MXPA00001888A - Protease variants and compositions - Google Patents

Abstract

Enzymes produced by mutating the genes for a number of subtilases and expressing the mutated genes in suitable hosts are presented. The enzymes exhibit improved wash performance in any detergent in comparison to their wild type parent enzymes.

Description

Variants and Protease Compositions TECHNICAL FIELD This invention relates to mutant, novel protease enzymes or enzyme variants useful in the formulation of detergent compositions and exhibiting improved washing ability in detergents; cleaning and detergent compositions containing the enzymes; mutated genes that are encoded for the expression of the enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing the variants of the enzyme.

BACKGROUND OF THE INVENTION In the detergent industry, enzymes have been implemented for more than 30 years in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. The most important commercial enzymes are proteases.

REF: 32788 A growing number of commercially used proteases are variants made from proteins of the naturally occurring non-cultured proteases, for example DURAZYM® (Novo Nordisk A / S), RELASE® (Novo Nordisk A / S), MAXAPEM® (Gist-Brocades NV), PURAFECT® (Genencor International, Inc.). In addition, a number of protease variants are described in the art, such as in European Patent No. 130756 (GENENTECH) (corresponding to reissued U.S. Patent No. 34,606 (GENENCOR)); European Patent No. 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); European Patent No. 260105 (GENENCOR); Thomas, Russell, and Fersht (1985) Na t ure 318 375-376; Thomas, Russell, and Fersht (1987) J. Mol. Bi ol. 193 803-813; Russell and Fersht Na t ure 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (A gene); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER &GAMBLE COMPANY); WO 95/30010 (PROCTER &GAMBLE COMPANY); WO 95/29979 (PROCTER &GAMBLE COMPANY); U.S. Patent No. 5,543,302 (SOLVAY S.A.); European Patent No. 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK A / S); WO 91/00345 (NOVO NORDISK A / S); European Patent No. 525 610 Al (SOLVAY); WO 94/02618 (GIST-BROCADES N.V.); and WO 96/34946 (NOVO NORDISK A / S).

However, although a number of useful protease variants have been described, there is still a need for new improved protease variants for a number of industrial uses. Therefore, an object of the present invention is to provide enhanced protease variants made from protein, especially for use in the detergent industry.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have intensively studied numerous of the possible combinations of residues N252, T255 and S259 of SAVINASE®, and have identified a number of variants with improved, improved washing capacity. Reference is made for additional details to the working examples in the present (vide infra). Accordingly, the present invention relates in its first aspect to a variant of the subtylase protease having an improved washing ability in detergents, comprising modification (s) in the position (s) 252, 255 and / or 259 Preferably, a subtyla variant according to the invention comprises modifications in both positions 252 and 255, and more preferred comprises modifications in all three positions 252, 255, and 259. In a second aspect, the invention relates to a variant of the subtylase enzyme having an improved washing ability in detergents, comprising at least one modification selected from the group comprising: 252L + 255I 252V + 255A 252M + 255C + 259H 252S + 255E + 259C ~ ~ 252K + 255S + 259C; or a variant comprising one or more conservative modifications in any of the variants mentioned above. { for example, a conservative modification of a variant 252L (a.a.hy drofóbi co) +2551 includes variants such as 2521 (a.a.hy drofóbi co) +2551, and 252V (a.a. hi drof óbi o) +2551. In a third aspect, the invention relates to an isolated DNA sequence encoding a subtyla variant of the invention.

In a fourth aspect, the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtyla variant of the invention. In a fifth aspect, the invention relates to a host cell, microbial, transformed with an expression vector according to the fourth aspect. In a further aspect, the invention relates to the production of the subtilisin enzymes of the invention by inserting an expression vector according to the fourth aspect in a suitable microbial host, culturing the host to express the desired subtylase enzyme, and recover the product of the enzyme. Still further, the invention relates to a composition comprising a subtyla variant of the invention. Finally, the invention relates to the use of the mutant enzymes for a number of pertinent, industrial uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially the detergent compositions comprising the mutant subtilisin enzymes.

DEFINITIONS Before discussing this invention in further detail, the next term will be defined first.

Nomenclature of Amino Acids to Ala Alanine V Val Valine L Leu Leucine I He Isoleucine P Pro Proline F Phe Phenylalanine W Trp Tryptophan M Met Methionine G Gly Glycine S Serine Thr Threonine C Cys Cysteine And Tyr Tyrosine N Asn Asparagine Q Gln Glutamine D Asp Acid Aspartic E Glu Glutamic Acid K Lys Lysine R Arg Arginine H = His = Histidine X = Xaa = Any amino acid Nomenclature of nucleic acids A Adenine G Guanine C Cytosine T Thymine (only in DNA) U Uracil (only in DNA) Nomenclature of variants In the description, the various variants of enzymes produced or contemplated according to the invention, the following nomenclatures have been adapted for ease of reference: Amino acid (s) substituted at the position (s) ) of the original amino acid (s). Accordingly, the replacement of glutamic acid by glycine at position 195 was designated as: Gly 195 Glu or G195E a glycine suppression at the same position is: Gly 195 * or G195 * and the insertion of an additional amino acid residue such as lysine is: Gly 195 GlyLys or G195GK Where a suppression is indicated compared to the sequence used for numbering, an insertion in such position is indicated as: * 36 Asp or * 36D for the insertion of an aspartic acid in the position 36 Multiple mutations are separated by pulses, ie: Arg 170 Tyr + Gly 195 Glu or R170Y + G195E representing mutations at positions 170 and 195 that substitute tyrosine and glutamic acid for arginine and glycine, respectively.

Proteases The enzymes that divide amide bonds in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzyma ti c Reacti on Mechanisms, W.H. Freeman and Company, San Francisco, Chapter 3).

Numbering amino acid positions / residues If no other is mentioned, the amino acid numbering used herein corresponds to that of the sequence BPN? (BASBPN) of subtilasa. For the additional description of the BPN sequence? see Siezen et al. reporters, Protein Engng. 4 (1991) 719-737 and Figure 1.

Serine Proteases A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue in the active site (White, Handler and Smith, 1973"Principi es de Bi ochemi s try ", Qinta Edition, McGraw-Hill Book Company, NY, pp. 271-272). Bacterial serine proteases have molecular weights in the range of 20,000 to 45,000 Daltons. These are inhibited by diisopropylfluorophosphate. These hydrolyze the terminal, simple esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more limited term, the alkaline protease, which covers a subgroup, reflects the high optimum pH of some of the serine proteases, from pH 9.0 to 11.0 (for review, see-Priest (1977) Bac teri ologlog! Rev. 41 711-753).

Subtilases A subgroup of the serine proteases by way of assay designated subtilases have been proposed by Siezen et al., Pro tein Engng. 4 (1991) 719-737. These are defined by the homology analysis of more than 40 amino acid sequences of the serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by gram-positive bacteria or fungi, and according to Siezen et al. Is now a subgroup of subtilases. A wide variety of subtylases have been identified, and the amino acid sequence of a number of subtypes "ace." For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. A subgroup of the subtilases, I-SI, comprises the "classical" subtilisins, such as subtilisin 168, subtilisin BPN ', subtilisin Carlsberg (ALCALASE®, NOVO N0RDT5CK A / S), and subtilisin DY An additional subgroup of subtilases, I-S2, is recognized by Siezen et al (supra). The proteases of subgroup I-S2 are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A / S), subtilisin 147 (ESPERASE®, NOVO NORDTSK A / S), and alkaline elastase YaB.

"SAVINASE®" The SAVINASE® is marketed by NOVO NORDISK A / S. This is subtilisin 309 of B. Lentus and differs from BABP92 only in that it has N87S (see figure 1 herein).

Subtilasa madre _ The term "subtilasa madre" is a subtilase defined according to Sienzen et al. (Protein Engineering 4: 719-737 (19991)). For additional details see the description of "SUBTILASAS" immediately before. A subtilasa mother can also be an subtyla isolated from a natural source, where a subsequent modification has been made while retaining the characteristics of a subtilasa. Alternatively, the term "subtylase mother" can be called "subtylase of the non-cultivated type".

Modification (s) of a subtilase variant The term "modification (s)" used in connection with the modification (s) of a subtilase variant as discussed herein is defined to include a chemical modification as well as a genetic manipulation. The modification (s) may be by substitution, deletion and / or insertions in the amino acid (s) of interest.

Subtylase variant In the context of this invention, the term variant mutated subtilasa or subtilasa means a subtilasa that has been produced by an organism which is expressing a mutant gene derived from a mother microorganism which possesses an original gene or mother and the which produces a corresponding mother enzyme, the mother gene that has been mutated in order to produce the mutant gene from which the mutant subtylase protease is produced when expressed in a suitable host.

Subtylase homologous sequences The specific amino acid residues of the SAVINASE® subtyla are identified for modification herein to obtain a subtyla variant of the invention. However, the invention is not limited to modifications of this particular subtilase, but extends to other mother subtilases (non-cultivated type), which have a primary structure homologous to that of SAVINASE®.

In order to identify other homologous subtilases, within the scope of this invention, an alignment of the subtylase (s) to a group of previously aligned subtilases is carried out keeping the previous alignment constant. A comparison is made to 18 highly conserved residues in the subtilases. The 18 highly conserved residues are shown in Table I (see Siezen et al. For additional details that relate to the conserved residues).

Table I 18 highly conserved residues in the subtilases Position: Residue conserved 23 G 32 D 34 G 39 H 64 H 65 G 66 T 70 G 83 G 127 G 146 G 154 G 155 N 219 G 220 T 221 S 225 P After the alignment that allows the insertions and deletions necessary in order to maintain the alignment, the appropriate homologous residues are identified. The homologous residues can then be modified according to the invention. Using the CLUSTALW computer alignment program (version 1.5, April 1995) (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) Nucleic Acids Research, 22: 4673-4680.), "With a GAP opening penalty of 10.0 and a GAP extension penalty of 0.1, using the weight matrix of the BLOSUM30 protein, the alignment of a subtyle given to a A group of prealigned subtilases is achieved using the option of profile alignments in the program For a given subtyla that is within the scope of the invention, preferably 100% of the 18 highly conserved residues must be retained. alignment greater than or equal to 17 out of the 18 residues, or as little as 16 of the conserved residues is also adequate to identify homologous residues.The conservation of the, in the subtilases, Asp32 / His trivalent, trivalent, catalytic The previously defined alignment is shown in Figure 1, where the identity of the percentage of the individual subtilases in this alignment to the 18 highly conserved residues is also shown. Based on this description, it is routine for a person skilled in the art to identify the appropriate homologous subtilases and corresponding homologous residues, which can be modified according to the invention. To illustrate, this table II below shows a limited list of homologous subtilases and the corresponding suitable residues to be modified according to the invention.

Table II Subtilases Homologous corresponding homologous residues, suitable to be modified according to the invention.

It is obvious that a similar or larger table covering other homologous subtilases can easily be produced by a person skilled in the art.

Washing capacity __ The ability of an enzyme to catalyze the degradation of several naturally occurring substrates present in the targets to be cleaned during eg washing is frequently referred to as its washing ability, washability, detergent power or washing capacity. . For all this request the term washing capacity will be used to include this property.

Isolated DNA sequence The term "isolated", when applied to a molecule of the DNA sequence, represents that the DNA sequence has been removed from its genetic, natural environment and thus is free of other foreign coding sequences or unwanted, and is in a form suitable for use within the production systems of genetically engineered proteins. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic es. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 'and 3' untranslated regions such as promoters and terminators. The identification of the associated regions will be apparent to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1_985). The term "an isolated DNA sequence" can alternatively be called "a ed DNA sequence".

Isolated protein When applied to a protein, the term "isolated" indicates that the protein is in a condition different from its natural environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homogeneous proteins (ie "homologous impurities" (see below)). It is preferred to provide the protein in a highly purified form, ie, greater than 40% pure, greater than 60% pure, greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE. The term "isolated protein" can be alternatively called "purified protein".

Homologous Impurities _ The term "homologous impurities" means any impurity (for example, a polypeptide different from the polypeptide of the invention) which originates from the homologous cell from which the polypeptide of the invention is originally obtained.

Obtained from The term "obtained from" as used herein in connection wa specific microbial source, means that the polynucleotide and / or polypeptide produced by the specific source, or by a cell into which a gene of the source .

Substrate The term "Substrate" used in connection wa substrate for a protease should be construed in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a protease of subtilisin.

Product The term "product" used in connection wa product derived from an enzymatic protease reaction should be interpreted in the context of this invention which includes the products of a hydrolysis reaction involving a subtylase protease. A product can be the substrate in a subsequent hydrolysis reaction.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows an alignment of a number of homologous subtilases, which align to the 18 highly conserved residues in the subtilases. The 18 highly conserved residues are highlighted in bold. All subtilases shown, except JP170, have 100% identity in conserved residues. The JP170 that has an "N" instead of "G" in the conserved residues G146.

DETAILED DESCRIPTION OF THE INVENTION Subtilase variants wimproved wash capacity: The present inventors have identified the improved wash capacity variants in BLS309 (SAVINASE®). Accordingly, one embodiment of the invention relates to a variant of the subtylase enzyme, wherein the modification is selected from the group comprising: N252L + T255I N252V + T255A N252M + T255C + S259H _7 N252S + T255E + S259C N252K + T255S + S259C; or a variant comprising one or more conservative modifications in any of the variants mentioned above (eg, a conservative modification of a variant N252L (aa hydrobbi co) + T255I include variants such as N252I (aa hydrophobic co) + T255I, and N252V (aa hydrophobic co) + T255I The numerous subtylase variants of the invention are discussed herein and show an improved washing ability in detergents (see working examples herein (see below)). in the art that the substitution of an amino acid to a similar conservative amino acid gives only a minor change in the characteristic of the enzyme, Table III below lists groups of conservative amino acids.

Table III Conservative amino acid substitutions Basic: argmma lysine histidine Acidic: glutamic acid aspartic acid Polar glutamine asparagine Hydrophobic leucine isoleucine valine Aromatic phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine Accordingly, subtylase variants such as 252L + 255I, 2521 + 2551, and 252V + 255I will have similar wash capacity improvement. In addition, subtylase variants such as N252L + T255I, N252I + T255I, and N252V + T255I will also have an improvement in similar wash capacity. Based on the subtyla variants described herein, it is routine work, for a person skilled in the art, to identify suitable, additional conservative substitutions in order to obtain a subtyla variant wan improved washing ability. In the embodiments of the invention, the subtilases of interest are those that belong to the subgroups I-SI and I-S2. In relation to the subgroup I-SI, the preferred subtylase is selected from the group comprising ABSS168, BASBPN, BSSDY and BLSCAR or functional variants thereof which have retained the characteristic of subgroup I-Sl. In relation to subgroup I-S2, the preferred subtylase is selected from the group comprising BLS147, BLS309, BAPB92, TVTHER and BYSYAB or functional variants thereof which retain the characteristic of subgroup I-S2. The present invention also comprises any one or more modifications at the positions mentioned above in combination with any other modification to the amino acid sequence of the mother enzyme. Especially, combinations with other modifications known in the art are considered to provide improved properties to the enzyme. The art describes a number of subtilase variants with different improved properties and a number of those are mentioned in the "background of the invention" section herein. { I saw upra). These references are described herein as references to identify a subtyla variant, which may be advantageously combined with a subtyla variant of the invention. Such combinations comprise the positions: 222 (improves oxidation stability), 218 (improves thermal stability), substitutions at the Ca binding sites that establish the enzyme, e.g. position 76, and many other apparent ones of the previous technique. In the additional embodiments, a subtyla variant of the invention can be advantageously combined with one or more modifications at any of the positions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 167, 170 , 206, 218, 222, 224, 235 and 274. Specifically, the following variants BLS309 and BAPB92 are considered appropriate for the combination: K27R, * 36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D , N123S, Y167A, Y167I, R170S, R170L, R170N, Q206E, N218S, M222S, M222A, T224S, K235L and T274A. In addition, variants comprising any of the variants V104N + S101G, K27R + V104Y + N123S + T274A or N76D + V104A or other combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with any one or more of the modifications mentioned above exhibit improved properties. Even more subtyla variants of (the) main aspect (s) of the invention are preferably combined with one or more modifications in any of positions 129, 131, 133 and 194, preferably as modifications 129K, 131H, 133P, 133D and 194P, and more preferably as modifications P129K, P131H, A133P, A133D and A194P. Any of these modifications may give a higher level of expression of a subtyla variant of the invention.

PRODUCTION OF MUTILATIONS IN SUBTILASE GENES _ Many methods for the cloning of a subtyla of the invention and for the introduction of mutations in genes (for example subtylase genes) are well known in the art. In general, normal methods for gene cloning and the introduction of mutations (random and / or site-directed) into the genes can be used to obtain a subtyla variant of the invention. For further description of suitable techniques, reference is made to the working examples in the present (see below) and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (Eds.) "Current protocols in Molecular Biology." John Wiley and Sons, 1995; Harwood, CR., And Cutting, SM (eds.) "Molecular Biological Methods for Bacillus." John Wiley and Sons , 1990); and WO 96/34946.

EXPRESSION VECTORS A recombinant expression vector comprising a DNA construct encoding the enzyme of the invention can be any vector which can be conveniently attached to recombinant DNA methods, and selection of the vector will often depend on the host cell in which must be entered. In this way, the vector can be a replication vector autonomously, that is, a vector which exists as an extrachromosomal entity, the replica of which is independent of the chromosomal replication, for example, a plasmid. Alternatively, the vetor may be one which, when introduced into a host cell, is integrated into the host cell genome in part or in its entirety and copied together with the chromosome (s) in the cells. which has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to the additional segments required for transcription of the DNA. In general, the expression vector is derived from the plasmid or viral DNA, or may contain elements of both. The term "operably linked" indicates that the segments are arranged so that they function in a concert for their intended purposes, for example the transcription initiates in a promoter and proceeds through the DNA sequence encoding the enzyme. The promoter can be any DNA sequence which shows transcriptional activity in the host cell of the selection and can be derived from the genes encoding the proteins either homologous or heterologous to the host cell. Examples of suitable promoters for use in bacterial host cells include the maltogenic amylase gene promoter Ba cill us stearo thermophilus, the alpha-amylase gene Bacill us li cheniformi s, the alpha-amylase gene Ba cill us amyl oli quefaci ens, the alkaline protease gene Bacill us subtilli or the xylosidase gene Bacill us pumil us, or the Lambda PR or P promoters of the bacteriophages or the lac, trp or tac promoters of E. coli . The DNA sequence encoding the enzyme of the invention can also, if necessary, be operably connected to a suitable terminator. The recombinant vector of the invention may additionally comprise a DNA sequence which makes it possible for the vector to be copied into the host cell in question. The vector may also comprise a selectable marker, for example, a gene the product of which complements a defect in the host cell, or a gene encoding resistance to, for example, antibiotics similar to kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycin or the like, or resistance to heavy metals or herbicides. To direct an enzyme of the present invention into the secretory pathway of host cells, a secretory signal sequence can be provided (also known as a leader sequence, prepro sequence or pre sequence) in the recombinant vector. The secretory signal sequence binds to the DNA sequence encoding the enzyme in the correct reading structure. The secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the enzyme. The sequence of the secretory signal may be that normally associated with the enzyme or it may be from a gene encoding another secreted protein. The methods used to ligate the DNA sequences encoding the present enzyme, the promoter and optionally the terminator and / or the sequence of the secretory signal, respectively, or to conjugate these sequences by suitable PCR amplification schemes and to insert them into suitable vectors containing the information necessary for copying or integration are well known to persons skilled in the art (see, for example, Sambrook et al., op.cit.).

GUEST CELL The DNA sequence encoding the present enzyme introduced into the host cell can be either homologous or heterologous to the host in question. If it is homologous to the host cell, i.e., produced by the host cell in nature, it will typically be operably linked to another promoter sequence or, if applicable, another sequence of the secretory signal and / or terminator sequence which in its natural environment. The term "homologue" is proposed to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is proposed to include a DNA sequence not expressed by the host cell in nature. In this way, the DNA sequence can be from another organism, or it can be a synthetic sequence. The host cell into which the construction of the DNA or the recombinant vector of the invention is introduced can be any cell which is capable of producing the present enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of bacterial host cells which, in the culture, are capable of producing the enzyme of the invention are gram-positive bacteria such as Bacillus strains, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans. B. circulans, B. lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram-negative bacteria such as Echerichia coli. The transformation of the bacteria can be carried out by protoplast transformation, electroporation, conjugation or by using competent cells in a manner known per se (see Sambrook et al., Supra). When the enzyme is expressed in bacteria such as E. coli, the enzyme can be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it can be directed to the periplasmic space by a sequence of bacterial secretion. In the above case, the cells are subjected to lysis and the granules are recovered and denatured after which the enzyme is refolded upon diluting the denaturing agent. In the latter case, the enzyme can be recovered from the periplasmic space by disrupting the cells, for example by disrupting high frequency sound waves or osmotic shock, to release the contents of the periplasmic space and recover the enzyme. When the enzyme is expressed in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme can be retained in the cytoplasm, or it can be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme can be recovered from the medium as described below.

METHOD OF PRODUCTION OF THE SUBTILASE ~ - The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions that allow the production of the enzyme, and the resulting enzyme is recovered from the culture. ~~ When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to allow the heterologous, recombinant production of the enzyme of the invention. ~ With which it is possible to make a subtyla composition. highly purified, characterized in that it is free of homologous impurities. In this context, homologous impurities means any impurity (for example polypeptides different from the enzyme of the invention) which originates from the homologous cell from which the enzyme of the invention is originally obtained. The medium used for the culture of the transformed host cells can be any conventional means suitable for culturing the host cells in question. The expressed subtilisa can conveniently be secreted into the culture medium and can be recovered therefrom by well known methods which include separating the cells from the medium by centrifugation or filtration, precipitating the protein components from the medium by means of a salt such as ammonium sulfate. , followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography or the like.

USE OF A SUBSTITUTE VARIANT OF I-TVENTION A variant of the subtylase protease of the invention can be used for a number of industrial applications, in particular within the detergent industry. In addition, the invention relates to an enzyme composition, which comprises a subtyla variant of the invention. A summary of preferred industrial applications corresponding to the preferred enzyme compositions are described below. This summary is not proposed in any way to be a complete list of suitable applications of a subtyla variant of the invention. A subtyla variant of the invention can be used in other industrial applications known in the art to include the use of a protease, in particular a subtylase.

DETERGENT COMPOSITIONS COMRINING MUTANT ENZYMES The present invention comprises the use of the mutant enzymes of the invention in cleaning and detergent compositions and such compositions comprising subtilisin mutant enzymes. Such cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions. - Additional reference is made to the working example (s) herein that show the improvements in washing ability for a number of subtyla variants of the invention.

DESCRIPTION OF THE DETERGENT AND EXAMPLES Surfactant system The detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant may be selected from nonionic and / or anionic and / or cationic and / or ampholytic surfactants and / or of amphoteric and / or semi-polar ions. The surfactant is typically present at a level of 0.1% to 60% by weight. The surfactant is preferentially formulated to be compatible with the enzyme components present in the composition. In liquid or gel compositions, the surfactant is formulated more preferably in such a way that it promotes, or at least does not degrade, the stability of any enzyme in these compositions. Preferred systems that are used in accordance with the present invention comprise as one surfactant one or more of the nonionic and / or anionic surfactants described herein. The polyethylene oxide, polypropylene and polybutylene oxide condensates of alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present invention, with the preferred polyethylene oxide condensates. These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight chain or chain configuration branched with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to about 2 to about 25 moles, more preferably about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include Igepal ™ CO-630, marketed by the GAF Corporation; and Triton ™ X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (eg, alkyl phenol ethoxylates). The condensation products of the primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the nonionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can be either straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. Preferred are the condensation products of the alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of sodium oxide. ethylene per mole of alcohol. Approximately 2 to about 7 moles of ethylene oxide and more preferably 2 to 5 moles of ethylene oxide per mole of alcohol are present in the condensation products. Examples of commercially available nonionic surfactants of this type include Tergitol ™ 15-S-9 (The linear alcohol condensation product of 11 to 15 carbon atoms with 9 moles of ethylene oxide), Tergitol ™ 24-L-6 NMW ( the condensation product of primary alcohol of 12 to 14 carbon atoms with 6 moles of ethylene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; Neodol ™ 45-9 (the linear alcohol condensation product of 14 to 15 carbon atoms with 9 moles of ethylene oxide), NeodolMR 23-3 (The linear alcohol condensation product of 12 to 13 carbon atoms with 3.0 moles of ethylene oxide), NeodolMR 45-7 (the condensation product of linear alcohol of 14 to 15 carbon atoms with 7 moles of ethylene oxide), NeodolMR 45-5 (the condensation product of linear alcohol of 14 to 15 carbon atoms with 5 moles of ethylene oxide) marketed by Shell Chemical Company, KyroM® EOB (the condensation product of 13 to 15 carbon atoms with 9 moles of ethylene oxide), marketed by Procter & Gamble Company, and Genapol LA 050 (the condensation product of alcohol of 12 to 14 carbon atoms with 5 moles of ethylene oxide) marketed by Hoechst. The preferred range of HLB in these products is 8-11 and most preferably 8-10. Also useful as the non-ionic surfactant of the surfactant systems of the present invention are the alkyl polysaccharides described in U.S. Patent No. 4,565,647, which has a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, for example a polyglycoside, a hydrophobic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 units of saccharide. Any reducing saccharide containing 5 or 6 carbon atoms can be used, for example, portions of glucose, galactose and galactosyl can be substituted for the glucosyl moieties (optionally, the hydrophobic group joins in the 2-, 3-, positions, 4-, etc. to thereby give a glucose or galactose as opposed to a glycoside or galactoside). The intersaccharide bonds can be, for example, between the position of the additional saccharide units and positions 2, 3, 4 and / or 6 in the preceding saccharide units. The preferred alkyl polyglycosides have the form R20 (C "H2nO) t (glycosyl) x wherein R2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 to approximately 10, preferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, much more preferably from about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is first formed and then reacted with glucose, or a source of glucose, to form the glucoside (linkage at position 1). The additional glycosyl units can then be linked between their position 1 and position 2, 3, 4 and / or 6 of the predefined glycosyl units, preferably predominantly position 2. The condensation products of ethylene oxide with a Hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional non-ionic surfactant systems of the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight of from about 1500 to about 1800 and will exhibit insolubility in water. The addition of polyoxyethylene portions to this hydrophobic portion tends to increase the solubility in the water of the molecule as a whole, and the liquid character of the product is retained to the point where the polyoxyethylene content is about 50% of the total product weight of condensation, which corresponds to condensation with up to about 40 moles of ethylene oxide. Examples of compounds of this type include certain of the commercially available Pluronic ™ surfactants marketed by BASF. Also suitable for use as the non-ionic surfactant of the nonionic surfactant system of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylene diamine. The hydrophobic portion of these products consists of the reaction product of ethylenediamine and excess propylene oxide, and in general has a molecular weight of about 2500 to about 3000. This hydrophobic portion is condensed with ethylene oxide to the extent that the condensation product it contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000. Examples of this type of ionic surfactant include certain of the commercially available Tetroniclffi compounds commercialized by BASF. - For the use as the non-ionic surfactant of the surfactant systems of the present invention, the polyethylene oxide condensates of alkyl phenols, the condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides and mixtures thereof. More preferred are alkyl phenol ethoxylates of 8 to 14 carbon atoms having from 3 to 15 ethoxy groups and alcohol ethoxylates of 8 to 18 carbon atoms (preferably an average of 10 carbon atoms) having 2 to 3 carbon atoms. to 10 ethoxy groups, and mixtures thereof. Highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the formula R2 - C - N - Z, O R1 - wherein R1 is H, or R1 is hydrocarbyl of 1 to 4 carbon atoms, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R2 is hydrocarbyl of 5 to 31 carbon atoms, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyl directly connected to the chain, or an alkoxylated derivative thereof. Preferably, R1 is methyl, R2 is alkyl of 11 to 15 carbon atoms or alkyl of 16 to 18 straight carbon atoms or an alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction. Highly preferred anionic surfactants include alkoxylated alkyl sulfate surfactants. Examples thereof are r-soluble salts or acids of the formula R0 (A) mS03M wherein R is an unsubstituted alkyl of 10 to 24 carbon atoms or a hydroxyalkyl group having an alkyl component of 10 to 24 carbon atoms. carbon, preferably an alkyl of 12 to 20 carbon atoms or hydroxyalkyl, more preferably alkyl of 12 to 28 carbon atoms or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3 and M is H or a cation which may be, for example, a metal cation (eg, sodium, potassium, lithium, calcium, magnesium, etc.). ), an ammonium or substituted ammonium cation. The alkyl ethoxylated sulfates as well as the alkyl propoxylated sulfates are contemplated herein. Specific examples of the substituted ammonium cations include methyl-, dimethyl-, trimethylammonium cations and quaternary ammonium cations such as tetramethylammonium and dimethyl piperidinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures of them, and similar. Exemplary surfactants are polyethoxylate (1.0) alkyl sulphate of 12 to 18 carbon atoms (E (1.0) M12C-? 8C), polyethoxylate sulfate (2.25) of alkyl of 12 to 18 carbon atoms ((2.25) M ? 2C-? 8C), polyethoxylate sulfate (3.0) of alkyl of 12 to 18 carbon atoms (E (3.0) M? 2C-? 8C), and polyethoxylate sulfate (4.0) of alkyl of 12 to 18 carbon atoms carbon (E (4.0) M12C-? 8C), wherein M is conveniently selected sodium and potassium. Suitable anionic surfactants which are used are the alkyl ester sulphonate surfactants which include linear esters of carboxylic acids of 8 to 20 carbon atoms (ie, fatty acids) which are sulfonated with gaseous SO 3 according to "The Journal of the American Oil Chemists Society ", 52 (1975), pp. 323-329. Suitable starting materials would include natural fatty substances such as tallow derivatives, palm oil, and so on. The preferred alkyl ester sulphonate surfactant, especially for laundry applications, comprises the alkyl ester sufates surfactants of the structural formula: 0 RJ - CH C OR " S03M wherein R3 is a hydroxycarbyl of 8 to 20 carbon atoms, preferably an alkyl, or combination thereof, R4 is a hydrocarbyl of 1 to 6 carbon atoms, preferably an alkyl, or combination thereof, and M is a cation which forms a water soluble salt with the alkyl ester sulfonate. Cations that form the suitable salt include metals such as sodium, potassium and lithium and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethanolamine and triethanolamine. Preferably, R3 is alkyl of 10 to 16 carbon atoms and R4 is methyl, ethyl or isopropyl. Especially preferred are the methyl ester sulfonates wherein R3 is alkyl of 10 to 16 carbon atoms. Other suitable anionic surfactants include the alkyl sulfate surfactants which are salts or water-soluble acids of the formula ROSO3M wherein R is preferably a hydrocarbyl of 10 to 24 carbon atoms, preferably an alkyl or hydroxyalkyl which has an alkyl component of 10 to 20 carbon atoms, more preferably an alkyl of 12 to 18 carbon atoms or hydroxyalkyl, and M is H or a cation, for example, an alkali metal cation (eg, sodium , potassium, lithium) or ammonium or substituted ammonium (for example methyl-, dimethyl- and trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and quaternary ammonium cations derived from alkylamines such as ethylamine , diethylamine, triethylamine, and mixtures thereof and the like). Typically, alkyl chains of 12 to 16 carbon atoms are preferred for the lower wash temperatures (for example below about 50 ° C) and alkyl chains of 16 to 18 carbon atoms are preferred for the higher wash temperature (for example above about 50 ° C). Other anionic surfactants useful for detersive purposes can also be included in the laundry detergent compositions of the present invention. These may include salts (including, for example, sodium, potassium, ammonium and substituted ammonium salts such as mono-, di- and triethanolamine salts) of soap, primary or secondary alkanesulfonates of 8 to 22 carbon atoms, olefinsulfonates of 8 at 24 carbon atoms, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, for example, as described in British Patent Specification No. 1,082,179, alkyl polyglycol ether sulfonates of 8 to 24 carbon atoms (containing up to 10 moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulphates, alkyl phenol ethylene oxide ether sulphates, paraffin sulphonates, alkyl phosphates, isethionates such as acyl isethionates, N-acyl taurates, succinamates of alkyl and sulfosuccinates, monoesters of sulphosuccinates (especially monoesters of 12 to 18 saturated and unsaturated carbon atoms) and diesters of sulfosuccinates (especially diesters of 6 to 12 saturated and unsaturated carbon atoms), acyl sarcosinates, sulfates of alkylpolysaccharides such as alkyl polyglucoside sulfates (the non-sulfate, nonionic compounds described below), primary alkyl, branched sulfates and alkyl polyethoxy carboxylates such as those of the formula RO (CH 2 CH 20) k-CH 2 COO-M + wherein R is a alkyl of 8 to 22 carbon atoms, k is an integer from 1 to 10, and M is a cation that forms the soluble salt. Resin acids and hydrogenated resin acids are also suitable, such as turpentine resin, hydrogenated turpentine resin and resin acids and hydrogenated resin acids present in or derived from the byproduct of wood pulp chemistry. Alkylbenzene sulfonates are highly preferred. Especially preferred are linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains 10 to 18 carbon atoms. _ Additional examples are described in "Surface Active Agents and Detergents" (Vol. I and II by Schwartz, Perry and Berch). A variety of such surfactants are also generally described in U.S. Patent No. 3,929,678, (Column 23, line 58 through Column 29, line 23, incorporated herein by reference). When included herein, the laundry detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants. The laundry detergent compositions of the present invention may also contain cationic, ampholytic, amphoteric and semi-polar ion surfactants, as well as nonionic and / or anionic surfactants other than those already described herein. The detersive, cationic surfactants suitable for use in the laundry detergent compositions of the present invention are those having a long chain hydrocarbyl group. Examples of such cationic surfactants include ammonium surfactants such as alkyltrimethylammonium halides, and those surfactants having the formula: [R2 (OR3) and] [R4 (OR3) and] 2R5N + X- wherein R is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R3 is selected from group consisting of -CH2CH2-, -CH2CH (CH3) -, -CH2CH (CH20H) -, -CH2CH2CH2-, and mixtures thereof: each R4 is selected from the group consisting of alkyl of 1 to 4 carbon atoms , hydroxyalkyl of 1 to 4 carbon atoms, benzyl ring structures formed by the joining of the two groups R4, -CH2CHOHCHOHCOR6CHOHCH2OH, wherein R6 is any of hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when and is not 0, R5 is the same or as R4 or is an alkyl chain, wherein the total number of carbon atoms or R2 plus R5 is not more than about 18, and y is from 0 to about 10, and the sum of the values of y is from 0 to about 15, and X is any compatible anion. Highly preferred thionics are the water-soluble quaternary ammonium compounds useful in the present composition having the formula: R1R2R3RN + X ~ (i) wherein Ri is alkyl of 8 to 16 carbon atoms, each of R2, R3 and R4 is independently alkyl of 1 to 4 carbon atoms, hydroxy alkyl of 1 to 4 carbon atoms, benzyl and - (C2H0) XH where x has a value of 2 to 5, and X is an anion. No more than one of R2, R3 or R4 must be benzyl.

The length of the preferred alkyl chain for Ri is 12 to 15 carbon atoms, particularly where the alkyl group is a mixture of chain lengths derived from coconut or palm kernel fat or is derived synthetically by the olefin increase or the synthesis of alcohols 0X0. Preferred groups for R2R3 and R4 are methyl or hydroxyethyl groups and the anion X can be selected from halide, methosulfate, acetate and phosphate ions. Examples of suitable quaternary ammonium compounds of the formulas (i) for use herein are: coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; dimethyl hydroxyethyl ammonium chloride or bromide 12 to 15 carbon atoms; coconut dimethyl hydroxyethyl ammonium chloride or bromide; methyl sulfate of myristyl trimethyl ammonium; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) ammonium chloride or bromide; Hill esters (compounds of the formula (i) wherein Ri is CH2-CH2-0-C-Calkyl of 12 to 14 carbon atoms O and R2R3R4 are methyl). di-alkyl imidazolines [compounds of the formula (i)]. Other cationic surfactants useful herein are also described in US Patent No. 4,228,044 and European Patent No. 000 224. When included herein, the laundry detergent compositions of the present invention typically comprise 0.2%. to about 25%, preferably from about 1% to about 8% by weight of such cationic surfactants. The ampholytic surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can also be broadly described as aliphatic derivatives of secondary and tertiary amines, or aliphatic derivatives of heterocyclic secondary or tertiary amines in which the aliphatic radical can be straight or branched chain. One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains a water-soluble, anionic group, for example carboxy, sulfonate, sulfate. See U.S. Patent No. 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants. When included herein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants. Amphoteric ion surfactants are also suitable for use in laundry detergent compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See U.S. Patent No. 3,929,678 (column 19, line 38 to column 22, line 48) for examples of amphoteric ion surfactants. When included herein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10%, by weight of such amphoteric ion surfactants. Non-ionic, semi-polar surfactants are a special category of non-ionic surfactants which include water-soluble amine oxides containing an alkyl portion of about 10 to about 18 carbon atoms and 2 portions selected from the group consisting of groups alkyl and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; the water-soluble phosphine oxides containing an alkyl portion of about 10 to about 18 carbon atoms and 2 portions selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water soluble sulfoxides containing an alkyl portion of about 10 to about 18 carbon atoms and a portion selected from the group consisting of alkyl and hydroxyalkyl portions of about 1 to about 3 carbon atoms. Semi-polar non-ionic detergent surfactants include amine oxide surfactants having the formula: O t R3 (OR4) x N: R5) wherein R3 is an alkyl, hydroxyalkyl or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms; R4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof; x is from 0 to about 3: and each R 5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups. The R5 groups can be linked together, for example, through an oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants include, in particular, alkyl dimethyl amine oxides of 10 to 18 carbon atoms and alkoxy ethyl dihydroxy ethyl amine oxides of 8 to 12 carbon atoms. When included herein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such non-ionic, semi-polar surfactants.

Adjuvant system _. The compositions according to the present invention may additionally comprise an adjuvant system. Any conventional adjuvant system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion inhibitors such as aminopolyphosphonates, particularly ethylene diamine tetramethylene phosphonic acid and diethylene pentamethylene phosphonic acid. triamine. Although less preferred for obvious environmental reasons, phosphate adjuvants can also be used herein.

Suitable adjuvants can be an organic ion exchange material, commonly an hydrous, inorganic aluminosilicate material, more particularly a synthetic, hydrated zeolite such as hydrated zeolite A, X, B, HS or MAP. Other suitable adjuvant, inorganic material is layered silicate, for example SKS-6 (Hoechst). SKS-6 is a stratified, crystalline silicate consisting of sodium silicate (Na2Si205). Suitable polycarboxylates containing a carboxy group include lactic acid, glycolic acid and ether derivatives thereof as described in Belgian Patents Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of succinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in Offenle-enschrift German Nos. 2,446,686 and 2,446,487, "U.S. Patent No. 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623 Polycarboxylates containing three carboxy groups include, in particular, water-soluble citrates, aconitrates and citraconates as well as succinate derivatives such as the carboxymethyloxysuccinates described in British Patent No. 1,379,241, the lactoxysuccinates described in the Netherlands Application No. 7205873, and the oxypolycarboxylate materials such as 2-oxa-1, 1 tricarboxylates, 3-propane described in British Patent No. 1, 387, 447. Polycarboxylate s containing four carboxy groups include oxydisuccinates described in British Patent No. 1,261,829, 1,1,1,2,2-ethane tetracarboxylates, 1,1,3,3-propane tetracarboxylates containing sulfo substituents include the sulfosuccinate derivatives described in British Patents Nos. 1,39,421 and 1,398,422 and in US Pat. No. 3,936,448 and the pyrolyzed, sulfonated citrates described in British Patent No. 1,082,179, while polycarboxylates containing phosphono substituents are described in US Pat. British Patent No. 1439,000. - The alicyclic and heterocyclic polycarboxylates include cyclopentan-cis, cis-cis-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydro-furan-cis, cis, cis-tetracarboxylates, 2,5-tetrahydro-furan-cis , discarboxylates, 2, 2, 5, 5, -tetrahydrofuran tetracarboxylates, 1, 2, 3, 4, 5, 6-hexane-hexacarboxylates and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include methyl acid, pyromellitic acid and the phthalic acid derivatives described in British Patent No. 1,425,343. From the foregoing, preferred polycarboxylates are hydroxy carboxylates containing up to three carboxy groups per molecule, more particularly citrates. Preferred adjuvant systems for use in the present compositions include a mixture of a water-insoluble aluminosilicate adjuvant such as zeolite A or a layered silicate (SKS-6), and a water-soluble carboxylate chelating agent. such as citric acid. A chelating agent suitable for inclusion in the detergent compositions according to the invention is ethylenediamine-N, N'-disuccinic acid (EDDS) or the alkali metal, alkaline earth metal, ammonium or substituted ammonium salts thereof, or mixtures thereof. The preordained EDDS compounds are the free acid form and the sodium or magnesium salt thereof. Examples of such preferred sodium salts of EDDS include Na2EDDS and Na4EDDS. Examples of such preferred magnesium salts of EDDS include MgEDDS and Mg2 EDDS. Magnesium salts are most preferred for inclusion in the compositions according to the invention. Preferred adjuvant systems include a mixture of a water-insoluble aluminosilicate adjuvant such as zeolite A, and a water-soluble carboxylate chelating agent such as citric acid. Other adjuvant materials that can form part of the adjuvant system for use in granular compositions include inorganic materials such as alkali metal carbonates, biocarbonates, silicates, and organic materials such as organic phosphonates, polyalkylene amino phosphonates and amino polycarboxylates. . Other suitable water-soluble organic salts are homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Polymers of this type are described in GB-A-1,596,756. Examples of such salts are polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, such copolymers having a molecular weight of 20,000 to 70,000, especially about 40,000. The salts of the detergent adjuvant are usually included in amounts of 5% to 80% by weight of the composition. Preferred levels of adjuvant for liquid detergents are from 5% to 30%.

Enzymes Preferred detergent compositions, in addition to the preparation of enzymes of the invention, comprise other enzyme (s) that provide a cleansing ability and / or benefits in tissue care. Such enzymes include other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g., laccases).

Proteases: Any other protease suitable for use in alkaline solutions can be used. Suitable proteases include those of animal, plant or microbial origin. The microbial origin is preferred. Chemically or genetically modified mutants are included. The protease may be a serine protease, preferably a microbial, alkaline protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 209, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (for example of porcine or bovine origin) and the Fusarium protease described in WO 89/06270. Preferred commercially available protease enzymes include those expired under the trade names Alcalase, Savinase, Primase, Durazym and Esperase by Novo Nordisk A / S (Denmark), those sold under the trade names Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the trade name Opticlean and Optimase by Solvay Enzymes. Protease enzymes can be incorporated in the compositions according to the invention at a level of 0.00001% to 2% protein of the enzyme by weight of the composition, preferably at a level of 0.0001% to 1% protein of the invention. the enzyme by weight of the composition, more preferably at a level of 0.001% to 0.5% protein of the enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% " the enzyme by weight of the composition.

Lipases: Any suitable lipase can be used for use in alkaline solutions. Suitable lipases include those of bacterial or fungal origin. Chemically and genetically modified mutants are included. Examples of useful lipases include a Humicola lanuginosa lipase, for example, as described in European patents Nos. 258 068 and 305 216, a Rhizomucor miehei lipase, for example, as described in European Patent No. 238 023, a lipase. Candida, such as a C. antarctic lipase, for example, lipase C. Antarctic A or B described in European Patent No. 214 761, a Pseudomonas upase such as a P lipase. alcaligenes and P. pseudoalcaligenes, for example, as described in European Patent No. 218 272, a lipase P. cepacia, for example, as described in European Patent No. 331 376, a lipase P. stutzeri, for example , as described in British Patent No. 1,372,034, a lipase P. fluorescens, a Bacillus lipase, for example, a lipase B. subti1is (Dartois et al., (1993), Biochemica et Biophysica act 1131, 253-260), a lipase B. stearothermophilus (JP 64/744992) and a lipase B. pumilus (WO 91 / 16422J). number of cloned lipases, including the lipase Penicillium camembertii described by yamaguchi et al., (1991), Gene 103, 61-67), the lipase Geotricum candidum (Schimada, Y. et al., (1989), J. Biochem., 106 , 383-388), and several Rhizopus lipases such as a R. delemar lipase (Hass, MJ et al. (1991), Gene 109, 117-113), a R. niveus lipase (Kugimiya et al., (19.92), Biosci , Biotech, Biochem 56, 716-719) and a R. oryzae lipase. Other types of lipolytic enzymes such as cutinases may also be useful, for example, a cutinase derived from Pseudomonas mendocin as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (for example described in WO 90/09446). - Especially suitable lipases are lipases such as Ml Lipase ™, Luma fast ™ and Lipomax ™ (Genencor), Lipolase ™ and Lipolase UltraMR (Novo Nordisk A / S), and Lipase P "Amano" (Amano Pharmaceutical Co.

Ltd.). Lipases are normally incorporated in the detergent composition at a level of 0.00001% to 2% protein of the enzyme by weight of the composition, preferably at a level of 0.0001% to 1% protein of the enzyme by weight of the composition. the composition, more preferably at a level of 0.001% to 0.5% protein of the enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein of the enzyme by weight of the composition.

Amylases: Any amylase _ (a and / or ß) suitable for use in alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. Chemically and genetically modified mutants are included. Amylases include, for example, α-amylases obtained from a special strain of B. licheniformis, described in more detail in British Patent No. 1,296,839. The commercially available amylases are Duramyl ™, Termamyl ™, Fungamyl ™ and BAN ™ (available from Novo Nordisk A / S) and Rapidase ™ and Maxamyl PM ™ (available from Genencor). The amylases are normally incorporated in the detergent composition at a level of 0.00001% to 2% protein of the enzyme by weight of the composition, preferably at a level of 0.0001% to 1% protein of the enzyme by weight of the composition. the composition, more preferably at a level of 0.001% to 0.5% protein of the enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein of the enzyme by weight of the composition.

Cells: Any cellulase suitable for use in alkaline solutions can be used. Suitable cellulases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cells are described in U.S. Patent No. 4,435,307, which describes fungal cells produced from Humicola insolens. Cellulases that are especially suitable are cellulase that have benefits in the care of color. Examples of such cellulases are the cellulases described in European Patent Application No. 0 495 257. Commercially available cells include Celluzyme ™ produced by a strain of Humicola insolens, (Novo Nordisk A / S) and KAC-500 (BJ MR (Kao Corporation). The cellulases are normally incorporated in the detergent composition at a level of 0.00001% to 2% protein of the enzyme by weight of the composition, preferably at a level of 0.0001% to 1% protein of the enzyme by weight of the composition, more preferably at a level of 0.001% to 0.5% of the enzyme's enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% of the enzyme protein by weight of the composition.

Peroxidases / Oxidases: Enzymes of peroxidases are used in combination with hydrogen peroxide or a source thereof (eg, a percarbonate, perborate or persulfate). Oxidases enzymes are used in combination with oxygen. Both types of enzymes are used for "bleaching by dissolution", ie to prevent the transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor, preferably together with an agent of increase as described for example in WO 94/12621 and WO 95/01426. Suitable peroxidases / oxidases include those of vegetable, baterian or fungal origin. They include - chemically or genetically modified mutants. The enzymes of peroxidase and / or oxidase are normally incorporated in the detergent composition at a level of 0.00001% to 2% protein of the enzyme by weight of the composition, preferably at a level of 0.0001% to 1% protein of the enzyme by weight of the composition, more preferably at a level of 0.001% to 0.5% protein of the enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein the enzyme by weight of the composition.

Mixtures of the enzymes mentioned above are included herein, in particular a mixture of a protease, an amylase, a lipase and / or a cellulose. The enzyme of the invention, or any other enzyme incorporated in the detergent composition, is normally incorporated in the detergent composition at a level of 0.00001% to 2% protein of the enzyme by weight of the composition, preferably in a level of 0.0001% to 1% protein of the enzyme by weight of the composition, more preferably at a level of 0.001% to 0.5% protein of the enzyme by weight of the composition, even more preferably in a level of 0.01% to 0.2% protein of the enzyme in weight of the composition.

Bleaching agents: Additional optional detergent ingredients that can be included in the detergent compositions of the present invention include bleaching agents such as PB1, PB4 and percarbonate with a particle size of 400-800 microns. These components of the bleaching agents may include one or more oxygen bleaching agents and, depending on the bleaching agent selected, one or more bleach activators.

When the present oxygen bleaching compounds will typically be present at levels of from about 1% to about 25%. In general, bleaching compounds are optional added components in non-liquid formulations, for example granular detergents. The bleach agent component for use herein may be any of the bleaching agents useful for detergent compositions including oxygen bleach as well as others known in the art. The bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent. One category of the oxygen bleaching agent that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloroperbenzoic acid, 4-nonylamino-4-oxyperoxybutyric acid and diperoxydecanedioic acid. Such bleaching agents are described in U.S. Patent No. 4,483,781, U.S. Patent No. 740,446, European Patent No. 0 133 354 and U.S. Patent No. 4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Patent No. 4,634,551. Another category of bleaching agents that can be used encompasses halogen bleaching agents. Examples of hypohalite bleaching agents, for example, include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulfonamides. Such materials are usually added in 0.5-10% by weight of the finished product, preferably 1-5% by weight. The hydrogen peroxide release agents can be used in combination with bleaching activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in US Pat. No. 4, 412, 934), 3,5-trimethyl -hexsanoloxybenzenesulfonate (ISONOBS, described in European Patent No. 120,591) or pentaacetylglucose (PAG), which are perhydrolyzed to form a peracid as the active bleaching species, leading to an improved bleaching effect. In addition, the decolorization activators of C8 (6-octanamido-caproyl) oxybenzenesulfonate, C9 (6-nonanamido caproyl) oxybenzenesulfonate and CIO oxybenzenesulfonate (6-decanamido caproyl) or mixtures thereof are very suitable. Also suitable activators are acylated citrate esters such as described in European Patent Application No. 91870207.7. Useful bleaching agents, including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleach compounds for use in cleaning compositions according to the invention, are described in the application USSN 08 / 136,626. Hydrogen peroxide can also be present by adding an enzyme system (ie an enzyme and a substrate accordingly) which is capable of generating hydrogen peroxide at the start or during the washing and / or rinsing process. Such enzyme systems are described in European Patent Application No. 0 537 381. - Bleaching agents other than oxygen bleaching agents are also known in the art and can be used herein. One type of oxygen-free bleaching agent of particular interest includes photoactivated bleaching agents such as sulfonated zinc and / or aluminum phthalocyanines. These materials can be deposited on the substrate during the washing process. With irradiation with light, in the presence of oxygen, such as when the fabrics are hung out to dry in daylight, the sulfonated zinc phthalocyanine is activated and, consequently, the substrate is bleached. The preferred zinc phthalocyanine and a photoactivated bleaching process are described in U.S. Patent No. 4,033,718. Typically, the detergent composition will contain about 0.025% to about 1.25% by weight, of sulfonated zinc phthalocyanine. The bleaching agents may also comprise a manganese catalyst. The manganese catalyst can, for example, be one of the described compounds - in the "Efficien anganese catalysts for low-temperature bleaching" Nature 369, 1994, pp. 637-639.

Soap suppressors: Another optional ingredient is a suds suppressor, exemplified by silicones, and silica-silicone mixtures. Silicones can be represented in general by alkylated polysiloxane materials, while silica is normally used in finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. These materials can be incorporated as particles, in which the suds suppressor or suds is advantageously incorporated advantageously into a waterproof, water-dispersible, substantially non-surfactant detergent-impermeable carrier. Alternatively, the suds suppressor can be dissolved or dispersed in a liquid carrier and applied by spraying on one or more of the other components. A preferred silicone suds control agent is described in U.S. Patent No. 3,933,672. Other particularly useful suds suppressors are self-emulsifying silicone suds suppressors, described in the German patent application DTOS 2,646,126. An example of such a compound is CD-544, commercially available from Dow Corning, which is a siloxane-glycol copolymer. The particularly preferred suds control agent is the suds suppressor system comprising a mixture of silicone oils and 2-alkyl alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R. Such suds suppressor system is described in European Patent Application No. 0 593 841.

Especially preferred silicone suds control agents are described in European patent application No. 92201649.8. The compositions may comprise a silicone / silica mixture in combination with non-porous, smoked silica such as Aerosil®. The suds suppressors described above are usually employed at levels of 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.

Other components: Other components used in detergent compositions such as soil suspending agents, soil release agents, optical brighteners, abrasives, bactericides, stain inhibitors, coloring agents, and / or encapsulated or non-encapsulated perfumes may be employed. . Especially suitable encapsulation materials are water soluble capsules which consist of a polysaccharide matrix and polyhydroxy compounds as described in GB 1,464,616. Other suitable water-soluble encapsulation materials comprise dextrins derived from acid esters of degelatinized starch of substituted dicarboxylic acids as described in U.S. Patent No. 3,455,838. These acid-ester dextrins are, preferably, starch preparations such as waxy maize, waxy sorghum, sorghum, tapioca and potato. Suitable examples of encapsulation materials include N-Lok manufactured by National Starch. The N-Lsk encapsulation material consists of a modified corn starch and glucose. The starch is modified by adding substituted, monofunctional groups such as octenyl succinic acid anhydride. Suitable antiredeposition and soil suspending agents herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or co-polymeric polycarboxylic acids or salts thereof. Polymers of this type include the polyacrylates and copolymers of maleic anhydride-acrylic acid previously mentioned as adjuvants, as well as copolymers of maleic anhydride with ethylene, methylvinyl ether or methacrylic acid, maleic anhydride constituting at least 20 mole percent of the copolymer. These materials are normally used at levels of 0.5% to 10% by weight, more preferably from 0.75% to 8%, much more preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic in character, examples of which are 4,4'-bis- (2-diethanolamino-4-anilono-s-triazin-6-ylamino) stilbene-2: 2'disulfonate disodium , 4, -4'-bis- (2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2: 2'-disodium disulfonate, 4,4'-bis- (2, 4-dianilino) -s-triazin-6-ylamino) stilbene-2: 2'-disodium disulfonate, 4 ', 4"-bis- (2,4-dianilino-s-tri-azin-6-ylamino) stilbene-2-sulfonate of monosodium, 4,4'-bis- (2-anilin-4- (N-methyl-N-2-hydroxyethylamino) -2-triazin-6-ylamino) stilbene-2,2'-disulfonate disodium, 4, 4 'bis- (4-phenyl-2, 1, 3-triazol-2-yl) -stilben-2, 2'-di-sodium disulfonate, 4,4' bis (2-anilin-4- (1- methyl-2-hydroxyethylamino) -s-triazin-6-ylamino) stilbene-2, 2 'disodium disulfonate, 2 (stilbe-4"- (naphtho-1', 2 ': 4, 5) -1 , 2, 3, - thiozol-2"-sodium sulphonate and 4,4'-bis (2-sulphotrisyl) biphenyl Other useful polymeric materials are polyethylene glycols , particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and more preferably about 4000. These are used at levels of 0.20% to 5%, more preferably 0.25% to 2.5% by weight. These polymers and the aforementioned homo- or co-polymeric polycarboxylate salts are valuable for improving the maintenance of whiteness, deposition of ash in the fabric, and cleaning capacity in the clay, proteinaceous and oxidizable soils in the presence of Transition metal impurities. The soil release agents useful in the compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and / or propylene glycol units in various arrangements. Examples of such polymers are described in U.S. Patent Nos. 4,116,885 and 4,711,730 and European Patent Application No. 0 272 033. A preferred, particular polymer according to European Patent No. 0 272 033 has the formula: (CH3 (PEG) 43) 0.75 (POH) 0.25 [T-PO] 2.β (T-PEG) 0.4] T (POH) 0.25 ((PEG) 43CH3) 0.75 where PEG is - (OC2H4) 0-, PO is (OC3H60) and T is (pOOC6H4CO).

- Modified polyesters are also very useful as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol, the final groups consisting primarily of sulfobenzoate and secondly of ethylene glycol mono esters and / or 1,2-propanediol. The objective is to obtain a polymer capped at both ends by the sulfobenzoate groups, "firstly", in the present context most of the copolymers herein will be capped at the ends by the sulfobezoate groups. However, some copolymers will be less than completely capped, and therefore their end groups may consist of ethylene glycol monoester and / or 1,2-propanediol, thereof being "second" in such species. The polyesters selected herein will contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of 1,2-propanediol, about 10% by weight of ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of sulfoisophthalic acid and have a molecular weight of about 3,000. The polyesters and their method of preparation are described in detail in European Patent No. 311 342.

Softening agents: The softening agents of. Fabrics can also be incorporated into laundry detergent compositions according to the present invention. These agents can be inorganic or organic in their type. Inorganic softening agents are exemplified by the smectite clays described in British Patent Application No. A-1 400898 and US Patent No. 5,019,292. Organic fabric softening agents include water insoluble tertiary amines as described in British Patent Application No. Al 514 276 and European Patent No. 0 011 340 and their combination with mono quaternary salts of 12 to 14 carbon atoms. they are described in European Patent No. B-0 026 528 and long chain diamides as described in European Patent No. 0 242 919. Other organic ingredients, fabric softening system tools include high molecular weight polyethylene oxide materials as described in European Patent No. 0 299 575 and 0 313 146. Esmectic clay levels are usually in the range of 5% to 15% , more preferably from 8% to 12% by weight, with the material being added as a mixed, dry component to the rest of the formulation. Organic fabric softening agents such as water-insoluble tertiary amines or long-chain diamine materials are incorporated at levels of 0.5% to 5% by weight, usually from 1% to 3% by weight while the materials of High molecular weight polyethylene oxide and water soluble cationic materials are added at levels from 0.1% to 2%, usually from 0.15% to 1.5% by weight. These materials are usually added to the spray-dried portion of the composition, although in some examples it may be more convenient to add them as a mixed, dried particulate material or spray them as a molten liquid over other solid components of the composition.

Polymeric Dye Transfer Inhibition Agents: The detergent compositions according to the present invention may also comprise from 0.001% to 10%, preferably from 0.01% to 2%, more preferably from 0.05% to 1% by weight of polymeric dye transfer inhibition agents. Polymeric dye transfer inhibiting agents are normally incorporated in the detergent compositions in order to inhibit the transfer of dyes from colored fabrics into fabrics washed therewith. These polymers have the ability to make complexes or adsorb the flushing temples washed out of the dyed fabrics before the dyes have the opportunity to become attached to other articles in the wash.

Especially suitable polymeric dye transfer inhibiting agents are polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polymers of polyvinylpyrrolidone, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. The addition of such polymers also increases the capacity of the enzymes according to the invention. The detergent composition "according to the invention may be in liquid, paste, gel, bar or granular form." Granules other than the powder may be produced, for example, as described in U.S. Patent Nos. 4,106,991 and 4,661,452. (both from Novo Industri A / S) and may optionally be coated by methods known in the art Examples of waxy coating materials are poly (ethylene oxide) (polyethylene glycol, PEG) products with average molecular weights of 1000 to 1000. 20,000, ethoxylated nonylphenols having from 16 to 50 ethylene oxide units, fatty alcohols, ethoxylates in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units, fatty alcohols; fatty acids, and mono- and di- and triglycerides of fatty acids Examples of suitable film-forming coating materials for application by fluidized-bed techniques are given in British Patent No. 1483591. The granular compositions according to the present invention can also be in "compact form", ie they can have a relatively higher density than conventional granular detergents, ie they form 550 to 950 g / 1; in such a case, the granular detergent compositions according to the present invention will contain a lower amount of "inorganic filler salt", compared to conventional granular detergents; the typical filler salts are alkaline earth metal salts of sulfates and chlorides, typically sodium sulfate; "Compact" detergent typically comprise no more than 10% filler salt. The liquid compositions according to the present invention may also be in the "concentrated form", in such case, the liquid detergent compositions according to the present invention will contain a lower amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergent is less than 30%, preferably less than 20%, much more preferably less than 10% by weight of the detergent compositions. The compositions of the invention can, for example, be formulated as laundry detergent compositions by hand and machine including laundry additive compositions and compositions suitable for use in the pretreatment of soiled fabrics, fabric softening compositions added to the rinse , and compositions for use in cleaning hard surfaces of the house, general and dishwashing operations. The following examples are proposed to exemplify the compositions for the present invention, but are not necessarily intended to limit or otherwise define the scope of the invention. In detergent compositions, the abbreviated identifications of the components have the following meanings: LAS: Sodium benzene sulfonate with 12 carbon atoms, linear, sodium TAS Tallow sodium sulfate XYAS Sodium alkyl sulfate of? XC-? YC SS: Secondary soap surfactant of the formula of 2-butyl octanoic acid 25EY: A primary, predominantly linear alcohol of 12 to 15 carbon atoms condensed with an average of Y moles of ethylene oxide 45EY: A predominantly linear primary alcohol of 14 to 15 carbon atoms with an average of Y moles of ethylene oxide XYEZS: Sodium alkyl sulfate of ?? C-? YC condensed with an average of Z moles of ethylene oxide per mole Non-ionic ethoxylated / propoxylated fatty alcohol, mixed, of 13 to 15 carbon atoms with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5, sold under the trade name Plurafax LF404 by BASF Gmbh CFAA: Alkyl N-methyl glucamide of 12 to 14 carbon atoms TFAA: Alkyl N-methyl glucamide of 16 to 18 carbon atoms Silicate: Amorphous Sodium Silicate (ratio of Si02: Na20 = 2.0) NaSKS-6 Stratified, crystalline silicate of the formula d-Na2Si20s Carbonate: Anhydrous sodium carbonate Phosphate Sodium Tripolyphosphate MA / AA: Maleic / acrylic acid copolymer 1: 4, weight. average molecular approximately 80,000 Polyacrylate Polyacrylate homopolymer with an average molecular weight of 8,000 sold under the trade name PA30 by BASF Gmgh Zeolite A: Hydrated Sodium aluminosilicate of the formula Na12 (A102Si02) 12.27H20 having a primary particle size in the range of 1 to 10 micrometers Citrate tri-sodium citrate dihydrate Citric acid Perborate citric acid Anhydrous sodium perborate monohydrate bleach substance, empirical formula NaB02.H202 PB4 Anhydrous sodium perborate tetrahydrate Percarbonate: Anhydrous sodium percarbonate bleaching substance of the empirical formula 2Na2C03.3H202 TAED; Tetraacetyl ethylene diamine CMC: Carboxymethyl cellulose sodium DETPMP Penta (methylene phosphonic acid) of diethylene triamine, marketed by Monsanto under the trade name Dequest 2060 PVP: Polyvinylpyrrolidone polymer EDDS Ethylenediamine-N, N'-disuccinic isomer [S, S] acid in the form of the sodium salt Suds 25% paraffin wax Pf 50 ° C, 17% hydrophobic silica, 58% Suppressor: Suds Granular paraffin oil: 12% Silicone / silica, 18% stearyl alcohol, 70% suppressant: starch in form granular Sulfate Sodium sulfate anhydrous HMWPEO: High molecular weight polyethylene oxide TAE 25 Tallow Alcohol Ethoxylate (25) Example of Detergent I _ A granular tissue cleaning composition according to the invention can be prepared as follows: Alkyl benzene sulfonate of 12 6.5 carbon atoms linear sodium Sodium sulfate 15.0 Zeolite A 26.0 Sodium Nitrilotriacetate 5.0 Enzyme of the invention 0.1 PVP 0.5 TAED 3.0 Boric acid 4.0 Perborate 18.0 Phenol sulfonate 0.1 Minors up to 100 Detergent Example II A granular, compact tissue cleaning composition (density 800 g / 1) according to the invention can be prepared as follows: 45AS 8.0 25E3S 2.0 25E3 3.0 25E3 3.0 TFAA 2.-5 Zeolite A 17.0 NaSKS-6 12.0 Citrus acid 3.0 Carbonate 7.0 MA / AA 5.0 CMC 0.4 Enzyme of the invention 0.1 TAED 6.0 Percarbonate 22.0 EDDS 0.3 Suppressor of granular suds 3.5 Water / minors Up to 100% Detergent Example III Granular fabric cleaning compositions according to the invention which are especially useful in the laundry of colored fabrics were prepared as follows: LAS 10.7 - TAS 2.4 - TFAA - 4.0 45AS 3.1 10.0 45E7 4.0 _ 25E3S 3.0 68E11 1. 25E5 - 8.0 Citrate 15.0 7.0 Carbonate - 10 Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 - 9.0 MA / AA 5.0 5.0 DETPMP 0.2 0.8 Enzyme of the invention 0.10 0.05 Silicate 2.5 -Sulfate 5.2 3.0 PVP 0.5 Poly (4-vinylpyridin) -N- 0.2 Oxide / copolymer of vinylimidazole and vinylpyrrolidone Perborate 1.0 Phenol sulfonate 0.2 Water / Minors Up to 100% Detergent Example IV The fabric cleaning compositions, granular according to the invention which provide a "softness through washing" capability, can be prepared as follows: 45AS 10.0 LAS 7.6 68AS 1.3 45E7 4.0 25E3 5.0 Coconut-alkyl-1.4 1.0 dimethyl hydroxy-ethyl ammonium citrate 5.0 3.0 Na-SKS-6 11.0 Zeolite A 15.0 15.0 MA / AA 4.0 4.0 DETPMP 0.4 0.4 Perborate 15.0 - Percarbonate - 15.0 TAED 5.0 5.0 Esmectic Clay 10.0 10.0 HMWPEO - 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0 Carbonate 10.0 10.0 Suppressor of granular suds 1.0 4.0 CMC 0.2 0.1 Water / Minors Up to 100% jara DETERGENT EXAMPLE V Heavy-duty liquid fabric cleaning compositions according to the invention can be prepared as follows: I II LAS acid form - 25.0 Citric acid 5.0 2.0 Acid form of 25AS 8.0 - Acid form of 25AE2S 3.0 - 25AE7 8.0 - CFAA 5 - DETPMP 1.0 1.0 Fatty acid Oleic acid - 1.0 Ethanol 4.0 6.0 Propanediol 2.0 6.0 Enzyme the invention 0.10 0.05 Coconut-alkyl _ 3.0 dimethyl hydroxy ethyl ammonium Smelic clay 5.0 PVP 2.0 Water / Children Up to 100% APPLICATIONS IN THE LEATHER INDUSTRY - A subtyla of the invention can be used in the leather industry, in particular for use in hair removal.

In said application, a subtyla variant of the invention is preferably used in an enzyme composition which also comprises another protease. For a more detailed description of other suitable proteases see the section referring to the enzymes suitable for use in a detergent composition (vi of supra).

APPLICATIONS IN THE WOOL INDUSTRY A subtyla of the invention can be used in the wool industry, in particular for use in the cleaning of fabrics comprising wool. In said application, a subtyla variant of the invention is preferably used in an enzyme composition which also comprises another protease. For a more detailed description of other suitable proteases see the section referring to enzymes suitable for use in a detergent composition (vi of s upra). The invention is described in further detail in the following examples which can not be proposed in any way to limit the scope of the invention as claimed.

MATERIALS AND METHODS Strains: B. subtilis DN1885 (Diderichsen et al., 1990). B. The 309 and 147 are specific strains of Bacill us l ent, deposited with the NCIB and granted accession numbers NCIB 10309 and 10147, and described in US Pat. No. 3,723,250 incorporated by reference herein. E. coli MC 1000 (M.J. Casadaban and S.N. Cohen (1980); J. Mol. Bi ol. , 138 179-207), was made r ", m + by conventional methods and is also described in the US patent application Serial No. 039,298.

Plasmids pJS3: shuttle vector E. coli - B. subtilis containing a synthetic gene encoding subtylase 309. (described by Jacob Schi0dt et al in Protein and Peptide letters 3: 39-44 (1996)). pSX222: expression vector of B. subtilis (described in WO 96/34946).

General Molecular Biology Methods: Unless otherwise mentioned, DNA manipulations and transformations were performed using normal methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (Eds.) "Current Protocols in Molecular Biology." John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Bilogical Methods for Bacillus." John Wiley and Sons, 1990). Enzymes for DNA manipulations were used according to the specifications of the suppliers.

Enzymes for DNA manipulations Unless otherwise mentioned, all enzymes mentioned for DNA manipulations, such as for example restriction endonucleases, ligases, etc., are obtained from New England Biolabs, Inc.

Proteolytic Activity In the context of this invention, the proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU). The activity is determined relatively to a normal enzyme (SAVINA? EÓ), and the determination is based on the digestion of a solution of dimethyl casein (DMC) by the proteolytic enzyme under normal conditions, ie 50 ° C, pH 8.3, 9 min. reaction time, 3 min. measurement time. A leaflet AF 220/1 is available with the request to Novo Nordisk A / S, Denmark, a leaflet which is included by reference. A GU is a Glycine Unit, defined as the activity of the proteolytic enzyme which, under normal conditions, during an incubation of 15 minutes at 40 degrees C, with N-acetyl casein as a substrate, produces an amount of the NH2 equivalent group to 1 mmol of glycine. - The activity of the enzyme can also be measured using the PNA enzyme, according to the reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in the Journal of American Oil Chemists Society, Rothgeb, TM, Goodlander, BD, Garrison, PH, and Smith, LA, (1988).

Fermentation The fermentation of subtylase enzymes was carried out at 30 ° C on a rotating shaking table (300 r.p.m.) in Erlenmeyer flasks with 500 ml baffles containing 100 ml of BPX medium for 5 days. Consequently, in order to make a broth of, for example, 2 liters, 20 Erlenmeyer flasks were fermented simultaneously.

Means: BPX: Composition (per liter) Potato starch 100g Ground barley 50g Soybean meal 20g Na2HP04 X 12 H20 9g Pluronic 0. lg Sodium caseinate lOg The starch in the medium is liquefied with α-amylase and the medium is sterilized on heating at 120 ° C for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by the addition of 0.1 M NaHCO3.

EXAMPLES EXAMPLE 1 Construction and Expression of Enzyme Variants: Mutagenesis is directed to the self o Variants directed to the site of subtilasa 309 were made through the "elimination of the single site (USE) "or the" Uracil-USE "technique described respectively by Deng et al. (Anal. Biochem. 200: 81-88 (1992)) and Markvardsen et al.

(BioTechniques 1 ((3): 371-372 (1995)) The template plasmid was pJS3, or an analog thereof containing a variant of Subtilase 309, for example the USE mutagenesis was performed on the analog of pJS3 which contains a gene encoding the N252L variant with an oligonucleotide targeted to the construction of a T255I variant resulting in a variant of Subtilase 309 N252L + final T255I Subtilase 309 variants constructed of pJS3 were then subcloned into the expression plasmid of B. subtili s pSX222, using the restriction enzymes Kpnl and Mlul. Mu randomized taguanescalized: The total category used to perform random mutagenesis, localized was: a mutagenic primer (oligonucleotide) was synthesized, which corresponds to the part of the DNA sequence to be mutagenized except for the nucleotide (s) corresponding to the codon (s) of amino acids to be mutagenized (s). The resulting mutagenic was used in a PCR reaction with an appropriate opposite primer. The resulting PCR fragment was purified and digested and cloned in a shuttle vector E. coli -B. subtili s. Alternatively and if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a second opposing primer, suitable to allow digestion and cloning of the mutagenized region in the shuttle vector. The PCR reactions were performed under normal conditions. Following this strategy, a random library was constructed, located in SAVINASE where positions N252, T255 and S259 were completely randomized. An oligonucleotide was synthesized with 25% of each of the four bases (N) in the first and second bases in the desired amino acid codons to be mutagenized. The third nucleotide (the unstable base) in the codons was synthesized with 50% G / 50% C (S) to avoid two (TAA, TGA) of the three stop codons.

The mutagenic primer (5-C TTC TGC GTT AAC AAG TCC GCT TCC ATA CAA GTT CGT SNN TCC TAA ACT SNN TGC CGT SNN CTT TAG ATG ATT-3 (anti-sense)) was used in a PCR reaction with a primer opposite, suitable (for example 5 'GAA CTC GAT CCA GCG ATT TC 3' (sense) and the plasmid pJS3 as template This resulting PCR product was cloned into the shuttle vector pJS3 by using the restriction enzymes Xhol and Hpal The randomized, localized library constructed of pJS3 was then subcloned into the expression plasmid B. subtilis pSX22, using the restriction enzymes Kpnl and Mlul The prepared library contained approximately 100,000 individual clones / library. serially ordered to confirm the designated mutations In order to purify a subtylase variant of the invention, the expression plasmid B. subtilis pSX222 comprising a variant of the invention was transformed into a B subtil strain. The component was fermented as described above in a medium containing 10 μg / ml Chloramphenicol (CAM).

EXAMPLE 2 Purification of the Enzyme Variants: This process refers to the purification of a 2-liter scale fermentation of the enzyme Subtilisin 147, the enzyme Subtilisin 309 or mutants thereof. About 1.6 liters of fermentation broth was centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The supernatants were adjusted to pH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filter plates. The filtrates were concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate was centrifuged and filtered before absorption at room temperature in a Bacitracin affinity column at pH 7. The protease was eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1M sodium chloride in a buffer solution with 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7. Fractions with the protease activity of the Bacitracin purification step were combined and applied to a 750 ml Sephadex G25 column (5 ml). cm in diameter) balanced with a buffer containing 0.01 dimethylglutaric acid, 0.2 M boric acid and 0.002 m calcium chloride adjusted to pH 6.5. The fractions with proteolytic activity of the Sephadex G25 column were combined and applied to a CM Sepharose CL 6B cation exchange column of 150 ml (5 cm diameter) equilibrated with a buffer containing dimethylglutaric acid 0.01, boronic acid 0.2 M, and Calcium chloride 0.002 M adjusted to pH 6.5. The protease was eluted using a linear gradient of 0-0.1 M sodium chloride in 2 liters of the same buffer (0-0.2 M sodium chloride in the case of Subtilisin 147). In a final purification step, the protease containing the fractions from the CM Sepharose column were combined and concentrated in an "Amicon" ultrapurification cell equipped with a GR81PP membrane (from the Danish Sugar Factories Inc.). techniques of Example 1 for the construction and the previous isolation process, the following Subtilisin 309 variants were produced and isolated: N252L + T255I N252V + T255A N252M + T255C + S259H N252S + T255E + S259C; and N252K + T255S + S259C.

EXAMPLE 3 Washing Capacity of the Detergent Compositions Comprising the Enzyme Variants The following examples provide the results of a number of wash tests that were conducted under the indicated conditions EXPERIMENTAL CONDITIONS Table VI: Experimental conditions for the evaluation of Subtilisin 309 variants The detergent used is a simple model formulation. The pH is adjusted to 10.5 which is within the normal range for a powder detergent. The composition of the model 95 detergent is as follows: % STP (Na5P3O10) 25% Na2S04 10% Na2C03 20% LAS (Nansa 8 OS) 5.0% Nonionic surfactant (Dobanol 25-7) 5.0% Na2S i205 0.5% Carboxymethylcellulose (CMC) 9.5% Water The hardness of the water was adjusted by adding CaCl2 and MgCl2 (Caz +: Mg2 + = 2: 1) to deionized water (see also Surfactants in Consumer Products - Theory, Technology and Application, Springer Verlag 1986). The pH of the detergent solution was adjusted to pH 10.5 by the addition of HCl. The measurement of the reflectance (R) in the test material was made at 460 nm using a Macbeth ColorEye 7000 photometer (Macbeth, Division of Kollmorgen Instruments Corporation, Germany). The measurements were made according to the manufacturers protocol. The washing capacity of Subtilisin 309 variants were evaluated when calculating a capacity factor: "• vari before False P = ^ Savinase" -Tx-Fal so P: Ranand capacity factor 'Rel ßctanci a of the test material with the variety Rsa in se: Ref l ectance of the washed test material Savinas e® RFaiso • Reflectance of the washed test material without enzyme All the subtilisin 309 variants claimed have improved the washing capacity compared to Savinase® - ie P > 1. The variants were divided into improved classes designated with capital letters: Class A 1 < P < 1.5 Class B 1.5 < P < 2 Class C P > 2 Table V: Subtilisin 309 variants and improved classes It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property.

Claims (24)

1. A subtylase enzyme variant having an improved washing ability in detergents, characterized in that it comprises modification (s) in the position (s) 252, 255 and 259 (in the BASBPN numbering).

2. A variant of subtylase enzyme having an improved washing ability in detergents, characterized in that it comprises at least one modification selected from the group comprising (in the BASBPN numbering): 252L + 255I 252V + 255A 252M + 255C + 259H 252S + 255E + 259C 252K + 255S + 259C; or a variant comprising one or more conservative modification (s) in any of the variants mentioned above (for example, a conservative modification of a variant 252L (aa hi drofóbi co) +2551 includes variants such as 2521 (aa hydrophobic) co) + 2551, and 252V (aa hi drofóbi co) +2551.

3. The subtylase enzyme variant according to claim 2, characterized in that the modification is selected from the group comprising (in the BASBPN numbering): N252L + T255I N252V + T255A N252M + T255C + S259H N252S + T255E + S259C N252K + T255S + S259C; or a variant comprising one or more conservative modification (s) in any of the variants mentioned above (for example, a conservative modification of a N252L variant (a.a. hi drof óbi co) +12551 includes variants such as N252I (a.a. hi drof óbi co) +12551, and N252V (a.a. hi drofóbico) + T255I.

4. The variant of any of claims 1 to 3, characterized in that the mother subtilase is selected from the sub-group I-SI.

5. The variant according to claim 4, characterized in that the mother subtylase is selected from the group comprising ABSS168, BASBPN, BSSDY and BLSCAR or functional variants thereof which have retained the characteristic of subgroup I-SI.

6. The variant according to any of claims 1 to 3, characterized in that the mother subtylase is selected from the sub-group I-S2.

7. The variant according to claim 6, characterized in that the mother subtylase is selected from the group comprising BLS147, BLS309, BAPB92, TVTHER and BYSYAB or functional variants thereof which have retained the characteristic of sub-group I-S2.

8. The variant according to any of the preceding claims, wherein the modification (s) are / are combined with one or more modification (s) in any other position.

9. The variant according to claim 8, characterized in that the modification (s) are / are combined (s) with the modification (s) in one or more of the positions 27, 36, 57, 76, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224, 235 and 274.

10. The variant according to claim 9, characterized in that the subtilase belongs to sub-group I-S2 and the additional change is selected from the group comprising K27R, * 36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Y167A, Y167I, R170S, R170L, R170N, Q206E, N218S, M222S, M22_A, T224S, K235L, and T274A.

11. The variant according to claim 10, characterized in that it comprises any of the variants V104N + S101G, K27R + V104Y + N123S + T274A, or N76D + V104a, or other combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with any one or more of the substitutions, deletions and / or insertions mentioned in any of claims 1 to 10.

12. The subtilase variant of any of the preceding claims, wherein the modification (s) are / are combined (s) with the modification (s) in one or more of the positions 129, 131, 133 and 194

13. The variant according to claim 2, characterized in that the subtilase belongs to the sub-group I-S2 and the additional change is selected from the group comprising P129K, P131H, A133P, A133D and A194P.

14. An isolated DNA sequence encoding a subtilase variant of any of claims 1 to 13.

15. An expression vector, characterized in that it comprises an isolated DNA sequence of claim 14.

16. A host cell, microbial, transformed with an expression vector of claim 15.

17. The microbial host according to claim 16, characterized in that it is a bacterium, preferably a Bacillus, especially B. l enthus

18. The microbial host according to claim 16, characterized in that it is a fungus or yeast, preferably a filamentous fungus, especially an Aspergillus.

19. A method for producing a variant according to any of claims 1 to 13, characterized in that a host of any of claims 16 to 18 is cultured under conductive conditions for the expression and secretion of the variant, and the variant is recovered.

20. A composition, characterized in that it comprises a subtilase variant according to any of claims 1 to 13.

21. The composition according to claim 20, characterized in that it additionally comprises a cellulose, lipase, cutinase, oxidoreductase, another protease or an amylase.

22. The composition according to claim 20 or 21, characterized in that the composition is a detergent composition.

23. The use of a subtilase variant according to any one of claims 1 to 13 or an enzyme composition according to any of claims 20 to 22 in a laundry detergent and / or for dish washing.

24. A process for the identification of a protease variant that exhibits improved washing ability in detergents, characterized in that it comprises effecting a mutation in the DNA encoding a subtylase enzyme or its pre- or preproenzyme in one or more of the corresponding positions. an amino acid (in the BASBPN numbering): N252L + T255I N252V + T255A N252M + T255C + S259H N252S + T255E + S259C N252K + T255S + S259C; or a variant comprising one or more conservative modification (s) in any of the variants mentioned above; transform a strain of Bacillus with the mutated DNA; select strains that produce such protease variants; ferment / cultivate such strain; recover the protease variant, and examine the improved washing ability in detergents.