acid Q is similar to the amino acids E; K; R,
The amino acid R is similar to the amino acids K; Q,
The amino acid S is similar to the amino acids A, N, and T,
The amino acid T is similar to the amino acid S,
Amino acid V is similar to amino acids I; L; M;
The amino acid W is similar to the amino acid F; Y,
The amino acid Y is similar to the amino acids F; H; and W.

Preferably, the present invention relates to an amylase variant comprising, compared to the parent sequence, two or more amino acid substitutions selected from the group consisting of X25H, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3, wherein the variant has amylase activity. Preferably, the parent amylase for the amylase variant of the present invention is the amylase according to SEQ ID NO: 1 or any amylase having at least 60% sequence identity with SEQ ID NO: 1, and most preferably, the parent amylase for the amylase variant is the amylase according to SEQ ID NO: 1.

Preferably, in this embodiment, the amino acid residues in the parent amylase at the above-listed positions (i.e., X) correspond to the amino acid residues shown in SEQ ID NO: 1 at each position according to the numbering of SEQ ID NO: 3.

Thus, preferably, the present invention relates to an amylase variant comprising amino acid substitutions at two or more positions corresponding to positions selected from the group consisting of N25, W116, R176, R181, G186, N195, I206, T225, R320, and Y482 according to the numbering of SEQ ID NO: 3.

In a preferred embodiment, the present invention relates to an amylase variant comprising amino acid substitutions at two or more positions corresponding to positions selected from the group consisting of N25H, W116K, R176K, R181T, G186E, N195F, I206Y, T225A, R320K, and Y482W according to the numbering of SEQ ID NO: 3.

The present invention relates to amylase variants comprising amino acid substitutions at one or more positions corresponding to position 25 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482.

Preferably, the present invention relates to an amylase variant comprising the amino acid substitution X25H according to the numbering of SEQ ID NO: 3 and one or more amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W.

Preferably, the present invention relates to an amylase variant comprising, compared to the parent sequence, the amino acid substitutions X25H according to the numbering of SEQ ID NO: 3 and one or more amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W, wherein the variant has amylase activity. Preferably, the parent amylase for the amylase variant of the present invention is the amylase according to SEQ ID NO: 1 or any amylase having at least 60% sequence identity with SEQ ID NO: 1, and most preferably, the parent amylase for the amylase variant is the amylase according to SEQ ID NO: 1.

Preferably, in this embodiment, the amino acid residues in the parent amylase at the above-listed positions (i.e., X) correspond to the amino acid residues shown in SEQ ID NO: 1 at each position according to the numbering of SEQ ID NO: 3.

Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at one or more positions corresponding to amino acid position N25 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of W116, R176, R181, G186, N195, I206, T225, R320, and Y482.

In a preferred embodiment, the present invention relates to an amylase variant comprising the amino acid substitution N25H according to the numbering of SEQ ID NO: 3 and an amino acid substitution at one or more positions corresponding to positions selected from the group consisting of W116K, R176K, R181T, G186E, N195F, I206Y, T225A, R320K, and Y482W.

The present invention relates to amylase variants preferably comprising amino acid substitutions at one or more positions corresponding to positions 25 and 195 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of 116, 176, 181, 186, 206, 225, 320, and 482.

Preferably, the present invention relates to an amylase variant comprising the amino acid substitutions X25H and X195F according to the numbering of SEQ ID NO: 3 and one or more amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X206Y, X225A, X320K, and X482W.

Preferably, in this embodiment, the amino acid residues in the parent amylase at the above-listed positions (i.e., X) correspond to the amino acid residues shown in SEQ ID NO: 1 at each position according to the numbering of SEQ ID NO: 3.

Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at one or more positions corresponding to amino acid positions N25 and N195 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of W116, R176, R181, G186, I206, T225, R320, and Y482.

In a preferred embodiment, the present invention relates to an amylase variant comprising amino acid substitutions at one or more positions corresponding to the amino acid substitutions N25H and N195F according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of W116K, R176K, R181T, G186E, I206Y, T225A, R320K, and Y482W.

The present invention relates to amylase variants comprising amino acid substitutions at one or more positions corresponding to positions 25 and 206 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of 116, 176, 181, 186, 195, 225, 320, and 482.

Preferably, the present invention relates to an amylase variant comprising the amino acid substitutions X25H and X206Y according to the numbering of SEQ ID NO: 3 and one or more amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X225A, X320K, and X482W.

Preferably, in this embodiment, the amino acid residues in the parent amylase at the above-listed positions (i.e., X) correspond to the amino acid residues shown in SEQ ID NO: 1 at each position according to the numbering of SEQ ID NO: 3.

Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at one or more positions corresponding to amino acid positions N25 and I206 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of W116, R176, R181, G186, N195, T225, R320, and Y482.

In a preferred embodiment, the present invention relates to an amylase variant comprising amino acid substitutions at one or more positions corresponding to the amino acid substitutions N25H and I206 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of W116K, R176K, R181T, G186E, N195F, T225A, R320K, and Y482W.

Preferably, the amylase variant comprises an amino acid substitution at one or more of the following amino acid positions (according to the numbering of SEQ ID NO: 3) compared to the parent amylase. Preferably, the parent amylase for the amylase variant is an amylase according to any of SEQ ID NOs: 1, 3, 4, or any of SEQ ID NOs: 15-41, and most preferably, the parent amylase for the amylase variant is an amylase according to SEQ ID NO: 1. Preferably, the amino acid residues of the parent amylase at the listed positions (i.e., X) correspond to the amino acid residues shown in SEQ ID NO: 1 at the respective positions (according to the numbering of SEQ ID NO: 3).

The amylase variant comprises an amino acid substitution at position 25 (according to the numbering of SEQ ID NO: 3), preferably the substitution X25H. Alternatively, the amino acid substitution at position 25 is X25Y or X25D.

Preferably, the amylase variant comprises an amino acid substitution at position 116 (according to the numbering of SEQ ID NO: 3), preferably the substitution X116K.

Preferably, the amylase variant comprises an amino acid substitution at position 176 (according to the numbering of SEQ ID NO: 3), preferably the substitution X176K.

Preferably, the amylase variant comprises an amino acid substitution at position 181 (according to the numbering of SEQ ID NO: 3), preferably the substitution X181T.

Preferably, the amylase variant comprises an amino acid substitution at position 186 (according to the numbering of SEQ ID NO: 3), preferably the substitution X186E.

Preferably, the amylase variant comprises an amino acid substitution at position 195 (according to the numbering of SEQ ID NO: 3), preferably the substitution X195F.

Preferably, the amylase variant comprises an amino acid substitution at position 206 (according to the numbering of SEQ ID NO: 3), preferably the substitution X206Y.

Particularly preferably, the amylase variant comprises an amino acid substitution at position 195 or 206 (according to the numbering of SEQ ID NO: 3), preferably the substitution X195F or X206Y.

Preferably, the amylase variant comprises an amino acid substitution at position 225 (according to the numbering of SEQ ID NO: 3), preferably the substitution X225A.

Preferably, the amylase variant comprises an amino acid substitution at position 320 (according to the numbering of SEQ ID NO: 3), preferably the substitution X320K.

Preferably, the amylase variant comprises an amino acid substitution at position 482 (according to the numbering of SEQ ID NO: 3), preferably the substitution X482W.

The amylase variant of the present invention is
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:3; and (ii) amino acid substitutions at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO:3.

In a further preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:3, and (ii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 181, 225, and 320 according to the numbering of SEQ ID NO:3.

In a further preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:3, and (ii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 176 and 186 according to the numbering of SEQ ID NO:3.

In a preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:3, and (ii) an amino acid substitution at position 195 or 206 according to the numbering of SEQ ID NO:3, preferably at either position 195 or 206.

In a preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206, and (iii) amino acid substitutions at one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 225, 320, and 482 according to the numbering of SEQ ID NO:3.

In a further preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:3, and (ii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 181, 225, and 320 according to the numbering of SEQ ID NO:3.

In a further preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206, and (iii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 181, 225, and 320 according to the numbering of SEQ ID NO:3.

In a further preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206, and (iii) an amino acid substitution at one or more or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 176 and 186 according to the numbering of SEQ ID NO:3.

In a further preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206;
(iii) amino acid substitutions at one or more or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 176 and 186 according to the numbering of SEQ ID NO:3, and (iv) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 181, 225, and 320 according to the numbering of SEQ ID NO:3.

In a particularly preferred embodiment, the amylase variant of the present invention comprises:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206;
(iii) amino acid substitutions at one or more or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 176 and 186 according to the numbering of SEQ ID NO: 3;
(iv) an amino acid substitution at one or more, two or more, three or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 181, 225, and 320 according to the numbering of SEQ ID NO:3, and (v) an amino acid substitution at the amino acid position corresponding to amino acid position 482 according to the numbering of SEQ ID NO:3.

Preferably, the amylase variants of the present invention contain amino acid substitutions at one of the following combinations of amino acid positions (according to the numbering of SEQ ID NO: 3):

From the table above, combinations of amino acid substitutions at amino acid positions including either 195 or 206 are particularly preferred.

Furthermore, from the table above, combinations of amino acid substitutions at amino acid positions containing at least one amino acid position selected from the group consisting of 116, 181, 225, and 320 are particularly preferred.

Even more preferred from the table above are combinations of amino acid substitutions at amino acid positions including either 195 or 206 and at least one amino acid position selected from the group consisting of 116, 181, 225, and 320.

More preferred from the table above are combinations of amino acid substitutions at amino acid positions including either 195 or 206, and at least one of 176 and 186.

Most preferred from the table above are combinations of amino acid substitutions at amino acid positions including either 195 or 206, at least one of 176 and 186, and at least one amino acid position selected from the group consisting of 116, 181, 225, and 320.

The amylase variant of the present invention is
and (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of: (i) the amino acid substitution X25H according to the numbering of SEQ ID NO:3; and (ii) X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO:3.

In a preferred embodiment, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO:3, preferably either X195F or X206Y, and (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO:3.

In a preferred embodiment, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) one or more or all of the amino acid substitutions selected from the group consisting of X176K and X186E according to the numbering of SEQ ID NO:3, or (iii) one or more, two or more, three or more, or all of the amino acid substitutions selected from the group consisting of X116K, X181T, X225A, and X320K according to the numbering of SEQ ID NO:3.

In a preferred embodiment, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) the amino acid substitution X195F or X206Y, preferably either X195F or X206Y;
(iii) one or more or all of the amino acid substitutions selected from the group consisting of X176K and X186E according to the numbering of SEQ ID NO:3, and (iv) one or more, two or more, three or more, or all of the amino acid substitutions selected from the group consisting of X116K, X181T, X225A, and X320K according to the numbering of SEQ ID NO:3.

In a preferred embodiment, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 3, preferably either X195F or X206Y;
(iii) one or more or all of the amino acid substitutions selected from the group consisting of X176K and X186E according to the numbering of SEQ ID NO: 3;
(iv) one or more, two or more, three or more, or all of the amino acid substitutions selected from the group consisting of X116K, X181T, X225A, and X320K according to the numbering of SEQ ID NO: 3, and (v) the amino acid substitution X482W.

Preferably, the amylase variant of the present invention contains one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

Particularly preferred are combinations of amino acid substitutions containing one or more, two or more, three or more, or all of the substitutions selected from the group consisting of X116K, X181T, X225A, and X320K from the table above.

X25H+X176K+X186E,
X25H+X116K+X181T+X225A+X320K,
X25H+X116K+X176K+X181T+X225A+X320K,
X25H+X116K+X181T+X186E+X225A+X320K,
X25H+X116K+X181T+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X225A+X320K+X482W,
Preferred are amylase variants which comprise a combination of mutations selected from the group consisting of: X25H + X116K + X181T + X186E + X225A + X320K + X482W, and X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K + X482W, and preferably further comprise either the substitution X195F or the substitution X206Y, all according to the numbering of SEQ ID NO: 3.

Furthermore, from the table above, combinations of amino acid substitutions containing either the substitution X195F or the substitution X206Y are particularly preferred.

Particularly preferred are combinations of amino acid substitutions containing at least one, preferably two, and more preferably all, of the amino acid substitutions selected from the group consisting of X176K and X186E from the table above.

From the table above, particularly preferred are combinations of amino acid substitutions containing one or more, two or more, three or more, or all of the substitutions selected from the group consisting of X116K, X181T, X225A, and X320K, and either X195F or X206Y.

Preferably, the amylase variant of the present invention contains one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

Preferably, the amylase variant of the present invention contains one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

In particular, combinations of amino acid substitutions containing at least one, at least two, at least three, or all of the amino acid substitutions selected from the group consisting of X116K, X181T, X225A, and X320K from the table above are particularly preferred.

Preferably, the amylase variant of the present invention contains one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

Furthermore, from the table above, a combination of amino acid substitutions containing at least one amino acid substitution selected from the group consisting of X176K and X186E is particularly preferred.

Thus, preferred are combinations of amino acid substitutions containing at least one, at least two, at least three, or all of the amino acid substitutions selected from the group consisting of the substitution X195F, at least one of the substitutions X176K and X186E, and X116K, X181T, X225A, and X320, in particular from the table above.

Most preferably, the amylase variant of the present invention comprises the following combination of amino acid substitutions: X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W (according to the numbering of SEQ ID NO: 3).

Preferably, the amylase variant of the present invention contains one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

Furthermore, from the table above, a combination of amino acid substitutions containing at least one amino acid substitution selected from the group consisting of X176K and X186E is particularly preferred.

Thus, preferred are combinations of amino acid substitutions, particularly those containing at least one, at least two, at least three, or all of the amino acid substitutions selected from the group consisting of the substitution X206Y, at least one of the substitutions X176K and X186E, and X116K, X181T, X225A, and X320 from the table above.

Particularly preferably, the amylase variant of the present invention comprises the following combination of amino acid substitutions: X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K + X482W (according to the numbering of SEQ ID NO: 3).

Preferably, the amylase variant further comprises a deletion of one or more, preferably two or more, amino acids corresponding to positions selected from the group consisting of 181, 182, 183, and 184. Preferably, the amylase variant of the invention having one or more amino acid substitutions as described herein comprises a deletion of one or more amino acids corresponding to positions 183 and 184, preferably a deletion of both amino acids corresponding to positions 183 and 184 (according to the numbering of SEQ ID NO: 3). Preferably, the amylase variant of the invention having one or more amino acid substitutions as described herein comprises a deletion of one or more, preferably two or more, most preferably two amino acids corresponding to positions selected from the group consisting of R181, G182, D183, and G184, preferably D183* and G184*, the numbering being according to the amino acid sequence set forth in SEQ ID NO: 3.

Preferably, the amylase variant comprises a deletion of two amino acids corresponding to positions selected from the group consisting of 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 deletions D183* and G184*, the numbering being according to the amino acid sequence set forth in SEQ ID NO: 3.

Amylase variants according to the present invention having one or more amino acid substitutions as described herein preferably have an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, or at least Both have 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity, but less than 100%.

Preferably, the amylase variant according to the present invention having one or more of the amino acid substitutions described herein preferably has a similar sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 4, or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4, more preferably SEQ ID NO: 1 or 3, and most preferably SEQ ID NO: 1, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, 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 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110. at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

Preferably, amylase variants according to the present invention having one or more of the amino acid substitutions described herein have at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, sequence identity to the amino acid sequence set forth in 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, and most preferably SEQ ID NO: 1.

In one embodiment, the amylase variant according to the present invention having one or more of the amino acid substitutions described herein preferably has a similar sequence to the amino acid sequence set forth in SEQ ID NO: 1 by at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, At least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

In another embodiment, the amylase variant according to the present invention having one or more of the amino acid substitutions described herein preferably has a similar sequence to the amino acid sequence set forth in SEQ ID NO: 3 by at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, or at least 90% of the amino acid sequence set forth in SEQ ID NO: 3. %, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

In another embodiment, the amylase variant according to the present invention having one or more of the amino acid substitutions described herein preferably has a similar sequence to the amino acid sequence set forth in SEQ ID NO:4 by at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, or at least 90%. %, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

In another embodiment, an amylase variant according to the present invention having one or more of the 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 with the amino acid sequence set forth in SEQ ID NO: 15.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 16.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 17.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 18.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 19.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 20.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:21.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:22.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:23.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:24.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:25.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:26.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:27.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:28.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:29.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 30.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 31.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 32.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 33.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 34.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 35.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 36.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 37.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 38.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 39.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 40.

In another embodiment, amylase variants according to the present invention having one or more of the amino acid substitutions described herein 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%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:41.

Preferably, the amylase variant according to the present invention having one or more of the amino acid substitutions described herein has a similar sequence to the amino acid sequence set forth in SEQ ID NO:4 by at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, At least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

Preferably, the amylase variant according to the present invention having one or more of the amino acid substitutions described herein has a similar sequence to the amino acid sequence set forth in SEQ ID NO: 3 by at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, or At least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

More preferably, the amylase variant according to the present invention having one or more of the amino acid substitutions described herein preferably has a similar sequence to the amino acid sequence set forth in SEQ ID NO: 1 by at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, At least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

The amylase variant according to the present invention having amylase activity preferably has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, or at least The sequence identity or similarity is at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, preferably sequence identity.

Preferably, the amylase variant of the present invention having amylase activity comprises:
(a) an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% 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 (i) the coding sequence of SEQ ID NO:1; or (ii) the complement of the polynucleotide set forth 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 further differs from SEQ ID NO:2 solely by the degeneracy of the genetic code); or (e) a fragment of (a), (b), (c), or (d) that has amylase activity.

Preferably, the amylase variants according to the present invention having 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% sequence identity or similarity, preferably sequence identity, to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, but less than 100%. Particularly preferably, amylase variants according to the invention having 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 with the amino acid sequence set forth in SEQ ID NO: 1. More preferably, amylase variants according to the invention having amylase activity have at least 95.0% but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

The structure of alpha-amylases comprises three separate domains, A, B, and C; see, e.g., Machius et al., 1995, J. Mol. Biol. 246:545-559. The alpha-amylase variants described herein may further comprise one or more non-catalytic CBMs (carbohydrate-binding modules, also called carbohydrate-binding domains or, particularly for amylases, starch-binding domains). CBMs can improve enzyme-substrate association. The CBMs are attached to the C-domain. Preferably, amylases of the present invention do not comprise a carbohydrate-binding domain. Preferably, alpha-amylase variants of the present invention consist of only three domains: A, B, and C.

As used herein, the "A and B domains" or "AB domain" of an alpha-amylase correspond to the 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 the amino acids aligned with amino acids 400-485 of SEQ ID NO:3.

Preferably, amylase variants comprising one or more of the amino acid modifications, preferably insertions, deletions, substitutions, or combinations thereof, preferably substitutions, as described above, are hybrid amylases comprising domains thereof from different parent amylases, in particular the AB domain and the C domain. Amylase variants can be generated by replacing the C domain or a portion thereof of one amylase with the C domain or a portion thereof of another amylase. Preferably, the A and B domains of the amylase variants described herein have at least 75% identity, e.g., 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 A and B domains of the amylase of SEQ ID NO: 3.

Preferably, the A and B domains of the amylase variants described herein have at least 75% identity to SEQ ID NO:6, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or at least at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity. This means that the amino acid sequences forming the A and B domains have at least 75% identity to SEQ ID NO:6, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, or at least "Sequence identity" means having at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

Preferably, the C domain of the amylase variants described herein has at least 75% identity, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or at least The C domain has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% identity.

Preferably, the C domain of the amylase variants described herein has at least 75% identity to SEQ ID NO:8, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or at least or has 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% identity.

In one embodiment of the present 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 90%, or at least 91% identity to the amino acid sequence of SEQ ID NO: 6. 9.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but not 100% The amino acid sequence forming the C domain has an identity of less than 75% to SEQ ID NO: 8, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%. , at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% sequence identity.

In one embodiment of the present 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, or at least 89% identity to SEQ ID NO:8. They all have 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity, but less than 100% identity.

In one embodiment of the present 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 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, or at least 90.5% identity to the amino acid sequence of SEQ ID NO:6. , at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity, and the amino acid sequence forming the C domain has 100% identity with SEQ ID NO:8.

In one embodiment, the present invention provides a hybrid derived from at least two different amylases (the hybrid comprises A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, 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 99%, at least 99.5%, at least 99.5%, at least 99.5%, at least 99.5%, at least 99.5%, at least 99.5%, at least 99.5%, at least 10 ... 8.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical; The amino acid sequence of the C domain has at least 75%, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 139%. The present invention relates to a method for producing an amylase variant, comprising the steps of: generating a hybrid of an amylase that is at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical; and introducing into the hybrid one or more amino acid modifications as described herein.

The amylase variants according to the present invention having amylase activity have a similar structure to the amino acid sequence set forth in SEQ ID NO: 1 by at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94% .5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, and comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89.5%, at least 90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95.5%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99.5%, or at least 99.5%, but less than 100%, with the amino acid sequence of SEQ ID NO:6. 9%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical to the amino acid sequence of SEQ ID NO:8, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% identical.

In one embodiment, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with one or more of the amino acid modifications listed herein.

Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with the amino acid substitutions X25H and preferably either X195F or X206Y according to the numbering of SEQ ID NO: 3, and 1 to 7, preferably 2 to 7 or 3 to 7, for example 1, 2, 3, 4, 5, 6, or 7, amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3, and preferably with deletions of one or more, preferably two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183, and 184 according to the numbering of SEQ ID NO: 3 as described herein.

In another embodiment, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with one or more of the amino acid modifications listed above, and has 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8, or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15 It further comprises 3 to 10, 3 to 8, or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 8 conservative amino acid exchanges, and preferably comprises one or more, preferably two or more, amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183, and 184 according to the numbering of SEQ ID NO: 3 as described herein.

Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with 1 to 7, preferably 2 to 7 or 3 to 7, for example 1, 2, 3, 4, 5, 6, or 7, amino acid substitutions selected from the group consisting of the amino acid substitutions X25H and preferably either X195F or X206Y according to the numbering of SEQ ID NO: 3, and X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3, and 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15 It further comprises 2 to 10, 2 to 8, or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 8 conservative amino acid exchanges, preferably comprising one or more, preferably two or more, amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183, and 184 according to the numbering of SEQ ID NO: 3 as described herein.

Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 with 1 to 7, preferably 2 to 7 or 3 to 7, e.g., 1, 2, 3, 4, 5, 6, or 7, amino acid substitutions selected from the group consisting of the amino acid substitutions X25H and preferably either X195F or X206Y according to the numbering of SEQ ID NO: 3, and X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3, and further comprises 1 to 10, preferably 1 to 5, conservative amino acid exchanges.

Conservative amino acid substitutions may occur throughout the entire sequence of the amylase variant. In one embodiment, such mutations do not involve functional domains of the amylase variant. In one embodiment, conservative mutations do not involve the catalytic center of the amylase variant.

In one embodiment, the amylase variants of the present invention preferably exhibit one or more improved properties compared to the parent amylase, preferably compared to the amylase set forth in 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, and most preferably SEQ ID NO: 1.

Preferably, the improved properties are expressed as an improvement factor (IF) of >1.0. Preferably, for improved thermal stability and improved cleaning performance, the improvement is expressed as an improvement factor. Preferably, the improvement factor is 1.1 or greater, 1.2 or greater, 1.3 or greater, 1.4 or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 or greater, 1.9 or greater, or 2.0 or greater. Preferably, the IF for cleaning performance is 1.1 or greater, 1.2 or greater, or 1.3 or greater. Preferably, the IF for thermal stability is 1.1 or greater, 1.2 or greater, 1.3 or greater, 1.5 or greater, or 2.0 or greater.

Alternatively, the improvement in amylase properties is expressed as a percentage improvement compared to the parent amylase. Preferably, the amylase variants of the present invention exhibit improved properties of 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 as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

Alternatively, particularly with regard to improved stability, the improved amylase properties are expressed as residual activity after a 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 a laundry or dishwashing detergent, preferably a laundry detergent). Preferably, the residual activity of the amylase variant is increased compared to the parent amylase. Preferably, the residual activity of the 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 an amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1. Residual activity compared to the parent amylase can also be converted into an improvement by forming a ratio of the residual activity of the variant to the residual activity of the parent amylase.

Preferably, the improved property is one or more properties selected from the group consisting of increased expression, activity, stability, 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, 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+ dependence, cleaning performance, laundry detergent cleaning performance, and/or ADW detergent cleaning performance, and cleaning performance at low temperatures (preferably below 40°C, more preferably below 30°C, and even more preferably below 25°C). Preferably, the improved stability is improved thermal stability, stability in a detergent composition (preferably a laundry or dishwashing detergent composition, preferably a laundry detergent composition), pH stability, oxidative stability, chemical stability, stability under storage conditions, substrate stability, and/or thermal activity. Preferably, the improved property is improved expression, improved solubility, improved thermal stability, improved thermal stability in a detergent composition, improved stability under storage in a detergent composition, and/or improved cleaning performance. Preferably, the improved property is improved thermal stability, preferably improved thermal stability in a detergent composition, improved stability under storage in a detergent composition, and/or improved cleaning performance. Preferably, the improved property is improved thermal stability, improved thermal stability in a detergent composition, improved stability under storage conditions, preferably improved stability under storage in a detergent composition, preferably improved stability under storage in a laundry detergent and/or improved stability under storage in an ADW detergent, improved cleaning performance, preferably improved cleaning performance in a laundry detergent, improved cleaning performance in an ADW detergent, and/or cleaning performance at low temperatures (preferably below 40°C, more preferably below 30°C, even more preferably below 25°C). Preferably, the improved property is improved storage stability and/or improved cleaning performance. Most preferably, the improved performance is improved storage stability, preferably improved stability in a detergent composition, preferably compared to the amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 or relative to the amylase as set forth in SEQ ID NO: 33.

Preferably, the present invention relates to an amylase variant of a parent amylase, said variant comprising:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO: 3;
(iii) any of SEQ ID NOs: 1, 3, 4, or SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% identity but less than 100% sequence identity to SEQ ID NO: 1 (preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1); and (iv) one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the present invention relates to an amylase variant of a parent amylase, said variant comprising:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO: 3;
(iii) comprises any of SEQ ID NOs: 1, 3, 4 or SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 80%, preferably at least 91% identity but less than 100% sequence identity to SEQ ID NO: 1 (preferably the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1); and (iv) said amylase variant invention comprises one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3;
(iii) SEQ ID NO: 1, 3, 4, or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, or at least and (iv) one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO:3, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO:3).

Preferably, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3;
(iii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, or at least 95% of SEQ ID NO: 1 , at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, and (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the present invention relates to an amylase variant of a parent amylase, said variant comprising:
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) an amino acid substitution at either position 195 or 206 according to the numbering of SEQ ID NO:3;
(iii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 225, 320, and 482 according to the numbering of SEQ ID NO: 3;
(iv) SEQ ID NO: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% identity but less than 100% sequence identity to SEQ ID NO: 1, and (v) one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the present invention relates to an amylase variant of a parent amylase, said variant comprising:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) either an amino acid substitution of X195F or X206Y according to the numbering of SEQ ID NO: 3;
(iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3;
(iv) SEQ ID NO: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% identity but less than 100% sequence identity to SEQ ID NO: 1, and (v) one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the present invention relates to an amylase variant of a parent amylase, said variant comprising:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) either an amino acid substitution of X195F or X206Y according to the numbering of SEQ ID NO: 3;
(iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3;
(iv) at least 60%, preferably at least 91% but less than 100% sequence identity with SEQ ID NO: 1; and (v) one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the amylase variant of the present invention comprises:
(i) (according to the numbering of SEQ ID NO: 3):
X25H+X116K+X181T+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K,
X25H+X116K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X181T+X206Y+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,
X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W,
X25H+X116K+X181T+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K,
X25H+X116K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X181T+X195F+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,
a combination of amino acid substitutions selected from the group consisting of: X25H + X116K + X181T + X186E + X195F + X225A + X320K + X482W, and X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W;
(ii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the amylase variant of the present invention comprises:
(i) (according to the numbering of SEQ ID NO: 3):
X25H+X116K+X181T+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K,
X25H+X116K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X181T+X195F+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,
a combination of amino acid substitutions selected from the group consisting of: X25H + X116K + X181T + X186E + X195F + X225A + X320K + X482W, and X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W;
(ii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the amylase variant of the present invention comprises:
(i) (according to the numbering of SEQ ID NO: 3):
X25H+X116K+X181T+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K,
X25H+X116K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X181T+X206Y+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,
a combination of amino acid substitutions selected from the group consisting of: X25H + X116K + X181T + X186E + X206Y + X225A + X320K + X482W, and X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K + X482W;
(ii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Preferably, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3;
(iii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably a homologue of SEQ ID NO: 1 at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, sequence identity; and (iv) one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 3), and (v) the amylase variant preferably exhibits one or more improved properties relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably relative to the parent amylase set forth in SEQ ID NO: 1 or relative to the amylase as set forth in SEQ ID NO: 33, preferably The amylase variant has increased stability, thermostability, storage stability, storage stability in detergent compositions, cleaning performance, cleaning performance in laundry detergents, and/or cleaning performance in dishwashing detergents, preferably the improved property is improved storage stability and/or cleaning performance, preferably improved cleaning performance on laundry, preferably the improved property is improved storage stability, preferably said improved property is expressed as an Improvement Factor (IF) of >1.0, preferably the improvement factor is 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more, preferably the amylase variant has increased stability, thermostability, storage stability in detergent compositions, cleaning performance, cleaning performance in laundry detergents, and/or cleaning performance in dishwashing detergents, preferably the improved property is improved storage stability and/or cleaning performance, preferably improved cleaning performance on laundry, preferably the improved property is improved storage stability, preferably said improved property is expressed as an Improvement Factor (IF) of >1.0, preferably the improvement factor is 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more, preferably the amylase variant has increased stability in detergent compositions, preferably against a reference amylase, preferably against said parent amylase, preferably against SEQ ID NO: 1 or SEQ ID NO: 3, preferably against the parent amylase as set forth in SEQ ID NO: 1 or against the amylase as set forth in SEQ ID NO: 33 and/or preferably the amylase variant exhibits improved washing performance on laundry, preferably at 40°C, preferably relative to an amylase having an amino acid sequence as set forth in SEQ ID NO: 33.

Preferably, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) either an amino acid substitution of X195F or X206Y according to the numbering of SEQ ID NO: 3;
(iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3;
(iv) SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or at least at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity;
(v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3, preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3); and (vi) the amylase variant is preferably set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, relative to said parent amylase. and wherein the amylase variant exhibits one or more improved properties relative to the parent amylase or relative to the amylase as set forth in SEQ ID NO: 33, preferably the amylase variant has increased stability, thermostability, storage stability, storage stability in a detergent composition, cleaning performance, cleaning performance in a laundry detergent, and/or cleaning performance in a dishwashing detergent, preferably the improved property is improved storage stability and/or cleaning performance, preferably improved cleaning performance on laundry, preferably the improved property is improved storage stability, preferably said improved property is expressed as an Improvement Factor (IF) of > 1.0, preferably an improvement Preferably, the amylase variant exhibits improved storage stability in detergent compositions relative to the reference amylase, preferably relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably relative to the parent amylase as set forth in SEQ ID NO: 1 or as set forth in SEQ ID NO: 33, preferably relative to the reference amylase, preferably relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably relative to the parent amylase as set forth in SEQ ID NO: 1 or and/or preferably the amylase variant exhibits improved washing performance, preferably on laundry at 40°C, relative to an amylase as described in No. 33, preferably after 14, 24, 56 or 84 days of storage, preferably 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more, preferably as determined in an ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP); and/or preferably the amylase variant exhibits improved washing performance, preferably on laundry at 40°C, relative to an amylase having the amino acid sequence as set forth in SEQ ID NO: 33.

Particularly preferably, the amylase variant of the present invention is
(i) (according to the numbering of SEQ ID NO: 3):
X25H+X116K+X181T+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K,
X25H+X116K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X181T+X206Y+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,
X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W,
X25H+X116K+X181T+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K,
X25H+X116K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X181T+X195F+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,
a combination of amino acid substitutions selected from the group consisting of: X25H + X116K + X181T + X186E + X195F + X225A + X320K + X482W, and X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W;
(ii) a sequence identical to SEQ ID NO: 1, with at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, or at least or at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, sequence identity; and (iii) a deletion of two amino acids corresponding to positions selected from the group consisting of 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 deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Particularly preferred are:
(i) Combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):
X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W, X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K, or X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K + X482W
(ii) at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity with SEQ ID NO: 1; and (iii) an amylase variant comprising a deletion of two amino acids corresponding to positions selected from the group consisting of 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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3).

Most preferred are amylase variants comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 with the amino acid modifications X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+D183*+G184*, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W according to the numbering of SEQ ID NO: 3.

Most preferred is an amylase variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 with the amino acid modifications X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W according to the numbering of SEQ ID NO: 3.

Nucleic Acid Constructs The present invention also refers to polynucleotides encoding the amylase variants of the present invention. Preferably, the polynucleotides are codon-optimized for improved expression in certain host cells, preferably Bacillus cells.

Therefore, the present invention also provides a method for producing a pharmaceutical composition comprising:
(a) a sequence identical to SEQ ID NO: 2 by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, or at least 91.5% , at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity to a nucleic acid sequence (wherein the nucleic acid encodes an amylase variant having amylase activity as described herein);
(b) a sequence identical to SEQ ID NO: 1 by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least a nucleic acid sequence encoding a polypeptide with 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity (wherein the nucleic acid encodes an amylase variant having amylase activity as described herein);
(c) under high stringency conditions, preferably very high stringency conditions,
(i) the coding sequence of SEQ ID NO:1 or the coding sequence of any amylase variant having amylase activity described herein; or (ii) a polynucleotide that hybridizes to the complement of the polynucleotide set forth in SEQ ID NO:2;
(d) a fragment of (a), (b), or (c), which fragment encodes a polypeptide having amylase activity; or (e) a nucleic acid sequence that is completely complementary to any of (a) through (d).
(f) also refers to nucleic acids, preferably isolated, synthetic and/or recombinant nucleic acids, including polynucleotides that differ from any of the nucleic acid sequences set forth in (a) through (e) solely due to the degeneracy of the genetic code.

The present invention also refers to nucleic acid constructs, preferably expression cassettes, comprising the polynucleotides described herein.

Typically, an expression cassette contains three elements: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. Additional regulatory elements may include transcriptional and translational enhancers. Intron sequences may also be added to the 5' untranslated region (UTR) or within the coding sequence to increase the amount of mature message that accumulates in the cytosol. Expression cassettes can be part of a vector, or can be integrated into the genome of a host cell and replicated along with the host cell's genome. Expression cassettes are typically capable of increasing or decreasing expression.

The present invention also refers to an expression vector comprising a polynucleotide or nucleic acid construct as described herein. The expression vector may be a low copy number vector or a high copy number vector.

As used herein, a vector may provide segments for the transcription and translation of an exogenous polynucleotide upon transformation into a host cell or host cell organelle. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication required for its maintenance and/or replication in a particular cell type, one or more selectable markers, a polyadenylation signal, a multiple cloning site, or other suitable sites for insertion of the exogenous coding sequence. One example is when the vector needs to be maintained in a bacterial cell as an episomal genetic element (e.g., a plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include the f1-ori and colE1.

A vector can replicate in a bacterial host cell without integration into the host cell's genome, for example as a plasmid, or it can integrate some or all of its DNA into the host cell's genome, thereby resulting in replication and expression of that DNA.

A polynucleotide encoding an amylase variant can be introduced into a vector by standard recombinant DNA techniques. Once in the vector, the polynucleotide containing the coding sequence can be suitable for introduction (transformed, transduced, transfected, etc.) into a host cell or host cell organelle. A cloning vector suitable for expression of the polynucleotide sequence in the host cell or host cell organelle can be selected.

Host Cell The present invention also refers to a host cell comprising a polynucleotide encoding an amylase variant as described herein, a nucleic acid construct as described herein, or an expression vector as described herein. In one embodiment of the invention, the vector is used for transformation of the host cell.

Polynucleotides encoding amylase variants as described herein can be transiently or stably introduced into host cells and can be maintained non-integrated, for example, as a plasmid. Stable transformation is typically by chromosomal integration of the nucleic acid comprising the exogenous coding sequence or as an episome (a separate piece of nuclear DNA). Transient transformation is typically by non-chromosomal integration of the nucleic acid comprising the exogenous nucleic acid sequence or as an episome. Alternatively, polynucleotides encoding amylase variants as described herein can be integrated into the host genome.

Introduction of nucleic acids into host cells can be achieved, 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 Spizizen, 1961, Journal of Bacteriology 81:823-829 or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), or by electroporation (see, e.g., Shigekawa and Transformation can be carried out by conjugation (see, e.g., 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 are known in the art for various types of host cells (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580 for E. coli protoplast transformation).

A variety of host cells can be used to express the nucleic acid constructs described herein. Host cells containing the genetic constructs described herein can be obtained by one of the methods described herein for introducing a polynucleotide into such a host cell. The host cells of the present invention do not naturally express the amylase variant. Thus, the host cells are recombinant host cells; the nucleic acid constructs described herein are heterologous to the host cell.

In one embodiment, the host cell is a prokaryotic or eukaryotic organism. In another embodiment, the host cell is a bacterium, archaea, 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 can be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to, the genera Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, and Gluconobacter. In certain embodiments, the bacterial host cell is an Escherichia coli cell. In one embodiment, the host cell is a bacterial cell. In certain embodiments, the host cell is of the genus Escherichia or Bacillus.

Preferably, the bacterial host cell is a Bacillus cell. The bacterial host cell can be any Bacillus cell. Bacillus cells useful in the practice of the present invention include Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lotus, Bacillus arginine ... Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus, Bacillus paralicheniformis Examples of suitable Bacillus species include, but are not limited to, Bacillus paralicheniformis, Bacillus subtilis, and 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 a preferred embodiment, 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.

Alternatively, the bacterial host cell may be Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus reuteri, Escherichia coli, Staphylococcus aureus, or the like. aureus), Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetacidophilum, Corynebacterium callunae, Corynebacterium ammoniagenes, Corynebacterium thermoaminozene thermoaminogenes, Corynebacterium melassecola, Corynebacterium effiziens, Corynebacterium efficiens, Corynebacterium desertii, Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum divarecatum, Pseudomonas putida, Pseudomonas syringae, Streptomyces coelicolor, Streptomyces lividans, Streptomyces albus, Streptomyces avermitilis, Gluconobacter oxydans, Gluconobacter morbifera, Gluconobacter oxydans, Gluconobacter morbifera, morbifer), Gluconobacter thailandicus, Acetobacter aceti, Clostridium acetobutylicum, Clostridium saccharobutylicum, Clostridium beijerinckii, Streptococcus equisimilis, Streptococcus pyogenes, The strain may be Streptococcus pyogenes, Streptococcus uberis, Streptococcus equi subsp., Zooepidemicus, or Basfia succiniciproducens.

Additional alternative host cells include Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Pichia pastoris (also known as Komagataella phaffii), Myceliophthora thermophila, and the like. thermophile (C1), Thermothermophila, Schizosaccharomyces pombe, Trichoderma, preferably Trichoderma reesei, and Saccharomyces, preferably Saccharomyces cerevisiae or Rhizomucor, but are not limited to these.

In another embodiment, the bacterial host cell may further contain modifications, e.g., deletions or disruptions, of other genes that may be undesirable for the production, recovery, or application of the polypeptide of interest.

Another embodiment of the present invention is a method for obtaining an amylase variant of a parent amylase, comprising the steps of:
a) a parent amylase, preferably any of SEQ ID NOs: 1, 3, 4, or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1;
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) introducing amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO: 3;
b) introducing deletions at two or more positions in the parent amylase, preferably selected from positions 181, 182, 183, and 184 according to the numbering of SEQ ID NO: 3;
thereby providing an amylase variant of said parent amylase,
The method comprises the steps of: the variant having at least 60% but less than 100% sequence identity with the amino acid sequence of the polypeptide of SEQ ID NO: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1; and the amylase variant having amylase activity, preferably the amylase variant having improved properties relative to the parent.

A preferred embodiment of the present invention is a method for obtaining an amylase variant of a parent amylase, comprising the steps of:
a) a parent amylase, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) introducing amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO: 3;
b) introducing deletions at two or more positions in the parent amylase, preferably selected from positions 181, 182, 183, and 184 according to the numbering of SEQ ID NO: 3;
thereby providing an amylase variant of said parent amylase,
The method includes the steps of: the variant having at least 80% but less than 100% sequence identity with the amino acid sequence of the polypeptide of SEQ ID NO: 1; the amylase variant having amylase activity; and preferably the amylase variant having improved properties relative to the parent.

Methods for introducing amino acid modifications, e.g., substitutions or deletions, into protein sequences are well known in the art. Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

The resulting amylase variant can be produced on an industrial scale and then purified. Industrial production of enzymes is typically achieved by culturing host cells that express the enzyme (also known as fermentation). Suitable host cells are described herein. A nucleic acid sequence encoding an amylase variant described herein can be transformed into a host cell, which is then cultured under conditions suitable for the host cell to produce the amylase variant. In a preferred embodiment, the amylase variant is purified from the host cell.

Thus, in yet another embodiment, the present invention provides a method for producing an amylase variant, comprising the steps of:
(a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding an amylase variant as described herein by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding an amylase variant as described herein;
(b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and (c) optionally recovering the amylase variant encoded by the polynucleotide.

Cultivation of the host cells is typically carried out in a suitable nutrient medium that allows the recombinant cells to grow and express the desired protein. At the end of fermentation, the fermentation broth is recovered and may be further processed; the fermentation broth contains a liquid fraction and a solid fraction. The enzyme of interest may be further purified from the fermentation broth.

The amylase variants described herein may be secreted (into the liquid fraction of the fermentation broth) or may not be secreted from the microbial cells (and thus be contained within the cells in the fermentation broth). Accordingly, the amylase variants may be recovered from the liquid fraction of the fermentation broth or from the cell lysate. Preferably, the amylase variants are secreted from the cells into the fermentation broth by a secretory signal peptide, preferably added to the end of the amino acid sequence of the amylase variant. Recovery of the amylase variants may be achieved by methods known to those skilled in the art. Suitable methods for recovering proteins from the fermentation broth include, but are not limited to, harvesting, centrifugation, filtration, extraction, and precipitation. If the product of interest precipitates or crystallizes in the fermentation broth or is at least partially bound to particulate matter in the fermentation broth, additional processing steps may be required to release the protein of interest from the biomass or to solubilize the crystals and precipitates of the protein of interest. WO 0043502 A1, WO 2008110498 A1, and WO 2017097869 A1 describe methods for recovering a protein of interest that precipitates and/or crystallizes from a fermentation broth during fermentation. When the desired protein is contained within the cells of the fermentation broth, it may be necessary to release the product of interest from the cells. Release from the cells can be achieved by, for example, but not limited to, cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, sonication, a French press, or a combination thereof.

Amylase variants can be purified from the fermentation broth by methods known in the art. For example, amylase variants can 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 can then be purified by various procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing (IEF)), solubility differences (e.g., ammonium sulfate precipitation), or extraction (see, e.g., *Protein Purification*, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). The purified polypeptide can then be concentrated by procedures known in the art, including, but not limited to, ultrafiltration and evaporation, particularly thin-film evaporation.

Preparation of Amylases Purified solutions of the amylase variants described herein can be further processed to form amylase-containing compositions. Accordingly, compositions comprising the amylase variants described herein and at least one additional component are also claimed herein.

Therefore, the present invention provides
Reference is also made to a method for making a composition comprising mixing: a) an amylase variant as described herein; and b) one or more components described herein.

Furthermore, the present invention therefore provides
Reference is also made to a method for improving amylase stability in a composition comprising the steps of combining: a) an amylase variant as described herein; and b) one or more components described herein.

The composition may be a non-complex formulation, e.g., an amylase variant formulation, or a complex formulation, e.g., a detergent composition.

In one embodiment of the present invention, the amylase variant is formulated as an amylase variant formulation, preferably a concentrated amylase variant formulation. The amylase variant formulation can be either solid or liquid. The protein formulation can be obtained by using techniques known in the art. For example, but not limited to, a solid enzyme formulation can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described, for example, in WO 94/19444 A1 and WO 97/43482 A1.

Preferably, the enzyme formulation contains the enzyme of the present invention in an amount of 2 to 120 mg of active enzyme per gram of enzyme formulation, preferably 6 to 80 mg/g, 6 to 60 mg/g, or 10 to 40 mg/g.

In one embodiment, the amylase variant formulation, particularly a liquid enzyme formulation, additionally comprises one or more additional compounds selected from the group consisting of solvents, salts, pH adjusters, preservatives, enzyme stabilizers, and thickeners. Preferably, the amylase variant formulation lacks surfactants. The solvent may be water and/or an organic solvent. Aqueous amylase variant formulations of the present invention may comprise water in an amount greater than about 50 wt%, greater than about 60 wt%, greater than about 70 wt%, or greater than about 80 wt%, based on the total weight of the enzyme formulation. Amylase variant containing formulations of the present invention may comprise an organic solvent in an amount greater than about 30 wt%, greater than 40 wt%, greater than about 50 wt%, greater than about 60 wt%, greater than about 70 wt%, or greater than about 80 wt%, based on the total weight of the enzyme formulation. The organic solvent may be a water-miscible solvent. The organic solvent may be one or more selected from the group consisting of glycerol, propanediol, polypropylene glycol, and polyethylene glycol.

In one embodiment, the amylase variant formulation includes at least one preservative. Preferably, preservative refers to a substance added to a liquid composition for preservation purposes; more preferably, the term preservative excludes compounds known to have a preservative function that are contained in the liquid composition formed during the production process. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, formic acid in its acid form or as a salt thereof, and 4,4'-dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention includes at least one preservative in an amount of less than 10 ppm, such as an amount ranging from 2 ppm to 5 wt. % based on the total weight of the liquid composition. Alternatively, the amylase variant formulation does not include a preservative, meaning that the preservative is included in an amount of less than 1 ppm, preferably 0 ppm.

Preferably, the amylase variant formulations include an enzyme stabilization system. Preferably, the amylase variant formulations described herein include from about 0.001% to about 10%, from about 0.005% to about 8%, or from about 0.01% to about 6% of an enzyme stabilization system by weight of the composition. The enzyme stabilization system can be any stabilization system compatible with the amylase.

Preferably, the enzyme stabilization system comprises at least one compound selected from the group consisting of a polyol (preferably 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol or sorbitol), an inorganic salt (preferably CaCl2, MgCl2 or NaCl), a short-chain (preferably _C1 - _C3 ) carboxylic acid or salt thereof (preferably formic acid, formate (preferably sodium formate), acetic acid, acetate or lactate), borate, boric acid, a boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), a peptide aldehyde (preferably Z-VAL-H or Z-GAY-H), a peptide acetal, and a peptide aldehyde bisulfite adduct. Preferably, the enzyme stabilization system comprises a combination of at least two compounds selected from the group consisting of salts, polyols, and short-chain carboxylic acids, and preferably one or more compounds selected from the group consisting of borate, boric acid, boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), peptide aldehyde, peptide acetal, and peptide aldehyde hydrosulfite adduct. In a preferred embodiment, the stabilization system comprises a protease inhibitor, preferably selected from borate, boric acid, boronic acid (preferably 4-FPBA), peptide aldehyde (preferably a peptide aldehyde such as Z-VAL-H or Z-GAY-H), peptide acetal, and peptide aldehyde hydrosulfite adduct, such that no proteases are present; preferably the protease inhibitor is a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H. In one embodiment, the stabilization system does not comprise a protease inhibitor. Preferably, the composition does not comprise boron. Preferably, the amylase variant formulation comprises a calcium salt, preferably calcium chloride.

Preferably, the liquid amylase variant formulation comprises or consists of an amylase variant, a solvent, an enzyme stabilization system, and optionally a preservative and optionally a second enzyme different from the amylase variant as described herein. Preferably, the amylase variant formulation lacks a surfactant.

Accordingly, the present invention provides a method for producing an amylase variant preparation, preferably a concentrated amylase variant preparation, comprising the steps of:
Reference is also made to a method comprising the step of mixing a) an amylase variant as described herein; and b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the amylase variant.

Further, therefore, the present invention provides a method for improving amylase stability in a formulation, comprising:
Reference is also made to a method comprising the step of mixing a) an amylase variant as described herein; and b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the amylase variant.

Second Enzyme In another embodiment, the composition comprising an amylase variant as described herein further comprises one or more second enzymes different from the amylase variant. Preferably, the second enzyme is a protease, a second amylase, a lipase, a cellulase, a mannanase, a hemicellulase, a phospholipase, an esterase, a pectinase, a lactase, a peroxidase, a xylanase, a cutinase, a pectate lyase, a keratinase, a reductase, an oxidase, a phenoloxidase, a lipoxygenase, a ligninase, a pullulanase, a tannase, a pentosanase, a malanase, a beta-glucanase, an arabinosidase, a hyaluronidase, a chondroitinase, a laccase, a nuclease, a DNase, a phosphodiesterase, a phytase, a caprylic/capsule enzyme, a caprylic/capsule enzyme, a caprylic/capsule enzyme, a caprylic/capsule enzyme, a caprylic/capsule enzyme, a capsular ... The second enzyme is preferably selected from the group consisting of ribohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, ribonuclease, transglutaminase, and dispersin, and combinations of at least two of the foregoing types. More preferably, the second enzyme is selected from the group consisting of protease, lipase, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase, and cutinase, and combinations of at least two of the foregoing types. Most preferably, the second enzyme is a protease, preferably a subtilisin protease.

The compositions of the present invention may contain one enzyme or more than one enzyme of different types, for example, an amylase and a protease, or more than one enzyme of the same type, for example, two or more different proteases, or a mixture thereof, for example, an amylase and two different proteases.

Proteases Proteases are active proteins that exert "protease activity" or "proteolytic activity." Proteolytic activity relates to the rate of degradation of a protein by a protease or proteolytic enzyme over a given period of time.

Preferably, the second enzyme different from the amylase variant is a protease having at least 40-100% 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 NOs: 10, 11, 12, 13, or 14, preferably SEQ ID NO: 10.

Preferably, the protease used in combination with the 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 includes at least one 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 (according to the BPN numbering) and have proteolytic activity. In one embodiment, such proteases are not mutated at positions Asp32, His64, and Ser221 (according to the BPN′ numbering). Preferably, the protease used in combination with the 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 is further characterized by having 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), at position 101 (according to BPN numbering), and has proteolytic activity. Most preferred are proteases having at least 80%, but less than 100%, sequence identity to SEQ ID NO: 10, and characterized by having the amino acid glutamic acid (E) at position 101 (according to BPN numbering), and having proteolytic activity. The protease can comprise an amino acid substitution 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 (according to BPN numbering) and have proteolytic activity. In one embodiment, the protease comprises one or more additional substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine or arginine at position 63 (63A, 63T or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid or glycine at position 217 (217D, 217E or 217G), or (i) a combination of two or more amino acids from (a) to (h). A suitable protease may be at least 80% identical to SEQ ID NO: 10 and is characterized by comprising one amino acid (according to (a) to (h)) or a combination according to (i) together with amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (according to BPN numbering), and has proteolytic activity. In one embodiment, the protease is at least 80% identical to SEQ ID NO: 10 and is characterized by comprising the mutations (according to BPN numbering) 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 further characterized by comprising S3T+V4I+S9R+A15T+V68A+D99S+R101S+A103S+I104V+N218D (according to BPN numbering), and has proteolytic activity. In another embodiment, the protease is at least 80% identical to SEQ ID NO: 10 and comprises R101E and S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 The antibody may have an amino acid sequence further characterized by including one or more substitutions selected from the group consisting of L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D, and L262N,Q,D (according to BPN numbering), and have proteolytic activity.

Lipases "Lipase", "lipolytic enzyme", "lipid esterase" all refer to enzymes in EC class 3.1.1 ("carboxyl ester hydrolases"). Lipase means 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 those of bacterial or fungal origin.

In one aspect of the invention, a suitable lipase (component (b)) is selected from: a lipase from the genus Humicola (synonym Thermomyces) as described in EP 258068, EP 305216, WO 92/05249 and WO 2009/109500, for example from H. lanuginosa (T. lanuginosus) or H. lanuginosa as described in WO 96/13580. lipases from H. insolens; lipases from Rhizomucor miehei as described in WO 92/05249; lipases from strains of Pseudomonas (some of which have now been renamed Burkholderia), such as P. alcaligenes or P. pseudoalcaligenes (EP 218272, WO 94/25578, WO 95/30744, WO 95/35381, WO 96/00292), P. Examples of P. cepacia include P. cepacia (EP 331376), P. stutzeri (GB 1372034), P. fluorescens, Pseudomonas sp. strain SD705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), Pseudomonas mendocina (WO 95/14783), P. lipases from P. glumae (WO 95/35381, WO 96/00292); lipases from Streptomyces griseus (WO 2011/150157) and S. Lipases from S. pristinaespiralis (WO 2012/137147), GDSL-type Streptomyces lipases (WO 2010/065455); lipases from Thermobifida fusca as disclosed in WO 2011/084412; Geobacillus stearothermophilus as disclosed in WO 2011/084417 lipases derived from Bacillus stearothermophilus; for example, Bacillus lipases as disclosed in WO 00/60063, B. subtilis as disclosed in Dartois et al. (1992), Biochemica et Biophysica Acta, 1131, 253-360 or WO 2011/084599, B. stearothermophilus (JP 64-074992 A) or B. Lipases from B. pumilus (WO 91/16422); lipases from Candida antarctica as disclosed in WO 94/01541; cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536; WO 88/09367); cutinase from Magnaporthe grisea (U.S. Pat. No. 5,389,536); cutinase from Fusarium grisea (WO 2010/107560); cutinase from Fusarium solani pisi as disclosed in WO 90/09446, WO 00/34450 and WO 01/92502; and cutinase 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 97/07202, WO 00/60063, WO 2007/087508, EP 407225 and EP 260105.

Commercially available lipase enzymes include, but are not limited to, those sold under the trade names Lipolase™, Lipex™, Lipolex™, and Lipoclean™ (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 class 3.1.1.3). The fungal triacylglycerol lipase may be selected from Thermomyces lanuginosus lipases. In one embodiment, the Thermomyces lanuginosa lipase is selected from triacylglycerol lipases according to amino acids 1-269 of SEQ ID NO: 2 in U.S. Pat. No. 5,869,438 and variants thereof having lipolytic activity.

The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity that is 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% identical when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 in U.S. Pat. No. 5,869,438.

The Thermomyces lanuginosa lipase may be selected from variants with lipolytic activity that contain only conservative mutations that do not belong to the functional domain of amino acids 1-269 of SEQ ID NO:2 of U.S. Patent No. 5,869,438. The lipase variant of this embodiment with lipolytic activity may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of U.S. Patent No. 5,869,438.

The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity that contain the following amino acid substitutions: T231R and N233R when compared to amino acids 1-269 of SEQ ID NO:2 in U.S. Pat. No. 5,869,438. The lipase variant may further contain one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, and P256K when compared to amino acids 1-269 of SEQ ID NO:2 in U.S. Pat. No. 5,869,438.

The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity that contain the amino acid substitutions T231R, N233R, Q4V, V60S, A150G, L227G, and P256K within the polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 in U.S. Pat. No. 5,869,438, and are at least 95%, at least 96%, or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 in U.S. Pat. No. 5,869,438.

The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity that contain the amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID NO:2 of U.S. Patent No. 5,869,438, and are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of U.S. Patent No. 5,869,438.

The Thermomyces lanuginosa lipase may be a variant of amino acids 1 to 269 of SEQ ID NO: 2 in U.S. Patent No. 5,869,438 that has lipolytic activity, and the variant of amino acids 1 to 269 of SEQ ID NO: 2 in U.S. Patent No. 5,869,438 is characterized in that it contains the amino acid substitutions T231R and N233R.

The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity that preferably contain at least one, preferably two or more, and more preferably all of the following substitutions within the polypeptide sequence of amino acids 1 to 269 of SEQ ID NO: 1 of WO 2015/010009: N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, and T189Q, and is at least 95%, at least 96%, or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1 to 269 of SEQ ID NO: 1 of WO 2015/010009.

Amylases different from the amylases described herein include those of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase is selected from the group of alpha-amylases (EC 3.2.1.1).

The amylase may be derived from Bacillus licheniformis having SEQ ID NO: 2 as described in WO 95/10603, and at least 95% variants thereof. Suitable variants are described in WO 95/10603 and contain one or more substitutions at 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, and have amylolytic activity. The variant is described in SEQ ID NO: 4 of WO 94/02597, WO 94/018314, WO 97/043424 and WO 99/019467.

The amylase may also be derived from B. stearothermophilus having SEQ ID NO: 6 as disclosed in WO 02/10355, or may be an amylase optionally having a C-terminal truncation beyond the wild-type sequence. Preferred variants of SEQ ID NO: 6 include those containing deletions at positions 179 and/or 181 and/or 182 and/or substitutions at position 193.

The amylase may further be derived from Bacillus sp. 707 having SEQ ID NO: 6 as disclosed in WO 99/19467, and at least 95% variants thereof. Preferred variants of SEQ ID NO: 6 have substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269.

The amylase may further be derived 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.

The amylase may further be derived from Bacillus sp. DSM 12649 having SEQ ID NO: 4 as disclosed in WO 00/22103, and at least 95% variants thereof.

The amylase may further be derived from Bacillus sp. A 7-7 (DSM 12368) having an amino acid sequence at least 95% identical to SEQ ID NO:2 as disclosed in WO 02/10356, in particular spanning the region of amino acids 32 to 516 according to SEQ ID NO:2.

The amylase may further be derived from Bacillus strain TS-23 having SEQ ID NO:2 as disclosed in WO 2009/061380 and variants thereof.

The amylase may further be derived from Cytophaga sp. having SEQ ID NO: 1 as disclosed in WO 2013/184577, and at least 95% variants thereof.

The amylase may further be derived from Bacillus megaterium DSM 90 having SEQ ID NO: 1 as disclosed in WO 2010/104675, and at least 95% variants thereof.

The amylase may further be derived from a Bacillus sp. comprising amino acids 1-485 of SEQ ID NO:2 as described in WO 00/60060, and at least 95% variants thereof.

The amylase may further be derived from Bacillus amyloliquefaciens or a variant thereof, preferably selected from the amylase according to SEQ ID NO: 3 as described in WO 2016/092009.

The amylase may have SEQ ID NO: 12 as described in WO 2006/002643 or an amylase variant thereof comprising the substitutions Y295F and M202LITV within SEQ ID NO: 12.

The amylase may have SEQ ID NO: 6 as described in WO 2011/098531, or an amylase variant comprising a substitution at one or more positions within SEQ ID NO: 6 selected from the group consisting of 193 [G, A, S, T or M], 195 [F, W, Y, L, I or V], 197 [F, W, Y, L, I 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].

The amylase may have SEQ ID NO: 1 as described in WO 2013/001078 or an amylase variant containing modifications at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 within SEQ ID NO: 1.

The amylase may have SEQ ID NO:2 as described in WO 2013/001087, or an amylase variant comprising a deletion of positions 181+182, or 182+183, or 183+184 within SEQ ID NO:2, optionally including one or more modifications at any of the positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476, and G477 within SEQ ID NO:2.

The amylase may be a hybrid alpha-amylase derived from the amylases described above, for example, as described in WO 2006/066594.

The hybrid amylase may be according to WO 2014/183920, having A and B domains 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.

The hybrid amylase may be according to WO2014/183921 having A and B domains having at least 75% identity to SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:32 and SEQ ID NO:39 as disclosed in WO2014/183921 and a C domain having at least 90% identity to SEQ ID NO:6 of WO2014/183921, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO:30 as disclosed in WO2014/183921 and has amylolytic activity;
The hybrid amylase may be according to WO2021/032881, comprising A and B domains derived from an alpha-amylase derived from Bacillus sp. A 7-7 (DSM 12368) and a C domain derived from an alpha-amylase derived from Bacillus cereus; preferably, the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44 (both sequences as disclosed in WO2021/032881); more preferably, the hybrid amylase is at least 80% identical to SEQ ID NO: 54 as disclosed in WO2021/032881.

In one embodiment, the at least one amylase is selected from the group consisting of amylases sold under the trade names Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™, Amplify™, and Amplify Prime™ (Novozymes). A/S) and commercially available amylases, including, but not limited to, products sold under the names Rapidase™, Purastar™, Powerase™, Effectenz™ (M100 from DuPont), Preferenz™ (S1000, S110 and F1000; DuPont), PrimaGreen™ (ALL; DuPont), and Optisize™ (DuPont).

Mannanase A "mannanase" as described herein is an enzyme selected from the group of mannan-degrading enzymes. The mannan-degrading enzymes may be selected from β-mannosidases (EC 3.2.1.25), endo-1,4-β-mannosidases (EC 3.2.1.78) and 1,4-β-mannobiosidases (EC 3.2.1.100). Preferably, the mannan-degrading enzyme is selected from the group of endo-1,4-β-mannosidases (EC 3.2.1.78), a group of enzymes that may be referred to herein as endo-β-1,4-D-mannanases, β-mannanases or mannanases.

The mannanase may be selected from alkaline mannanases of family 5 or 26 (i.e., GH5 or GH26). The term "alkaline mannanase" is intended to encompass mannanases having an enzymatic activity of at least 40% of its maximum activity at a given pH in the range of 7 to 12, preferably 7.5 to 10.5.

Mannanase is described in Japanese Patent Application Laid-Open No. 0304706 (beta-mannanase derived from Bacillus sp.), Japanese Patent Application Laid-Open No. 63056289 (alkali- and thermostable beta-mannanase), Japanese Patent Application Laid-Open No. 63036774 (Bacillus microorganism FERM P-8856 producing beta-mannanase and beta-mannosidase at alkaline pH), Japanese Patent Application Laid-Open No. 08051975 (alkaline beta-mannanase derived from alkaliphilic Bacillus sp. AM-001), and International Publication No. 97/11164 (Bacillus amyloliquefaciens amyloliquefaciens], WO 91/18974 [mannanases active at extremes of pH and temperature], WO 97/11164 [mannanases from Bacillus amyloliquefaciens], WO 2014/100018 [mannanases from Bacillus circulans or Bacillus lentus], The mannanase may be selected from mannanases derived from Bacillus organisms, such as those described in endo-(3-mannanase 1 (Bleman 1; see U.S. Pat. No. 5,476,775) cloned from Bacillus lentus strain CMG1240. Suitable mannanases are described in WO 99/064619.

The mannanase may be selected from mannanases derived from Trichoderma organisms, such as those disclosed in WO 93/24622.

The mannanase may be selected from commercially available mannanases such as Mannaway® (Novozymes A/S) or Preferenz® (M100) (DuPont).

A "cellulase" is an enzyme capable of hydrolyzing cellulose. Cellulases can be selected from cellobiohydrolases (1,4-P-D-glucan cellobiohydrolases, EC 3.2.1.91), endo-ss-1,4-glucanases (EC 3.2.1.4), and ss-glucosidases (EC 3.2.1.21). Endoglucanases of EC class 3.2.1.4 can be named endoglucanases, endo-1,4-ss-D-glucan 4-glucanohydrolases, endo-1,4-beta-glucanases, carboxymethylcellulases, and beta-1,4-glucanases.

Endoglucanases can be classified by amino acid sequence similarity under Family 5, which contains over 20 endoglucanases of EC 3.2.1.4 (Henrissat, B. UniProt accessed 10/26/2011). T.-M. Enveri, "Microbial Cellulases," W. M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, pp. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, p. 200-391 (edited by Wood, W.A. and Kellogg, S.T.); Beguin, P. , “Molecular Biology of Cellulose Degradation”, Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219248; Begun, P. and Aubert, J-P. , “The biological degradation of cellulose”, FEMS Microbiology Reviews 13 (1994) p. 25-58; Henrissat, B. See also "Cellulases and their interaction with cellulose," Cellulose (1994), Vol. 1, pp. 169-196.

Preferably, the at least one cellulase is selected from glycosyl hydrolase family 7 (GH7, pfam00840), which is preferably selected from endoglucanases (EC 3.2.1.4).

Preferably, alkaline cellulase is used, and "alkaline cellulase" is intended to include cellulases that have enzymatic activity at a given pH in the range of 7 to 12, preferably 7.5 to 10.5.

In one embodiment, the cellulase is selected from cellulases that contain a cellulose-binding domain. In another embodiment, the cellulase contains a catalytic domain but does not contain a cellulose-binding domain.

In one embodiment, the at least one endoglucanase in the EC class 3.2.1.4 is
Bacillus species such as Bacillus sp. CBS 670.93 and CBS 669.93
Melanocarpus, such as Melanocarpus albomyces, as disclosed in WO 97/14804
Clostridium, for example Clostridium thermocellum
Humicola species such as Humicola insolens (DSM 1800) as disclosed in EP 0 495 257, EP 0 531 315, EP 0 531 372, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,776,757, WO 89/09259, WO 91/17244, WO 94/07998 (the human variant of sequence 43 kd shown in Figure 1), WO 95/24471, WO 96/11262 and WO 98/12307.
Fusarium species such as Fusarium oxysporum, for example strain J79 (DSM 2672), as disclosed in EP 0 495 257, EP 0 531 315, EP 0 531 372, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,776,757, WO 89/09259, WO 91/17244, WO 95/24471 and WO 96/11262.
Thielavia, such as Thielavia terrestris or Myceliophthora thermophila strain CBS 11765 as disclosed in EP 0 531 315, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,776,757, WO 89/09259, WO 91/17244, WO 95/24471, WO 96/11262, WO 96/29397 (SEQ ID NO: 9 and variants thereof), and WO 98/12307.
Trichoderma species such as Trichoderma reesei, Trichoderma longibrachiatum or Trichoderma harzianum as disclosed in EP 1 305 432, EP 1 240 525, WO 92/06165, WO 94/21801, WO 94/26880, WO 95/02043, WO 95/24471 and WO 02/099091.
Aspergillus, such as Aspergillus aculeatus, as disclosed in WO 93/17244
Erwinia species, such as Erwinia chrysanthermi, as described by M. H. Boyer et al. in European Journal of Biochemistry, vol. 162, pp. 311-316 (1987)
Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, as disclosed in WO 96/11262 and WO 96/29397 (SEQ ID NO: 5 and variants thereof), Acremonium species such as Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, and Acremonium furatum
Cellvibrio mixtus DSM 11683, Cellvibrio mixtus DSM 11684, Cellvibrio mixtus DSM 11685, Cellvibrio mixtus ACM 2601, Cellvibrio mixtus DSM 1523, and Cellvibrio gilvus DSM as disclosed in WO 98/08940 Cellvibrio such as 11686
- Derived from the genus Cephalosporium, such as Cephalosporium sp. RYM-202 as disclosed in WO 96/11262.

Suitable cellulases also include variants of the above-described cellulases that have cellulolytic activity. In one embodiment, the cellulase variants include variants that have at least 40-100% identity when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above. In one embodiment, the cellulase variants that have cellulolytic activity are 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% similar and/or identical to the full-length polypeptide sequence of the parent enzyme as disclosed above.

The cellulase may be Humicola insolens DSM 1800 cellulase complex, which has endoglucanase, cellobiohydrolase, and beta-glucosidase activity.

The cellulase may preferably be Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4) having a polypeptide sequence according to positions 21 to 435 of SEQ ID NO: 2 as disclosed in WO 2018/224544, or a variant at least 95% identical thereto.

The cellulase may preferably be a Humicola insolens endoglucanase (EC 3.2.1.4) having a 43 kDa conforming to the polypeptide sequence as disclosed in Figure 1a of WO 94/07998 ("43 kD hum"), or a variant thereof preferably at least 90% identical thereto, preferably one disclosed in WO 94/07998.

The cellulase may be a Bacillus sp. cellulase (EC 3.2.1.4) selected from polypeptides at least 80% similar and/or identical to the amino acid sequence of positions 1 to 773 of SEQ ID NO: 2 of WO 2004/053039, or catalytically active fragments thereof. In one embodiment, the cellulase is a mature polypeptide at least 95% identical to SEQ ID NO: 1 of WO 2018/224544.

The cellulase may be a Thielavia terrestris cellulase (EC 3.2.1.4) having a polypeptide at least 80% similar and/or identical to the amino acid sequence from positions 1 to 299 of SEQ ID NO: 4 of WO 2004/053039, or a catalytically active fragment thereof. In one embodiment, the cellulase is a mature polypeptide at least 95% identical to SEQ ID NO: 4 of WO 2018/224544.

The cellulase may preferably be a mature Sordaria fimicola cellulase having a polypeptide sequence according to SEQ ID NO: 5 of WO 2018/224544 or a variant at least 95% identical thereto.

The at least one cellulase may be selected from Renozyme®, Celluzyme®, Celluclean®, Endolase®, and Carezyme® (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor Int. Inc.), and KAC-500(B)™ (Kao Corporation).

Detergent Compositions In one embodiment, the present invention relates to the use of an amylase variant in a detergent composition. Accordingly, the present invention also relates to a detergent composition comprising an amylase variant as described herein and one or more detergent components.

Therefore, the present invention provides
Reference is also made to a method for making a detergent composition comprising the steps of mixing: a) an amylase variant as described herein; and b) one or more detergent components described herein.

Further, therefore, the present invention provides a method for making a detergent composition with improved amylase stability and/or providing a detergent composition with improved cleaning performance, comprising the steps of:
c) an amylase variant as described herein; and d) one or more detergent components as described herein.

The one or more detergent components may be selected from the group consisting of additional enzymes different from the amylase variant, enzyme stabilizing systems, surfactants, antifoaming agents, builders, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, drying agents, brighteners, buffers, preservatives, anticorrosion additives, dyes, and fragrances.

Preferably, at least one component of the detergent is selected from the group consisting of surfactants, builders, polymers, preservatives, and a second enzyme different from the amylase variant.

Preferably, one or more of the detergent components, preferably surfactants and/or builders, are biodegradable and/or bio-based.

A detergent component may have more than one function in the final application of the detergent composition; therefore, any detergent component referred to herein in the context of a particular function may also have another function in the final application of the detergent composition. The function of a particular detergent component in the final application of the detergent composition typically depends on its amount in the detergent composition, i.e., the effective amount of the detergent component. Detergent components vary in type and/or amount in a detergent composition depending on the desired application, such as washing white fabrics, colored fabrics, and wool. The components selected further depend on the physical form of the detergent composition (liquid, solid, gel, provided in a pouch or as a tablet, etc.). For example, the components selected for a laundry formulation further depend on the wash temperature used, washing machine configuration (vertical axis machines versus horizontal axis machines), aspects such as water consumption per wash cycle, and local practices, which themselves relate to geographic characteristics such as average water hardness.

In one embodiment, the detergent composition is a blend of three or more detergent components, at least one component effective in stain removal, at least one component effective in providing optimal cleaning conditions, and at least one component 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, which may be compressed into tablets.

The detergent composition may be a unit dose or multi-unit 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 or dishwashing detergent composition suitable for home care and/or industrial and institutional (I&I) cleaning. Both laundry and dishwashing compositions may be in the form of hand wash or automatic cleaning compositions. Preferably, the dishwashing composition is automatic dishwashing (ADW).

The detergent pouch can be of any form, shape, and material suitable for holding the composition, e.g., preventing release of the composition from the pouch prior to contact with water. The pouch is made from a water-soluble film that encloses an interior volume. The interior volume can be divided into pouch compartments. Preferred films are polymeric materials, preferably polymers that can be formed into films or sheets, such as polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). The pouch can contain solid laundry detergent compositions or portions of components and/or liquid detergent compositions or portions of components separated by a water-soluble film. The compartment for the liquid components can be different in composition from the compartment containing the solids (see, e.g., U.S. Patent Application Publication No. 2009/0011970).

Preferably, the amylase variant according to the present invention may be added to the detergent composition in an amount corresponding to 0.002 to 6 mg of active enzyme variant per g of detergent composition, preferably 0.005 to 5 mg/g, 0.005 to 3 mg/g, 0.01 to 2 mg/g, or 0.05 to 2 mg/g.

In one embodiment, the detergent composition has a pH in the range of 5 to 12, preferably in the range of 6 to 11, and more preferably in a range selected from 6 to 10, 7 to 9, and 7.5 to 8.5. In one embodiment, the formulation is a detergent composition, preferably a liquid detergent composition.

In one embodiment, the detergent composition according to the present invention comprises one or more surfactants. Depending on their ionic charge, surfactants are called nonionic, anionic, cationic or amphoteric.

The detergent compositions of the present invention may contain one or more surfactants, which may be anionic, and/or cationic, and/or nonionic, and/or semi-polar, and/or zwitterionic, or mixtures thereof. In preferred embodiments, the detergent compositions of the present invention contain at least one surfactant. In certain embodiments, the detergent compositions of the present invention contain a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant is typically present at a level of about 0.1 to 60% by weight, e.g., 1 to 40% by weight, 3 to 20% by weight, or 3 to 10% by weight. The surfactant is selected based on the desired cleaning application and includes any conventional surfactant known in the art. Any surfactant known in the art for use in detergents may be utilized. Non-limiting examples of surfactants are disclosed in McCutcheon's 2016 Detergents and Emulsifiers and McCutcheon's 2016 Functional Materials, both North American and International editions, MC Publishing Co., 2016 edition. Further useful examples are disclosed in earlier editions of the same publications known to those skilled in the art.

When included therein, the detergent will typically contain about 1 to 40% by weight, e.g., 5 to 30% by weight, 5 to 15% by weight, or 20 to 25% by weight, of anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates, and the like. phosphates (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 glycerol esters, alpha-sulfofatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonates (MES), alkyl- or alkenyl succinic acids, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soaps, and combinations thereof.

When included, detergents will typically contain from about 0 to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quats (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

When present, the detergent will typically contain about 0.2 to 40% by weight of anionic surfactant, for example 0.5 to 30% by weight, especially 1 to 20% by weight, 3 to 10% by weight, 3 to 5% by weight or 8 to 12% by weight. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated fatty acid alkyl esters and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), N-acyl N-alkyl derivatives of polyhydroxyalkyl fatty acid amides or glucosamine (glucosamide, GA or fatty acid glucamide, FAGA), and products available under the trade names SPAN and TWEEN®, and combinations thereof.

When included, the detergent will typically contain from about 0 to about 10% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs) such as alkyl dimethyl amine oxide, N-(cocoalkyl)-N,N-dimethyl amine oxide, and N-(tallow-alkyl)-N,N-bis-(2-hydroxy-ethyl) amine oxide, fatty acid alkanolamides, and ethoxylated fatty acid alkanolamides, and combinations thereof.

When included, the detergent will typically contain from about 0 to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines, alkyldimethylbetaines, sulfobetaines, and combinations thereof.

Detergent compositions according to the present invention may contain one or more compounds selected from complexing agents (chelants, sequestrants), precipitating agents, and ion-exchange compounds capable of forming water-soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building agents," although this is not meant to limit such compounds to this function in the ultimate application of the detergent composition.

In one embodiment, the detergent compositions of the present invention comprise at least one builder selected from non-phosphate builders such as sodium gluconate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphonate, sodium aminocarboxylate, sodium polycarboxylate, sodium polysulfonate, and sodium polyphosphonate. In one embodiment, the detergent compositions of the present invention comprise a strong metal ion-trapping builder. Preferably, the detergent compositions of the present invention are phosphate-free, meaning essentially free of phosphate builders. As used herein, "essentially phosphate-free" should be understood to mean that the phosphate and polyphosphate content, determined by gravimetric measurement, is in the range of 10 ppm to 1% by weight in total, with reference to the respective detergent composition of the present invention. In another preferred embodiment, the detergent compositions comprise a phosphonate, preferably DTPMP and/or HEDP.

In one embodiment, the detergent composition of the present invention comprises at least one "citrate" selected from monoalkali and dialkali metal salts of citric acid, and particularly the monosodium and preferably trisodium salts, ammonium or substituted ammonium salts of citric acid, and such citric acids. The citrate may be used as an anhydrous compound or as a hydrate, for example, sodium citrate dihydrate. The citrate may be present in a total amount ranging from 0% to about 20% by weight, from about 0.5% to about 10% by weight, or from 1 to 5% by weight, based on the total weight of the detergent composition. In one embodiment, the detergent composition of the present invention comprises a total amount of citrate ranging from about 1 to 3% based on the total weight of the detergent composition.

The detergent compositions of the present invention may comprise one or more silicates. "Silicate" in the context of the present invention includes in particular aluminosilicates such as sodium disilicate and sodium metasilicate, sodium aluminosilicates such as Zeolite A (i.e. _Na12 ( _AlO2 ) ₁₂ ( _SiO2 ) ₁₂ * _{27H2O ) and} layered silicates, in particular those of _the formulae alpha- _Na2Si2O5 , _{beta - Na2Si2O5} and _delta - _Na2Si2O5 .

The detergent compositions of the present invention may comprise one or more carbonate salts. The term "carbonate" includes alkali metal carbonates and alkali metal bicarbonates, with sodium salts being preferred. Sodium _carbonate ( _Na2CO3 ) is particularly preferred.

The detergent compositions of the present invention may contain one or more phosphate salts. "Phosphonates" include 2-phosphonium butane-1,2,4-tricarboxylic acid (PBTC); ethylenediaminetetra(methylenephosphonic acid) (EDTMPA); 1-hydroxyethane-1,1-diphosphonic acid (HEDP), CH ₂ C(OH)[PO(OH) ₂ ] ₂ ; aminotris(methylenephosphonic acid) (ATMP), N[CH ₂ PO(OH) ₂ ] ₃ ; aminotris(methylenephosphonate), sodium salt (ATMP), N[CH ₂ PO(ONa) ₂ ] ₃ ; 2-hydroxyethyliminobis(methylenephosphonic acid), HOCH ₂ CH ₂ N[CH ₂ PO(OH) ₂ ] ₂ ; diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), (HO) ₂ POCH ₂ N[CH ₂ Examples of suitable phosphonic acid salts include, but are not limited to, CH ₂ N[CH ₂ PO(OH) ₂ ] ₂ ] ₂ ; diethylenetriaminepenta(methylenephosphonate), sodium salt, C ₉ H _(28-x) N ₃ Na _x O ₁₅ P ₅ (x=7); hexamethylenediamine(tetramethylenephosphonate), potassium salt, C ₁₀ H _(28-x) N ₂ K _x O ₁₂ P ₄ (x=6); and bis(hexamethylene)triamine(pentamethylenephosphonic 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 aminocarboxylates. Non-limiting examples of suitable "aminocarboxylates" include, but are not limited to, diethanolglycine (DEG), dimethylglycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethylaminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), N-(2hydroxyethyl)iminodiacetic acid (HEIDA), hydroxyethylenediaminetriacetic acid, N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA), hydroxyethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid (DTPA), and methylglycinediacetic acid (MGDA), glutamic acid-diacetic acid (GLDA), iminodisuccinic acid (IDS), hydroxyiminodisuccinic acid, ethylenediaminedisuccinic acid (EDDS), aspartic acid-diacetic acid, and alkali metal or ammonium salts thereof. More preferred 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-sulfomethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfomethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), Examples of suitable diacetic acids include alpha-alanine-N,N-diacetic acid (alpha-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), as well as alkali metal or ammonium salts thereof. MGDA or EDDS are preferred. The term "ammonium salt" as used in this context refers to a salt with at least one cation having a permanently or temporarily quaternized nitrogen atom. Examples of cations having at least one permanently quaternized nitrogen atom include tetramethylammonium, tetraethylammonium, dimethyldiethylammonium, and n- _C10 - _C20 -alkyltrimethylammonium. Examples of cations having at least one nitrogen atom that is temporarily quaternized include monomethylammonium, dimethylammonium, trimethylammonium, monoethylammonium, diethylammonium, triethylammonium, n-C ₁₀ -C _{20 -alkyl} dimethylammonium 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, tris(2-hydroxyethyl)ammonium, N-methyl 2-hydroxyethylammonium, N,N-dimethyl-2-hydroxyethylammonium and especially protected amines and ammonia, such as NH ₄ ⁺ .

In one embodiment, the detergent composition of the present invention contains two or more builders. Preferably, the detergent composition of the present invention contains less than 0.2 wt. % nitrilotriacetic acid (NTA) or 0.01 to 0.1 wt. % NTA, based on the total weight of the detergent composition.

In one embodiment, the detergent composition of the present invention comprises at least one aminocarboxylic acid salt selected from methylglycine diacetate (MGDA), glutamic acid diacetate (GLDA) and their corresponding salts, such as alkali (e.g., sodium) salts, in an amount ranging from 0.1% to 25.0% by weight, 1.0% to 18.0% by weight, 3.0% to 15.0% by weight, 3.0% to 10.0% by weight, or 5.0% to 8.0% by weight, based on the total weight of the detergent composition.

The detergent compositions of the present invention may include one or more hydrotropes. The one or more hydrotropes may be selected from organic solvents known in the art, such as ethanol, isopropanol, ethylene glycol, 1,2-propylene glycol, and additional organic solvents that are water-miscible under standard, unrestricted conditions. In one embodiment, the detergent compositions of the present invention include 1,2-propylene glycol in a total amount ranging from 5 to 10% by weight, preferably about 6% by weight, based on the total weight of the detergent composition. Further non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

In one embodiment, the detergent composition includes at least one preservative. Preferably, preservative refers to a substance added to a liquid composition for preservation purposes; more preferably, the term preservative excludes compounds known to have a preservative function that are included in the liquid composition formed during the production process. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, formic acid in its acid form or as a salt thereof, and 4,4'-dichloro-2-hydroxydiphenyl ether. Typically, the liquid compositions of the present invention include at least one preservative in an amount of less than 10 ppm, such as an amount ranging from 2 ppm to 5 wt. % based on the total weight of the liquid composition. Alternatively, the detergent composition does not include a preservative, meaning that the preservative is included in an amount of less than 1 ppm, preferably 0 ppm.

In one embodiment, the detergent composition comprising an amylase variant as described herein further comprises one or more second enzymes different from the amylase variant. Preferably, the second enzyme is a protease, a second amylase, a lipase, a cellulase, a mannanase, a hemicellulase, a phospholipase, an esterase, a pectinase, a lactase, a peroxidase, a xylanase, a cutinase, a pectate lyase, a keratinase, a reductase, an oxidase, a phenoloxidase, a lipoxygenase, a ligninase, a pullulanase, a tannase, a pentosanase, a malanase, a beta-glucanase, an arabinosidase, a hyaluronidase, a chondroitinase, a laccase, a nuclease, a DNase, a phosphodiesterase, a phytase, a caprylic acid esterase, a phosphodiesterase, a phospholip ... The second enzyme is selected from the group consisting of ribohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, ribonuclease, transglutaminase, and dispersin, and combinations of at least two of the above types. More preferably, the second enzyme is selected from the group consisting of protease, lipase, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase, and cutinase, and combinations of at least two of the above types. Most preferably, the second enzyme is a protease, preferably a subtilisin protease.

Particularly preferred additional enzymes are disclosed elsewhere herein, the disclosures of which are incorporated by reference into this section of the specification.

The compositions of the present invention may contain one type of enzyme, or two or more enzymes of different types, such as an amylase and a protease, or two or more enzymes of the same type, such as two or more different proteases, or a mixture thereof, such as an amylase and two different proteases.

The detergent composition may include a water-soluble source of calcium and/or magnesium ions. In one embodiment, the detergent composition includes an enzyme stabilization system as described herein. Preferably, particularly in the case of liquid detergent compositions, the detergent composition may include at least one protease inhibitor as described herein, preferably selected from a boronic acid derivative, preferably 4-FPBA, and a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H. Preferably, the detergent composition does not contain boron.

In one embodiment, the present invention relates to a method of providing a detergent composition, preferably a liquid detergent composition, more preferably a liquid laundry detergent composition, comprising mixing in one or more steps: (a) at least one amylase variant according to the present invention (preferably the amylase is provided in an amylase variant formulation as described herein); and (b) at least one detergent component selected from surfactants, builders, polymers, preservatives, and a second enzyme different from the amylase variant, preferably present in an amount effective in cleaning performance and/or maintaining the physical characteristics of the detergent.

In one embodiment, the present invention provides
a) an amylase variant as described herein;
b) one or more surfactants, preferably at a concentration of 0.2 to 65%, preferably 0.2 to 40%,
c) preferably at a concentration of 0.01 to 25% of one or more builders, and d) optionally one or more additional compounds selected from the group consisting of an additional enzyme different from the amylase under a) above, an antifoaming agent, a polymer, a bleaching system (bleach), a rheology modifier, a hydrotrope, a softener, a drying agent, a brightener, a buffer, a preservative, an anticorrosion additive, a dye, and a fragrance;
Preferably, the detergent composition is a liquid, powder, pouch, or capsule detergent composition.

Preferably, the detergent composition, preferably a powder detergent composition, of the present invention comprises, in addition to the amylase variant as described herein, one or more compounds selected from the group consisting of alcohol ethoxylate 7 EO, coco fatty acid C12-18, C12-C14-fatty alcohol ether sulfate (1-3 EO, preferably 2 EO), linear alkylbenzene sulfonic acid, sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, HEDP, MGDA, sodium sulfate, sodium chloride, optical brighteners, and polymers, and optionally bleach activators and percarbonates.

Preferably, the detergent composition of the present invention, preferably a powder detergent composition, comprises in addition to the amylase variant as described herein:
b) one or more surfactants selected from the group consisting of alcohol ethoxylate 7 EO, coco fatty acid C12-18, C12-C14-fatty alcohol ether sulfate (1-3 EO, preferably 2 EO), linear alkyl benzene sulfonic acid, preferably in a concentration of 0.2 to 65%;
c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA, and DTPMP, preferably at a concentration of 0.01 to 25%, and d) one or more compounds selected from the group consisting of sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, sodium sulfate, sodium chloride, optical brighteners, and polymers, and optionally bleach activators and percarbonates.

Preferably, the detergent compositions, preferably liquid detergent compositions, of the present invention comprise, in addition to the amylase variants described herein, one or more compounds selected from the group consisting of alcohol ethoxylate 7EO, coco fatty acid C12-18, C12-C14-fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, sulfonic acid, 1,2 propanediol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymers.

Preferably, the detergent compositions of the present invention, preferably liquid detergent compositions, comprise in addition to the amylase variants as described herein:
b) one or more surfactants selected from the group consisting of alcohol ethoxylate 7 EO, coco fatty acid C12-18, C12-C14-fatty alcohol ether sulfate (1-3 EO, preferably 2 EO), linear alkyl benzene sulfonic acid, preferably in a concentration of 0.2 to 65%;
c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA, and DTPMP, preferably at a concentration of 0.01 to 25%, and d) one or more compounds selected from the group consisting of sulfonic acid, 1,2 propanediol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymers.

In one embodiment, the amylase variants described herein are included in a detergent composition comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w/w):
A formulation comprising 0.05% to 1.0% of an amylase variant described herein;
8% to 15% anionic cleansing surfactants (such as alkyl benzene sulfonates, alkyl ethoxylated sulfates, and mixtures);
0.5% to 4% of a non-ionic cleansing surfactant (such as an alkyl ethoxylated alcohol);
0-4% cationic cleansing surfactants (e.g., quaternary ammonium compounds);
0% to 4% of other cleansing surfactants (such as zwitterionic cleansing surfactants, amphoteric surfactants, and mixtures thereof);
1% to 4% of a carboxylic acid polymer (such as a copolymer of maleic and acrylic acid);
0.5% to 4% polyethylene glycol polymer (such as a polyethylene glycol polymer containing polyvinyl acetate side chains);
0.1-2% polyester soil release polymer (such as Repel-o-tex and/or Texcare polymer);
0.5% to 2% cellulose polymers (such as carboxymethylcellulose, methylcellulose, and combinations thereof);
0% to 4% of other polymers (such as amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof);
0% to 4 wt % of a zeolite builder and a phosphate builder (such as Zeolite 4A and/or sodium tripolyphosphate);
0% to 3% other builders (such as sodium citrate and/or citric acid);
15% to 30% carbonate (such as sodium carbonate and/or sodium bicarbonate);
0% to 10% silicate (such as sodium silicate);
10% to 40% filler (such as sodium sulfate and/or biofiller);
a source of 10% to 20% available oxygen (such as sodium percarbonate);
2% to 8% bleach activator (such as tetraacetylethylenediamine (TAED) and/or nonanoyloxybenzenesulfonate (NOBS));
0% to 0.1% bleach catalyst (such as an oxaziridinium-based bleach catalyst and/or a transition metal bleach catalyst);
0% to 10% other bleaching agents (such as reduced bleaches and/or preformed peracids);
0.2% to 1% of a chelant (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and/or hydroxyethanediphosphonic acid (HEDP));
0% to 0.1% photobleaching agent (such as zinc and/or aluminum sulfonated phthalocyanine);
0% to 1% of a hueing agent (such as Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof);
0.1% to 0.4% brightener (such as Brightener 15 and/or Brightener 49);
0% to 4% fabric softener (such as montmorillonite clay and/or polydimethylsiloxane (PDMS));
0% to 1% flocculating agent (such as polyethylene oxide);
0% to 0.1% foam suppressor (such as silicone and/or fatty acid);
0.1% to 1% fragrance (such as fragrance microcapsules, spray fragrance, starch-encapsulated fragrance harmonizers, fragrance-loaded zeolites, and any combination thereof);
0% to 1% aesthetic agents (such as colored soap rings and/or colored speckles/noodles); and optionally about 0.05 wt% to about 0.2 wt% protease (such as Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, Excellenz P, etc.);
Optionally, about 0.05 wt % to about 0.2 wt % of an additional amylase different from the amylase variants described herein (such as Termamyl®, Termamyl Ultra®, Natalase®, Optisize HT Plus®, Purastar, Powerase®, Stainzyme®, Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S, and any combination thereof);
Optionally, 0.05% to 0.2% cellulase (such as Carezyme, Celluclean, Puradax, Biotouch, Whitezyme, Revitalenz, and combinations thereof);
Optionally, 0.05% to 0.2% lipase (such as Lipex, Lipolex, Lipoclean, Preferenz L, and any combination thereof);
Optionally, 0.05% to 0.2% of other enzymes (such as xyloglucanase, cutinase, pectate lyase (such as Xpect), mannanase (such as Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F, and combinations thereof), bleaching enzymes, and combinations thereof);

In another embodiment, the amylase variants described herein are included in a detergent composition comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w/w):
A formulation comprising 0.05% to 1.0% of an amylase variant described herein;
about 0.5 wt % to about 1.5 wt % of a carboxyl group-containing polymer (comprising about 60 wt % to about 70 wt % of a monomer (A) based on acrylic acid; and about 30 wt % to about 40 wt % of a sulfonic acid group-containing monomer (B); and the average molecular weight is preferably about 23,000 to about 50,000, in the range of about 25,000 to 38,000 as described in WO2014032269;
about 8 wt % to about 15 wt % of an anionic detersive surfactant (such as alkyl benzene sulfonates, alkyl ethoxylated sulfates, and mixtures thereof);
about 0.5 wt % to 4 wt % of a non-ionic cleansing surfactant (such as an alkyl ethoxylated alcohol);
about 0 wt % to about 4 wt % of a cationic cleansing surfactant (such as a quaternary ammonium compound);
about 0 wt % to 4 wt % of other cleansing surfactants (such as zwitterionic cleansing surfactants, amphoteric surfactants, and mixtures thereof);
about 1 wt % to about 4 wt % of a carboxylic acid polymer (such as a copolymer of maleic acid and acrylic acid);
about 0 wt % to about 4 wt % of a polyethylene glycol polymer (such as a polyethylene glycol polymer containing polyvinyl acetate side chains);
about 0.1 wt % to about 2 wt % of a polyester soil release polymer (such as Repel-O-Tex® and/or Texcare® polymer);
about 0.5 wt % to about 2 wt % of a cellulose polymer (such as carboxymethyl cellulose, methyl cellulose, and combinations thereof);
from about 0 wt % to about 4 wt % of other polymers (such as amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof);
about 0 wt % to about 4 wt % of a zeolite builder and a phosphate builder (such as Zeolite 4A and/or sodium tripolyphosphate);
about 0 wt % to about 3 wt % of other builders (such as sodium citrate and/or citric acid);
about 15 wt % to about 30 wt % carbonate (e.g., sodium carbonate and/or sodium bicarbonate);
about 0 wt % to about 10 wt % silicate (such as sodium silicate);
about 10 wt % to about 40 wt % of a filler (such as sodium sulfate and/or a biofiller);
about 10 wt % to about 20 wt % of a source of available oxygen (such as sodium percarbonate);
about 2 wt % to about 8 wt % of a bleach activator (such as tetraacetylethylenediamine (TAED) and/or nonanoyloxybenzenesulfonate (NOBS));
about 0 wt % to about 0.1 wt % of a bleach catalyst (such as an oxaziridinium-based bleach catalyst and/or a transition metal bleach catalyst);
about 0 wt % to about 10 wt % of other bleaching agents (such as reduced bleaching agents and/or preformed peracids);
about 0.2 wt % to about 1 wt % of a chelant (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and/or hydroxyethanediphosphonic acid (HEDP));
about 0 wt % to about 0.1 wt % of a photobleaching agent (such as zinc and/or aluminum sulfonated phthalocyanine);
about 0 wt % to about 0.5 wt % of a hueing agent (such as Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof);
about 0.1 wt % to about 0.4 wt % of a brightener (such as Brightener 15 and/or Brightener 49);
0 wt % to 15 wt % of a fabric softener (such as montmorillonite clay and/or polydimethylsiloxane (PDMS));
0 wt % to 1 wt % of a flocculant (such as polyethylene oxide);
0 wt % to 0.1 wt % of a foam suppressor (such as a silicone and/or a fatty acid);
0.1 wt % to 1 wt % of a fragrance (such as fragrance microcapsules, spray fragrance, starch-encapsulated fragrance harmonizers, fragrance-loaded zeolites, and any combination thereof);
0 wt % to 1 wt % aesthetic agents (such as colored soap rings and/or colored speckles/noodles);
Optionally, about 0.05 wt% to about 0.2 wt% of a protease (such as Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, Excellenz P, etc.);
Optionally, about 0.05 wt % to about 0.2 wt % of an additional amylase different from the amylase variants described herein (such as Termamyl®, Termamyl Ultra®, Natalase®, Optisize HT Plus®, Purastar, Powerase®, Stainzyme®, Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S, etc.);
Optionally, about 0.05 wt% to 0.5 wt% cellulase (typically, such as Carezyme®, Celluzyme®, Puradax®, Celluclean®, Biotouch, Whitezyme, Revitalenz, and combinations thereof, having an enzymatic activity of about 10-50 mg active enzyme/g)
Optionally, about 0.2 wt% to about 1 wt% lipase (typically, such as Lipex®, Lipolex®, Lipoclean®, Preferenz L, and any combination thereof, having an enzyme activity of about 10 mg to about 50 mg active enzyme/g)
Optionally, 0 wt % to 2 wt % of other enzymes (typically xyloglucanases (e.g., Whitezyme®) having an enzymatic activity of about 10 mg to about 50 mg active enzyme/g, cutinases, pectate lyases (e.g., Xpect), mannanases (e.g., Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F, and combinations thereof), bleaching enzymes, etc.).

Further preferred detergent compositions comprise the components listed below (all percentages are w/w):
- water, alcohol ethoxy sulfate, alcohol ethoxylate, amino oxide, citric acid, C12-18 palm kernel cap fatty acid, ethanol, 1,2 propanediol, sodium formate, calcium chloride, sodium hydroxide, silicone emulsion, trans-sulfated EHDQ, amylase variant as described herein;
- Sodium linear alkylbenzene sulfonate 8.8%. Ethoxylated fatty alcohol C12-18 (7 EO) 4.7%. Sodium soap 3.2%. Antifoaming agent DC2-4248S 3.9%. Sodium aluminum silicate Zeolite 4A 28.3%. Sodium carbonate 11.6%. Sodium salt of copolymer derived from acrylic acid and maleic acid (Sokalan CP5) 2.4%. Sodium silicate 3.0%. Carboxymethylcellulose 1.2%. Dequest 2066 2.8%. Optical brightener 0.2%. Sodium sulfate 6.5%. 0.4% of a formulation comprising an amylase variant as described herein;
- 12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG (monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soy soap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTM PA and 0.2% PCA, amylase variant as described herein;
-5-15% anionic surfactants; <5% nonionic surfactants, phosphonates, soaps; enzymes, optical brighteners, benzisothiazolinones, methylisothiazolinones, fragrances, alpha-isomethyl ionone, citronellol, geraniol, linalool, amylase variants as described herein;
Water, Sodium Dodecyl Benzene Sulfonate, C14-C15 Pareth-7, Sodium Citrate, Propylene Glycol, Sodium Palm Kernel Oil, Sodium Laureth Sulfate, MEA Dodecyl Benzene Sulfonate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Cumene Sulfonate, Fragrance, PEG/Vinyl Acetate Copolymer, Sodium Formate, Hydrogenated Castor Oil, Sodium Diethylenetriamine Pentamethylene Phosphonate, PEG/PPG-10/2 Propylheptyl ether, buthiophenyl methylpropional, polyvinylpyridine-N-oxide, sorbitol, glycerin, ethanolamine, sodium hydroxide, alpha-isomethylonone, calcium chloride, geraniol, linalool, citronellol, tripropylene glycol, benzisothiazolinone, dimethicone, sodium acetate, cellulase, colorant, glyceryl stearate, hydroxyethylcellulose, silica, amylase variant as described herein;
Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Fatty Acid, Alcohol, Sodium Formate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Hydroxide, Fragrance, Polyvinylpyridine-N-oxide, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Colorant, Cellulase, Amylase Variant as described herein;
Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Fatty Acid, Alcohol, Sodium Formate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Hydroxide, Fragrance, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Colorant, Cellulase, Amylase Variant as described herein;
Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Fatty Acid, Alcohol, Sodium Formate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Hydroxide, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Cellulase, Silica, Amylase Variant as described herein;
Water, Sodium Dodecyl Benzene Sulfonate, C14-C15 Pareth-7, Sodium Citrate, Propylene Glycol, Sodium Palm Kernel Oil, Sodium Laureth Sulfate, MEA Dodecyl Benzene Sulfonate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Cumene Sulfonate, Fragrance, PEG/Vinyl Acetate Copolymer, Sodium Formate, C12-C14 Pareth-7, Hydrogenated Castor Oil, Sodium Diethylenetriamine Pentamethylene Phosphonate, PEG/PPG-10/2 Propylheptyl ether, buthiophenyl methylpropional, fluorescent brightener, sorbitol, glycerin, ethanolamine, sodium hydroxide, alpha-isomethylonone, calcium chloride, geraniol, linalool, citronellol, tripropylene glycol, sodium chloride, benzisothiazolinone, dimethicone, sodium acetate, cellulase, colorant, glyceryl stearate, hydroxyethylcellulose, silica, amylase variant as described herein;
-15-30% anionic surfactants, non-ionic surfactants, 5-15% soaps, <5% polycarboxylates, fragrances, phosphates, optical brighteners, amylase variants as described herein;
-15-30% anionic surfactant, 5-15% nonionic surfactant, soap, benzisothiazolinone, methylisothiazolinone, fragrance, amylase variant as described herein;
-11% LAS, 2% AS/AEOS, 2% soap, 3% AEO, 15.15% sodium carbonate, 3% sodium silicate, 18.75% zeolite, 0.15% chelant, 2% sodium citrate, 1.65% AA/MA copolymer, 2.5% CMC and 0.5% SRP, amylase variant as described herein;
- 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodium carbonate, 1% socalan, 35.5% sodium sulfate, amylase variant as described herein;
-15-30% anionic surfactants, <5% nonionic surfactants, phosphonates, polycarboxylates, zeolites; enzymes, fragrances, hexyl cinnamal, amylase variants as described herein;
-15-30% of the following: anionic surfactants, oxygen-based bleaches and zeolites, less than 5% of the following: nonionic surfactants, phosphonates, polycarboxylates, soaps, additional ingredients: fragrance, hexyl cinnamal, benzyl salicylate, linalool, optical brighteners, enzymes and citronellol, amylase variants as described herein;
- Water, alcohol ethoxysulfate, diethylene glycol, alcohol ethoxylate, ethanolamine, linear alkylbenzene sulfonate, sodium fatty acid, polyethyleneimine, ethoxylate, citric acid, sodium cumene sulfonate, propylene glycol, DTPA, disodium diaminostilbene disulfonate, dipropylethyltetramine, sodium hydroxide, sodium formate, calcium formate, dimethicone, Liquitint™, hydrogenated castor oil, fragrance, amylase variant as described herein;
Linear alkylbenzene sulfonate, propylene glycol, citric acid, sodium hydroxide, ethanolamine, ethanol, alcohol sulfate, polyethyleneimine ethoxylate, sodium fatty acid, diquaternium ethoxysulfate, diethylene glycol, laureth-9, alkyldimethylamine oxide, fragrance, disodium diaminostilbene disulfonate, DTPA, sodium formate, calcium formate, polyethylene glycol 4000, mannanase, Liquitint™ Blue, dimethicone, amylase variant as described herein;
- water, sodium alcohol ethoxy sulfate, propylene glycol, ethanol, sodium linear alkylbenzene sulfonate, salt, polyethyleneimine ethoxylate, diethylene glycol, trans sulfated and ethoxylated hexamethylene diamine, alcohol ethoxylate, linear alkylbenzene sulfonate, MEA salt, sodium formate, sodium alkyl sulfate, DTPA, amine oxide, calcium formate, disodium diaminostilbene, disulfonate, dimethicone, benzisothiazolinone, amylase variant as described herein;
Water, alcohol ethoxysulfate, linear alkylbenzene sulfonate, diethylene glycol, propylene glycol, ethanolamine, citric acid, alcohol sulfate, sodium hydroxide, polyethyleneimine, ethoxylate, sodium fatty acid, ethanol, laureth-9, diquaternium ethoxysulfate, lauramine oxide, sodium cumene, sulfonate, fragrance, DTPA, disodium, diaminostilbene, disulfonate, sodium formate, disodium distyrylbiphenyl, disulfonate, calcium formate, polyethylene glycol 4000, mannanase, pectinase, Liquitint™ Blue, dimethicone, amylase variant as described herein;
Water, alcohol ethoxy sulfate, propylene glycol, sodium fatty acid, lauryltrimonium chloride, ethanol, sodium hydroxide, sodium cumene sulfonate, citric acid, ethanolamine, diethylene glycol, silicone polyether, fragrance, polyethylene-imine ethoxylate, laureth-9, DTPA, polyacrylamide quaternium chloride, disodium diaminostilbene disulfonate, sodium formate, Liquitint™ Orange, dipropylethyltetraamine, dimethicone, cellulase, amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, Sodium Alkyl Sulfate, MEA Citrate, Linear Alkyl Benzene Sulfonate, MEA Salt, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Sodium Fatty Acids, Ethanolamine, Lauramine Oxide, Laureth-9, DTPA, Sodium Cumene Sulfonate, Sodium Formate, Calcium Formate, Linear Alkyl Benzene Sulfonate, Sodium Salt, Alcohol Sulfate, Sodium Hydroxide, Diquaternium Ethoxy Sulfate, Fragrance, Mannanase, Pectinase, Disodium Diaminostilbene Disulfonate, Benzisothiazolinone, Liquitint™ Blue, Dimethicone, Dipropylethyltetraamine, Amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, MEA Citrate, Sodium Alkyl Sulfate, Alcohol Ethoxylate, Linear Alkyl Benzene Sulfonate, MEA Salt, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Diethylene Glycol, Propylene Glycol, Diquaternium Ethoxy Sulfate, Polyethyleneimine, Ethoxylate Propoxylate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Sodium Formate, Calcium Formate, Lauramine Oxide, Liquitint™ Blue, Dimethicone/Polydimethyl Silicone, Amylase Variant as described herein;
Water, alcohol ethoxysulfate, linear alkylbenzene sulfonate, alcohol ethoxylate, citric acid, ethanolamine, sodium fatty acid, diethylene glycol, propylene glycol, sodium hydroxide, polyethyleneimine ethoxylate, silicone polyether, ethanol, sodium cumene sulfate, diquaternium ethoxysulfate, laureth-9, fragrance, DTPA, disodium diaminostilbene disulfonate, disodium distyrylbiphenyl disulfonate, sodium formate, calcium formate, mannanase, Liquitint™ orange, dimethicone, polyacrylamide quaternium chloride, cellulase, dipropylethyltetraamine, amylase variant as described herein;
- Water, alcohol ethoxy sulfate, diethylene glycol, monoethanolamine citrate,
Sodium formate, propylene glycol, linear alkylbenzene sulfonate, ethanolamine, ethanol, polyethyleneimine ethoxylate, benzisothiazoline, calcium formate, citric acid, sodium diethylenetriaminepentaacetate, dimethicone, diquaternium ethoxysulfate, disodium diaminostilbene disulfonate, laureth-9, mannanase, sodium cumene sulfonate, sodium fatty acids, amylase variant as described herein;
Water, alcohol ethoxy sulfate, MEA citrate, alcohol sulfate, alcohol ethoxylate, linear alkylbenzene sulfonate MEA, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternium ethoxy sulfate, polyethyleneimine ethoxylate propoxylate, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodium diaminostilbene disulfonate, mannanase, cellulase, sodium formate, calcium formate, lauramine oxide, Liquitint™ Blue, dimethicone, amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, MEA Citrate, Linear Alkyl Benzene Sulfonate: Sodium Salt, Alcohol Ethoxylate, Linear Alkyl Benzene Sulfonate: MEA Salt, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Diethylene Glycol, Propylene Glycol, Diquaternium Ethoxy Sulfate, Polyethyleneimine Ethoxylate Propoxylate, Ethanol, Sodium Cumene Sulfonate, Citric Acid, DTPA, Disodium Diaminostilbenedisulfonate, Sodium Formate, Calcium Formate, Dimethicone, Amylase variant as described herein;
- Water, Alcohol Ethoxylate Sulfate, Sodium Linear Alkylbenzene Sulfonate/Mea Salt, Propylene Glycol, Diethylene Glycol, Sodium Formate, Ethanol, Sodium Fatty Acids, Fragrance, Lauramine Oxide, DTPA, Polyethyleneamine Ethoxylate, Calcium Formate, Disodium Diaminostilbene Disulfonate, Dimethicone, Tetramine, Liquitint™ Blue, Amylase Variant as described herein;
linear alkylbenzene sulfonate, C12-16 Pareth-9, propylene glycol, alcohol ethoxysulfate, water, polyethyleneimine ethoxylate, glycerin, fatty acid salts, PEG-136 polyvinyl acetate, ethylenediamine disuccinate, monoethanolamine citrate, sodium bisulfite, sodium diethylenetriaminepentaacetate, disodium distyrylbiphenyldisulfonate, calcium formate, mannanase, exiloglucanase, sodium formate, hydrogenated castor oil, dyes, subtilisin, benzisothiazoline, fragrance, amylase variant as described herein;
- deionized water, dipropylene glycol butyl ether, sodium alkyl sulfate, hydrogen peroxide, ethanol, magnesium sulfate, alkyldimethylamine oxide, citric acid, sodium hydroxide, trimethoxybenzoic acid, fragrance, amylase variant as described herein;
- Water, alkyl ethoxylate, linear alkyl benzene sulfonate, hydrogen peroxide, diquaternium ethoxysulfate, ethanolamine, disodium distyryl biphenyl disulfonate, tetrabutyl ethylidine bisphenol, F&DC Yellow 3, fragrance, amylase variant as described herein;
- sodium percarbonate, sodium sulfate, sodium carbonate, sodium aluminosilicate, nonanoyloxybenzenesulfonate, sodium polyacrylate, water, sodium alkylbenzenesulfonate, DTPA, polyethylene glycol, sodium palmitate, modified starch, FD&C Blue 1, fragrance, amylase variant as described herein;
- Water, alkyl ethoxylate, MEA borate, linear alkyl benzene sulfonate, propylene glycol, diquaternium ethoxysulfate, calcium chloride enzyme, ethanolamine, benzisothiazolinone, sodium citrate, sodium hydroxide, fragrance, amylase variant as described herein;
- water, alkylamine oxide, dipropylene glycol phenyl ether, hydrogen peroxide, citric acid, ethylenediamine, disuccinic acid sodium salt, sodium alkyl sulfate, fragrance, amylase variant as described herein;
- sodium bicarbonate, sodium carbonate, sodium percarbonate, alcohol ethoxylate, sodium chloride, maleic acid/acrylic acid copolymer, nonanoyloxybenzenesulfonate, sodium sulfate, colorants, diethylenetriaminepentaacetic acid sodium salt, hydrated aluminosilicate (zeolite), polyethylene glycol, sodium alkylbenzenesulfonate, sodium palmitate, starch, water, fragrance, amylase variant as described herein;
- Polyvinyl alcohol pouch film (with compressed liquid and powder portions): Liquid component: dipropylene glycol, diquaternium ethoxysulfate, water, glycerin, Liquitint™ Orange, amylase variant as described herein;
- Active ingredients: sodium percarbonate, nonanoyloxybenzenesulfonate, sodium carbonate, sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodium alkylbenzenesulfonate, maleic/acrylic acid copolymer, water, polyethylene glycol, sodium palmitate, modified starch, glycerin, DTPA, fragrance, amylase variant as described herein;
- Water, Sodium Alcohol Ethoxy Sulfate, Linear Alkylbenzene Sulfonate, Sodium/MEA Salt, MEA Citrate, Propylene Glycol, Polyethyleneimine, Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Sodium Fatty Acids, Sodium Cumene Sulfonate, DTPA, Fragrance, Disodium Diaminostilbene Disulfonate, Calcium Formate, Sodium Formate, Gluconase, Dimethicone, Liquitint™ Blue, Mannanase, Amylase variant as described herein;
- sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, bentonite, water, sodium percarbonate, sodium polyacrylate, silicates, alkyl sulfates, nonanoyloxybenzenesulfonate, DTPA, polyethylene glycol 4000, silicones, ethoxylates, fragrances, polyethylene oxide, palmitic acid, disodium diaminostilbene disulfonate, Liquitint™ Red, FD&C Blue 1, cellulase, amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium/MEA Salt, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine, Propoxyethoxylate, Diquaternium Ethoxysulfate, Alcohol Sulfate, Dimethicone, Fragrance, Sodium Fatty Acids, DTPA, Sodium Bisulfite, Disodium Diaminostilbene Disulfonate, Glucanase, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Sodium Formate, Liquitint™ Blue, Amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium/MEA Salt, Propylene Glycol, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Polyethyleneimine Ethoxylate, Alcohol Sulfate, Dimethicone, Fragrance, Sodium Fatty Acids, DTPA, Sodium Bisulfite, Disodium Diaminostilbene Disulfonate, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Propanaminium Propanamide, Gluconase, Sodium Formate, Liquitint™ Blue, Amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium/MEA Salt, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Diquaternium Ethoxy Sulfate, Alcohol Sulfate, Dimethicone, Fragrance, Sodium Fatty Acids, DTPA, Sodium Bisulfite, Disodium Diaminostilbene Disulfonate, Glucanase, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Propanaminium Propanamide, Sodium Formate, Liquitint™ Blue, Amylase variant as described herein;
- sodium carbonate, sodium aluminosilicate, alcohol sulfate, sodium sulfate, linear alkylbenzene sulfonate, water, sodium polyacrylate, silicates, ethoxylates, sodium percarbonate, polyethylene glycol 4000, disodium diaminostilbene disulfonate, silicones, cellulase, amylase variants as described herein;
- sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, alkyl sulfate, sodium percarbonate, water, sodium polyacrylate, silicates, nonanoyloxybenzenesulfonate, ethoxylates, polyethylene glycol 4000, fragrance, DTPA, disodium diaminostilbene disulfonate, palmitic acid, silicone, cellulase, amylase variant as described herein;
- sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, water, nonanoyloxybenzenesulfonate, alkyl sulfate, sodium polyacrylate, silicate, sodium percarbonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, silicone, cellulase, amylase variant as described herein;
- sodium carbonate, sodium aluminosilicate, sulfuric acid, sodium percarbonate, alkyl sulfate, linear alkylbenzene sulfonate, water, nonanoyloxybenzene sulfonate, sodium polyacrylate, silicate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, palmitic acid, disodium diaminostilbene disulfonate, FD&C Blue 1, silicone, cellulase, alkyl ether sulfate, amylase variant as described herein;
- sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicone, sodium polyacrylate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, amylase variant as described herein;
- sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, sodium percarbonate, alkyl sulfate, water, sodium polyacrylate, silicate, nonanoyloxybenzenesulfonate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, cellulase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, MEA Citrate, Linear Alkyl Benzene Sulfonate, Sodium Salt, Linear Alkyl Benzene Sulfonate:MEA Salt, Alcohol Ethoxylate, Sodium Fatty Acid, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate Propoxylate, Diquaternium Ethoxy Sulfate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Sodium Bisulfate, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Sodium Formate, Calcium Formate, Lauramine Oxide, Liquitint™ Blue, Dimethicone/Polydimethyl Silicone, Amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, Linear Alkylbenzene Sulfonate: Sodium/MEA Salt, MEA Citrate, Propylene Glycol, Polyethyleneimine Ethoxylate, Fragrance, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Alcohol Sulfate, Sodium Fatty Acids, DTPA, Disodium Diaminostilbene Disulfonate, MEA, Mannanase, Gluconase, Sodium Formate, Dimethicone, Liquitint™ Blue, Tetramine, Amylase variant as described herein;
Water, Sodium Alcohol Ethoxy Sulfate, MEA Citrate, Linear Alkyl Benzene Sulfonate, Sodium Salt, Linear Alkyl Benzene Sulfonate:MEA Salt, Alcohol Ethoxylate, Sodium Fatty Acid, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate Propoxylate, Diquaternium Ethoxy Sulfate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Sodium Bisulfate, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Sodium Formate, Calcium Formate, Lauramine Oxide, Liquitint™ Blue, Dimethicone/Polydimethyl Silicone, Amylase variant as described herein;
- sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicone, sodium polyacrylate ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, amylase variant as described herein; or - water, dodecylbenzenesulfonate, laureth-11, PEG-75 lanolin, propylene glycol, alcohol denat., potassium soyate, potassium hydroxide, disodium cocoamphodiacetate, ethylenediamine triacetate cocosalkyl acetamide, fragrance, zinc ricinoleate, sodium chloride, benzisothiazolinone, methylisothiazolinone, ci 16255, benzyl alcohol, amylase variant as described herein.

Preferably, the above-listed compositions containing a protease further contain 4-FPBA and/or a peptide aldehyde protease inhibitor, most preferably Z-GAY or Z-VAL.

The amylase variants described herein may be included in one of the following detergent compositions:

Methods of Use The present invention also relates to the use of the amylase variants as described herein in cleaning processes such as laundry or hard surface cleaning, preferably for home care or I&I cleaning, including manual and automatic dishwashing and medical device cleaning. Thus, the present invention also refers to the use of an amylase variant as described herein to provide a detergent composition with improved enzyme stability, preferably amylase stability, and/or to provide a detergent composition with improved cleaning performance, preferably on amylase-sensitive stains.

The present invention therefore also refers to a method for cleaning, preferably laundry or hard surface cleaning, comprising the step of contacting an object, preferably a textile or hard surface, with a composition comprising an amylase variant as described herein, wherein the composition preferably comprises at least one additional detergent ingredient, preferably a surfactant and/or builder.

Furthermore, the present invention also refers to a method for improving amylase stability in a detergent composition and/or for improving the cleaning performance of a detergent composition, preferably on amylase-sensitive stains, comprising the step of incorporating an amylase variant as described herein into the detergent composition.

Preferred Embodiments Particularly preferred herein are the following:
1. An amylase variant of a parent amylase, wherein the amylase variant comprises:
an amylase variant comprising: (i) amino acid substitutions at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO:3; and (ii) at least 60%, preferably at least 91% identity, but less than 100% sequence identity, to any of SEQ ID NOs:1, 3, 4, or SEQ ID NOs:15-41, preferably SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1 (preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1).

2. The amylase variant is
(i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) amino acid substitutions at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO: 3;
(iii) An amylase variant according to embodiment 1, comprising at least 60%, preferably at least 91% identity but less than 100% sequence identity to SEQ ID NO: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 (preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1).

3. An amylase variant according to any of the preceding embodiments, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of 116, 181, 225, and 320.

4. An amylase variant according to any of the preceding embodiments, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of 195 or 206, preferably wherein the amylase variant comprises an amino acid substitution at either position 195 or 206.

5. An amylase variant according to any of the preceding embodiments, wherein the amylase comprises an amino acid substitution at position 482.

6. An amylase variant according to any of the preceding embodiments, wherein the amino acid substitution at position 25 is X25H or an amino acid substitution similar to X25H.

7. An amylase variant according to any of the preceding embodiments, wherein the amino acid substitution at position 116 is X116K, the amino acid substitution at position 176 is X176K, the amino acid substitution at position 181 is X181T, the amino acid substitution at position 186 is X186E, the amino acid substitution at position 195 is X195F, the amino acid substitution at position 206 is X206Y, the amino acid substitution at position 225 is X225A, the amino acid substitution at position 320 is X320K, and the amino acid substitution at position 482 is X482W, or an amino acid substitution similar to these substitutions.

8. An amylase variant according to any of the preceding embodiments, wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

9. In one embodiment, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO:3; and (iii) at least 60%, preferably at least 91%, but less than 100% sequence identity to any of SEQ ID NOs:1, 3, 4, or SEQ ID NOs:15-41, preferably SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1.

10. In one embodiment, the amylase variant of the present invention comprises:
(i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 3;
(ii) either an amino acid substitution of X195F or X206Y according to the numbering of SEQ ID NO: 3;
(iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO:3; and (iv) at least 60%, preferably at least 91%, but less than 100% sequence identity to any of SEQ ID NOs:1, 3, 4, or SEQ ID NOs:15-41, preferably SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1.

11. An amylase variant according to any of the preceding embodiments, wherein the amylase variant has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO:1.

12. The amylase variant is
a) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and one or more substitutions selected from 176 and 186; preferably one or more substitutions selected from X176K and X186E; or b) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and one or more substitutions selected from 116, 181, 225, and 320, preferably X116K, X225A, and X320K. or c) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and one or more substitutions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A, and X320K.

13. An amylase variant according to any of the preceding embodiments, wherein the variant comprises the amino acid substitution X195F and substitutions at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and substitutions at one or more positions selected from 116, 181, 225, and 320, preferably one or more substitutions selected from X116K, X225A, and X320K.

14. An amylase variant according to any of the preceding embodiments, wherein the variant comprises the amino acid substitution X206Y and substitutions at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and substitutions at one or more positions selected from 116, 181, 225, and 320, preferably one or more substitutions selected from X116K, X225A, and X320K.

15. The variant is:
X25H+X176K+X186E,
X25H+X176K+X186E+X195F,
X25H+X116K+X181T+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K,
X25H+X116K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X181T+X195F+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,
10. The amylase variant according to any of the preceding embodiments, comprising a combination of amino acid substitutions selected from the group consisting of: X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W.

16. The variant is:
X25H+X176K+X186E,
X25H+X176K+X186E+X206Y,
X25H+X116K+X181T+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K,
X25H+X116K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X181T+X206Y+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,
10. The amylase variant according to any of the preceding embodiments, comprising a combination of amino acid substitutions selected from the group consisting of: X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, and X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W.

17. An amylase variant according to any of the preceding embodiments, comprising the amino acid substitutions X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 3).

18. An amylase variant according to any of the preceding embodiments, further comprising a deletion of two amino acids corresponding to positions selected from the group consisting of 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 numbering is according to the amino acid sequence set forth in SEQ ID NO: 3.

19. An amylase variant according to any of the preceding embodiments, wherein the amylase variant has at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, sequence identity to any of SEQ ID NOs: 1, 3, or 4, preferably SEQ ID NO: 1.

20. An amylase variant according to any of the preceding embodiments, wherein the variant comprises A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75% but less than 100% identical to the amino acid sequence of SEQ ID NO: 6, and wherein 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.

21. An amylase variant according to any of the preceding embodiments, wherein the amylase variant comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4, or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with 1 to 7, preferably 2 to 7 or 3 to 7, e.g., 1, 2, 3, 4, 5, 6, or 7, amino acid substitutions selected from the group consisting of the amino acid substitution X25H and either of the amino acid substitutions X195F or X206Y, and X116K, X176K, X181T, X186E, X225A, X320K, and X482W, and preferably 1 to 10, preferably 1 to 5 additional conservative amino acid substitutions, and preferably a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183, and 184.

22. An amylase variant according to any of the preceding embodiments, wherein the amylase variant further comprises 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8, or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 8 conservative amino acid exchanges, preferably wherein the conservative mutations do not involve the catalytic center of the amylase variant.

23. The amylase variant is selected from the group consisting of the modifications X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W, X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K, and X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K according to the numbering of SEQ ID NO: 3. 0K+X482W, preferably X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, and optionally 1 to 10, preferably 1 to 5, additional conservative amino acid substitutions.

24. An amylase variant according to any of the preceding embodiments, wherein the substitutions are selected from N25H, W116K, R176K, R181T, G186E, N195F, I206Y, T225A, R320K, and Y482W.

25. The amylase variant preferably exhibits one or more improved properties relative to said parent amylase, preferably relative to the parent amylase set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably relative to the parent amylase set forth in SEQ ID NO: 1 or relative to the amylase set forth in SEQ ID NO: 33; preferably the amylase variant has increased stability, thermostability, storage stability, storage stability in a detergent composition, cleaning performance, cleaning performance in a laundry detergent, and/or cleaning performance in a dishwashing detergent; preferably the improved property is improved storage stability and/or improved cleaning performance, preferably improved cleaning performance on laundry; most preferably the improved property is improved storage stability; preferably the improved property is expressed as an improvement factor (IF) of >1.0; preferably the improvement factor is 1.1 or greater, preferably 1.2 or greater, more preferably 1.3 or greater; preferably the amylase variant exhibits one or more improved properties relative to the reference amylase, preferably relative to said parent amylase, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably An amylase variant according to any of the preceding embodiments, which exhibits improved storage stability in a detergent composition relative to the parent amylase set forth in SEQ ID NO: 1 or relative to the amylase as set forth in SEQ ID NO: 33, preferably having an improvement of at least 1.1, preferably at least 1.2, more preferably at least 1.3 relative to a reference amylase, preferably relative to said parent amylase, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably relative to the parent amylase set forth in SEQ ID NO: 1 or relative to the amylase as set forth in SEQ ID NO: 33, preferably after 14, 24, 56 or 84 days, preferably 84 days of storage in a detergent composition, preferably at 40°C, as preferably determined in an ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP); and/or preferably, the amylase variant exhibits improved cleaning performance on laundry, preferably at 40°C, preferably relative to an amylase having the amino acid sequence set forth in SEQ ID NO: 33.

26. A polynucleotide encoding an amylase variant according to any of the preceding embodiments.
27. A formulation comprising an amylase variant according to any one of embodiments 1 to 25 and at least one additional component, preferably a solvent.

28. The composition according to embodiment 27, wherein the composition comprises one or more second enzymes different from the amylase variant referred to in any of the preceding claims, preferably selected from the group consisting of proteases, second amylases, lipases, cellulases, hemicellulases, mannanases, xylanases, DNases, dispersins, pectinases, oxidoreductases, and cutinases, preferably one or more second enzymes selected from proteases, mannanases, and lipases, most preferably proteases.

29. A formulation according to embodiment 27 or 28, comprising an enzyme stabilization system, preferably comprising a calcium salt, and optionally a protease inhibitor, such that proteases are absent.

30. A detergent composition comprising an amylase variant according to any one of embodiments 1 to 25, preferably a laundry detergent composition or a hard surface cleaning detergent composition, most preferably a laundry detergent composition.

31. A detergent composition according to embodiment 30, wherein the composition comprises one or more surfactants and/or one or more builders, preferably one or more strong metal ion-scavenging builders.

32. A detergent composition according to either embodiment 30 or 31, wherein the detergent composition comprises a component selected from the group consisting of an enzyme stabilizing system, a surfactant, an antifoaming agent, a builder, a polymer, a bleaching system (bleach), a rheology modifier, a hydrotrope, a softener, a drying agent, a brightener, a buffer, a suds suppressor, a preservative, an anticorrosion additive, a dye, and a fragrance.

33. The detergent composition according to any of embodiments 30 to 32, comprising a builder, wherein the builder is selected from MDGA, GLDA, DTPMP, HEDP, and EDDS, preferably MDGA or EDDS.

34. The detergent composition according to embodiment 33, wherein the builder is a non-phosphate builder.
35. The detergent composition according to any of embodiments 30-34, comprising a surfactant, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof.

36. The detergent composition according to any of embodiments 30-35, wherein the detergent composition is devoid of anionic surfactants.
37. The detergent composition according to any of embodiments 30-36, wherein the surfactant and/or builder is biodegradable and/or bio-based.

38. The detergent composition according to any of embodiments 30-37, wherein the detergent composition is a liquid or a solid.
39. The detergent composition according to any of embodiments 30-38, wherein the detergent composition is in the form of a pouch.

40. The detergent composition according to any of embodiments 30-39, wherein the detergent composition is a liquid laundry detergent composition.
41. The detergent composition according to any of embodiments 30-40, wherein the detergent composition does not contain boron.

42. The detergent composition according to any of embodiments 30-41, wherein the detergent composition does not comprise a preservative.
43. A method for cleaning comprising contacting a textile or a hard surface, preferably a textile, with an amylase variant according to any of embodiments 1 to 25, or a formulation according to any of embodiments 27 to 29, or a detergent composition according to any of embodiments 30 to 42, preferably at low temperature, preferably at 30°C or 40°C, preferably at 30°C.

44. Use of an amylase variant according to any of embodiments 1 to 25 or a formulation according to any of embodiments 27 to 29 or a detergent composition according to any of embodiments 30 to 42 for improving amylase stability in a detergent composition and/or improving the cleaning performance of a detergent composition, preferably for amylase-sensitive stains, preferably for an amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, or for an amylase as set forth in SEQ ID NO: 33, preferably at 30°C or 40°C, preferably 40°C, as preferably determined in an ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP).

Common methods:
Amylase Expression: The amylase-encoding gene was cloned into a Gram-positive expression vector containing a promoter sequence, a sequence encoding a secretory signal peptide, and a ribosome binding site by a standard protocol based on restriction ligation. Following the reaction, the plasmid construction mixture was transformed into B. subtilis PY79 by an established natural competent transformation method. Successful transformants were selected by plating onto LB agar plates supplemented with 20 μg/ml kanamycin sulfate and incubating overnight at 37°C. After overnight selection, individual colonies were obtained and subsequently grown in rich medium (e.g., LB broth) with 20 μg/ml kanamycin sulfate overnight at 37°C with shaking at 250 rpm. The following morning, cells were pelleted by centrifugation, and plasmid DNA was isolated by alkaline lysis using a Macherey-Nagel NucleoSpin kit. The isolated DNA could then be transformed into electrocompetent Bacillus licheniformis cells.

B. licheniformis cells were made electrocompetent using standard Gram-positive electrocompetent cell preparation techniques. For example, cells were prepared by growing a B. licheniformis strain in an osmolyte-rich medium (e.g., LB broth with 0.5 M D-sorbitol) and harvesting the cells in early exponential growth phase. The cells were chilled on ice and harvested by pelleting via centrifugation. After harvest, the cells were washed to remove salts using an osmolyte-rich wash buffer (e.g., 10% glycerol with 0.5 M D-sorbitol and 0.5 M D-mannitol) with three cycles of suspension and pelleting via centrifugation. Finally, the cells were concentrated by resuspending in wash buffer at 1-10% of the original culture volume.

Once prepared, the plasmid DNA was added to electrocompetent B. licheniformis cells in a 0.2 cm Bio-Rad electroporation cuvette. Cells were electroporated using a Bio-Rad Gene Pulser Xcell according to the manufacturer's instructions. Cells were immediately rescued after shock by the addition of 1 ml of osmolyte-rich, high-density medium. After recovering for 2 hours at 37°C, successful transformants were selected by plating on LB agar plates supplemented with 20 μg/ml kanamycin sulfate and incubating overnight at 37°C.

Amylase Expression and Protein Quantification. Single colonies of the expression strains were picked into 60 μL of rich medium (e.g., LB broth) supplemented with 20 μg/mL kanamycin sulfate in a 96-well plate (GE Life Sciences part 28403943). The cultures were grown at 37°C for 16 hours, after which 15 μL of the culture was used to inoculate 600 μL of defined glucose-mineral medium and 20 μg/mL kanamycin sulfate in a 96-well plate. The cultures were grown at 37°C for 48 hours, after which the supernatant was collected by centrifugation to pellet the cells and remove the remaining culture medium. Larger quantities were made by combining supernatants from the same clone. The expression level of alpha-amylase activity (concentration in mg/mL) was determined by LabChip (LC) analysis to allow for comparable dosage in laundry detergents.

Amylase Activity Measurement: α-Amylase activity can be determined by a method utilizing ethylidene-4-nitrophenyl-α-D-maltoheptaoside (EPS) as a substrate. D-Maltoheptaside is a block oligosaccharide that can be cleaved by endo-amylases. After cleavage, the α-glucosidase included in the kit digests the substrate, liberating free PNP molecules that have a yellow color, which can be measured by visible spectrophotometry at 405 nm. Kits containing the EPS substrate and α-glucosidase are manufactured by Roche Costum Biotech (cat. no. 10880078103) or Thermo Scientific (TR25421). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity per mg of enzyme) of the α-amylase of interest under a given set of conditions. Residual activity was calculated by dividing the activity after storage time by the activity of the sample at time zero. Residual activity was compared to the residual activity of a reference given in the examples below.

Example 1: Storage stability in builder-containing detergents for up to 84 days at 40°C.

Amylases were formulated into a liquid model detergent (ES1-C) and tested for their residual activity after 84 days of storage at 40°C. The final dosage was 0.1 g/L amylase. To assess amylase stability in the presence of strong chelators, HEDP and/or _CaCl2 were supplemented as indicated in the table. No additional Ca2 ⁺ was supplemented with the enzyme.

After the designated time points, samples were taken and the residual activity was measured using EPS as a substrate as described above. The residual activity was calculated by dividing the activity after the storage time by the activity of the sample at time zero. The residual activity was compared to the residual activity of a reference amylase (SEQ ID NO: 33).

As can be deduced from the table above, the amylase variants of the present invention are more stable than the reference amylase.

Example 2: Improved Amylase Cleaning Performance Determination of cleaning performance was carried out using a Launder-o-meter:
Several soiled swatches were washed in a liquid laundry formulation at 40°C with a cotton ballast fabric and 20 steel balls. After washing, the fabrics were rinsed, spun, and air-dried. Wash performance was determined by measuring the CIELab values of the soiled fabrics before and after washing using a MACH5 multi-area color measurement. For data evaluation, ΔE was calculated between the unwashed stain and the washed stain, and ΔΔE was calculated between the detergent with enzymes and the detergent without enzymes. Data represent the average of two separate experiments.

As can be derived from the table above, the amylase variants of the present invention provide better cleaning results compared to the reference amylase.

Determination of washing performance in a full-scale washing machine:
Multiple stain monitors were washed in a full-scale washing machine in the presence of cotton ballast fabric at 40°C in liquid laundry formulations with different amylases. Each stain was washed in quadruplicate (two units with two strips per stain per unit). After washing, the stain monitors were allowed to air dry. Wash performance was determined by measuring the CIELab values of the stained fabrics before and after washing using a MACH5 multi-area color measurement. For data evaluation, mean values were determined, and ΔY was calculated between unwashed and washed stains, and ΔΔY was calculated between detergents with and without enzymes.

As can be derived from the table above, the amylase variants of the present invention provide better cleaning results compared to the reference amylase.

Claims (15)

1. An amylase variant of a parent amylase, the amylase variant comprising:
(i) comprising an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3;
(ii) comprising amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO:3; and (iii) having at least 60% but less than 100% sequence identity with any of SEQ ID NOs:1, 3, 4, or SEQ ID NOs:15-41, preferably SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1. The amylase variant of claim 1, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of 116, 181, 225, and 320. The amylase variant according to claim 1 or claim 2, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of 195 or 206, preferably, the amylase variant comprises an amino acid substitution at either position 195 or 206. The amylase variant according to any one of claims 1 to 3, wherein the amylase variant comprises an amino acid substitution at position 482. The amylase variant according to any one of claims 1 to 4, wherein the amino acid substitution at position 25 is X25H. The amylase variant according to any one of claims 1 to 5, wherein the amino acid substitution at position 116 is X116K, the amino acid substitution at position 176 is X176K, the amino acid substitution at position 181 is X181T, the amino acid substitution at position 186 is X186E, the amino acid substitution at position 195 is X195F, the amino acid substitution at position 206 is X206Y, the amino acid substitution at position 225 is X225A, the amino acid substitution at position 320 is X320K, and the amino acid substitution at position 482 is X482W. The amylase variant is
a) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and one or more substitutions selected from 176 and 186; preferably one or more substitutions selected from X176K and X186E; or b) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and one or more substitutions selected from 116, 181, 225, and 320, preferably X116K, X225A, and X320K. or c) one or more substitutions selected from amino acid position 195, preferably X195F, or amino acid position 206, preferably X206Y, and one or more substitutions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and one or more substitutions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K. wherein the variant is
X25H+X176K+X186E,
X25H+X176K+X186E+X195F,
X25H+X176K+X186E+X206Y,
X25H+X116K+X181T+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K,
X25H+X116K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X181T+X195F+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,
X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,
X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W,
X25H+X116K+X181T+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K,
X25H+X116K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X181T+X206Y+X225A+X320K+X482W,
X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,
X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,
8. The amylase variant of any one of claims 1 to 7, comprising a combination of amino acid substitutions selected from the group consisting of: X25H + X116K + X181T + X186E + X206Y + X225A + X320K + X482W, and X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K + X482W. The amylase variant according to any one of claims 1 to 8, further comprising a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, wherein the numbering is according to the amino acid sequence set forth in SEQ ID NO: 3. The amylase variant according to any one of claims 1 to 9, wherein the amylase variant has at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, sequence identity to SEQ ID NO: 1, 3, 4, or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1. A polynucleotide encoding the amylase variant described in any one of claims 1 to 10. A composition comprising the amylase variant described in any one of claims 1 to 10 and at least one additional component. The composition according to claim 12, wherein the composition comprises one or more second enzymes different from the amylase variant referred to in any one of claims 1 to 12, preferably selected from the group consisting of proteases, second amylases, lipases, cellulases, hemicellulases, mannanases, xylanases, DNases, dispersins, pectinases, oxidoreductases, and cutinases, preferably one or more second enzymes selected from proteases, mannanases, and lipases, most preferably proteases. The composition according to claim 12 or claim 13, wherein the composition is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition. The composition according to any one of claims 12 to 14, wherein the composition contains one or more surfactants and/or one or more builders, preferably one or more strong metal ion-trapping builders.