MXPA99004108A - Subtilase variants and compositions - Google Patents

Abstract

Enzymes produced by mutating the genes for a number of subtilases at positions 167 and 170 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 SUBSTITUTE COMPOSITIONS FIELD OF THE INVENTION This invention relates to new mutant protease enzymes or enzymatic variants useful for formulating detergent compositions and exhibiting improved performance under washing conditions in detergents; cleaning compositions and detergents containing these enzymes; genes that have undergone mutation that code for the expression of enzymes when they are inserted into a suitable host cell or organism; and the host cells transformed with these and capable of expressing the enzymatic variants. BACKGROUND OF THE INVENTION In the detergent industry for more than 30 years enzymes have been applied in washing formulations. The enzymes used in these formulations comprise proteases, lipases, amylases, cellulases as well as other enzymes or mixtures thereof. From a commercial point of view, the most important enzymes are proteases. An increasing number of proteases for commercial use are variants of engineered proteins (genetically modified) of proteases that occur naturally, for example DURAZYM® (Novo Nordisk A / S), RELASE® (Novo Nordisk A / S), MAXAPEM ® P1269 / 99MX (Gist Brocades, NV), PURAFECT® (Genecor International, Inc.) In addition, various protease variants are described in the art, as in EP 130756 (GENETECH) (which corresponds to the U.S. Pat. Re-published No. 34,606 (GENENCOR), EP 214435 (HENKEL), WO 87/04461 (AMGEN), WO 87/05050 (GENEX), EP 260105 (GENENCOR), Thomas, Russell and Fersht (1985) Nature 318 375- 376; Thomas, Russell and Fersht (1987) J. Mol. Biol. 193 803-813; Rusell and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); 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 5,543,302 (SOLVAY S.A) EP 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK A / S) WO 91/00345 (NOVO NORDISK A / S); EP 525 610 Al (SOLVAY) and WO 94/02618 (GIST BROCADES N.V.). However, although various variants of useful proteases have been described, there is still a need for new variants of improved proteases for various industrial uses. Therefore, an object of the present invention is to provide improved variants of proteases of genetically modified proteins, especially for use in the detergent industry. ni. r a / a t? SUMMARY OF THE INVENTION Recently it has been identified that the subtilase variant that has improved behavior under washing conditions can be obtained by substituting one or more amino acid residues located in a hydrophobic domain of the subtyla of origin or in the vicinity thereof for a amino acid residue more hydrophobic than the original residue, the hydrophobic domain includes the residues corresponding to residues 1165, Y167, Y171 of BLS309 and the residues in the neighborhood thereof comprise residues corresponding to residues E136, G159, S164, R170 , A194 and G195 of BLS309 (WO / 34946). Based on this information, the inventors of the present have intensively studied several of the possible combinations of residues Y167 and R170 of SAVINASE® and identified several of the variants with improved performance and surprisingly increased in washing. For more details reference is made to the working examples (see below). Accordingly, the present invention relates in its first aspect to a subtylase protease variant having improved wash conditions in detergents, which comprises modifications in both position 167 and 170. In a second aspect the invention is refers to a variant of subtylase enzyme which has improved washing conditions in detergents, comprising at least one modification selected from the group comprising (in the BASBPN numbering): 167 (G, A, S or T.}. +170 (G, A, S or T.}. 167 { G, A, S or T.}. +170 { L, I or V.}. 167 (G, A, S or T.) }. +170 (Q or N.}. 167P 167P + 170 (L, I or V.}. 167 { L, I, or V.}. +170 (G, A, S or T.}. 167 {L, I or V.}. +170 { Q or N.}. 167P + 170 { G, A, S or T.}. 167 { L, I, or V} +170 { L, I, or V.}. 167 { F, W or Y.}. +170 (G, A, S or T.}. 167 { F, W or Y .}. + .170 { E or D.}. 167 (F, W or Y.}. +170 { R, K or H.}. 167 { F, W or Y.}. + 170 (L, I, or V.}. 167 { L, I, or V.}. 170H 167 { G, A, S or T.} The previous nomenclature, for example, the variant "167 { G, A, S or T.}. +170 { G, A, S or T.}. "Is a matrix nomenclature, where the term" 167 (G, A, S or T) + 170 (G, A, S or T.}. "Comprises the following variants: 167G + 170G, 167G + 170A, 167G + 170S, 167G + 170T, 167A + 170G, 167A + 170A, 167A + 170S, 167A + 170T, 167S + 170G, 167S + 170A, 167S + 170S, 167S + 170T, 167T + 170G, 167T + 170A, 167T + 170S or 167T + 170T For more details related to the nomenclature of a subtile variant in the present, see the "Definitions" section in the present (see below). 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 microbial host cell transformed with an expression vector according to the fourth aspect. In another aspect the invention relates to the production of subtilisin enzymes of the invention by means of inserting an expression vector according to the fourth aspect into a suitable microbial host, cultivating the host to express the desired subtylase enzyme and recovering the enzyme product. . Moreover, the invention relates to a composition comprising a subtyla variant of the invention. Finally, the invention relates to the use of mutant enzymes for various industrially important uses, in particular for use in cleaning compositions comprising mutant enzymes, in particular, detergent compositions comprising the mutant subtilisin enzymes.

DEFINITIONS Before discussing this invention in more detail, the following terms will be defined first.

Amino Acid Nomenclature A = Ala = Alanine V = Val = Valine L = Leu = Leucine I = lie = Isoleucine P = Pro = Proline F = Phe = Phenylalanine W = Trp = Tryptophan M = Met = Methionine G = Gly = Glycine S = Ser = Serine T = Thr = Threonine C = Cys = Cysteine Y = Tyr = Tyrosine N = Asn = Asparagine Q = Gln = Glutamine D = Asp = Aspartic Acid E = Glu = Glutamic Acid K = Lys = Lysine R = Arg = Arginine H = His = Histidine X = Xaa = Any amino acid Nucleic acid nomenclature A = Adenine G = Guanine C = Cytosine T = Thymine (only in DNA) U = Uracil (only in RNA) Nomenclature of variants To describe the various enzymes produced or contemplated according to the invention, the following nomenclatures have been adapted for easy reference: Original amino acid (s) (s) position (s) amino acid (s) substituent (s) According to this substitution glycine for acid Glutamic at position 195 would be designated as: Glyl95Glu or G195E a deletion of glycine at the same position is: Glyl95 * or G195 * and the insertion of an additional amino acid residue such as lysine is: Glyl95GlyLys or G195GK Where a deletion is indicated compared to the sequence used for numbering, an insertion at that position is indicated as: * 36Asp or * 36D for the insertion of an aspartic acid at position 36. Multiple mutations are separated by plus signs, for example: Argl70Tyr + Glyl95Glu or R170Y + G195E that represent mutations in positions 170 and 195 that substitute arginine and glycine for tyrosine and glutamic acid, respectively. When the original amino acid (s) can comprise any amino acid, then only position (s) and amino acid (s) are mentioned as substituent (s), for example: 170Ser. This nomenclature is of particular importance in relation to the modification (s) according to the invention in homologous subtilases (see below). 170Ser then comprises, for example, both a Lysl70Ser modification in BASBPN and an Argl70Ser modification in BLSSAVI. See Figure 1 in relation to the examples. When the amino acid (s) substituent (s) can (n) comprise any amino acid, then only the original amino acid (s) and the position (s) are mentioned, for example: Argl70.

When both the original amino acid (s) and the amino acid (s) substituent (s) can comprise any amino acid, then only the position (s) is mentioned, for example: 170. When the amino acid (s) and / or the amino acid (s) substituent (s) can comprise more than one and not all of the amino acids, then the selected amino acids are enclosed by a " {..}. ", for example: Argl70. { Gly, Ala, Ser or Thr} comprising the variants Argl70Gly, Argl70Ala, Argl70Ser or Argl70Thr; or for example Tyrl67. { Gly, Ala, Ser or Thr} + Argl70. { Gly, Ala, Ser or Thr} comprising the Tyrl67Gly + Argl70Gly variants, Tyrl67Gly + Argl70Ala, Tyrl67Gly + Argl70Ser Tyrl67Gly + Argl70Thr, Tyrl67Ala + Argl70Gly, Tyrl67Ala + Argl70Ala, Tyrl67Ala + Argl70Ser, Tyrl67Ala + Argl70Thr, Tyrl67Ser + Argl70Gly, Tyrl67Ser + Argl70Ala, Tyrl67Ser + Argl70Ser, Tyrl67Ser + Argl70Thr , Tyrl67Thr + Argl70Gly, Tyrl67Thr + Argl70Ala, Tyrl67Thr + Argl70Ser, or Tyrl67Thr + Argl70Thr. This nomenclature is of particular importance with respect to a modification (s) of a conservative amino acid of preferred subtylase variants of the invention.

For example, one (s) modification (s), for example, of a preferred variant Tyrl67Ala + Argl70Ser are Tyrl67. { Gly, Ala, Ser or Thr} + Argl70. { Gly, Ala, Ser or Thr} , which substitute here a small amino acid for another small amino acid. See the section "Detailed description of the invention" for more details.

Proteases The enzymes that unfold amide bonds in protein substrates are classified as proteases or (indistinctly) peptidases (see Walsh, 1979, In zyma t i c Rea c t i on Mechani sms, W. H. Freeman and Company, San Francisco, Chapter 3).

Numbering of amino acid positions / residues If not stated otherwise, the amino acid numbering used herein corresponds to that of the BPN 'subtylase sequence (BASBPN). For further description of the BPN1 sequence see Siezen et al., Protein Engng. 4 (1991) 719-737 and Figure 1.

Serine proteases A serine protease is an enzyme that catalyzes the hydrolysis of peptide bonds and in which there is an essential serine residue in the active site (White, Handler and Smith, 1973"Prin cipi es of Bi ochemi s try, "Fífth Edition, McGraw-Hill Book Company, NY, pp. 271-272). The bacterial serine proteases have molecular weights in the range between 20,000 and 40,000 Daltones. They are inhibited with diisopropyl fluorophosphate. They hydrolyse simple terminal esters and are similar in activity to eukaryotic chymotrypsin, which is also a serine protease. A more restricted term, alkaline protease, which covers a subgroup, reflects the high optimum pH of some serine proteases, between pH 9.0 and 11.0 (for review, see Priest (1977) Ba cteri ol olog i ca! Rev. 41 711 -753).

Subtilases A sub-group of serine proteases designated tentatively as subtilases has been proposed by Siezen et al., Pro tein Engn. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously called proteases analogous to subtilisin. A subtilisin was previously defined as a serine protease produced by Gram positive or fungal bacteria and according to Siezen et al. currently it is a sub-group of subtilasas. A wide variety of subtilases have been identified and the amino acid sequence of several subtilases has been determined. For a more detailed description of these subtilases and their amino acid sequences, reference is made herein to Siezen et al. and to Figure 1. A subgroup of subtilases, I-SI, comprises the "classical" subtilisins, such as subtilisin 168, subtilisin BPN1, subtilisin Carlsberg (ALCALASE®, NOVO NORDISK A / S) and subtilisin DY. Another sub-group of the subtilases I-S2, is recognized by Siezen et al. (above). The proteases of subgroup I-S2 are described as very alkaline subtilisins and comprise enzymes such as BP92 (MAXACAL®, Gist Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK a / s), subtilisin 147 (ESPERASE®, NOVO NORDISK A / S) and alkaline elastase YaB.

"SAVINASE®" The SAVINASE® is marketed by NOVO NORDISK A / S. It is subtilisin 309 of B.Lentus and is distinct from BABP92 only because it has N87S (see Figure 1 in this).

Subtilase precursor The term "subtilasa precursor" is a subtyla defined by Siezen et al. (Protein Engineering 4: 719-737 (1991)). For more details see the description of "SUBTILASAS" in the foregoing. A subtyla precursor may also be a subtylase isolated from natural sources, in which subsequent modification has been made while retaining the characteristic of a subtylase. Alternatively, the term "subtilasa precursor" can be called "subtylase type natural".

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

Subtylase variant In the context of this invention, the term subtylase or subtylase variant that has undergone mutation is applied to a subtyla that has been produced by an organism which expresses a mutant gene derived from a precursor microorganism that possesses an original gene or precursor and which produces the corresponding precursor enzyme, the precursor gene that has undergone mutation to produce the mutant gene from which subtylase protease with mutation is produced occurs when expressed in a suitable host.

Subtylase homologous sequences SAVINASE® subtyla specific amino acid residues are identified herein by modification to obtain a subtyla variant of the invention. However, the invention is not limited to modifications of this particular subtilase, but extends to other precursor subtilases (natural type), which have a primary structure homologous to that of SAVINASE®. To identify other homologous subtilases, within the scope of this invention, an alignment of this subtylase (s) was performed to a previously aligned group of subtilases. A comparison was made of the 18 most conserved residues in subtilases. The 18 most conserved residues are shown in Table I (see Siezen et al. For more details regarding the conserved residues).

Table I 18 more conserved residues in subtilases Position Residue conserved 23 G 32 D 34 G 39 H 64 H 65 G 66 T 70 G 83 G 125 S 127 G 146 G 154 G 155 N 219 G 220 T 221 S 225 P After alignment allowing the insertions and deletions necessary to maintain the proper alignment, identify the homologous residues. The homologous residues can then be modified according to the invention. By using CLUSTALW (version 1.5, April 1995) computerized alignment program (Thompson, JD, Higgins, DG and Gibson, TJ (1994) Nuecleic Acids Research, 22: 4673-4680), with an open GAP penalty of 10.0 and a GAP extension penalty of 0.1, using the BLOSUM30 protein weight matrix, achieves the alignment of a given subtyle with a group of subtilases previously aligned by the option Profile alignments of the program. For a given subtyla that is within the scope of the invention, preferably 100% of the 18 most conserved residues must be conserved. However, an alignment greater than or equal to 17 of the 18 residues or as low as 16 of those conserved residues is also adequate to identify the homologous residues. In subtilases, the conservation of the catalytic triad Asp32 / His64 / Ser221 must be maintained. The previously defined alignment is shown in Figure 1, where the percent identity of the individual subtilases is also shown in this alignment with respect to the 18 most conserved residues. In addition, in the process for identifying a homologous precursor subtyla (natural type) within the scope of the invention, the 18 residues conserved above are related to the primary precursor sequence (wild type) of the homologous precursor subtylase. In other words, if a subtyla precursor has been modified in any of the 18 most conserved residues mentioned above, that is the original natural type precursor sequence in the 18 conserved residues, which determines whether both the original precursor subtylase and a possible variant of that subtylase precursor, whether or not they are a homologous subtilase, according to the scope of the present invention. Based on this description it is routine for a person skilled in the art to identify suitable 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 Homologous subtilases and corresponding homologous residues, suitable for modification according to the invention It is evident that a similar or larger table covering other homologous subtilases can be easily generated by a person skilled in the art field.

Behavior in washing conditions The ability of an enzyme to catalyze the degradation of various substrates that occur naturally in the objects to be cleaned during washing, for example, is often referred to as its washing ability, Washing ability, detergency or behavior in washing conditions. Throughout this application the term behavior in the washing conditions will be used to encompass this property.

Isolated DNA sequence The term "isolated", when applied to a DNA sequence molecule, indicates that the DNA sequence has been removed from its natural environment and thus has been released from other foreign or undesirable coding sequences and it is in a form suitable for use in systems for the production of proteins obtained by genetic engineering. These isolated molecules are those that are separated from their natural environment and include gnenomic clones cDNA. The isolated DNA molecules of the present invention are free of other genes with which they are normally associated, but may include 5 'and 3' untranslated regions that occur naturally as promoters and terminators. The identification of associated regions will be apparent to one skilled in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). The term "an isolated DNA sequence" can alternatively be referred to as "a cloned DNA sequence".

Isolated protein When applied to a protein, the term "isolated" indicates that the protein is in a state different from its native environment. In a preferred form, the isolated protein is practically free of other proteins, in particular of other homologous proteins (ie, "homologous impurities" (see below)). It is preferred to provide the protein in a fairly purified form, for example, with more than 40% purity, more than 60% purity, more than 80% purity, more preferably more than 95% purity and even more preferably more than 99% purity, as determined by SDS-PAGE. The term "isolated protein" can be alternatively called "purified protein".

Homologous Impurities The term "homologous impurities" means any impurity (e.g., another polypeptide than the polypeptide of the invention) that originates from the homologous cell from which the polypeptide of the invention is originally obtained.

Obtained from The term "obtained from" in the sense in which it is used herein in relation to a specific microbial source, is applied to the polynucleotide and / or the polypeptide produced by the specific source or by a which has been inserted a gene from the source.

Substrate The term "Substrate" used in relation to a substrate for protease should be interpreted in its broadest sense since it comprises a compound that contains at least one peptide bond capable of being hydrolyzed by a protease subtilisin.

Product The term "product" used in relation to the product derived from an enzymatic protease reaction in the context of this invention should be interpreted as a term that 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 DRAWINGS Figure 1 shows an alignment of several homologous subtilases, which are aligned with the 18 most conserved residues in the subtilases. The 18 most conserved residues are highlighted with bold. All subtilases shown, except JP170, have 100% identity in the conserved residues. JP170 has an "N" instead of a "G" in the conserved residues G146.

DETAILED DESCRIPTION OF THE INVENTION Subtylase variants with improved wash conditions: Numerous variants of subtilas of the invention are analyzed herein and show improved performance under washing conditions in detergents (see examples of operation herein (see below) )). Accordingly, one embodiment of the invention relates to a variant of subtylase enzyme according to the second aspect of the invention, wherein the modification is selected from the group comprising (in BASBPN numbering): 167A + 170S 167A + 170L 167A + 170N 167P 167P + 170L 167V + 170T 167I + 170T 167V + 170Q 167S + 170Q 167T + 170N 167A + 170A 167T + 170L 167T + 170A 167P + 170S 167I + 170L 167F + 170T 167F + 170E 167F + 170H 167F + 170T 167F + 170L 167L 170H 167S The present inventors have identified variants with improved wash performance in BLS309 (SAVINASE®), which in its primary natural type precursor sequence includes Y167 and R170 as the original natural type amino acids (see Figure 1).

Accordingly, another embodiment of the invention relates to a variant of subtylase enzyme according to the second aspect of the invention, wherein the modification is selected from the group comprising (in BASBPN numbering): Y167. { G, A, S or T.}. + R170. { G, A, S or T.}. Y167 { G, A, S or T.}. + R170. { L, I, or V.}. Y167 { G, A, S or T.}. + R170. { Q or N.} Y167P Y167P + R170. { L, I, or V.}. Y167 { L, I, or V.}. + R170. { G, A, S or T.}. Y167 { L, I, or V.}. + R170. { Q or N.} Y167P + R170 (G, A, S or T.} .Y167 {L, I, or V.}. + R170 {L, I, or V.} .Y167 {F, W or Y .}. + R170 { G, A, S or T.} .Y167 { F, W or Y.}. + R170 { E or D.} .Y167 { F, W or Y .}. + R170 (R, K or H.} .Y167 (F, W or Y.). + R170 (L, I, or V.} .Y167 {L, I, or V.} .R170H Y167 {G, A, S or T.}., Or more preferably a subtylase enzyme variant according to the immediate previous modality, wherein the modification is selected from the group comprising (in BASBPN numbering): Y167A + R170S Y167A + R170L Y167A + R170N Y167P Y167P + R170L Y167V + R170T Y167I + R170T Y167V + R170Q Y167S + R170Q Y167T + R170N Y167A + R170A Y167T Y167T + R170A Y167P + R170S Y167I + R170L Y167F + R170T Y167F + R170E Y167F + R170H Y167F + R170T Y167F + R170L Y167L R170H Y167S It is well known in the art that replacing an amino acid with a similar conservative amino acid only produces a minor change in the characteristics of the enzyme Table III below lists g rupos of conservative amino acids.

Table III Conservative amino acid substitutions Basics: R = arginine K = lysine H = histidine Acids: E = glutamic acid D = aspartic acid Polar: Q = glutamine N = asparagine Hydrophobic: L = leucine I = isoleucine V = valine M = methionine Aromatics: F = phenylalanine W = tryptophan Y = tyrosine Small: G = glycine A = alanine S = serine T = threonine Accordingly, subtylase variants such as 167A + 170S, 167G + 170S, 167S + 170S and 167T + 170S, will have similar improvements in the behavior in washing conditions. In addition, the subtilase variants such as Y167G + R170S, Y167S + R170S and Y167T + R170S, will have an improvement in performance under washing conditions similar to the variant Y167A + R170S. See, for example, the examples of operation herein for a performance test under specific wash conditions of the variant Y167A + R170S. Based on the foregoing and in particular on the numerous subtyla variants exemplified herein, it is routine work, for one skilled in the art, to identify other suitable conservative modification (s), in particular those of the art. exemplified variants, to obtain a subtilase variant with improved washing conditions, according to all aspects and modalities of a subtyla variant of the invention. In embodiments of the invention, the subtilases of interest are those that belong to subgroups I-SI and I-S2. With regard to the subgroup I-SI, the preferred subtyla precursor is selected from the group comprising ABSS168, BASBPN, BSSDY and BLSCAR or functional variants thereof which have retained the characteristic of subgroup I-SI. In relation to subgroup I-S2, the preferred precursor subtylase is selected from the group comprising BLS147, BLS309, BAPB92, TVTHER AND BYSYAB or functional variants thereof which have retained the characteristic of subgroup I-S2. In particular, the subtyla precursor is BLS309 (SAVINASE® NOVO NORDISK A / S).

The present invention also comprises any one or more modifications in the aforementioned positions in combination with any other modification to the amino acid sequence of the precursor enzyme. In particular, combinations are conceived with other modifications known in the art to impart improved properties to the enzyme. Various subtylase variants with different improved properties are described in the field of the art and several of them are mentioned in the "Background of the invention" section herein (see above). These references are set forth herein as references to identify a subtyla variant of the invention. Such combinations include positions 222 (improves stability to oxidation), 218 (improves thermal stability) substitutions at the Ca-binding sites that stabilize the enzyme, eg, position 76 and many others evident from the prior art. In other embodiments, a subtilase variant of the invention can advantageously be combined with one or more modifications in any of the positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 206, 218, 222 , 224, 235 and 274. Specifically, the following variants BLS309 and BAPB92 are considered suitable for the combination: K27R, * 36D, S57P, N76D, S87N, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Q206E, N218S, M222S, M222A, T224S, K235L and T274A. In addition, variants comprising any of the variants S101G + V104N, S87N + S101G + V104N, 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 of one or more of the modifications mentioned above exhibit improved properties. Still other subtilase 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 give a higher level of expression to a subtyla variant of the invention. For more details reference is made to working examples in the present (see below). Accordingly, still another embodiment of the invention relates to a variant according to the invention, wherein the modification is selected from the group comprising: Y167A + R170S + A194P Y167A + R170L + A194P Y167A + R170N + A194P Y167A + R170S + P129K Y167A + R170L + P129K Y167A + R170N + P129K Y167A + R170S + P131H Y167A + R170L + P131H Y167A + R170N + P131H Y167A + R170S + A133P Y167A + R170L + A133P Y167A + R170N + A133P Y167A + R170S + A133D Y167A + R170L + A133D Y167A + R170N + A133D PRODUCTION OF MUTILATIONS IN SUBTILASE GENES Many methods for cloning a subtyla of the invention and for introducing mutations in the genes (for example, subtylase genes) are well known in the art. In general, standard procedures for cloning genes and introducing mutations (random and / or site-directed) into these genes can be used to obtain a suitable variant of the invention. For further description of suitable techniques reference is made to working examples herein (see below) and (Sambrook et al.) (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F.M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Hardwood, C.R., and Cutting, S.M. (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 that conveniently can be subjected to recombinant DNA procedures and the selection of the vector will often depend on the host cell in which it is entered. In this way, the vector can be an autonomous replication vector, ie a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector can be that which, when introduced into a host cell, integrates into the host cell genome in part or in its entirety and replicates together with the chromosome (s) in which It has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is functionally linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA or may contain elements of both. The term "functionally linked" indicates that the segments are positioned in such a way that they function harmoniously for their intended purposes, for example, transcription is initiated in a promoter and follows through the DNA sequence encoding the enzyme. The promoter can be any sequence of DNA that shows transcriptional activity in the selected host cell and can be derived from genes that code for proteins either homologous or heterologous to the host cell. Examples of suitable promoters for use in bacterial host cells include the maltogenic amylase gene gene from Bacillus stearothermophilus, the alpha-amylase gene from Bacillus licheniformis, the alpha-amylase gene from Bacillus amyloliquefaciens, the alkaline protease gene from Bacillus subtilis, the gene from Bacillus pumilus xylosidase or the Lambda PR or PL phage promoters or the E.coli lac, trp or tac promoters. The DNA sequence encoding the enzyme of the invention, if necessary, can also be functionally connected to a suitable terminator.

The recombinant vector of the invention can further comprise a DNA sequence that allows the vector to replicate in the host cell in question. The vector may also comprise a selectable marker, for example a gene whose product complements a defect in the host cell or a gene which codes for resistance, for example, to antibiotics such as kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycin or the like or resistance to heavy metals or herbicides. To direct an enzyme of the present invention to the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) can be provided to the recombinant vector. The secretory signal sequence is linked to a DNA sequence encoding the enzyme in the correct reading frame. Sequences of secretory signals are generally placed at the 5 'position of the DNA sequence encoding the enzyme. The secretory signal sequence may be one that is normally associated with the enzyme or may be derived from a gene encoding another secreted protein. The methods used to ligate the DNA sequences encoding the enzyme of the present, the promoter and optionally the terminator and / or secretory signal, respectively or to assemble these sequences by suitable PCR amplification schemes and to insert them into suitable vectors which contain the information necessary for replication or integration are well known to those skilled in the art (see, for example, Sambrook et al., op.cit.).

HOSPITAL CELL The DNA sequence encoding the present enzyme that is introduced into the host cell can be homologous or heterologous to the cell in question. If it is homologous to the host cell, that is, produced by the host cell naturally, it will typically be functionally connected to another promoter sequence or, if applicable, to another secretory signal sequence and / or terminator sequence apart from its natural environment . The term "homologous" is used to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is used to include a DNA sequence not expressed in nature by the host cell. In this way, the DNA sequence can be derived from another organism or can be a synthetic sequence. The host cell in which the DNA construct or the recombinant vector of the invention is introduced can be any cell that is capable of producing the present enzyme and includes bacteria, yeasts, fungi and larger eukaryotic cells. Examples of bacterial host cells which, in culture, are capable of producing the enzyme of the invention are gram-positive bacteria such as strains of Bacillus, 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 J9. thuringiensis or strains of Streptomyces, such as S. lividans or S. murinus or gram-negative bacteria such as Escherichia coli. The transformation of the bacterium can be effected by protoplast transformation, electroporation, conjugation or using competent cells in a manner that is known per se (see Sambrook et al., (Above) .When the enzyme is expressed in bacteria 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 first case, the cells are used and the granules are recovered and denatured after which the enzyme is reconcentrated by dilution of the denaturing agent. In the latter case, the enzyme can be recovered from the periplasmic space by rupturing the cells, for example, by sonication 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 strains of Bacillus or Streptomyces, the enzyme can be retained in the cytoplasm or can be directed to the extracellular medium by a sequence of bacterial secretion. In the latter case, the enzyme can be recovered from the medium in the same manner as described above.

METHOD FOR PRODUCING SUBTILASE The present invention provides a method for 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 heterologous recombinant production of the enzyme of the invention.

It is therefore possible to make a highly purified subtyla composition, characterized in that it is free of homologous impurities. In this context, homologous impurities means any impurity (eg, other polypeptides than the enzyme of the invention) that originate from the homologous cell from which the enzyme of the invention is originally obtained. The medium used to culture the transformed host cells can be any conventional means suitable for developing the host cells in question. The expressed subtylase can be conveniently secreted into the culture medium and can be recovered from it by well-known methods, including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components from the medium using a salt such as sulfate. of ammonium, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography or the like.

USE OF A SUBTILASE VARIANT OF THE INVENTION A subtylase protease variant of the invention can be used for various industrial applications, in particular the detergent industry.

Additionally the invention relates to an enzymatic composition, which comprises a subtyla variant of the invention. A summary of the preferred industrial applications and the corresponding preferred enzyme compositions is described below. This summary is by no means intended to be a complete list of suitable applications of a subtyla variant of the invention. The subtilase variants 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 COMMENTS COMPRISING MUTATING ENZYMES The present invention comprises the use of the mutant enzymes of the invention in cleaning and detergent compositions and these compositions comprise the mutant subtilisin enzymes. Cleaning and detergent compositions are well described in the field and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of these cleaning compositions and suitable detergents. Further reference is made to the working examples herein that show the improvements in washing behavior for several of the subtyla variants of the invention.

EXPECTATION AND EXAMPLES OF DETERGENTS Surfactant system The detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant can be selected from nonionic and / or anionic and / or cationic and / or ampholytic and / or zwitterionic surfactants and / or semi polar. The surfactant is typically present at levels between 0.1% and 60% by weight. The surfactant is preferably formulated to be compatible with the enzyme components present in the composition. In liquid or gel compositions the surfactant is preferably formulated in such a way as to promote or at least not degrade the stability of any enzyme in these compositions. Preferred systems for use in accordance with the present invention comprise as the surfactant one or more of the nonionic and / or anionic surfactants described herein. Polylene, polypropylene and polybutylene oxide condensates and alkylphenols are suitable for use as nonionic surfactants of the surfactant systems of the present invention and are the preferred polylene oxide condensates. These compounds include the condensation products of alkyl phenols having an alkyl group containing between about 6 and 14 carbon atoms, preferably between about 8 and 14 carbon atoms, either a straight chain or branched chain configuration with the alkylene oxide. In a preferred embodiment, the lene oxide is present in an amount between about 2 and about 25 moles, more preferably between about 3 and about 15 moles, of lene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include Igepal ™ CO-630, marketed by GAF Corporation and Triton ™ X-45, X-114, X-100 and X-102, all marketed by Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (eg, alkyl phenol xylates). The condensation products of primary and secondary aliphatic alcohols with about 1 to 25 moles of lene oxide are suitable for use as a nonionic surfactant of the nonionic surfactant system of the present invention. The alkyl chain of the aliphatic alcohol may be linear or branched, primary or secondary and generally contains between about 8 and about 22 carbon atoms. Preferred are condensation products of alcohols having an alkyl group containing between about 8 and about 20 carbon atoms, more preferably between about 10 and 18 carbon atoms, with about 2 to 10 moles of lene oxide per mole of alcohol. Approximately between 2 and about 7 moles of lene oxide and preferably superlative between 2 and 5 moles of lene oxide per mole of alcohol are present in these condensation products. Examples of commercially available nonionic surfactants of this type include Tergitol ™ 15-S-9 (The condensation product of a Cn-C15 linear alcohol with 9 moles of lene oxide), Tergitol ™ 24-L-6 NMW (the product of condensation of a C12-C14 primary alcohol with 6 moles of lene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; Neodol ™ 45-9 (condensation product of a C14-C15 linear alcohol with 9 moles of lene oxide), Neodol ™ 23-3 (condensation product of a C12-C13 linear alcohol with 3.0 moles of lene oxide), Neodol ™ 45-7 (condensation product of a CX4-C15 linear alcohol with 7 moles of lene oxide), Neodol ™ 45-5 (condensation product of a C14-C15 linear alcohol with 5 moles of lene oxide) marketed by Shell Chemical Company, Kyro ™ EOB (condensation product of a C13-C15 alcohol with 9 moles of lene oxide), marketed by Procter & Gamble Company and Genapol LA 050 (condensation product of a C12-C14 alcohol with 5 moles of lene oxide) marketed by Hoechst. The preferred HBL range in these products is 8-11 and preferably superlative 8-10. Also useful as nonionic surfactants of the surfactant systems of the present invention are the alkylpolysaccharides disclosed in U.S. Pat., 565,647, having a hydrophobic group containing between about 6 and 30 carbon atoms, preferably between about 10 and 16 carbon atoms and a polysaccharide, for example, a polyglycoside, a hydrophilic group containing between about 1.3 and 10, preferably between about 1.3 and 3, preferably superlative between about 1.3 and about 2.7 units of saccharide. Any reducing saccharide containing between 5 or 6 carbon atoms can be used, for example, glucose, galactose and galactosyl moieties can substitute for the glucosyl moieties (optionally the hydrophobic group is attached in the 2-, 3-, 4- positions -, etc, leaving, in this way, a glucose or galactose as opposed to a glucoside or galactoside). The intersaccharide bonds may be, for example, between a position of the additional saccharide units and positions 2-, 3-, 4- and / or 6- of the preceding saccharide units. The alkyl polyglycosides that are preferred have the formula R20 CnH2nO) t (glycosyl) wherein R is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl and mixtures thereof in which the alkyl groups contain between about 10 and 18, preferably between about 12 and 14, carbon atoms; n is 2 or 3, preferably 2; t is between 0 and approximately 10, preferably 0; and x is between about 1.3 and 10, preferably between about 1.3 and 3, more preferably between about 1.3 and 2.7. The glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or the alkylpolyethoxy alcohol is first formed and then reacted with glucose or a glucose source, to form the glucoside (attached at position 1). The additional glycosilic units can then be linked between their position 1 and positions 2-, 3-, 4-, and / or 6 of the preceding glycosilic units, preferably predominantly in position 2. The oxide condensation products of ethylene with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as additional non-ionic surfactants of the nonionic surfactant systems of the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight between about 1500 and 1800 and will exhibit insolubility in water. The addition of polyoxyethylene fractions to this hydrophobic portion tends to increase the water solubility 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 some of the commercially available Pluronic ™ surfactants marketed by BASF. Also suitable for use as nonionic surfactants 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 fraction of these products is constituted by the reaction product of ethylenediamine and an excess of propylene oxide and in general have a molecular weight between about 2500 and 3000. This hydrophobic fraction is condensed with ethylene oxide to a point in the that the condensation product contains between about 40% and 80% by weight of polyoxyethylene and has a molecular weight between about 5,000 and 11,000. Examples of this type of nonionic surfactants include some of the commercially available Tetronic ™ compounds marketed by BASF. Preferred for use as nonionic surfactants of the surfactant systems of the present invention are the condensates of polyethylene oxide and alkyl phenols, the condensation products of primary and secondary aliphatic alcohols with about 1 to 25 moles of ethylene oxide, the alkyl polysaccharides. and the mixtures thereof. Most preferred are C8-C14 alkyl phenol ethoxylates having between 3 and 15 ethoxy groups and C8-C18 alcohol ethoxylates (preferably on average C10) having between 2 and 10 ethoxy groups and mixtures thereof. Most preferred nonionic surfactants are the polyhydroxy fatty acid amide surfactants of the formula R2 - C - N - Z, II II, O R wherein R1 is H or R1 is C1-4 hydrocarbyl, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R is C5_31 hydrocarbyl and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyl directly attached to the chain or an alkoxylated derivative thereof. Preferably R is methyl, R is linear C 1 - C 2 -C 6 alkyl or a C 16 -C 18 alkyl or alkenyl chain such as an alkyl derived from coconut or mixtures thereof and Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose in a reductive amination reaction. Most preferred anionic surfactants include alkyl alkoxylated sulfate surfactants. Examples of these are the salts or water-soluble acids of the formula RO (A) mS03M wherein R is an unsubstituted C 10 -C 24 -alkyl or a hydroxyalkyl group having a C 10 -C 24 alkyl component, preferably an alkyl or a hydroxy C12-C20 alkyl, more preferably an alkyl or a C12-C18 hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and 6, more preferably between about 0.5 and 3 and M is H or a cation that can be, for example, a metal cation (for example, sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or a cation substituted with ammonium. The alkyl ethoxylated sulfates as well as the alkyl propoxylated sulfates are contemplated herein. Specific examples of ammonium substituted cations include methyl-, dimethyl-, tri-ethylammonium cations and quaternary ammonium cations such as tetramethyl ammonium and dimethyl piperidinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof and the like. Surfactants to be exemplified are C12-C18 alkyl sulphide polyethoxylate (1.0) (C12-Clß E (10) M), C12-C18 alkyl sulphonate polyethoxylate (2.25) (C12-C18 (2.25) M), C12 alkyl sulfate C18 polyethoxylate (3.0) (C12-C18E (3.0) M) and C12-C18 alkyl sulphate polyethoxylate (4.0) (C12-C18E (4.0) M), wherein M is conveniently selected from sodium and potassium. Suitable anionic surfactants for use are the alkyl sulfonate ester surfactants which include linear esters of C8-C20 carboxylic acids (eg, fatty acids) which are sulfonated with S03 according to "The Journal of the American Oil Chemists Society", 52 (1975). ), pp. 323-329. Suitable raw materials could include natural fatty substances such as tallow derivatives, palm oil, etc. Alkyl sulphonate ester surfactants, especially for laundry applications, comprise alkyl sulfonate ester surfactants of the structural formula: OR R - CH - C - OR S03M wherein R is a C8-C20 hydrocarbyl, preferably an alkyl or a combination thereof, R4 is a hydrocarbyl CL-Cg, preferably an alkyl or a combination thereof, and M is a cation which forms a salt soluble in water with the alkyl sulfonate ester. Suitable cations to form the salt include metals such as sodium, potassium and lithium and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethanolamine and triethanolane. Preferably, R is C10-C16 alkyl and R4 is methyl, ethyl or isopropyl. Especially preferred are methyl ester sulfonates wherein R is C 10 -C 16 alkyl. Other suitable anionic surfactants include the alkyl sulfate surfactants which are salts or water soluble acids of the formula ROS03M wherein R is preferably a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl component, more preferably a C12-C18 alkyl or hydroxyalkyl and M is H or a cation, for example an alkali metal cation (for example, 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 piperidinium cations and quaternary ammonium cations derived from alkylamines such as ethylamine, diethylamine, triethylamine and mixtures thereof and the like). Typically, C12-C16 alkyl chains are preferred for low wash temperatures (e.g., below about 50 ° C) and lower alkyl chains are preferred for higher wash temperatures (e.g., above about 50). ° C). Other anionic surfactants useful for detergency purposes may 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 C8-C22 primary or secondary alkanesulfonates, C8-C24 olefinsulfonates, sulfonated polycarboxylic acids. prepared by sulfonation of the pyrolyzed product of alkali metal citrates, for example, those described in the description of British Patent No. 1,082,179, C8-C24 alkyl polyglycol ether sulfates (containing up to 10 moles of ethylene oxide); alkyl glycerol sulfonates, glycerol fatty acid acyl sulfonates, glycerol fatty acid oleyl sulphates, ethylene oxide ether sulphates and alkyl phenol, parafin sulfonates, alkyl phosphates, isethionates such as acyl isethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated C 12 -C 18 monoesters) and diesters of sulphosuccinates (especially saturated and unsaturated diesters C 6) -C12), acyl sarcosinates, alkylpolysaccharide sulfates such as the alkyl polyglycoside sulphates (the non-sulfated nonionic compounds are described below), branched primary alkyl sulphates and alkyl polyethoxy carboxylates as those of the formula RO (CH2CH20) k-CH2-COO- M + where R is a C 8 -C 22 alkyl, k is an integer between 1 and 10 and M is a soluble salt forming cation. Resin acids and hydrogenated resin acids are also suitable, such as rosin, hydrogenated rosin and resin acids and hydrogenated resin acids present in tall oil or derivatives thereof. The alkylbenzene sulfonates are quite preferred. Especially preferred are linear alkylbenzene sulphonates (straight chain) (LAS) wherein the alkyl group preferably contains between 10 and 18 carbon atoms. Other examples are described in "Surface Active Agents and Detergents" (Vol. I and II by Schwartz, Perry and Berch). A variety of surfactants are also generally described in U.S. Patent 3,929,678 (Column 23, from line 58 to Column 29, line 23, which is considered part of the present, as a reference). When included herein, the laundry detergent compositions of the present invention typically comprise between about 1% and 40%, preferably between about 3% and 20% by weight, of anionic surfactants. The laundry detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic and semi-polar surfactants, as well as non-ionic and / or anionic surfactants other than those already described herein. Cationic detergent surfactants suitable for use in the laundry detergent compositions of the present invention are those having a long chain hydrocarbyl group. Examples of these cationic surfactants include ammonium surfactants such as alkyltrimethylammonium halides and surfactants having the formula: [R (OR3) [R4 (OR3) and] 2R5N + X- wherein R2 is an alkyl or alkylbenzyl group having 8 to 18 carbon atoms in the alkyl chain, each R3 is selected from the group consisting of -CH2CH2 -, -CH2CH (CH3) -, CH2CH (CH2OH) -, -CH2CH2CH2-, and mixtures thereof, each R is selected from the group consisting of C1-C4 alkyl, hydroxyalkyl C -C ^ benzyl ring structures formed at join the two groups R4, -CH2CHOHCHOHCOR6CHOHCH2OH, wherein R6 is any hexose or hexose polymer having a molecular weight less than 1000 and hydrogen when and is not 0; R is equal to R4 or is an alkyl chain, wherein the number total of carbon atoms or R plus R is not greater than about 18, each y is between 0 and about 10, and the sum of the values of y is between 0 and about 15, and x is any compatible anion, and quite preferred cationic surfactants are water-soluble quaternary ammonium compounds useful in the present composition which They have the formula: R1R2R3R4N + X "(i) wherein R x is C 8 -C 16 alkyl, each of R 2, R 3, and R 4 is independently C 1 -C 4 alkyl hydroxy C 1 -C 4 alkyl, benzyl and (C 2 H 40) X H wherein x has a value between 2 and 5 and X is an anion No more than one of R2, R3 or R4 should be benzyl. The preferred length in the alkyl chain for Rj is C12-C1S, particularly when the alkyl group is a mixture of chain lengths derived from coconut oil or palm kernel oil or synthetically derived by olefin formation or synthesis of alcohols 0X0. Preferred groups for R2, R3 and R4 are methyl and hydroxyethyl groups and the anion X can be selected from the halide, methosulfate, acetate and phosphate ions. Examples of suitable quaternary ammonium compounds of formula (i) to be used herein are: alkyl chloride (derived from coconut oil) trimethyl ammonium chloride or bromide; alkyl chloride or bromide (derived from coconut oil) methyl dihydroxyethyl ammonium; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C12-C1S dimethyl hydroxyethyl ammonium chloride or bromide; alkyl chloride or bromide (derived from coconut oil) dimethyl hydroxyethyl ammonium; Methyl Trimethyl Ammonium Methyl Sulfate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide; choline esters (compounds of formula (i) wherein R is CH2-CH2 -0-C-C12.14 alkyl and R3R4 are methyl) or di-imidazolines [compounds of the formula (i)]. Other cationic surfactants useful herein are also disclosed in U.S. Patent 4,228,044 and EP 000 224. When included herein, the laundry detergent compositions of the present invention typically comprise between 0.2% and about 25%, preferably between about 1% and 8% by weight of the cationic surfactants. Ampholytic surfactants are also suitable for use in the detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines or aliphatic derivatives of heterocyclic secondary and 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 between about 8 and 18 carbon atoms and at least one contains an anionic group which is solubilized in water, for example carboxy, sulfonate, sulfate. See U.S. Patent 3,929,678 (column 19, lines 18-35) for examples of these ampholytic surfactants. When included herein, the detergent compositions of the present invention typically comprise between 0.2% and about 15%, preferably between about 1% and 10% by weight of these ampholytic surfactants. Zwitterionic surfactants are also suitable for the detergent compositions of the present invention. 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 compound, quaternary phosphonium or tertiary sulfonium. See U.S. Patent 3,929,678 (column 19, line 38 to column 22, line 48) for examples of zwitterionic surfactants. When included herein, the detergent compositions of the present invention typically comprise between 0.2% and about 15%, preferably between about 1% and 10% by weight of these zwitterionic surfactants. Semi-polar nonionic surfactants are a special category of non-ionic surfactants which include water-soluble amine oxides containing an alkyl fraction with approximately 10 to 18 carbon atoms and 2 fractions selected from the group consisting of alkyl and hydroxyalkyl groups containing between about 1 and 3 carbon atoms; water-soluble phosphine oxides containing an alkyl fraction with approximately 10 to 18 carbon atoms and 2 fractions selected from the group consisting of alkyl and hydroxyalkyl groups containing between 1 and 3 carbon atoms and water soluble sulfoxides containing an alkyl fraction with approximately between 10 and 18 carbon atoms and a fraction selected from the group consisting of alkylamide and hydroxyalkylene fractions with approximately between 1 and 3 carbon atoms. Semi-polar non-ionic detergent surfactants include the amine oxides having the formula: O t R3 (OR4) xN (R5) 2 wherein R3 is an alkyl, hydroxyalkyl or alkyl phenyl group or mixtures thereof containing between about 8 and 22 carbon atoms; R 4 is an alkylene or hydroxyalkylene group containing between about 2 and 3 carbon atoms or mixtures thereof; x is between 0 and about 3: and each R is an alkyl or hydroxyalkyl group containing between about 1 and 3 carbon atoms or a polyethylene oxide group containing between about 1 and 3 ethylene oxide groups. The R groups can be linked together, for example, through an oxygen or nitrogen atom, to form a ring structure. These amine oxide surfactants in particular include C10-C1 alkyl oxides dimethyl amine and C8-C12 alkoxy ethyl dihydroxy ethyl amine oxides. When included herein, the laundry detergent compositions of the present invention typically comprise between 0.2% and about 15%, preferably between about 1% and 10% by weight of these semi-polar nonionic surfactants.

Fortifier System The compositions according to the present invention may further comprise a fortifier system. Any conventional fortifier system suitable for use herein include aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphosphonates, in particular ethylenediamine tetramethylene phosphonic acid and diethylene triamine pemtamethylene phosphonic acid. Although with less preference for obvious environmental reasons, phosphate fortifiers can also be used here. Suitable fortifiers may also be an inorganic ion exchange material, usually an inorganic hydrated aluminosilicate material, in particular a hydrated synthetic zeolite such as the hydrated zeolite A, X, B, HS or MAP. Another inorganic fortifier material is layered silicate, for example SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na2Si205). Suitable polycarboxylates containing a carboxy group including lactic acid, glycolic acid etherified derivatives thereof are set forth 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) acetic acid, maleic acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid as well as the ether carboxylates described in Germán Offenle- enschrift 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 carboxymethyloxysuccinates described in British Patent No. 1,379,241, Lactoxysuccinates described in Dutch Application 7205873 and oxypolycarboxylate materials such as 2-oxa-1,1,3-propanedicarboxylates described in British Patent No. 1,387,447. Polycarboxylates containing four carboxy groups include oxydisuccinates disclosed in British Patent No. 1,261,829, 1,1,2,2-ethane tetracarboxylates, 1, 1,3, 3-propaned tetracarboxylates containing sulfo substituents include the sulfosuccinate derivatives disclosed in British Patents Nos. 1,398,421 and 1,398,422 and in U.S. Patent No. 3,936,448 and the sulfonated pyrolysed citrates described in British Patent No. 1,082,179, while polycarboxylates containing phosphonium substituents are disclosed in British Patent No. 1,439,000. Alicyclic and heterocyclic polycarboxylates include cyclopentan-cis, cis, cis-tetracarboxylates, cyclopentadienido pentacarboxylates, 2,3,4, 5- tetrahydrofuran-cis, cis, cis-tetracarboxylates, 2,5-tetrahydrofuran-cis, dicarboxylates, 2,2,5,5-tetrahydrofuran tetracarboxylates, 1, 2, 3, 4, 5, 6-hexan-hexacarboxylates and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include pyromellitic acid and phthalic acid derivatives set forth in British Patent No. 1,425,343. Of the above, the polycarboxylates that are preferred are the hydroxy carboxylates which contain up to three carboxy groups per molecule, in particular, the citrates. Fortifier systems that are preferred for use in the present compositions include a water-insoluble silicate alumina fortifier mixture such as zeolite A or a layered silicate (SKS-6) and a water soluble carboxylate chelating agent such as citric acid. A suitable chelator for inclusion in the detergent compositions according to the invention is ethylenediamine-N, '-disuccinic acid (EDDS) or the alkali metal, alkaline earth, substituted ammonium salts or mixtures thereof. The EDDS compounds that are preferred are the acid form and salts of sodium or magnesium thereof. Examples of the EDDS sodium salts that are preferred include Na2EDDS and Na4EDDS. Examples of the magnesium salts of EDDS that are preferred include MgEDDS and Mg2EDDS. Magnesium salts are the most preferred to be included in the compositions according to the invention. Preferred fortifier systems include a mixture of a water insoluble aluminosilicate fortifier such as zeolite A and a soluble carboxylate chelating agent such as citric acid. Other fortifier materials that can be part of the fortifier system for use in granular compositions include inorganic materials such as carbonates, bicarbonates, alkali metal silicates and organic materials such as organic phosphonates, amino polyalkylene phosphonates and amino polycarboxylates. Other suitable water-soluble organic salts are homo- or copolymeric 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 disclosed in GB-A-1,596,756. Examples of these salts are the polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, these copolymers have a molecular weight between 20,000 and 70,000, especially about 40,000. Builder salts for detergency are usually included in amounts between 5% and 80% by weight of the composition. Preferably the fortifier levels for liquid detergents are between 5% and 30%.

Enzymes Preferred detergent compositions, in addition to the enzyme preparation of the invention, comprise other enzyme (s) that impart cleaning qualities and / or benefits in the care of the fabric. These enzymes include other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (for example, laccases).

Proteases: Any other suitable protease can be used for use in alkaline solutions. Suitable proteases include those of plant or microbial origin. Those of microbial origin are preferred. Chemically or genetically modified mutants are included. The protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are the subtilisins, especially those derived from Bacillus, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, 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 marketed under the trade names Alcalase, Savinase, Primase, Durazym and Esperase from Novo Nordisk A / S (Denmark), which are marketed under the tradenames Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genecor International and those marketed under the Opticlean and Optimase trade names of Solvay Enzymes. The protease enzymes can be incorporated in the compositions according to the invention at a level between 0.00001% and 2% enzyme protein per composition weight, preferably at a level between 0.0001% and 1% enzyme protein per weight of the composition, with greater preference at a level between 0.001% and 0.5% of enzymatic protein per weight of the composition and even more preferably at a level between 0.01% and 0.2% of enzyme protein per 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 or genetically modified mutants are included. Examples of useful lipases include a Humicola lanuginosa lipase, for example, such as that described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, for example, such as that described in EP 238 023, a Candida lipase, as a lipase C. Antarctic, for example the A or B Acarbatic lipase described in EP 214 761, a Pseudomonas lipase such as the lipase P. alcaligenes and P. pseudoalcaligenes, for example, as described in EP 218 272, a lipase P. cepacia, for example, as described in EP 331 376, a lipase P. stutzeri, for example, as disclosed in GB 1,372,034, a lipase P. fluorescens, a Bacillus lipase, for example, a lipase B. subtilis (Dartois et al., (1993), Biochemica et Biophysica act 1131, 253-260), a lipase B. stearothermophilus (JP 64/744992) and a lipase B pumilus (WO / 91/16422). In addition, several cloned lipases may be useful, 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 the lipase R. dele ar (Hass, MJ et al., (1991), Gene 109, 117-113), a lipase ^ niveus (Kugimiya et al., (1992), 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, the one described in WO 90/09446). Especially suitable lipases are lipases such as MI Lipase ™, Luma Fast ™ and Lipomax ™ (Genencor), Lipolase ™ and Lipolase Ultra ™ (Novo Nordisk A / S) and Lipase P "Amano" (Amano Pharmaceutical Co.

Ltd.).

The lipases are normally incorporated in the detergent composition at a level between 0.00001% and 2% of the enzymatic protein by weight of the composition, preferably at a level between 0.0001% and 1% of enzyme protein by weight of the composition, with greater preference is a level between 0.001% and 0.5% enzyme protein per weight of the composition, even more preferably at a level between 0.01% and 0.2% enzyme protein per 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 or genetically modified mutants are included. Amylases include, for example, α-amylases obtained from a special strain of B. licheniformis, described in more detail in GB 1,296,839. Commercially available amylases are Duramyl ™, Termamyl ™, Fungamyl ™ and BAN ™ (available from Novo Nordisk A / S) and Rapidase ™ and Maxamyl P ™ (available from Genencor). The amylases are usually incorporated in the detergent composition at a level between 0.00001% and 2% of the enzyme protein by weight of the composition, preferably at a level between 0.0001% and 1% of the enzyme protein by weight of the composition, with greater preference at a level between 0.001% and 0.5% of the enzyme protein per weight of the composition and even more preferably at a level between 0.01% and 0.2% of the enzyme protein per weight of the composition.

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

Peroxidases / Oxidases: The peroxidases enzymes are used in combination with hydrogen peroxide or a source thereof (for example, a percarbonate, perborate or persulfate). The oxidases enzymes are used in combination with oxygen. Both types of enzymes are used for "solution bleaching", that is, to avoid the transfer of a textile dye from a dyed fabric to another fabric when these fabrics are washed together in the same wash liquor, preferably together with a reinforcing agent as described, for example, in WO 94/12621 and WO 95/01426. Suitable peroxidases / oxidases include those of plant, bacterial and fungal origin. Chemically or genetically modified mutants are included. The peroxidases / oxidases enzymes are normally incorporated in the detergent composition at a level between 0.00001% and 2% of the enzyme protein by weight of the composition, preferably at a level between 0.0001% and 1% of the enzyme protein by weight of the enzyme. composition, more preferably at a level between 0.001% and 0.5% of the enzyme protein by weight of the composition and even more preferably at a level between 0.01% and 0.2% of the enzyme protein by weight of the composition.

Included here are mixtures of the enzymes mentioned above, in particular a mixture of a protease, an amylase, a lipase and / or a cellulase. The enzyme of the invention or any other enzyme incorporated in the detergent composition is usually incorporated in the detergent composition at a level between 0.00001% and 2% of the enzyme protein by weight of the composition, preferably at a level between 0.0001% and 1% of the enzyme protein by weight of the composition, more preferably at a level between 0.001% and 0.5% of the enzyme protein by weight of the composition and even more preferably at a level between 0.01% and 0.2% of the protein enzymatic by weight of the composition.

Bleaching agents: Additional optional ingredients for detergents 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 bleaching agent components can include one or more oxygenated bleaching agents and depending on the bleaching agent selected, one or more bleach activators. When oxygen is present, bleaching compounds will typically be present at levels between about 1% and 25%. In general, bleaching compounds are optional added components in non-liquid formulations, for example, granular detergents. The whitening agent component used herein can be any of the bleaching agents useful for detergent compositions including oxygenated bleach as well as others known in the art. The bleaching agent suitable for the present invention may be an activated or non-activated bleaching agent. A category of oxygenated bleaching agents that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydecandioic acid. These bleaching agents are set forth in U.S. Patent 4,483,781, U.S. Patent 740,446, EP 0 133 354, and U.S. Patent 4,412,934. Most preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Patent 4,634,551. Another category of bleaching agents that can be used includes halogenated 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 sulfonamides. These materials are usually added in a proportion of 0.5% -10% by weight of the finished product, preferably in the proportion of 1-5% by weight. Agents releasing hydrogen peroxide can be used in combination with bleach activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in U.S. Patent 4,412,934), 3,5-trimethyl-hexanoloxybenzenesulfonate (ISONOBS, described in EP 120,591) or pentaacetylglucose (PAG), which are perhydrolyzed to form a permeate as the active bleaching species, which leads to improve the bleaching effect. Also very suitable are the bleach activators C8 (6-octanamido caproyl) oxybenzene sulfonate, C9 (6-nanoamido caproyl) oxybenzenesulfonate and CIO (6-decanamido caproyl) oxybenzenesulfonate or mixtures thereof. Suitable activators are also acylated citrate esters such as those described in European Patent Application No. 91870207.7. Useful bleaching agents, including peroxy acids and bleaching systems comprising bleach activators and peroxygen bleach compounds for use in cleaning compositions according to the invention are described in USSN application 08/136, 626. Hydrogen peroxide may also be present by adding a system enzyme (that is, an enzyme and a substrate therefor) that is capable of generating hydrogen peroxide at the start or during the washing and / or rinsing process. These enzyme systems are disclosed in European Patent Application EP 0 537 381. Bleaching agents other than oxygenated bleaching agents are also known in the art and can be used herein. One type of non-oxygenated bleaching agents of particular interest includes photoactivated bleaching agents such as the sulphonated phthalocyanines of zinc and / or aluminum. These materials can be deposited on the substrate during the washing process. Under light irradiation, in the presence of oxygen, such as when clothes are d outside and in daylight, the sulphonated zinc phthalocyanine is activated and as a consequence, the substrate is bleached. The preferred sulfonated zinc phthalocyanine and a photoactivated bleaching process are described in U.S. Patent 4,033,718. Typically, the detergent composition will contain between about 0.025% and 1.25% by weight of the sulfonated zinc phthalocyanine. The bleaching agents may also comprise a manganese catalyst. The manganese catalyst may be, for example, one of the compounds described in "Efficient Manganese Catalysts for Low-Temperature Bleaching", Nature 369, 1994, p. 637-639.

Foam suppressors: Another additional ingredient is the suds suppressor, which is exemplified by silicones, and silica-silicone mixtures. Silicones are generally represented by the alkylated polysiloxane materials, while silica is normally used in finely divided forms exemplified by silica aerogels and xelogels and hydrophobic silicas of various types. These materials can be incorporated as particulates, in which the foam suppressor is freely incorporated with advantage in a detergent vehicle that is waterproof, water soluble or dispersible in water, practically non-surfactant. Alternatively, the foam suppressant can be dissolved or dispersed in a liquid vehicle and applied by spraying on one or more of the other components. A preferred foam controller silicone agent is disclosed in U.S. Patent 3,933,672. Other particularly useful foam suppressors are self-emulsifiable silicone-based foam suppressors, which are described in the German Patent Application DTOS 2,646,126. An example of these compounds is DC-544, commercially available from Dow Corning, which is a siloxane-glycol copolymer. Especially preferred foam controlling agents are the suds suppressor systems comprising a mixture of silicone oils and 2-alkyl-alkanols. Suitable 2-alkyl-alkanes are 2-butyl-octaneol which are commercially available under the trade name Isofol 12 R. These foam suppressor systems are described in European Patent Application EP 0 593 841. Foaming controlling agents based on silicone which are especially preferred are described in European Patent Application No. 92201649.8. These compositions may contain a mixture of silicone / silica in combination with calcined non-porous silica such as Aerosil. The foam suppressors described above are usually employed at levels between 0.001% and 2% by weight of the composition, preferably between 0.01% and 1% by weight.

Other components: Other components used in detergent compositions can be used as soil suspending agents, soil release agents, optical brighteners, abrasives, bactericides, fogging inhibitors, coloring agents and / or encapsulated or non-encapsulated perfumes. Particularly suitable encapsulating materials are water-soluble capsules consisting of a polysaccharide matrix and polyhydroxy compounds as described in GB 1,464,616. Other suitable water soluble encapsulating materials comprise dextrins derived from ungelatinized starch acid esters of substituted dicarboxylic acids as described in U.S. Patent 3,455,838. These acid-ester dextrins, preferably, are prepared from starches such as waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of these encapsulation materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material is constituted by a modified corn starch and glucose. The starch is modified by the addition of monofunctional substituent groups such as octenyl succinic acid anhydride. Suitable anti-re-deposit and soil-suspending agents herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose and homo- or copolymeric polycarboxylic acids or their salts. Polymers of this type include the polyacrylates and the maleic anhydride-acrylic acid copolymers previously mentioned as fortifiers, as well as maleic anhydride copolymers with ethylene, methyl vinyl ether or methacrylic acid, the maleic anhydride constituting at least 20 mole percent of the copolymer . These materials are normally used at levels between 0.5% and 10% by weight, more preferably between 0.75% and 8% by weight, preferably superlative between 1% and 6% by weight of the composition. The optical brighteners that are preferred are ionic, examples of these are 4,4'-bis- (2-diethanolamino-4-anilino-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-dianyl-s-triazin-6-ylamino) stilbene-2: 2 ' disodium disodium, 4 ', 4"-bis- (2,4-dianilino-s-triazin-6-ylamino) -stilben-2-sulfonate monosodium, 4,4'-bis (2-anilino-4 - (N -methyl-N-2-hydroxyethylamino) -s-triazin-6-ylamino) stilbene-2,2'-disodium disulfonate, 4,4 '-bis- (-phenyl-2,3,1-triazol-2-yl) ) -disilben-2,2 'disodium disulfonate, 4,4' bis (2-anilino-4- (1-ethyl-2-hydroxyethylamino) -s-triazin-6-ylamino) stilbene-2, 2'-disulfonate disodium , 2 (stilbene-4"- (naphtho-1, 2,: 4,5) -l, 2, 3-triazole -2" -sulphonate of sodium and 4,4 '-bis (2-sulphostyril) biphenyl. Other useful polymeric materials are polyethylene glycols, in particular those with a molecular weight of 1000-10000, more particularly between 2000 and 8. 000 and superlative preference of approximately 4000. These are used at levels between 0.20% and 5%, more preferably between 0.25% and 2.5% by weight. These polymers and the abovementioned homo- or copolymeric polycarboxylate salts are valuable for improving the preservation of whiteness, the deposition of ash on the fabric and the cleaning qualities of clays, dirt of protein origin and oxidisable in the presence of impurities of metals of transition. 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 of different structures. Examples of these polymers are set forth in U.S. Patents 4,116,885 and 4,711,730 and EP 0 272 033. A polymer that is particularly preferred according to EP 0 272 033 has the formula: (CH 3 (PEG) 43) 0.75 (POH) 0.25 [T-PO) 2.8 (T-PEG) 0.4] T (POH) 0.25 ((PEG) 43CH3) 0.75 where PEG is - (OC2H4) 0-, PO is (OC3H60) and T is (pOOC6H4CO). Also very useful are the modified polyesters such as the random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol, the terminal groups being composed primarily of sulfobenzoate and secondly of mono esters of ethylene glycol and / or 1,2-propanediol. The objective is to obtain a polymer blocked at the two ends by the sulfobenzoate groups, "first", in the present context most of these copolymers will be blocked at the ends by sulfobenzoate groups. However, some copolymers will not be completely blocked and therefore, their terminal groups can be constituted by ethylene glycol and / or 1,2-propanediol, in this the "second place" of these species consists. The polyesters selected herein 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. Sulfoisophthalic acid weight and have a molecular weight of about 3,000. The polyesters and their methods of preparation are described in detail in EP 311 342.

Softening agents: Fabric softening agents may also be incorporated into the laundry detergent compositions of the present invention. These agents can be of the organic or inorganic type. Inorganic softening agents are exemplified with the smectite clays set forth in GB-A-1 400898 and in U.S. Patent 5,019,292. Fabric softening agents include water-insoluble tertiary amines as disclosed in GB-A-1 514 276 and EP 0 011 340 and combinations thereof with C12-C14 mono quaternary ammonium salts are disclosed in EP-B-0 026 528 and the amides with two long chains are disclosed in EP 0 242 919. Other useful organic ingredients of the fabric softener systems include high molecular weight polyethylene oxide materials such as those described in EP 0 299 575 and 0 313 146 The smectite clay levels are usually in the range between 5% and 15%, more preferably between 8% and 12% by weight and the material is added as a dry mixed component to the rest of the formulation. Fabric-softening agents for organic fabrics such as water-insoluble tertiary amine materials or long-chain amides are incorporated at levels between 0.5% and 5% by weight, usually between 1% and 3% by weight, while the materials of High molecular weight polyethylene oxide and water soluble cationic materials are added at levels between 0.1% and 2%, usually between 0.15% and 1.5% by weight. These materials are normally added to the spray drying portion of the composition, although in some cases it is more convenient to add them as a dry blended particulate or to atomize them as molten liquid over other solid components of the composition.

Polymeric dye transfer inhibiting agents: The detergent compositions according to the present invention may also comprise between 0.001% and 10%, preferably between 0.01% and 2%, more preferably between 0.05% and 1% by weight of polymeric inhibitory agents of transfer of dyes. These polymeric dye transfer inhibiting agents are normally incorporated into the detergent compositions to inhibit the transfer of the dyes from the dyed fabrics to the fabrics that are washed therewith. These polymers have the ability to complex or absorb fugitive dyes that come out of the fabrics dyed with the wash before the dyes have the opportunity to anchor themselves to other articles in the wash. Especially suitable dye transfer inhibiting polymeric agents are polymers of polyamine N-oxide, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polymers of polyvinylpyrrolidone, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. The addition of these polymers also reinforces the behavior of the enzymes according to the invention. The detergent composition according to the invention can be in the liquid, paste, gel, bar or granular forms. Non-powdered granulates can be produced, for example, such as those disclosed in U.S. Patent 4,106,991 and 4,661,452 (both by Novo Industri A / S) and optionally can be coated by methods known in the art. Examples of waxy materials for coating are the products of poly (ethylene oxide) (polyethylene glycol, PEG) with average molecular weights between 1000 and 20,000; ethoxylated nonylphenols having between 16 and 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains between 12 and 20 carbon atoms and in which there are between 15 and 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di- and triglycerides of fatty acids. Examples of suitable film-forming coating materials for application by fluidized-bed techniques are given in GB 1483591. The granular compositions according to the present invention can also be in "compact form", that is, they can have a relatively higher density than that of conventional granular detergents, for example, between 550 and 950 g / 1; in this case, the granular detergent compositions according to the present invention will contain a smaller amount of "inorganic filler salt" as compared to conventional granular detergents; typical filler salts are alkaline earth metal salts of sulfates and chlorides, typically sodium sulfate; The "compact" detergent typically contains no more than 10% loading salt. The liquid compositions according to the present invention can also be in "concentrated form", in such case, the liquid detergent compositions according to the present invention will contain a smaller amount of water, in comparison with conventional liquid detergents. Typically, the water content of the liquid concentrated detergent is less than 30%, more preferably less than 20%, preferably superlative less than 10% by weight of the detergent compositions. The compositions of the invention, for example, can be formulated as hand and machine laundry detergent compositions and compositions suitable for use in pretreatment of soiled fabrics, fabric softening compositions that are added in the rinse and compositions to be used in general operations. domestic cleaning of compact surfaces and vajilla washing operations.

The following examples are intended to exemplify the compositions of the present invention, but do not necessarily mean that they limit or otherwise define the scope of the invention. In the detergent compositions, the abbreviated identifications of the component have the following meanings: LAS: Sodium C12 alkyl benzene sulfonate linear TAS: Alkyl (tallow derivative) sodium sulfate XYAS: Alkyl Clx-Clv sodium sulfate SS: Secondary surfactant soap of the formula 2-butyl octanoic acid 25EY: A predominantly linear C12-C15 primary alcohol condensed with an average of Y moles of ethylene oxide 45EY: A C14-C15 predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide XYEZS: Alkyl Clx-Cly sodium sulphate condensed with an average of Z moles of ethylene oxide per mole Nonionic: Mixture of C13-C1S ethoxylated / propoxylated fatty alcohols with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5 marketed under the trade name Plurafax LF404 by BASF Gmbh CFAA: C12 alkyl C14 N-methyl glucamide TFAA: Alqui lo C16 - C18 N-methyl glucamide Silicate: Amorphous sodium silicate (ratio Si02: Na20 = 2.0) NaSKS-6: Silicate crystal ino in sheets of formula d-Na2Si205 Carbonate Anhydrous sodium carbonate Phosphate: Sodium Tripolyphosphate MA / AA: Acrylic / maleic acid copolymer 1: 4, with average molecular weight of approximately 80,000. Poly- polyacrylate homopolymer with an average molecular weight of 8,000 acrylates marketed under the trade name PA30 by BASF Gmbh Zeolite A: Hydrated sodium aluminosilicate of formula Na12 (A102SÍ02) 12. 27H20 having a primary particle size in the range between 1 and 10 microns Citrate: Trisodium citrate dihydrate Citric: Citric acid Perborate Monohydrate sodium perborate anhydrous bleach, empirical formula NaB02. H202 PB4 Anhydrous sodium perborate tetrahydrate Percarbonate Anhydrous sodium percarbonate bleach of empirical formula 2Na2C03. 3H202 TAED: Tetraacetyl ethylene diamine CMC: Sodium carboxymethyl cellulose DETPMP: Diethylene triamine penta (methylene phosphonic acid), marketed by Monsanto under the trade name Dequest 2060 PVP: Polyvinylpyrrolidone polymer EDDS: Ethidylenediamine-N, N'-disuccinic acid, isomer [s, s] in the form of sodium salt % paraffin wax suppressant, mp50 ° C, 17% foam: hydrophobic silica, 58% paraffin oil Suppressor 12% silicone / silica, 18% alcohol stearyl foams, 70% starch in form granulate: granular Sulphate: anhydrous sodium sulfate HMWPEO: high molecular weight polyethylene oxide TAE 25: tallow alcohol ethoxylate (25) Detergent Example I A granular composition for cleaning fabrics according to the invention can be prepared in the following manner: Alkyl C12 benzene sulphonate 6.5 sodium linear 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 Minority Up to 100 Detergent Example II A composition for cleaning compact granulated fabrics (density 800 g / 1) according to the invention can be prepared as follows: 45AS 8.0 25E3S 2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0 Citric acid 3.0 Carbonate .0 MA / AA 5.0 CMC O .4 Enzyme of the invention 0.1 TAED 6. O Percarbonate 22.0 EDDS 0.3 Foam suppressor 3.5 water granules / minorities Up to 100% Detergent Example III Granular compositions for cleaning fabrics according to the invention which is especially useful in the washing of colored fabrics were prepared as follows: TAS 2.4 TFAA-4.0 45AS 3.1 10.0 45E7 4.0 25E3S-3.0 68E11 1.8 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 Sulphate 5.2 3.0 PVP 0.5 Poly (4-vinylpyridine) -N- - 0.2 Oxide / copolymer of vinyl imidazole and vinyl pyrrolidone Perborate 1.0 Phenol sulfonate 0.2 Water / Minority Up to 100% Detergent Example IV Granular compositions for cleaning fabrics according to the invention that impart capacity "Softener in washing" can be prepared in the following way: 45AS - 10.0 LAS 7.6 68AS 1.3 45E7 4.0 25E3 - 5.0 Coconut-alkyl-1.4- 1.0 dimethyl hydroxyethyl ammonium citrate 5.0 3.0 Na-SKS-6 - 11 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 Clay "Smectite 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 Foam suppressor 1.0 4.0 CMC granulate 0.2 0.1 Water / Miñoritarios Up to 100% Detergent Example V Heavy-duty liquid compositions for cleaning fabrics according to the invention can be prepared as follows: II The acid form 25 Citric acid 5.0 2.0 25AS acid form 8.0 25AE2S 3.0 25AE7 8.0 CFAA 5 DETPMP 1.0 1.0 Fatty acid 8 Oleic acid - 1.0 Ethanol 4.0 6.0 Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coconut alkyl chloride - 3.0 dimethyl hydroxyethyl ammonium Clay smectite - 5.0 PVP 2.0 Water / Minority Up to 100% APPLICATIONS IN THE PELLET INDUSTRY A subtyla of the invention can be used in the fur industry, in particular for use in hair removal. In this application a subtyla variant of the invention is preferably used in an enzyme composition which further comprises another protease. For a more detailed description of other suitable proteases see the section relating to the suitable enzymes that are used in a detergent composition (see above).

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 clothing comprising wool. In this application a subtylase variant of the invention is preferably used in an enzymatic composition that also contains another protease. For a more detailed description of other suitable proteases see the section related to the suitable enzymes that are used in a detergent composition (see above). The invention is described in more detail in the following examples which are in no way intended to limit the scope of the invention as requested.

MATERIALS AND METHODS Strains B. subtili s DN1885 (Diderichsen et al., 1990) B. l in your 309 and 147 are specific Baci llus len tus strains, deposited in the NCIB and to which the accession numbers NCIB 10309 and 10147 and described in the United States Patent No. 3,723,250 which is considered part of this, as a reference. E. col i MC 1000 (M.J. Casabadan and S.N. Cohen (1980); J. Mol. Bi ol. 138-177-207), r ", m + was made by conventional methods and is also described in U.S. Patent Application Serial Number 039,298.

Plasmids pJS3: E. col i - B. subti l i s shuttle vector containing a synthetic gene encoding subtylase 309. (Described by Jacob Schiodt et al in Protein and Peptide letters 3: 39-44 (1996)). pSX222: B. subti l i s expression vector (Described in WO 96/34946).

General molecular biology methods: Unless otherwise indicated, DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. 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.) Enzymes for DNA manipulations were used according to specifications of the suppliers.

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

Proteolytic Activity In the context of this invention, proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU). The activity is determined in relation to a standard enzyme (SAVINASE®) and the determination is based on the digestion of a solution of dimethyl casein (DMC) with the proteolytic enzyme under standard conditions, ie 50 ° C, pH 8.3, time reaction time 9 min, determination time 3 min. The brochure AF 220/1 is available upon request from Novo Nordisk A / S, Denmark, this brochure is considered part of this, as a reference. A GU is a Glycine Unit, defined as the proteolytic enzymatic activity, which under standard conditions, during 15 minutes of incubation at 40 degrees C, with N-acetyl casein as substrate, produces a quantity of NH2 groups equivalent to 1 mmol of glycine. Enzymatic activity can also be determined using the PNA assay, according to the reaction with the soluble succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol substrate, which is described in the Journal of the American Oil Chemists Society, Rothgeb, TM , Goodlander, BD, Garrison, PH and Smith, L.A. , (1988).

Fermentation: Fermentations of the subtylase enzymes were carried out at 30 aC on a rotary vibrating table (300 r.p.m.) in an Erlen eyer flask with a deviation of 500 ml containing 100 ml of BPX medium for 5 days. Consequently, to make a 2-liter broth, for example, 20 Erlenmeyer flasks were fermented simultaneously.

Medium: BPX: Composition (per liter) Potato starch 100g Shredded barley 50g Soybean meal 20g Na2HP04 X 12 H20 9g Pluronic 0. lg Sodium caseinate lOg The starch in the medium is hydrolysed with α-amylase and the medium is sterilized by heating at 120 ° C for 45 min. After sterilization the pH of the medium is adjusted to 9 by the addition of NaHC03 0. IM.

EXAMPLES EXAMPLE 1 Construction and Expression of Variants of Enzymes Mu tagénesi s directed to the si ti o: Subtylase 309 variants directed to the site were made through the technique "Unique site elimination (USE)" ("Elimination of single site") or "Uracil -USE "described respecti by Deng et al. (Anal Biochem 200: 81-88 (1992)) and Markvardsen et al. (BioTechniques 18 (3): 371-372 (1995)). The standard plasmid was pJS3 or an analogue thereof containing a variant of Subtilasa 309, for example, USE mutagenesis carried out on the pJS3 analog containing a gene encoding the Y167A variant with an oligonucleotide targeted at the construction variant R170L which resulted in a variant of Subtilasa 309 final Y167A + R170L. The Subtilasa 309 variants constructed in pJS3 were then subcloned into the expression plasmid B. subti l i s pSX222, using the restriction enzymes Kpnl and Mlul.

Mu tagénesi s Localized theory The general strategy used to carry out the localized random mutagenesis was: a mutagenic primer (oligonucleotide) was synthesized which corresponded to the part of the ez or / or DNA sequence that was to be mutagenized except for the nucleotide (s) corresponding to an amino acid codon that was to be mutagenized. Subsequently, the resulting mutagenic primer was used in the PCR reaction with a suitable counter primer. The resulting PCR fragment was purified and digested and cloned into an E. coli-B shuttle vector. subtilis. Alternati, if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a second counter-primer suitable to allow digestion and cloning of the mutagenized region in the shuttle vector. PCR reactions are carried out under normal conditions. Following this strategy, a random library located in SAVINASE was built, where both position Y167 and position R170 were completely randomized. An oligonucleotide was synthesized with 25% of each of the four bases (N) in the first and second bases in the amino acid codons that were to be mutagenized. The third nucleotide (the vacillating base) in codons was synthesized with 50% G / 50% C (S) to avoid two (TAA, TGA) of the three nonsense codons. Mutagenic primers (5'-GTT TGG ATC AGT AGC TCC GAC TGC CAT TGC GTT CGC ATA SNN CGC CGG SNN GCT GAT TGA GCC-3 '(anti sense)) were used in a PCR reaction with a suitable counter-primer (e.g. , 5 'GAA CTC GAT CCA GCG ATT TC 3' (sense)) and plasmid pJS3 as standard. This resulting PCR product was cloned into the shuttle vector pJS3 using the restriction enzymes Asp 718 and Bel I. The localized random library constructed in pJS3 was then subcloned into the expression plasmid B. subti l i s pSX222, using the restriction enzymes Kpnl and Mlul. The prepared library contained approximately 100,000 individual clones / library. Ten randomly selected colonies were sequenced to confirm the designed mutations. To purify a subtyla variant of the invention, the expression plasmid B. subti l i s pSX222 comprising a variant of the invention was transformed into a suitable strain of B. subti lis and fermented in the manner described above in a medium containing 10 μg / ml of Chloramphenicol (CAM).

EXAMPLE 2 Purification of Enzyme Variants: This procedure refers to the purification of a 2 liter scale fermentation of the enzyme Subtilisin 147, the enzyme Subtilisin 309 or mutants thereof. Approximately 1.6 liters of fermentation broth was centrifuged at 5000 rpm for 35 minutes in 1 liter glasses. The supernatants were adjusted to a pH of 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 being absorbed at room temperature in an affinity column of Bacitracin at pH 7. The proteases were eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1M sodium chloride in a buffer solution with 0.01 dimethyl glutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to a pH of 7. The fractions with protease activity from the Bacitracin purification step were combined and applied to a Sephadex G25 column. 750 ml (5 cm 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. Fractions with proteolytic activity from the Sephadex G25 column tp TÍÜ / a QMV combined and applied to a CM Sepharose CL 6B cation exchange column of 150 ml (5 cm diameter) balanced with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid and 0.002 M calcium chloride adjusted at 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 fractions containing protease from the CM Sepharose column were combined and concentrated in an Amicon ultrafiltration cell equipped with GR81PP membrane (from Danish Sugar Factories Inc.). Using the techniques of Example 1 for the above construction and isolation procedure, the following subtilysis variants were produced and isolated: Y167A + R170S Y167A + R170L Y167A + R170N Y167P Y167P + R170L Y167V + R170T Y167I + R170T Y167V + R170Q Y167S + R170Q Y167T + R170N Y167A + R170A Y167T Y167T-1R170A Y167P + R170S Y167I + R170L Y167F + R170T Y167F + R170E Y167F + R170H Y167F + R170T Y167F + R170L Y167L R170H Y167S EXAMPLE 3 Behavior in washing conditions of the Compositions Detergents that include Variants of Enzyme The following examples provide results of several washing tests that were performed under the conditions indicated. Experimental conditions Table IV: Experimental conditions for evaluation of subtilisin 309 variants.

After washing the clothes were rinsed with water from the pipe and dried with air. The detergent model above is a simple detergent formulation. The most characteristic features are that STP is used as a fortifier and the content of anionic surfactant (LAS) is quite high. In addition, the pH is adjusted to 10.4, which is within the normal range of a powder detergent.

Table V The composition of the model detergent is as follows % STP (Na5P3O10) 10% Zeolite (Wessalith P) 10% Na2S04 10% Na2C03 25% LAS (Nansa 80S) 5% NI (Dobanol 25-7) 5% Na2Si205 0.5% Carboxymethylcellulose (CMC) 9.5% Water Hardness Water is adjusted by adding CaCl2 and MgCl2 to deionized water. The pH of the detergent solution is modified to the desired value by the addition of acid. The measurement of the remission (R) in the test material was made at 460 nm using an Elrepho 2000 photometer (without UV). Opening: 10 mM. The behavior under washing conditions in XnM is calculated as: Rx - Ro Px Rx, ref - Ro Rx: is the effect of washing the enzyme in X nM (in units of remission) R0: is the effect of washing the enzyme in 0 nM (white value) Table VI: Variants and factors of improvement for SAVINASE® As can be seen in Table VI, the SAVINASE® variants of the invention show improvement in the behavior under washing conditions. In a subsequent similar wash test the SAVINASE® variants showed a performance value under washing conditions in the interval between the variant Y167I + R170L and Y167A + R170S: Y167P Y167P + R170L Y167V + R170T Y167I + R170T Y167V + R170Q Y167S + R170Q Y167T + R170N Y167A + R170A Y167T + R170L Y167T + R170A Y167P + R170S EXAMPLE 4 Behavior in Washing Conditions of Detergent Compositions Comprising Enzyme Variants The following examples provide results of various washing tests that were carried out under the conditions indicated EXPERIMENTAL CONDITIONS Table VII: Experimental conditions for 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 80S) 5.0% Nonionic Surfactant (Dobanol 25-7) 5.0% Na2Si205 0.5% Carboxymethylcellulose (CMC) 9.5% Water The hardness of the water was adjusted by adding CaCl2 and MgCl2 (Ca +: Mg + = 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 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 behavior under washing conditions of Subtilisin 309 variants were evaluated by calculating an operating factor: *, 'na n t - R White R Savtnase R White P: Function factor R V "a" r i, a3n "tt" e: 'Ref readiness of the test material washed with the Savinase variant' Reflectance of the test material washed with Savinase® R White Reflectance of washed test material without enzyme The claimed Subtilisin 309 variants have improved wash performance compared to SAVINASE® - ie, P > 1. The variants are divided into improvement classes designated with capital letters: Class A 1 < P < 1.5 Class B 1.5 < P < 2 Class C P > 2 Table VIII: Subtilisin 309 variants and breeding classes.

EXAMPLE 5 Comparative fermentation experiment with variant (s) of the main aspect of the invention combined with other modification (s) in the position (s) 129, 131, 133 and / or 194 The Savinase® variant Y167I + R170L + A194P was compared in a fermentation experiment with a Y167I + R170L variant that did not have the A194P substitution. The two variants were cloned into a pSX222 base expression vector and fermented in the manner described above in 100 ml of BPX medium containing 10 μg / ml CAM. After 5 days of fermentation 1.5 ml of the BPX fermentation medium was centrifuged and the supernatant was used to measure the activity Proteolytic (KPNU) in the manner described above. The fermentation medium containing the variant Y167I + R170L + A19 P had a significantly higher level of proteolytic activity compared to the fermentation medium containing the variant Y167I + R170L. The two variants have the same specific activity. Similar results were obtained with the variants Y167A + R170S + A194P, Y167A + R170L + A194P and Y167A + R170N + A194P compared with their corresponding variants without the A194P mutation. In addition, similar results were obtained with the variants A133P + Y167A + R170S, A133D + Y167A + R170S, P129K + Y167A + R170S and P131H + Y167A + R170S compared to their corresponding variants without the mutations A133P, A133D, P129K and P131H.

Claims (19)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A method to produce a variant of subtyla enzyme that has behavior under washing conditions improved in detergents, compared to the behavior in washing conditions of subtilisin 309 of B. lentus and that includes modifications in both position 167 and 170 (in BASBPN numbering) and where the method comprises: (i) constructing a expression vector comprising an isolated DNA sequence encoding the subtilase variant; (ii) transforming a microbial host cell with the expression vector of step i); (iii) culturing the host cell of step ii); in conditions conducive to the expression and secretion of the variant; and (iv) recovering the variant; with the exception of subtylase variants Y167L + R170L; Y167L + R170I; Y167I + R170L; Y167I + R170I; Y167F + R170L; Y167F + R170I; Y167V + R170L and Y167V + R170I subtyla subgroup I-S2. The method according to claim 1, wherein the modification is selected from the group comprising (in BASBPN numbering): 167 (G, A, S or T.}. +170 (G, A, S or T.). 167 { G, A, S or T.}. +170 { L, I or V.}. 167 (G, A, S or T.}. +170 { Q or N.}. 167P + 170 { L, I or V.}. 167 { L, I, or V.}. +170 { G, A, S or T.}. 167 { L, I or V.}. +170 { Q or N.}. 167P + 170 (G, A, S or T.}. 167 { L, I, or V.}. +170 (L, I, or V.}. 167 { F, W or Y.}. +170 { G, A, S or T.}. 167 { F, W or Y.}. +170 { E or D.}. 167 { F, W or Y.}. +170 { R, K or H.}. 167 (F, W or Y.}. +170 (L, I, or V.) 3. The method according to claim 2, wherein the modification is selected from the group comprising (in BASBPN numbering): 167A + 170S, 167A + 170L, 167A + 170N 167P + 170L 167V + 170T 167I + 170T 167V + 170Q 167S + 170Q 167T + 170N 167A + 170A 167T + 170L 167T + 170A 167P + 170S 167F + 170T 167F + 170E 167F + 170H 167F + 170T 4. The method according to claim 2, wherein the modification is elecciona of the group comprising (in BASBPN numbering): Y167. { G, A, S or T} + R170. { G, A, S or T.}. Y167 (G, A, S or T.}. + R170 (L, I or V.} .Y167 (G, A, S or T.}. + R170 (Q or N.} Y167P + R170. L, I or V.}. Y167 { L, I, or V.}. + R170 { G, A, S or T.} .Y167 { L, I or V.}. + R170 { Q or N.}. Y167P + R170 (G, A, S or T.} .Y167 { L, I, or V.}. + R170 (L, I, or V.} .Y167 { F, W or Y.}. + R170 { G, A, S or T.} .Y167 { F, W or Y.}. + R170 { E or D.} .Y167 { F, W or Y.}. + R170 { R, K or H.} .Y167 { F, W or Y.}. + R170 { L, I, or V.}. where the subtylase variants Y167L + R170L; Y167L + R170I; Y167I + R170L; Y167I + R170I; Y167F + R170L; Y167F + R170I; Y167V + R170L and Y167V + R170I are rejected. The method according to claim 4, wherein the modification is selected from the group comprising (in BASBPN numbering): Y167A + R170S Y167S + R170A + R170L Y167A + R170N Y167P + R170L Y167V + R170T Y167I + R170T Y167V + R170Q Y167S + R170Q Y167T + R170N Y167A + R170A Y167T + R170A Y167P + R170S Y167F + R170T Y167F + R170E Y167F + R170H Y167F + R170T. The method according to any one of claims 1 to 5, wherein the variant is a variant of a precursor subtyla that is selected from the subgroup I-SI. The method according to claim 6, wherein the precursor 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. The method according to any one of claims 1 to 5, wherein the variant is a variant of a precursor subtyla that is selected from subgroup I-S2. The method of claim 8, wherein the precursor subtylase is selected from the group comprising BLS147, BLS309, BAPB92, TVTHER and BYSYAB or functional variants thereof which have retained the characteristic of subgroup I-S2. The method according to any of the preceding claims, wherein the modification (s) is combined with one or more modifications in some other position (s). The method according to claim 10, wherein the modification (s) is combined with modification (s) in one or more of the positions 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and 274. The method according to claim 11, wherein the subtyla belongs to the subgroup I-S2 and the other change is selected from the group comprising K27R, * 36D, S57P, N76D, S87N , G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Q206E, N218S, M222S, M222A, T224S, K235L and T274A. The method according to claim 11, which comprises any of the variaS101G + V104N, S87N + S101G + V104N, K27R + V104Y + N123S + T274A or N76D + V104A or other combinations of these mutations (V104N, S101G, K27R, V104Y , N123S, T274A, N76D, V104A), in combination with one or more of the substitutions, deletions and / or insertions mentioned in any of claims 1 to 12. The method according to any one of the preceding claims, wherein the ( s) modification (s) are combined with modification (s) in one or more of positions 129, 131, 133 and 194. 15. The method according to claim 14, wherein the subtilase belongs to the subgroup I-S2 and the other modification is selected from the group comprising P129K, P131H, A133P, A133D and A194P. 16. The method according to claim 15, wherein the modification is selected from the group comprising: Y167A + R170S + A194P Y167A + R170L + A194P Y167A + R170N + A194P Y167A + R170S + P129K Y167A + R170L + P129K Y167A + R170N + P129K Y167A + R170S + P131H Y167A + R170L + P131H Y167A + R170N + P131H Y167A + R170S + A133P Y167A + R170L + A133P Y167A + R170N + A133P Y167A + R170S + A133D Y167A + R170L + A133D Y167A + R170N + A133D 17. The method according to any of claims 1 to 16, wherein the microbial host cell is a bacterium, preferably a Bacillus, especially B. len tus. 18. The method according to any of claims 1 to 16, wherein the microbial host cell is a fungus or a yeast, preferably a filamentous fungus, especially an Aspergillus. 19. The use of a subtilase variant produced according to any one of claims 1 to 18 in a laundry detergent and / or dishwashing detergent.