Title of Invention	A SUBTILASE ENZYME VARIANT AND COMPOSITIONS CONTAINING THE SAME
Abstract	A continuous process for the preparation of 2-hydroxy-4-methylthiobutanoic acid or a salt thereof which includes introducing an aqueous mineral acid into a nitrile hydrolysis reactor including a continuous stirred tank reactor and introducing 2-hydroxy-4-methylthiobutanenitrile into the nitrile hydrolysis reactor. 2-hydroxy-4-methylthiobutanenitrile is continually hydrolyzed within the nitrile hydrolysis reactor to produce a nitrile hydrolysis reactor product stream containing 2-hydroxy-4-methylthiobutanamide. The nitrile hydrolysis reactor product stream is continuously introduced into an amide hydrolysis flow reactor. 2-hydroxy-4-methylthiobutanamide is continually hydrolyzed within the amide hydrolysis flow reactor to produce an aqueous hydrolyzate product containing 2-hydroxy-4-methylthiobutanoic acid. 2-hydroxy-4-methylthiobutanoic acid is recovered from the aqueous hydrolyzate product.

Title of Invention

A SUBTILASE ENZYME VARIANT AND COMPOSITIONS CONTAINING THE SAME

Abstract

A continuous process for the preparation of 2-hydroxy-4-methylthiobutanoic acid or a salt thereof which includes introducing an aqueous mineral acid into a nitrile hydrolysis reactor including a continuous stirred tank reactor and introducing 2-hydroxy-4-methylthiobutanenitrile into the nitrile hydrolysis reactor. 2-hydroxy-4-methylthiobutanenitrile is continually hydrolyzed within the nitrile hydrolysis reactor to produce a nitrile hydrolysis reactor product stream containing 2-hydroxy-4-methylthiobutanamide. The nitrile hydrolysis reactor product stream is continuously introduced into an amide hydrolysis flow reactor. 2-hydroxy-4-methylthiobutanamide is continually hydrolyzed within the amide hydrolysis flow reactor to produce an aqueous hydrolyzate product containing 2-hydroxy-4-methylthiobutanoic acid. 2-hydroxy-4-methylthiobutanoic acid is recovered from the aqueous hydrolyzate product.

Full Text	Title: Protease Variants and Compositions TECHNICAL FIELD This invention relates tp novel mutant protease enzymes or enzyme variants useful in formulating detergent compositions and exhibiting improved wash performance in detergents; cleaning and detergent compositions containing said enzymes; mutated genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing said enzyme variants. BACKGROUND OF THE INVENTION In the: detergent industry enzymes have for more than 3 0 years been iiiplemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially mo t iikportant enzymes are proteases. An increasing number of commercially used proteases are protein engineered variants of naturally occurr'ing wild type proteases, e.g. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.). . Further a number of protease variants are describe in the art, such as in EP 130756 (GENENTECH) (corresponding to US Reissue Patent 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; Russel and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Alrtgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER Si GAMBLE COMPANY); WO 95/30010 (PROCTER & GAMBLE COMPANY); WO 95/29979 (PROCTER & GAMBLE COMPANY); US 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, even though a number of useful protease variants have been described, there is still a need for new improved protease variants for a number of industrial uses. Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry. SUMMARY OF THE INVENTION Recently it has been identified that subtilase variant with improved wash performance can be obtained by substituting one or more amino acid residues situated in, or in the vicinity of a hydrophobic domain of the parent subtilase for an amino acid residue more hydrophobic than the original residue, said hydrophobic domain comprising the residues corresponding to residues 1165, Y167, Y171 of BLS309, and said residues in the yicinity thereof comprises residues corresponding to the residues E136, G159, S164, R170, A194, and G195 of BLS309 (WO 96/34946). Based on this information the present inventors have intensively studied numerous of the possible combinations of the Y167 and R170 residues of SAVINASE®, and identified a number of variants with surprisingly increased improved wash performance. For further details reference is made to working examples herein {vide infra). Accordingly, the present invention relates in its first aspect to a subtilase protease variant having improved wash performance in detergents, comprising modifications in both position 167 and 170 In a second aspect the invention relates to a subtilase enzyme variant having improved wash performance in detergents, comprising at least one modification chosen 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 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} ,i^7{L,I, or V} 170H 167{G,A,S, or T}. a matrix The nomenclature above e.g. the "l67fG,A,S, or T}+170{G,A,S, or T}" variant (s) is nomenclature, wherein the term "167{G,A,S, or T}+170{G,A,S, or T-}" comprise following variants: 167G+170G, 167G+170A, 167G + 170S, 167G-4-170T, 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 further details relating to the nomenclature of a subtilase variant herein, see section "Definitions" herein (vide infra). In a third aspect the invention relates to an isolated DNA sequence encoding a subtilase variant of the invention. In a fourth aspect the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase 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 a further aspect the invention relates to the production of the subtilisin enzymes of the invention by inserting an expression vector according to the fourth aspect into a suitable microbial host, cultivating the host to express the desired subtilase enzyme, and recovering the enzyme product. Even further the invention relates to a composition comprising a subtilase variant of the invention. Finally the invention relates to the use of the mutant enzymes for a number of industrial relevant uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes. DEFINITONS Prior to discussing this invention in further detail, the following term will first be defined. Nomenclature of Amino Acids 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 = Gin = Glutamine D = Asp = Aspartic Acid E = Glu = Glutamic Acid K = Lys = Lysine R = Arg = Arginine H = His = Histidine X = Xaa = Any amino acid Nomenclature of nucleic acids A = Adenine G = Guanine C = Cytosine T = Thymine (only in DNA) U = Uracil (only in RNA) Nomenclature of variants In describing the various enzyme variants produced or con-templated according to the invention, the following nomenclatures have been adapted for ease of reference: Original amino acid(s) position(s) substituted amino acid(s) According to this the substitution of Glutamic acid for glycine in position 195 is designated as: Glyl95Glu or G195E a deletion of glycine in the same position is: Glyl95 or G195* and insertion of an additional amino acid residue such as lysine is: Glyl95GlyLys or G195GK ) Where a deletion in comparison with the sequence used for the numbering is indicated, an insertion in such a position is ihdicated as: * 36Asp or 36D for insertion of an aspartic acid in position 36 Multiple mutations are separated by pluses, e.g.: Argl70Tyr + Glyl95Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively. When the original amino acid(s) may comprise any amino acid, then are only position(s) and substituted amino acid(s) mentioned, e.g.: 170Ser. This nomenclature is particular relevant relating to modification(s) according to the invention in homologous subtilases (vide infra). 170Ser then comprise e.g. both a Lysl70Ser modification in BASBPN and Argl70Ser modification in BLSSAVI. See figure 1 in relation to said examples. When the substituted amino acid(s) may comprise any amino acid, then are only the original amino acid(s) and position(s) mentioned, e.g.: Argl70. When both the original amino acid(s) and substituted amino acid(s) may comprise any amino acid, then is only the position(s) mentioned, e.g.: 170. When the original amino acid(s) and/or substituted amino acid(s) may comprise more than one and not all amino acid(s) , then the selected amino acids are surrounded by a "{ }", e.g. Argl70{Gly,Ala,Ser, or Thr} comprising the variants Argl70Gly, Argl70Ala, Argl70Ser, or Argl70Thr; or e.g. Tyrl67{Glyi,Ala,Ser, or Thr} + Argl70{Gly,Ala,Ser, or Thr} comprising the variants Tyrl67Gly + Argl70Gly, Tyrl67Gly + Argl70Ala, Tyif'l67Gly + 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 particular relevant relating to a conservative amino acid modification(s) of a preferred subtilase variants of the invention. E.g. a conservative amino acid modification(s) of e.g. a preferred variant Tyrl67Ala + Argl70Ser are Tyrl67{Gly,Ala,Ser, or Thr} -f Argl70{Gly,Ala,Ser, or Thr}, which here substitute a small amino acid to another small amino acid. See section "Detailed description of the invention" for further details. Proteases Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3). i Numbering of amino acid positions/residues If ho other mentioned the amino acid numbering used herein correspond to that of the subtilase BPN" (BASBPN) sequence. For further description of the BPN sequence see Siezen et al., Protein Engng. 4 (1991) 719-737 and Figure 1. Serine proteases A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residiie at the active site (White, Handler and Smith, 1973 "Prindiples of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272) . The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753). Subtilases A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al,, Protein Engng. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases have been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. and figure' 1 herein. One subgroup of the subtilases, I-Sl, comprises the "classical" subtilisins, such as subtilisin 168, subtilisin BPN', subtilisin Carlsberg (ALCALASE, NOVO NORDISK A/S), and subtilisin DY. A further subgroup of the subtilases I-S2, is recognised by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprise I enzymes such as subtilisln PB92 (MAXACAL, Gist-Brocades NV) , subtilisin 309 (SAVINASE®, NOVO NORDISK A/S), subtillsin 147 (ESPERASE, NOVO NORDISK A/S), and alkaline elastase YaB. "SAVINASE®" SAVINASE® is marketed by NOVO NORDISK A/S. It is subtilisin 309 from B. Lentus and differs from BABP92 only in having N87S (see figure 1 herein). Parent subtilase The term "parent subtilase" is a subtilase defined according to Siezen et al. (Protein Engineering 4:719-737 (1991)). For further details see description of "SUBTILASES" immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modification have been made while retaining the characteristic of a subtilase. Alternatively the term "parent subtilase" may be termed "wild-type subtilase". Modification's) of a subtilase variant The term "modification(s)" used in connection with modification (s) of a subtilase variant as discussed herein is defined to include chemical modification as well as genetic manipulation. The modification(s) can be by substitution, deletion and/or insertions in or at the amino acid(s) of interest. Subtilase variant In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host. Homolocfous subtilase sequences Specific amino acid residues of SAVINASE® subtilase are identified for modification herein to obtain a subtilase variant of the invention. However, the invention is not limited to modifications of this particular subtilase, but extend to other parent (wild-type) subtilases, which have a homologous primary structure to that of SAVINASE®. In order to identify other homologous subtilases, within the scope of this invention, an alignment of said subtilase(s) to a group of previously aligned subtilases is performed keeping the previous alignment constant. A comparison to 18 highly conserved residues in subtilases is performed. The 18 highly conserved residues are shown in table I (see Siezen et al. for further details relating to said conserved residues). Table I 18 highly conserved residues in subtilases Position: Conserved residue 23 G 32 D 34 G t 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 aligning allowing for necessary insertions and deletions in order to maintain the alignment suitable homologous residues are identified. Said homologous residues can then be modified according to the invention. Using the CLUSTALW (version 1.5, April 1995) computer I alignment program (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (i994) Nucleic Acids Research, 22:4673-4680.), with GAP open penalty of 10.0 and GAP extension penalty of 0.1, using the BEOSUM30 protein weight matrix, alignment of a given subtilase to a group of previously aligned subtilases is achieved using the Profile alignments option in the program. For a given subtilase to be within the scope of the invention, preferably 100% of the 18 highly conserved residues should be conserved. However, alignment of greater than or equal to 17 out of the 18 residues, or as little as 16 of said conserved residues is also adequate to identify homologous residues. Conservation of the, in subtilases, catalytic triad Asp32/His64/Ser221 should be maintained. The previously defined alignment is shown figure 1, where the percent identity of the individual subtilases in this alignment to the 18 highly conserved residues are shown too. Further in said process to identify a homologous parent (wild-type) subtilase within the scope of the invention, the 18 conserved residues above relates to the parent (wild-type) primary sequence of said homologous parent subtilase- In order words, if a parent subtilase have been modified in any of said 18 conserved residues above, it is the original parent wild-type sequence in said 18 conserved residues, which determine whether or not both the original parent subtilase and a possible variant of said parent subtilase, which are modified in any of said 18 conserved residues above, are a homologous subtilase within 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 a homologous subtilases and corresponding suitable residues to be modified according to the invention• It is obvious that a similar or larger table covering other homologous subtilases may easily be produced by a person skilled in the art. Wash performance The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, wash-ability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property. Isolated DNA sequence The term "isolated", when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but; may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of or- dinaryi skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term "an isolated DNA sequence" may alternatively be termed "a cloned DNA sequence". Isolated protein When applied to a protein, the term "isolated" indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities" (see below)). It is preferred to provide the protein in a highly purified form, i.e., greater than 40% pure, greater than 60% pure, greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE. The term "isolated protein" may alternatively be termed "purified protein". Homologous impurities The term "homologous impurities" means any impurity (e.g. another polypeptide than the polypeptide of the invention) which originate from the homologous cell where the polypeptide of the invention is originally obtained from. Obtained from The term "obtained from" as used herein in connection witah a specific microbial source, means that the polynucleotide and/or polypeptide produced by the specific source, or by a cell in which a gene from the source have been inserted. Substrate The term "Substrate" used in connection with a substrate for a protease is should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease. Product The term "product" used in connection with a product derived from a protease enzymatic reaction should in the context of this invention be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction. •; BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows an alignment of a number of homologous subtilases, which are aligned to 18 highly conserved residues in subtilases. DETAILED DESCRIPTION OF THE INVENTION Subtilase variants with improved wash performance: Numerous subtilase variants of the invention is tested herein and showing improved wash-performance in detergents (see working examples herein (vide infra)). Accordingly, an embodiment of the invention relates to a subtilase enzyme variant according to the second aspect of the invention, wherein the modification is chosen 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 lft7F+170T 167F+170L 167L 17 OH 167S. The present inventors have identified the improved wash performance variants in BLS309 (SAVINASE®), which in its parent wild-type primary sequence comprise Y167 and R170 as the original wild-type amino acids (see figure 1). Accordingly, a further embodiment of the invention relates to a subtilase enzyme variant according to the second aspect of the invention, wherein the modification is chosen 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} Yi67{F,W or Y}+R170{E, or D} Y167{F,W or Y}+R170{R,K, or H} Yl67{F,W or Y}+R170{L,I, or V} Y167{L,I, or V} R170H Y167{G,A,S, or T}; or more preferred a subtilase enzyme variant according to the embodiment immediately above, wherein the modification is chosen from the group comprising (in BASBPN numbering): Y167A+R170S Y167A+R170L Y167A+R170N Y167P Y167P+R170L Y167V+R170T Y167I+R170T Yq67V+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 substitution of one amino acid to a similar conservative amino acid only give a minor change in the characteristic of the enzyme. Table III below list groups of conservative amino acids. Table III Conservative amino acid substitutions Basic: R = arginine K = lysine H = histidine Acidic: E = glutamic acid D = aspartic acid Polar: Q = glutamine N = asparagine Hydrophobic: L = leucine I = isoleucine V = valine M = methionine Aromatic: F = phenylalanine W = tryptophan Y = tyrosine Small: G = glycine A = alanine S = serine T = threonine Accordingly, subtilase variants such as 167A4-170S, 167G+170S, 167S+170S, and 167T+170S, will have a similar wash-performance improvement• Further, subtilase variants such as Y167G+R170S, Y167S+R170S, and Y167T+R170S, will have a similar wash-performance improvement as the variant Y167A+R170S. See e.g. working examples herein for a specific wash performance test of said Y167A+R170S variant. Based on the disclosed and in particular the numerous exemplified subtilase variants herein, it is routine work, for a person skilled in the art, to identify further suitable conservative modification(s) , of in particular said exemplified variants, in order to obtain a subtilase variant with improved wash-performance, according to all aspects and embodiment of a subtilase variant of the invention. In embodiments of the invention, the subtilases of interest are those belonging to the subgroups I-Sl and I-S2. Relating to subgroup I-Sl preferred parent subtilase is chosen from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR or functional variants thereof having retained the characteristic of sub-group I-Sl. Relating to subgroup I-S2 preferred parent subtilase is chosen from the group comprising BLS147, BLS309, BAPB92, TVTHER AND BYSYAB or functional variants thereof having retained the characteristic of sub-group I-S2. In particular said parent subtilase is BLS309 (SAVINASE® NOVO NORDISK A/S) . The present invention also comprises any one or more modifications in the above mentioned positions in combination with any other modification to the amino acid sequence of the parent enzyme. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged. The art describe a number of subtilase variants with differnt improved properties and a number of those are mentioiied in the "Background of the invention" section herein (vide supra). Those references are disclosed here as references to identify a subtilase variant, which advantageously can be combined with a subtilase variant of the invention. Such combinations comprise the positions: 222 (improve oxidation stability), 218 (improves thermal stability), substitutions in the Ca-binding sites stabilizing the enzyme, e.gr. position 76, and many other apparent from the prior art. In further embodiments a subtilase variant of the invention may advantageously be combined with one or more modification(s) 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 BLS309 and BAPB92 variants are considered appropriate for combination: K27R, 36D, S57P, N76D, S87N, G97N, SIOIG, V104A, V104N, V104Y, H120D, N123S, Q206E, N218S, M222S, M222A, T224S, K235L and T274A. Furthermore variants comprising any of the variants S101G+V104N, S87N4-S101G4-V104N, K27R+V104Y+N123S+T274A, or N7 6D+V104A or other combinations of these mutations (V104N, SIOIG, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with any one or more of the modification(s) mentioned above exhibit improved properties. Even further subtilase variants of the main aspect(s) of the invention are preferably combined with one or more modification(s) in any of the positions 129, ,131, 133 and 194, preferably as 129K, 131H, 133P, 133D and 194P modifications, and most preferably as P129K, P131H, A133P, A133D and A194P modifications. Any of those modification(s) give a higher expression level of a subtilase variant of the invention. For further details reference is made to working examples herein (vide infna) . Accordingly, an , even further embodiment of the invention relates to a variant according to the invention, wherein said modification is chosen from the group comprising: Y167A+R170S+A194P .Y167A+R170L+A194P Y167A+R170N+A194P Y167A+R170S+P129K Y167A+R170L+P129K Y167A+R170N+P129K Y167A+R170S+P13IH Y167A+R170L+P131H Y167A+R170N+P131H Y167A+R170S+A133P Y167A+R170L+A133P Y167A+R170N+A133P Y167A+R170S+A133D Y167A+R170L+A133D Y167A+R170N+A133D PRODUCING MUTATIONS IN SUBTILASE GENES Many methods for cloning a subtilase of the invention and for introducing mutations into genes (e.g. subtilase genes) are well known in the art. In general standard procedures for cloning of genes and introducing mutations (random and/or site directed) into said genes may be used in order to obtain a subtilase variant of the invention. For further description of suitable techniques reference is made to working examples herein {vide infra) 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; Harwood, 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 may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome(s) into which it has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to 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, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the enzyme. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable prompters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkalihe protease gen, or the Bacillus pumilus xylosidase gene, or the phage Lambda PR or PL promoters or the E. coli Lac, trp or tac promoters. I The DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides. To direct an enzyme of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein. The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.). HOST QELL The DNA sequence encoding the present enzyme introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence. TJae host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram-pbsitive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. jbrevls, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circularise B. lautus, B. megatbBrium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or iS. murinu, or gram-negative bacteria such as Echerichia coli. The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra). When expressing the enzyme in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme. When expressing the enzyme in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below. METHOD OF PRODUCING SUBTILASE The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting 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 enable heterologous recombinant production of the enzyme of the invention. Thereby it is possible to make a highly purified subtilase composition, characterized in being free from homologous impurities. In this context homologous impurities means any impurities (e.g. other polypeptides than the enzyme of the invention) which originate from the homologous cell where the enzyme pf the invention is originally obtained from. The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells iin question. The expressed subtilase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like. USE OF A SUBTILASE VARIANT OF THE INVENTION A subtilase protease variant of the invention may be used for a number of industrial applications, in particular within the detergent industry. Further the invention relates to an enzyme composition, which comprise a subtilase variant of the invention. An summary of preferred industrial applications and corresponding preferred enzyme compositions are described below. This summary is not in any way intended to be a complete list of suitable applications of a subtilase variant of the invention. A subtilase variants of the invention may be used in other industrial applications known in the art to include use of a protease, in particular a subtilase. DETERGENT COMPOSITIONS COMPRISING THE MUTANT ENZYMES The present invention comprises the use of the mutant enzymes of the invention in cleaning and detergent compositions and such compositions comprising the mutant subtilisin enzymes. Such cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions. Further reference is made to workings example(s) herein showing wash performance improvements for a number of subtilase variants of the invention. DETERGENT DISCLOSURE AND EXAMPLES Surfactant system The detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant can be selected from nonibnic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants. The surfactant is typically present at a level frqm 0.1% to 60% by weight. The surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or gel compositions the surfactant is most preferably formulated in such a way that it promotes, or at least does not degrade, the stability of any enzyme in these compositions. Preferred systems to be used according to the present Lnvention comprise as a surfactant one or more of the nonionic and/or anionic surfactants described herein. Polyethylene, polypropylene, and polybutylene oxide conden-sates of alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present inven-tion, with the polyethylene oxide condensates being preferred. These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight chain or branched-chain configuration' with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and Triton™ X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (e.g., alkyl phenol ethoxylates). The condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the nonionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. Preferred are the condensation products of alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcohol. About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available nonionic surfactants of this type include TergitolTM 15-S-9 (The condensation product of C11-C15 linear alcohol with 9 moles ethylene oxide), TergitolTM 24-L-6 NMW (the condensation product of C12-C14 primary alcohol with 6 naoles ethylene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; WeodolTM 45-9 (the condensation product of C14-C15 linear • TM alcohol with 9 moles of ethylene oxide), Neodol 23-3 (the condensation product of C12-C13 linear alcohol with 3.0 moles of ethylene oxide) , NeodolTM 45-7 (the condensation product of C14-C15 linear alcohol with 7 moles of ethylene oxide), Neodol™ 45-5 (the condensation product of C14-C15 linear alcohol with 5 mol6s of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C13-C15 alcohol with 9 moles ethylene oxide), marketed by The Procter & Gamble Company, arid Genapol LA 050 (the condensation product of C12-C14 alcohol with 5 moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB in these products is from 8-11 and most preferred from 8-10. Also useful as the nonionic surfactant of the surfactant systems of the present invention are alkylpolysaccharides disclosed in US 4,565,647, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e.g. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing saccharide containing 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside). The intersaccharide bonds can be, e.g., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6- positions on the preceding saccharide units. The preferred alkylpolyglycosides have the formula R20(CnH2nO)t(glycosyl)x wherein R2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to ibout 18, preferably from about 12 to about 14, carbon atoms; n Is 2 or 3, preferably 2; t is from 0 to about 10, pre-ferably 3; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the 1-position). The additional glycosyl units can then be attached between their 1-position and the preceding glycosyl units 2-, 3-, 4-, and/or 6-position, preferably predominantly the 2-position. The ciondensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional nonionic surfactant systems of the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility. The addition of polyoxyethylene moieties 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 up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide. Examples of compounds of this type include certain of the commercially available PluronicTM surfactants, marketed by BASF. Also suitable for use as the nonionic surfactant of the nonionic surfactant system of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene pxide and ethylenediamine. The hydrophobic moiety of these products consists of the reaction product of ethylenediamine and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000. This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product dontains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to abput 11,000. Examples of this type of nonionic surfactant include certain of the commercially available TetronicTM compounds, marketed by BASF. Preferred for use as the nonionic surfactant of the surfactant systems of the present invention are polyethylene oxide condensates of alkyl phianols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylen6oxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C8-C14 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C8-C18 alcohol ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof. Highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the formula 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 hydroxyIs directly connected to the chain, or an alkoxylated derivative thereof. Preferably, R1 is methyl, R2 is straight C11-15 alkyl or C16-18 alkyl or alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction. Highly preferred anionic surfactants include alkyl I alkoxylated sulfate surfactants. Examples hereof are water soluble salts or acids of the formula R0(A)mS03M wherein R is an unsubstituted C10-C-24 alkyl or hydroxyalkyl group having a C10-C24 alkyl component, preferably a C22-C20 alkyl or hydroxyalkyl, more preferably C12-C18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium. calC1um, magnesium, etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein. SpeC1fic examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like. Exemplary surfactants are C12-C18 alkyl polyethoxylate (1.0) sulfate (C12-C18E(1.0)M), C12-C18 alkyl polyethoxylate (2-25) sulfate (C12-C18(2.25)M, and C12-C18 alkyl polyethoxylate (3.0) sulfate (C12-C18E(3.0)M) , and C12-C18 alkyl polyethoxylate (4.0) sulfate (C12~C18E(4.0)M) , wherein M is conveniently selected from sodium and potassium. Suitable anionic surfactants to be used are alkyl ester sulfonate surfactants including linear esters of C8-C20 carboxylic acids (i.e., fatty acids) which are sulfonated with gaseous SO3 according to "The Journal of the American Oil Chemists SoC1ety", 52 (1975), pp. 323-329. Suitable starting materials would include natural fatty substances as derived from tallow, palm oil, etc. The preferred alkyl ester sulfonate surfactant, espQc:.ally for laundry applications, comprise alkyl ester sulfonate surfactants of the structural formula: wherein R3 is a C8-C20 hydrocarbyl, preferably an alkyl, or combination thereof, R is a C1-C5 hydrocarbyl, preferably an alkyl, or combination thereof, and M is a cation which forms a water soluble salt with the alkyl ester sulfonate. Suitable salt-forming cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethonolamine, and triethanolamine. Preferably, R3 is C10-C16 alkyl, and R4 is methyl, ethyl or isopropyl. EspeC1ally preferred are the methyl ester sulfonates wherein R3 is C1Q-C1G alkyl. Other suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO3M wherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C1Q-C20 alkyl component, more preferably a C12-C18 alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium), or ammonium or substituted ammonium (e.g. methyl-, dimethyl-, and trimethyl ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and quaternary ammonium cations derived from alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like). Typically, alkyl chains of C12-C16 are preferred for lower wash temperatures (e.g. below about 50oC) and C16-C18 alkyl chains are preferred for higher wash temperatures (e.g. above about 50oC) . Other anionic surfactants useful for detersive purposes can also be included in the laundry detergent compositions of the present invention. Theses can include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, Ca- C22 primary or secondary alkanesulfonates, C8-C24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal C1trates, e.g., as described in British patent speC1fication No. 1,082,179, C8-C24 alkylpolyglycolethersulfates (containing up to \|0 moles of ethylene oxide); alkyl glycerol sulfonates, fatty Acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as the acyl ' i isethibnates, N-acyl taurates, alkyl sucC1namates and sulfosucC1nates, monoesters of sulfosucC1nates (espeC1ally saturated and unsaturated C12-C18 monoesters) and diesters of sulfosucC1nates (espeC1ally saturated and unsaturated C6-C12 diesters), acyl sarcosinates, sulfates of aIkyIpolysaccharides such as the sulfates of alkylpolyglucoside (the nonionic noBsulfated compounds being described below), branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as those of the formula RO(CH2CH2O)k-CH2C00-M+ wherein R is a C8-C22 alkyl, k is an integer from 1 to 10, and M is a soluble salt forming cation. Resin acids and hydrogenated resin acids ar^ also suitable, such as rosin, hydrogenated rosin, and resin acids and hydrogenated iresin acids present in or derived from tall oil.A Alkylbenzene sulfonates are highly preferred. EspeC1ally preferred are linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms. Further examples are described in "Surface Active Agents and Detergents" (Vol. I and II by Schwartz, Perrry and Berch). A variety of such surfactants are also generally disclosed in US 3,9?9,678, (Column 23, line 58 through Column 29, line 23, herdinj incorporated by reference). When included therein, the laundry detergent compositions of th^l present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants. The laundry detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as well as the nonionic and/or anionic surfactants other than those already described herein. Cationic detersive surfactants suitable for use in the laundry detergent compositions of the present invention are those having one long-chain hydrocarbyl group. Examples of such I cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides, and those surfactants having the formula: wherein R^ is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R"^ is selected form the group consisting of -CH2CH2", -CH2CH(CH3)-, -CH2CH(CH20H)-, -CH2CH2CH2-/ and mixtures thereof; each R' is selected from the group consisting of C1-C4 alkyl, C1-C4 hydroxyalkyl, benzyl ring structures formed by joining the two R"^ groups, -CH2CHOHCHOHCOR^CHOHCM20H, wherein R6 is any hexose or hexose polymer having a molecular weight less than about 1000, andfthydrogen when y is not 0; R5 is the same as R4 or is an alkyl chain,wherein the total number of carbon atoms or R plus R^ is not more than about 18; each y is from 0 to about 10,and the sum of the y values is from 0 to about 15; and X is any compatible anion. Highly preferred cationic surfactants are the water soluble quaternary ammonium compounds useful in the present composition having the formula: RlR2R3R4N+X- (i) wherein R1 is Cg-C1g alkyl, each of R2, R3 and R4 is independently C1-C4 alkyl, C1-C4 hydroxy alkyl, benzyl, and -(C2H4o)xH where x has a value from 2 to 5, and X is an anion. r Not more than one of R2, R3 or R4 should be benzyl. The preferred alkyl chain length for Ri is C12"C15, particularly where the alkyl group is a mixture of chain length, derived from coconut or palm kernel fat or is derived synthetically by olefin build up or 0X0 alcohols synthesis. preferred groups for R2R3 and R4 are methyl and hydroxyethyl groups and the anion X may be selected from halide, methosulphate, acetate and phosphate ions. Examples of suitable quaternary ammonium compounds of formulae (i) for use herein are: coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C12-15 dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myrlstyd trlmethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy)4 ammonium chloride or bromide; choline esters (compounds of formula (i) wherein R1 is di-alkyl imidazolines [compounds of formula (i)]. Other cationic surfactants useful herein are also described in US 4,228,044 and in EP 000 224. When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 25%, preferably from about 1% to about 8% by weight of such cationic surfactants. Ampholytic surfactants are also suitable for use in the laundry 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 from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g. carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants. When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants. Zwitterionic surfactants are also suitable for use in laundry detergent compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosplionium or tertiary sulfonium compounds. See US 3,929,678 (column 19, line 38 through column 22, line 48) for examples of zwitterionic surfactants. When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such zwitterionic surfactants. Semi-polar nonionic surfactants are a speC1al category of nonionic surfactants which include water-soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; watersoluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms. Semi-polar nonionic detergent surfactants include the amine oxide surfactants having the formula: wherein H^ is an a],kyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms, R4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atom, or mixtures thereof; x is from 0 to about 3: and each R5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups. The R5 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure. These amine oxide surfactants in particular include C10-c18 alkyl dimethyl amine oxides and C8-C12 alkoxy ethyl dihydroxy ethyl amine oxides. When included therein, the laundry detergent compositions of the present invention typically comprise from 0,2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar nonionic surfactants. Builder system The compositions according to the present invention may further comprise a builder system. Any conventional builder system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphosphonates, particularly ethylenediamine tetramethylene phosphonic acid and diethylene triamine pentamethylenephosphonic acid. Though less preferred for obvious environmental reasons, phosphate builders can also be used herein. Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP. Another suitable inorganic builder material is layered silicate, e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na2Si2O5) . Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed in Belgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of sucC1nic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German Offenle-enschrift 2,446,686, and 2,446,487, US 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623. Polycarboxylates containing three carboxy groups include, in partiC1;ular, water-soluble C1trates, aconitrates and C1traconates as well as sucC1nate derivatives such as the carboxymethyloxysucC1nates described in British Patent No. 1,379,241, lactoxysucC1nates described in Netherlands Application 7205873, and the oxypolycarboxylate materials such as 2-oxa-l,1,3-propane tricarboxylates described in British Patent No, 1,387,447. Polycarboxylates containing four carboxy groups include oxydisucC1nates disclosed in British Patent No..1,261,829, 1,1,2,S,-ethane tetracarboxylates, 1,1,3,3-propane tetrackrboxylates containing sulfo substituents include the sulfosucC1nate derivatives disclosed in British Patent Nos. 1,398,421 and 1,398,422 and in US 3,936,448, and the sulfonated pyrolysed C1trates described in British Patent No. 1,082,179, while polycarboxylates containing phosphone substituents are disclosed in British Patent No. 1,439,000. Alicyclic and heterocyclic polycarboxylates include cyclopentane-C1s,C1s-C1s-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydro-furan - C1s, C1s, C1s-tetrpicarboxylates, 2,5-tetrahydro-furan-C1s, discarboxylates, 2,2,^,5,-tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6-hexane - hexacarboxylates and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent No. 1,425,343. Of the above, the preferred polycarboxylates are hydroxy-carboxylates containing up to three carboxy groups per molecule, more particularly C1trates. Preferred builder systems for use in the present compositions include a mixture of a water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6), and a water-soluble carboxylate chelating agent such as C1tric acid. A suitable chelant for inclusion in the detergent composi-ions in accordance with the invention is ethylenediamine-N,N'-disucC1nic acid (EDDS) or the alkali metal, alkaline earth metal, ammonium, or substituted ammonium salts thereof, or mixtures thereof. Preferred EDDS compounds are the free acid form and the sodium or magnesium salt thereof. Examples of such preferred sodium salts of EDDS include Na2EDDS and Na4EDDS. Examples of such preferred magnesium salts of EDDS include MgEDDS and Mg2EDDS. The magnesium salts are the most preferred for inclusion in it compositions in accordance with the invention. Preferred builder systems include a mixture of a water-insoluble aluminosilicate builder such as zeolite A, and a water soluble carboxylate chelating agent such as C1tric acid. Other builder materials that can form part of the builder system for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates, amino polyalkylene phosphonates and amino polycarboxylates. Other suitable water-soluble organic salts are the homo-or co-polymeric acids or their salts, in which the polyccarboxylic acid comprises at least two carboxyl radicals separated form each other by not more than two carbon atoms. Polymers of this type are disclosed in GB-A-1,596,756. Examples of such salts are polyacrylates of MW 2000-5000 and their idiopolymers with maleic anhydride, such copolymers having a molecular weight of from 20,000 to 70,000, espeC1ally about 40,000. Detergency builder salts are normally included in amounts of from 5% to 80% by v/eight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%. Enzymes Preferred detergent compositions, in addition to the enzyme preparation of the invention, comprise other enzyme(s) which provides cleaning performance and/or fabric care benefits. Such enzymes include other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g. laccases). Proteases; Any other protease suitable for use in alkaline sol--utions can be used. Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is 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 prpteases are subtilisins, espeC1ally those derived from BaC1llus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of pociline or bovine origin) and the Fusarium protease described in WO 89/06270. Preferred commerC1ally available protease enzymes include those sold under the trade names Alcalase, Savinase, Primase, Durazym, and Esperase by Novo Nordisk A/S (Denmark), those sold under the tradename Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the tradename Opticlean and Optimase by Solvay Enzymes. Protease enzymes may be incorporated into the compositions in accordance with the invention at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition. Lipases; Any lipase suitable for use in alkaline solutions can be used. 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, e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcalioenes and P. pseudoalcaliqenes lipase, e.g., as described in EP 218 272, a P. cepaC1a lipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., as disclosed in GB 1,372,034, a P. fluorescens lipase, a BaC1l- lus lipase, e.g., a B. subtilis lipase (Dartois et al., (1993), Biochemica et Biophysica acta 1131, 253-260), a B. stearo-thermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422). Furthermore, a number of cloned lipases may be useful, including the PeniC1llium camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. et al., (1989), J. Biochem., 106, 383-388) and various Rhizopus lipases such as a R. delemar lipase (Hass, M.J et al., (1991), Gene 109, 117-113), a R niveus lipase (Kugimiya et al., (1992), BiosC1. Biotech. Biochem. 5fe, 716-719) and a R. oryzae lipase. Other types of lipolytic enzymes such as cutinases may also be useful, e.g., a cutinase derived from Pseudomonas mendoC1na as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446) . EspeC1ally suitable lipases are lipases such as Ml LipaseTM, Luma fastTM and LipomaxTM (Genencor) , LipolaseTM and Lipolase UltraTM (Novo Nordisk A/S) , and Lipase P "Amano" (Amano Pharmaceutical Co. Ltd.). The lipases are normally incorporated in the detergent composition at a level of from 0-00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition. Amylases: Any amylase (a and/or P) 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, a-amylases obtained from a speC1al strain of B. licheniformis described in more detail in GB 1,296,839. CommerC1ally available amylases are Duramyl™, Termamyl™, Fungamyl™ and BANTM (available from Novo Nordisk A/S) and RapidaseTM and Maxamyl PTM (available from Genencor). The amylases are normally incorporated in the detergent composition at a level of from 0,00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0,2% of enzyme protein by weight of the composition. Cellulafees; Any qellulase suitable for use in alkaline solutions can be used. Suitable cellulases include those of bacterikl or fungal origin. Chemically or genetically modified mutants are inqluded. Suitable cellulases are disclosed in US 4,435,307, which discloses fungal cellulases produced from Humicola insolens. EspeC1ally suitable cellulases are the cellulases having colour care benefits. Examples of such cellulases are cellulases described in European patent application No. 0 495 257. CommerC1ally available cellulases include CelluzymeTM produced by a strain of Humicola insolens, (Novo Nordisk A/S), and KAC-500(B)™ (Kao Corporation). Cellulases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition. Peroxidases/Oxidases; Peroxidase enzymes are used in combination with hydrogen peroxide or a source thereof (e.g. a percarbonate, perborate or persulfate). Oxidase enzymes are used in combination with oxygen. Both types of enzymes are used for "solution bleaching", i.e. to prevent transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor, preferably together with an enhanC1ng agent as described in e.g. WO 94/12621 and WO 95/01426. Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included. I Peroxidase and/or oxidase enzymes are normally incorporated in the detergent composition at a level of from 0.0000i% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition. Mixtures of the above mentioned enzymes are encompassed herein, 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 normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01% to 0.2% of enzyme protein by weight of the composition. Bleaching agents; Additional optional detergent ingredients that can be included in the detergent compositions of the present invention include bleaching agents such as PBl, PB4 and percarbonate with a particle size of 400-800 microns. These bleaching agent components can include one or more oxygen bleaching agents and, depending upon the bleaching agent chosen, one or more bleach activators. When present oxygen bleaching compounds will typically be present at levels of from about 1% to about 25%. In genqjral, bleaching compounds are optional added components in non-liquid formulations, e.g. granular detergents. The bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygen bleaches as well as others known in the art. The bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent. One category of oxygen bleaching agent that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chlqro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents are disclosed^in US 4,483,781, US 740,446, EP 0 133 354 and US 4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in US 4,634,551. Another category of bleaching agents that can be used encompasses the halogen bleaching agents. Examples of hypohalite bleaching agents, for example, include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such materials are normally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight. The hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in US 4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG), which are perhydrolyzed to form a peracid as the active bleaching speC1es, leading to improved bleaching effect. In addition, very suitable are the bleach activators C8(6-octanamido-caproyl) oxybenzene-sulfonate, C9(6-nonanamido caproyl) oxybenzenesulfonate and C1O (6-decanamido caproyl) oxybenzenesulfonate or mixtures thereof. Also suitable activators are acylated C1trate esters such as disclosed in European Patent Application No. 91870207.7. Useful bleaching agents, including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleaching compounds for use in cleaning compositions according to the invention are described in application USSN 08/136,626. The hydrogen peroxide may also be present by adding an enzymatic system (i.e. an enzyme and a substrate therefore) which is capable of generation of hydrogen peroxide at the beginning or during the washing and/or rinsing process. Such enzymatic systems are disclosed in European Patent Application EP 0 537 381. Bleaching agents other than oxygen bleaching agents are also known in the art and can be utilized herein. One type of non-oxygen bleaching agent of particular interest includes photoactivated bleaching agents such as the sulfonated zinc and/or aluminium phthalocyanines. These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, in the presence of oxygen, such as by hanging cl6thes out to dry in the daylight, the sulfonated zinc phthalocyanine is activated and, consequently, the substrate is bleached. Preferred zinc phthalocyanine and a photoactivated bleaching process are described in US 4,033,718. Typically, detergent composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc phthalocyanine. Bleaching agents may also comprise a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "EffiC1ent manganese catalysts for low-temperature bleaching". Nature 369. 1994, pp. 637-639. Suds suppressors; Another optional ingredient is a suds suppressor, exemplified by silicones, and silica-silicone mixtures. Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used in finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. Theses materials can be i incorporated as particulates, in which the suds suppressor is advantageously releasably incorporated in a water-soluble or waterdjispersible, substantially non surface-active detergent imperm.eable carrier. Alternatively the suds suppressor can be dissolved or dispersed in a liquid carrier and applied by spraying on to one or more of the other components. A preferred silicone suds controlling agent is disclosed in US 3,933,672. Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors, described in German Patent Application DTOS 2,646,126. An example of such a compound is DC-544, commerC1ally available form Dow Corning, which is a siloxane-glycol copolymer. EspeC1ally preferred suds controlling agent are the suds suppressor system comprising a mixture of silicone oils and 2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol which are commerC1ally available under the trade name Isofol 12 R. Such suds suppressor system are described in European Patent Application EP 0 593 841. Esecially preferred silicone suds controlling agents are described in European Patent Application No. 92201649.8. Said compositions can comprise a silicone/ silica mixture in combinatioh with fumed nonporous silica such as Aerosil. The suds suppressors described above are normally employed at levels of from 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight. Other components; Other components used in detergent compositions may be employed such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bacteriC1des, tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes. EspeC1ally suitable encapsulating materials are water soluble capsules which consist of a matrix of polysaccharide and polyhydroxy compounds such as described in GB 1,464,616. Other suitable water soluble encapsulating materials comprise dextrirjs derived from ungelatinized starch acid esters of substituted c^icarboxylic acids such as described in US 3,455/838. These acid-ester dextrins are, preferably, prepared from ^uch starches as waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of said encapsulation materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material consists of a modified maize starch and glucose. The starch is modified by adding monofunctional substituted groups such as octenyl sucC1nic acid anhydride. Antiredeposition and soil suspension agents suitable herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or co-polymeric polycarboxylic acids or their salts. Polymers of particular preferred polymer in accordance with EP 0 272 033 has the formula: (CH3(PEG)43)o,75(POH)o.25[T-PO)2.8(T-PEG)o.4]T(POH) 0.25 ( (PEG) 43CH3) 0.75 where PEG is -(OC2H4)0-, PO is (OC3H6O) and T is (POOC6H4CO) . Also very useful are modified polyesters as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol, the end groups consisting primarily of sulphobenzoate and secondarily of mono esters of ethylene glycol and/or 1,2-propanediol. The target is to obtain a polymer capped at both end by sulphobenzoate groups, "primarily", in the present context most of said copolymers herein will be endcapped by sulphobenzoate groups. However, some copolymers will be less than fully capped, and therefore their end groups may consist of monoester of ethylene glycol and/or 1,2-propanediol, thereof consist "secondarily" of such speC1es. The selected polyesters herein contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of 1,2-propanediol, about 10% by weight ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, and have a molecular weight of about 3.000. The polyesters and their method of preparation are described in detail in EP 311 342. Softening agents: Fabric softening agents can also be incorporated into laundry detergent compositions in accordance with the present invention. These agents may be inorganic or organic in type. Inorganic softening agents are exemplified by the smectite clayg disclosed in GB-A-1 400898 and in US 5,019,292. Organic fabric softening agents include the water insoluble tertiary amines as disclosed in GB-Al 514 276 and EP 0 oil 340 and their combination with mono C12-C14 quaternary ammonium salts are disclosed in EP-B-0 026 528 and di-long-chain mides as disclosed in EP 0 242 919. Other useful organic ingredients of fabric softening systems include high molecular weight polyethylene oxide materials as disclosed in EP 0 299 575 and 0 313 146. Levels of smectite clay are normally in the range from 5% to 15%, more preferably from 8% to 12% by weight, with the material being added as a dry mixed component to the remainder of the formulation. Organic fabric softening agents such as the water-insoluble tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to 5% by weight, normally from 1% to 3% by weight whilst the high molecular weight polyethylene oxide materials and the water soluble cationic materials are added at levels of from 0.1% to 2%, normally from 0.15% to 1.5% by weight. These materials are normally added to the spray dried portion of the composition, although in some instances it may be more convenient to add them as a dry mixed particulate, or spray them as molten liquid on to other solid components of the composition. Polymeric dye-transfer inhibiting agents: The detergent compositions according to the present invention may also comprise from 0001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weight of polymeric dye-transfer inhibiting agents. Said polymeric dye-transfer inhibiting agents are normally incorporated into detergent compositions in order to inhibit the transfer of dyes from colored fabrics onto fabrics washed therewith. These polymers have the ability of complexing or adsorbing the fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become attached to other articles in the wash. EspeC1ally suitable polymeric dye-transfer inhibiting agents are polyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone and N-vinylimidazole, polyvinylpyrrolidone poilymers, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Addition of such polymers also enhances the performance op the enzymes according the invention. The detergent composition according to the invention can be in liquid, paste, gels, bars or granular forms. Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (poiyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 ta 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. form 550 to 950 g/1; in such case, the granular detergent compositions according to the present invention will contain a lower amount of "Inorganic filler salt", compared to conventional granular detergents; typical filler salts are alkaline earth metal salts of sulphates and chlorides, typically sodium sulphate; "Compact" detergent typically comprise not more than 10% filler 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 lower amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergent is less than 30%, more preferably less than 20%, most preferably less than 10% by weight of the detergent compositions. The compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations and dishwashing operations. C1trate: Tri-sodium C1trate dihydrate C1tric: C1tric acid Perborate: Anhydrous sodium perborate monohydrate bleach, empirical formula NaBO2.H2O2 PB4: Anhydrous sodium perborate tetrahydrate Percarbonate: Anhydrous sodium percarbonate bleach of empirical formula 2Na2O3.3H2O2 TAED: A Tetraacetyl ethylene diamine CMC: Sodium carboxymethyl cellulose DETPMP: .! Diethylene triamine penta (methylene phosphonic acid), marketed by Monsanto under the Tradename Dequest 2060 PVP: Polyvinylpyrrolidone polymer EDDS: Ethylenediamine-N, N'-disucC1nic acid, [S,S] isomer in the form of the sodium salt Suds 25% paraffin wax Mpt 50°C, 17% hydrophobic silica, 58% Suppressor: paraffin oil Granular Suds 12% Silicone/silica, 18% stearyl alcohol, 70% suppressor: starch in granular form Sulphate: Anhydrous sodium sulphate HMWPEO: High molecular weight polyethylene oxide TAE 25^ fallow alcohol ethoxylate (25) ► Detergent Example I jA granular fabric cleaning composition in accordance with the iriVentioh may be prepared as follows: Sodium linear C12 alkyl 6.5 benzene sulfonate 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 sulphonate 0.1 Minors Up to 100 Detergent Example II A compact granular fabric cleaning composition (density 800 g/1) in accord with the invention may be prepared as follows: 45AS 8.0 25E3S 2.0 ■i 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0 C1tric acid 3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of the invention 0.1 TAED i 6.0 Percarbonate 22.0 EDDS 0.3 Granular puds suppressor 3.5 water/minors Up to 100% Detergent Example III Granular fabric cleaning compositions in accordance with the invention which are espeC1ally useful in the laundering of coloured fabrics were prepared as follows: LAS 10.7 - TAS 2.4 - TFAA - 4.0 45AS 3.1 10.0 45E7 4.0 25E3S - 3.0 68E11 1.8 25E5 - 8.0 C1trate 15.0 7.0 Carbonate - 10 C1tric 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 j of the invention 0.10 0. 05 Silicate 2.5 Sulphate 5.2 3.0 PVP I 0.5 - Poly (4-vinylpyridine)-N- - 0.2 Oxide/copolymer of vinyl- imidazole and vinyl- pyrrol idone Perborate 1.0 Phenol sulfonate 0.2 - Water/Minors Up to 100% Detergent Example IV Granular fabric cleaning compositions in accordance with the invention which provide "Softening through the wash" capability may be prepared as follows: 45AS " 10.0 LAS 7.6 68AS 1.3 45E7 4.0 25E3 - 5.0 Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride C1trate 5.0 3.0 Na--SKS-6 - 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4 Perborate 15.0 Percarbonate - 15.0 TAED 5.0 5.0 Smectite clay 10.0 10.0 HMWPEO - 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0 Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1 Water/Minors Up to 100% ■J Detergent Example V Heavy duty liquid fabric cleaning compositions in accordance with the invention may be prepared as follows: I II LAS acid form - 25.0 C1tric acid 5.0 2.0 2 5AS acid.form 8.0 25AE2S,acid form 3.0 25AE7 I 8.0 CFAA 5 DETPMP. 1.0 1.0 Fatty acid 8 Oleic acid - 1.0 Ethanql 4.0 6.0 Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coco-alkyl dimethyl - 3.0 hydroxy ethyl ammonium chloride Smectite clay - 5.0 PVP 2.0 Water / Minors Up to 100% LEATHER INDUSTRY APPLICATIONS A subtilase of the invention may be used in the leather industry, in particular for use in depilation of sKins. In said application a subtilase variant of the invention is preferably used in an enzyme composition which further comprise another protease. For a more detailed description of suitable other proteases see section relating to suitable enzymes for use in a determent composition {vide supra). WOOL INDUSTRY APPLICATIONS A subtilase of the invention may be used in the wool industry, in particular for use in cleaning of clothes comprising wool. In said application a subtilase variant of the invention is preferably used in an enzyme composition which further comprise another protease. For a more detailed description of suitable other proteases see section relating to suitable enzymes for use in a detergent composition {vide supra). The invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention as claimed. MATERIALS AND METHODS Strains; B. subtilis DN1885 (Diderichsen et al., 1990). B. lentus 309 and 147 are speC1fic strains of BaC1llus lentus, deposited with the NC1B and accorded the accession numbers NC1B 10309 and 10147, and described in US Patent No. 3,723,250 incorporated by reference herein. E. coli MC 1000 (M.J. Casadaban and S.N. Cohen (1980); J. Mol. Biol. 138 179-207) , was made r- ,m+ by conventional methods and is also described in US Patent Application Serial No. 039,298. Plasmids: pJS3: E. call - B. snbtilis shuttle vector containing a synthetic gene encoding for subtilase 309. (Described by Jacob Schiodt et al. in Protein and Peptide letters 3:39-44 (1996)). pSX222: B. subtilis expression vector (Described in WO 96/34946). General molecular biology methods: Unless otherwise mentioned the 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, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for BaC1llus", John Wiley and Sons, 1990). Enzymes for DNA manipulations were used according to the speC1fications of the suppliers. Enzymes for DMA manipulations Unless otherwise mentioned all enzymes for DNA manipulations, such las e.g. restiction endonucleases, ligases etc., are ob- tained 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 relatively to an enzyme standard (SAVINASEO), and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50°C, pH 8.3, 9 min. reaction time, 3 min. measuring time. A folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference. A GU is a GlyC1ne Unit, defined as the proteolytic enzyme activity which, under standard conditions, during a 15-minutes' incubation at 40 deg C, with N-acetyl casein as substrate, produces an amount of NH2-group equivalent to 1 mmole of glyC1ne. Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate sucC1nyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in the Journal of American Oil Chemists SoC1ety, Rothgeb, T.M., Goodlander, B.D., Garrison, P.H., and Smith, L.A., (1988). Fermentation: Fermentation of subtilase enzymes were performed at 30°C on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days. Consequently in order to make an e.g. 2 liter broth 20 Erlenmeyer flasks were fermented simultaneously. Media: BPX: Composition (per liter) Potato starch 100g Ground barley 50g Soybean flour 20g Na2HPO4 X 12 H2O 9g Pluronic 0.1g Sodium caseinate 10g The starch in the medium is liquified with a-amylase and i the medium is sterilized by heating at 120°C for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by addition 6f NaHCOa to 0.1 M. EXAMPLES EXAMPLE 1 Construction and Expression of Enzyme Variants; Site-directed mutagenesis: Subtilase 309 site-directed variants was made by the "Unique site elimination (USE)" or the "UraCil-USE" technique described respectively by Deng et al. (Anal. Biochem. 200:81-88 (1992)) and Markvardsen et al. (BioTechniques 18(3):371-372 (1995)). The template plasmid was pJS3, or a analogue of this containing a variant of Subtilase 309, e.g. USE mutagenesis was performed on pJS3 analogue containing a gene encoding the Y167A variant with a oligonucleotide directed to the construct R170L variant resulting in a final yi67A+R170L Subtilase 309 variant. The in pJS3 constructed Subtilase 309 variants was then subcloned into the B.subtilis pSX222 expression plasmid, using the restriction enzymes Kpnl and Mlul. LocmlizBd Random mutagenesis: The overall strategy to used to perform localized random mutagenesis was: a mutagenic primer (oligonucleotide) was synthesized which corresponds to the part of the DNA sequence to be mutagenized except for the nucleotide(s) corresponding to amino acid codon(s) to be mutagenized. Subsequently, the resulting mutagenic primer was used in a PCR reaction with a suitable opposite primer. The resulting PCR fragment was purified and digested and cloned into a E.coli~B.subtil is shuttle vector. Alternatively and if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a second suitable opposite primer so as to allow digestion and cloning of the mutagenized region into the shuttle vector. The PCR reactions are performed under normal conditions. Following this strategy a localized random library was constructed in SAVINASE wherein both position Y167 and R170 was completely randomized. One oligonucleotide was synthesized with 25% of each of the four bases (W) in the first and the second base at amino acid codons wanted to be mutagenized. The third nucleotide (the wobble base) in codons were synthesized with 50%G/50%C (S) to avoid two (TAA, TGA) of the three stop-codons . The mutagenic primer (5'- GTT TGG ATC AGT AGC TCC GAC TGC CAT TGC GTT CGC ATA SNU CGC CGG SNN GCT GAT TGA GCC -3' (anti-sense)) were used In a PCR reaction with a suitable opposite primer (e.g. 5' GAA CTC GAT CCA GCG ATT TC 3' (sense)) and the plasmid pJS3 as template. This resulting PCR product was cloned into the pJS3 shuttle vector by using the restriction enzymes Asp 718 and Bel I. The in pJS3 constructed localized random library was then subclored into the B.subtilis pSX222 expression plasmid, using the restriction enzymes KphI and Mlul, The library prepared contained approximately 100,000 individual clones/library. Ten randomly chosen colonies were sequenced to confirm the mutations Resigned. In order to purify a subtilase variant of the invention the B. subtilis •'pSX222 expression plasmid comprising a variant of the invention was transformed into a competent B. subtilis strain and was fermented as described above in a medium containing 10 \|ig/ml Chloramphenicol (CAM) . EXAMPLE 2 Purification of Enzyme Variants; This procedure relates to purification of a 2 litre scale fermentation of the Subtilisin 147 enzyme, the Subtilisin 309 enzyme or mutants thereof. Approximately 1.6 litres of fermentation broth were centrifuged at 5000 rpm for 35 minutes in 1 litre beakers. The supernatants were adjusted to pH 6.5 using 10% acetic acid and filtered on Seitz Supra SlOO filter plates. The filtrates were concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon SIYIO UF cartridge. The UF concentrate was centrifuged and filtered prior to absorption at rpom temperature on a BaC1traC1n affinity column at pH 7. The protease was eluted from the BaC1traC1n column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethyl-glutaric acid, 0.1 M boric acid and 0.002 M calC1um chloride adjusted to pH 7. The fractions with protease activity from the BaC1traC1n purification step were combined and applied to a 750 ml Sephadex G25 column (5 cm dia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.2 M boric acid and 0.002 m calC1um chloride adjusted to pH 6.5. Fractions with proteolytic activity from the Sephadex G25 column were combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm dia.) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid, and 0.002 M calC1um chloride adjusted to pH 6.5. The protease was eluted using a linear gradient of 0-0.1 M sodium ,^^loride in 2 litres of the same buffer (0-0.2 M sodium chloride in case of Subtilisin 147). In a final purification step protease containing fractions from the CM Sepharose column were combined and concentrated in an Amicon ultrafiltration cell equipped with a GR81PP membrane (from the Danish Sugar Factories Inc.) By using the techniques of Example 1 for the construction and the above isolation procedure the following subtilisin 309 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+R170A Y167P+R170S Y167I+R170L Y167F+R170T Y167F+R170E Yr67F+R170H Y167F+R170T Y167F+R170L Y167L R170H Y167S EXAMPLE 3 Wash Performance of Detergent Compositions Comprising Enzyme Variants The following examples provide results from a number of washing tests that were conducted under the conditions indicated Experimental conditions Table IV: Experimental conditions for evaluation of Subtilisin 309 variants. Subsequent to washing the cloths were flushed in tap water and air-dried. The above model detergent is a simple detergent formulation. The most characteristic features are that STP is used as builder and the content of anionic tenside (LAS) is quite high. Further the pH is adjusted to 10.4, which is within the normal range of a powder detergent. Water hardness is adjusted by adding CaCl2 and MgCl2 to deionized water. pH of the detergent solution is modified to the desired value by addition of acid. Measurement of remission (i?) on the test material has been done at 460 nm using an Elrepho 2000 photometer (without UV). Aperture: 10 mM. The wash performance at XnM is calculated as: Rx: is the wash effect of the enzyme at X nM (in remission units). R90: is the wash effect of the enzyme at 0 nM (blank value) Table VI: Variants and improvement factors for SAVINASE®, I ■ ,\| • As it can be seen from Table VI SAVINASE® variants of the invention exhibits an improvement in wash performance, In a similar wash assay following SAVINASE® variants showed a wash performance valu6 in the range between the variant Y167I+R170L and Y167A+R170S: Y167P Y167P+R170L Y167V+R170T Y167H-R170T Y167V+R170Q Y167S+R170Q Y167T+R170N Y167A+R170A Y167T+R170L Y167T+R170A Y167P+R170S EXAMPLE 4 Wash Performance of Detergent Compositions Comprising Enzyme Variants The following examples provide results from a number of wash ing tests that were conducted under the conditions indicated EXPERIMENTAL CONDITIONS Table VII: Experimental conditions for evaluation of Subtilisin 309 variants. The detergent used is a simple model formulation. pH is adjusted to 10,5 which is within the normal range for a powder detergent: The composition of model detergent 95 is as follows: 25% STP (NaaPaO10) 25% Na2SO4 10% Na2SO4 20% LAS (Nansa SOS) 5.0% Nonionic tenside (Dobanol 25-7) 5.0% Na2SO4 0.5% Carboxymethylcellulose (CMC) 9.5% Water Water hardness was adjusted by adding CaCl2 and MgCl2 (Ca2 +:Mg2+ = 2:1) to deionized water (see also Surfactants in Consumer Products - Theory, Technology and Application, springer 1986). pH of the detergent solution was adjusted to pH 10.5 by addition of HCl. Measurement of reflectance (R) on the test material was done at 460 ,nm using a Macbeth ColorEye 7000 photometer (Macbeth, Division of Kollmorgen Instruments Corporation, Germany). The measurements were done according to the manufacturers protocol. The wash performance of the Subtilisin 309 variants was evaluated by calculating a performance factor: P: Performance factor Rvariant: Reflectance of test material washed with variant Rsavinase'- Reflectance of test material washed with Savinase® Reiank: Reflectance of test material washed with no enzyme The claimed Subtilisin 309 variants all have improved wash performance compared to Savinase® - i.e. P > 1. The variants are divided into improvement classes designated with capital letters: Class A: 1 Class B: 1.5 2 Table VIII: Subtilisin 309 variants and improvement classes. The Savinase® variant Y167I+R170L+A194P was in a fermentation experiment compared to a variant Y167I+R170L not having the A194P substitution. Both variants were cloned in a pSX222 expression vector background and fermented as described above in a 100 ml BPX medium containing 10 μg/ml CAM. After 5 days fermentation 1.5 ml of the BPX fermentation medium was centrifuged and the supernatant was used to measure the ProtdiSlytic activity (KPNU) as described above. The fermentation medium containing the Y167I+R170L+A194P variant had a significant higher level of proteolytic activity as compared to the fermentation medium containing the Y167I+R170L variant. Both variants have the same speC1fic activity. Similar results were obtained with the variants Y167A+;R170S+A194P, Y167A+R170L+A194P, and Y167A+R170N+A194P compared with their corresponding variant without the A194P mutation. Further, similar results were obtained with the variants A133P+Y167A+R170S, A133D+Y167A+R170S, P129K+Y167A+R170S, and P131H+Y167A+R170S compared with their corresponding variants without the A133P, A133D, P129K, and P131H mutations. WE CLAIM: 1. A process for the preparation of 2-hydroxy-4-methylthiobutanoic acid or a salt thereof comprising the steps of: a) introducing an aqueous mineral acid and 2-hydroxy-4-methylthiobutanenitrile into a nitrile hydrolysis reactor comprising a first continuous stirred tank reactor; b) continuously hydrolysing 2-hydroxy-4-methylthiobutanenitrile within said nitrile hydrolysis reactor to produce a nitrile hydrolysis reactor product stream containing 2-hydroxy-4-methylthiobutanamide; and c) continuously hydrolysing 2-hydroxy-4-methylthiobutanamide in an amide hydrolysis flow reactor to produce a finished aqueous hydrolysate product containing 2-hydroxy-4-methylthiobutanoic acid. 2. The process as claimed in claim 1, further comprising, in between step (b) and step (c), continuously introducing water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid into said amide hydrolysis flow reactor; and optionally diluting said nitrile hydrolysis reactor product stream with said water before said nitrile hydrolysis reactor product stream and said water are introduced into said amide hydrolysis flow reactor. 3. The process as claimed in claim 2, wherein said water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid are mixed to form a finishing reaction stream such that the hydrolysis of 2-hydroxy-4-methylthiobutanamide is substantially completed as said finishing reaction stream flows through said amide hydrolysis flow reactor. 4. The process as claimed in claim 1, further comprising, in between step (b) and step (c), continuously introducing water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid into a continuous amide hydrolysis reactor comprising a second continuous stirred tank reactor such that a substantial portion of 2-hydroxy-4-methylthiobutanamide contained in said nitrile hydrolysis reactor product stream is hydrolysed in said second continuous stirred tank reactor to form a finishing reaction stream; optionally diluting said nitrile hydrolysis reactor product stream with said water before said nitrile hydrolysis reactor product stream and said water are introduced into said continuous amide hydrolysis reactor; and continuously introducing said finishing reaction stream into said amide hydrolysis flow reactor. 5. The process as claimed in claim 4, wherein said water, said nitrile hydrolysis reaction product stream and optionally said aqueous mineral acid are mixed to form an amide hydrolysis mixture that is introduced into the continuous amide hydrolysis reactor. 6. The process as claimed in claim 4 or claim 5, wherein said continuous amide hydrolysis reactor is operated at a temperature ranging from 70°C to 120^C. 7. The process as claimed in any preceding claim, wherein the aqueous mineral acid introduced into said nitrile hydrolysis reactor is sulfuric acid in an acid stream having a strength of between 50% by weight and 70% by weight sulfuric acid. 8. The process as claimed in anyone of claims 1 to 6, wherein the aqueous mineral acid introduced into said nitrile hydrolysis reactor is sulfuric acid in an acid stream having a strength of between 70% by weight and 99% by weight sulfuric acid, and said acid stream is continuously introduced to said nitrile hydrolysis reactor concurrently with a water stream to form sulfuric acid having a strength of between 50% by weight and 70% by weight on an organic-free basis within said nitrile hydrolysis reactor. 9. The process as set further in claim 7 or 8, wherein the molar ratio of sulfuric acid to 2-hydroxy-4-methylthiobutanenitrile introduced into said nitrile hydrolysis reactor is between 1.0 and 2.0 during the period between start up of the process until steady state conditions are established in said amide hydrolysis flow reactor, and thereafter said molar ratio of sulfuric acid to 2-hydroxy-4-methylthiobutanenitrile is between 0.6 and 1.5. 10. The process as claimed in any preceding claim, wherein at least 90% of 2-hydroxy-4-methylthiobutanenitrile is converted to 2-hydroxy-4-methylthiobutanamide within said nitrile hydrolysis reactor. 11. The process as claimed in any preceding claim, wherein the ratio of the rate of mineral acid flow into said amide hydrolysis flow reactor to the rates of 2-hydroxy-4-methylthiobutanamide and 2-hydroxy-4-methylthiobutanenitrile flow into said amide hydrolysis flow reactor is controlled to provide an excess of at least 5% molar excess mineral acid than is stoichiometrically equivalent to 2-hydroxy-4-methylthiobutanamide and 2-hydroxy-4-methylthiobutanenitrile introduced into said amide hydrolysis flow reactor. 12. The process as claimed in any preceding claim, wherein said finished aqueous hydrolysate product produced upon start up of the process comprises up to 0.05 wt.% amide and up to 0.05 wt.% nitrile. 13. The process as claimed in anyone of claims 3 to 12, wherein said amide hydrolysis flow reactor is a plug flow reactor, and the flow of said finishing reaction stream through said plug flow reactor is turbulent. 14. The process as claimed in anyone of claims 3 to 12, wherein said amide hydrolysis flow reactor comprises a pipeline reactor and said finishing reaction stream moves through said pipeline reactor in turbulent flow. 15. The process as claimed in claim 14, wherein said amide hydrolysis flow reactor is operated at a Reynolds number greater than 3,000. 16. The process as claimed in anyone of claims 1 to 12, wherein said amide hydrolysis flow reactor is a plug flow reactor and is operated at a Peclet number of at least 50, a peak temperature of 90°C to 120°C and a residence time between 30 and 100 minutes. 17. The process as claimed in any preceding claim, wherein said amide hydrolysis flow reactor operates substantially adiabatically; or operates substantially isothermally; or operates adiabatically and autothermally. 18. The process as claimed in any preceding claim, wherein 2-hydroxy-4-methylthiobutanoic acid is recovered by extracting 2-hydroxy-4-methylthiobutanoic acid from said finished aqueous hydrolysate product. 19. The process as claimed in any preceding claim, wherein 2-hydroxy-4-methylthiobutanoic acid is recovered by neutralizing said finished aqueous hydrolysate product to form an organic phase containing 2-hydroxy-4-methylthiobutanoic acid and an aqueous phase, and separating said organic phase and said aqueous phase to recover 2-hydroxy-4-methylthiobutanoic acid. 20. The process as claimed in claim 1, wherein, in step (a), 2-hydroxy-4-methylthiobutanenitrile and aqueous mineral acid are introduced into an aqueous hydrolysis mixture comprising 2-hydroxy-4-methylthiobutanamide, mineral acid and unreacted 2-hydroxy-4-methylthiobutanenitrile; wherein the nitrile hydrolysis reactor comprises a back-mixed reaction zone and a circulation zone in fluid flow communication with said back-mixed reaction zone, said circulation zone comprising a circulating line; and wherein the aqueous hydrolysis mixture is continuously circulated in a circulating stream that is withdrawn from said back-mixed reaction zone, passed through said circulation zone and returned to said back-mixed reaction zone, said circulating stream as withdrawn from said back-mixed reaction zone containing unreacted 2-hydroxy-4-methylthiobutanenitrile. 21. The process as claimed in claim 20, further comprising, in between step (b) and step (c), removing a portion of said circulating aqueous hydrolysis mixture as said nitrile hydrolysis reactor product stream from a forward flow port in said circulation zone; transferring said nitrile hydrolysis reactor product stream, water and optionally aqueous mineral acid to said amide hydrolysis flow reactor; and diluting said nitrile hydrolysis reactor product stream with said water at a point downstream of said forward flow port; wherein the sum of the residence time of said circulating stream in said circulation zone upstream of said forward flow port and the residence time of said nitrile hydrolysis reactor product stream downstream of said forward flow port prior to dilution is sufficient to substantially extinguish residual 2-hydroxy-4-methylthiobutanenitrile prior to the dilution of said nitrile hydrolysis reactor product stream. 22. The process as claimed in claim 21, wherein said water, said nitrile hydrolysis reaction product stream and optionally said aqueous mineral acid are mixed to form a finishing reaction stream that is introduced into the amide hydrolysis flow reactor. 23. The process as claimed in claim 20, further comprising, in between step (b) and step (c), removing a portion of said circulating aqueous hydrolysis mixture as said nitrile hydrolysis reactor product stream from a forward flow port in said circulation zone; transferring said nitrile hydrolysis reactor product stream, water and optionally aqueous mineral acid to a continuous amide hydrolysis reactor; and diluting said nitrile hydrolysis reactor product stream with said water at a point downstream of said forward flow port to provide an amide hydrolysis mixture, a substantial portion of 2-hydroxy-4-methylthiobutanamide contained in said amide hydrolysis mixture being hydrolysed in said continuous amide hydrolysis reactor to form a finishing reaction stream; and transferring said finishing reaction stream to said amide hydrolysis flow reactor; wherein the sum of the residence time of said circulating stream in said circulation zone upstream of said forward flow port and the residence time of said nitrile hydrolysis reactor product stream downstream of said forward flow port prior to dilution is sufficient to substantially extinguish residual 2-hydroxy-4-methylthiobutanenitrile prior to the dilution of said nitrile hydrolysis reactor product stream. 24. The process as claimed in claim 23, wherein the flow regimes and residence times are such as to provide the equivalent of at least one sequential back-mixed reaction zone in a nitrile extinction reaction region comprising the portion of said circulation zone upstream of said forward flow port and a forward flow reaction zone within which said nitrile reactor product stream flows between said forward flow port and said point of dilution or said continuous amide hydrolysis reactor. 25. The process as claimed in claim 23, wherein the flow regime in a forward flow reaction zone within which said nitrile reactor product stream flows between said forward flow port and said point of dilution or said continuous amide hydrolysis reactor is laminar, and the flow regime and residence time are such as to provide the equivalent of at least one sequential back-mixed reaction zone in said forward flow reaction zone. 26. The process as claimed in anyone of claims 21 to 25, wherein the flow regime in said circulating line is substantially turbulent, the temperature of said circulating stream is at least 50°C throughout said circulation zone, and the residence time of said circulating stream in said ciculation zone upstream of said forward flow port is at least 3 seconds. 27. The process as claimed in anyone of claims 21 to 26, wherein between said forward flow port and said point of dilution the temperature of said nitrile hydrolysis reactor product stream is at least 50°C and the residence time is at least 30 seconds. 28. The process as claimed in claim 23, wherein the sum of the residence time of said circulating stream in said circulation zone upstream of said forward flow port and the residence time of said nitrile hydrolysis reactor product stream downstream of said forward flow port prior to dilution is at least 20 seconds. 29. The process as claimed in anyone of claims 21 to 28, wherein said nitrile hydrolysis reactor product stream is transferred to said point of dilution via a vertical downcomer line. 30. The process as claimed in anyone of claims 20 to 29, wherein said finished aqueous hydrolysate product comprises up to 0.05 wt.% amide and up to 0.05 wt.% nitrile. 31. The process as claimed in claim 1, wherein at least 90% of 2-hydroxy-4-methylthiobutanenitrile is converted to 2-hydroxy-4-methylthiobutanamide within said nitrile hydrolysis reactor and the process further comprises continuously removing heat of nitrile hydrolysis from the nitrile hydrolysis reactor during step (b); and, in between step (b) and step (c), continuously introducing water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid into said amide hydrolysis flow reactor. 32. The process as claimed in claim 4, wherein at least 90% of 2-hydroxy-4-methylthiobutanenitrile is converted to 2-hydroxy-4-methylthiobutanamide within said nitrile hydrolysis reactor and the process further comprises continuously removing heat of nitrile hydrolysis from the nitrile hydrolysis reactor during step (b); and diluting the nitrile hydrolysis reactor product stream with said water before said nitrile hydrolysis reactor product stream and said water are introduced into said amide hydrolysis flow reactor for preventing liquid phase separation. 33. An apparatus for use in a process for the preparation of 2-hydroxy-4-methylthiobutanoic acid, comprising: a nitrile hydrolysis reactor (10) comprising a first continuous stirred tank reactor for the continuous hydrolysis of 2-hydroxy-4-methylthiobutanenitrile to produce an aqueous hydrolysis mixture containing 2-hydroxy-4- methylthiobutanamide, said nitrile hydrolysis reactor being adapted to receive aqueous mineral acid and 2-hydroxy-4-methylthiobutanenitrile feed streams and having an exit for the aqueous hydrolysis mixture; a circulating line (14) for circulating the aqueous hydrolysis mixture from said exit of said nitrile hydrolysis reactor through said circulating line and back to said nitrile hydrolysis reactor; a forward flow port (26) in said circulating line for removing a portion of the aqueous hydrolysis mixture to form a nitrile hydrolysis reactor product stream; and an amide hydrolysis flow reactor (16) for the continuous hydrolysis of 2- hydroxy-4-methylthiobutanamide in the nitrile hydrolysis reactor product stream to produce a finished aqueous hydrolysate product containing 2- hydroxy-4-methylthiobutanoic acid. 34. The apparatus as claimed in claim 33, wherein said amide hydrolysis flow reactor is insulated for adiabatic operation. 35. The apparatus as claimed in claim 33 or claim 34, further comprising a transfer line (28) for transporting the nitrile hydrolysis reactor product stream to a point of dilution (30), said circulating line and said transfer line providing additional residence time for substantially extinguishing residual 2-hydroxy-4-methylthiobutanenitrile prior to dilution of the nitrile hydrolysis reactor product stream. 36. The apparatus as claimed in any one of claims 33 to 35, further comprising a second continuous stirred tank reactor (36) which is located upstream of said amide hydrolysis flow reactor and is adapted to receive water and the aqueous hydrolysis mixture exiting said nitrile hydrolysis reactor and to transfer a finishing reaction stream exiting from said second continuous stirred tank reactor to said amide hydrolysis flow reactor. 37. The apparatus as claimed in any one of claims 33 to 36, further comprising means for removing heat generated by dilution of mineral acid and reaction of 2-hydroxy-4-methylthiobutanenitrile and water in order to maintain a reaction temperature for hydrolysis of 2-hydroxy-4-mehtylthiobutanenitrile. 38. A process for the preparation of 2-hydroxy-4-methylthiobutanoic acid or a salt thereof substantially as herein described with reference to the accompanying drawings. 39. An apparatus for use in a process for the preparation of 2-hydroxy-4- methylthiobutanoic acid substantially as herein described with reference to the accompanying drawings. Dated this 3 day of June 1996

Full Text

Title: Protease Variants and Compositions TECHNICAL FIELD
This invention relates tp novel mutant protease enzymes or enzyme variants useful in formulating detergent compositions and exhibiting improved wash performance in detergents; cleaning and detergent compositions containing said enzymes; mutated genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing said enzyme variants.
BACKGROUND OF THE INVENTION
In the: detergent industry enzymes have for more than 3 0 years been iiiplemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially

mo

t iikportant enzymes are proteases.

An increasing number of commercially used proteases are protein engineered variants of naturally occurr'ing wild type proteases, e.g. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).
. Further a number of protease variants are describe in the art, such as in EP 130756 (GENENTECH) (corresponding to US Reissue Patent 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; Russel and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Alrtgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER Si GAMBLE COMPANY); WO 95/30010 (PROCTER & GAMBLE COMPANY); WO 95/29979 (PROCTER & GAMBLE COMPANY); US 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, even though a number of useful protease variants have been described, there is still a need for new improved protease variants for a number of industrial uses.
Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry.
SUMMARY OF THE INVENTION
Recently it has been identified that subtilase variant with improved wash performance can be obtained by substituting one or more amino acid residues situated in, or in the vicinity of a hydrophobic domain of the parent subtilase for an amino acid residue more hydrophobic than the original residue, said hydrophobic domain comprising the residues corresponding to residues 1165, Y167, Y171 of BLS309, and said residues in the yicinity thereof comprises residues corresponding to the residues E136, G159, S164, R170, A194, and G195 of BLS309 (WO 96/34946).
Based on this information the present inventors have intensively studied numerous of the possible combinations of
the Y167 and R170 residues of SAVINASE®, and identified a number of variants with surprisingly increased improved wash performance.
For further details reference is made to working examples herein {vide infra).
Accordingly, the present invention relates in its first aspect to a subtilase protease variant having improved wash performance in detergents, comprising modifications in both position 167 and 170
In a second aspect the invention relates to a subtilase enzyme variant having improved wash performance in detergents, comprising at least one modification chosen 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
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} ,i^7{L,I, or V} 170H
167{G,A,S, or T}.

a matrix
The nomenclature above e.g. the "l67fG,A,S, or T}+170{G,A,S, or T}" variant (s) is nomenclature, wherein the term
"167{G,A,S, or T}+170{G,A,S, or T-}" comprise following
variants:
167G+170G, 167G+170A, 167G + 170S, 167G-4-170T,
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 further details relating to the nomenclature of a subtilase variant herein, see section "Definitions" herein (vide infra).
In a third aspect the invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a fourth aspect the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase 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 a further aspect the invention relates to the production of the subtilisin enzymes of the invention by inserting an expression vector according to the fourth aspect into a suitable microbial host, cultivating the host to express

the desired subtilase enzyme, and recovering the enzyme product.
Even further the invention relates to a composition comprising a subtilase variant of the invention.
Finally the invention relates to the use of the mutant enzymes for a number of industrial relevant uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes.
DEFINITONS Prior to discussing this invention in further detail, the following term will first be defined.

Nomenclature of Amino Acids
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 = Gin = Glutamine
D = Asp = Aspartic Acid
E = Glu = Glutamic Acid
K = Lys = Lysine
R = Arg = Arginine
H = His = Histidine
X = Xaa = Any amino acid
Nomenclature of nucleic acids
A = Adenine
G = Guanine
C = Cytosine
T = Thymine (only in DNA)
U = Uracil (only in RNA)
Nomenclature of variants
In describing the various enzyme variants produced or con-templated according to the invention, the following nomenclatures have been adapted for ease of reference:
Original amino acid(s) position(s) substituted amino acid(s)

According to this the substitution of Glutamic acid for glycine in position 195 is designated as:
Glyl95Glu or G195E
a deletion of glycine in the same position is:
Glyl95 or G195*
and insertion of an additional amino acid residue such as lysine is:
Glyl95GlyLys or G195GK
) Where a deletion in comparison with the sequence used for the numbering is indicated, an insertion in such a position is ihdicated as:
* 36Asp or *36D for insertion of an aspartic acid in position 36
Multiple mutations are separated by pluses, e.g.:
Argl70Tyr + Glyl95Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.
When the original amino acid(s) may comprise any amino acid, then are only position(s) and substituted amino acid(s) mentioned, e.g.: 170Ser.
This nomenclature is particular relevant relating to modification(s) according to the invention in homologous subtilases (vide infra). 170Ser then comprise e.g. both a Lysl70Ser modification in BASBPN and Argl70Ser modification in BLSSAVI. See figure 1 in relation to said examples.
When the substituted amino acid(s) may comprise any amino acid, then are only the original amino acid(s) and position(s) mentioned, e.g.: Argl70.
When both the original amino acid(s) and substituted amino acid(s) may comprise any amino acid, then is only the position(s) mentioned, e.g.: 170.

When the original amino acid(s) and/or substituted amino acid(s) may comprise more than one and not all amino acid(s) ,
then the selected amino acids are surrounded by a
"{ }", e.g.
Argl70{Gly,Ala,Ser, or Thr}
comprising the variants
Argl70Gly, Argl70Ala, Argl70Ser, or Argl70Thr; or e.g.
Tyrl67{Glyi,Ala,Ser, or Thr} + Argl70{Gly,Ala,Ser, or Thr}
comprising the variants
Tyrl67Gly + Argl70Gly, Tyrl67Gly + Argl70Ala, Tyif'l67Gly + 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 particular relevant relating to a
conservative amino acid modification(s) of a preferred
subtilase variants of the invention.
E.g. a conservative amino acid modification(s) of e.g.
a preferred variant Tyrl67Ala + Argl70Ser are
Tyrl67{Gly,Ala,Ser, or Thr} -f Argl70{Gly,Ala,Ser, or Thr},
which here substitute a small amino acid to another small amino
acid. See section "Detailed description of the invention" for
further details.
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3).
i Numbering of amino acid positions/residues
If ho other mentioned the amino acid numbering used herein
correspond to that of the subtilase BPN" (BASBPN) sequence. For

further description of the BPN sequence see Siezen et al., Protein Engng. 4 (1991) 719-737 and Figure 1.
Serine proteases
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residiie at the active site (White, Handler and Smith, 1973 "Prindiples of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272) .
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al,, Protein Engng. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases have been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. and figure' 1 herein.
One subgroup of the subtilases, I-Sl, comprises the "classical" subtilisins, such as subtilisin 168, subtilisin BPN', subtilisin Carlsberg (ALCALASE*, NOVO NORDISK A/S), and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognised by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprise

I
enzymes such as subtilisln PB92 (MAXACAL*, Gist-Brocades NV) , subtilisin 309 (SAVINASE®, NOVO NORDISK A/S), subtillsin 147 (ESPERASE*, NOVO NORDISK A/S), and alkaline elastase YaB.
"SAVINASE®"
SAVINASE® is marketed by NOVO NORDISK A/S.
It is subtilisin 309 from B. Lentus and differs from BABP92 only in having N87S (see figure 1 herein).
Parent subtilase
The term "parent subtilase" is a subtilase defined according to Siezen et al. (Protein Engineering 4:719-737 (1991)). For further details see description of "SUBTILASES" immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modification have been made while retaining the characteristic of a subtilase. Alternatively the term "parent subtilase" may be termed "wild-type subtilase".
Modification's) of a subtilase variant
The term "modification(s)" used in connection with modification (s) of a subtilase variant as discussed herein is defined to include chemical modification as well as genetic manipulation. The modification(s) can be by substitution, deletion and/or insertions in or at the amino acid(s) of interest.
Subtilase variant
In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
Homolocfous subtilase sequences

Specific amino acid residues of SAVINASE® subtilase are
identified for modification herein to obtain a subtilase variant of the invention.
However, the invention is not limited to modifications of this particular subtilase, but extend to other parent (wild-type) subtilases, which have a homologous primary structure to
that of SAVINASE®.
In order to identify other homologous subtilases, within the scope of this invention, an alignment of said subtilase(s) to a group of previously aligned subtilases is performed keeping the previous alignment constant. A comparison to 18 highly conserved residues in subtilases is performed. The 18 highly conserved residues are shown in table I (see Siezen et al. for further details relating to said conserved residues).
Table I
18 highly conserved residues in subtilases
Position: Conserved residue
23 G
32 D
34 G
t
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 aligning allowing for necessary insertions and
deletions in order to maintain the alignment suitable
homologous residues are identified. Said homologous residues
can then be modified according to the invention.
Using the CLUSTALW (version 1.5, April 1995) computer I alignment program (Thompson, J.D., Higgins, D.G. and Gibson,
T.J. (i994) Nucleic Acids Research, 22:4673-4680.), with GAP
open penalty of 10.0 and GAP extension penalty of 0.1, using
the BEOSUM30 protein weight matrix, alignment of a given
subtilase to a group of previously aligned subtilases is
achieved using the Profile alignments option in the program.
For a given subtilase to be within the scope of the invention,
preferably 100% of the 18 highly conserved residues should be
conserved. However, alignment of greater than or equal to 17
out of the 18 residues, or as little as 16 of said conserved
residues is also adequate to identify homologous residues.
Conservation of the, in subtilases, catalytic triad
Asp32/His64/Ser221 should be maintained.
The previously defined alignment is shown figure 1, where the percent identity of the individual subtilases in this alignment to the 18 highly conserved residues are shown too.
Further in said process to identify a homologous parent (wild-type) subtilase within the scope of the invention, the 18 conserved residues above relates to the parent (wild-type) primary sequence of said homologous parent subtilase- In order words, if a parent subtilase have been modified in any of said 18 conserved residues above, it is the original parent wild-type sequence in said 18 conserved residues, which determine whether or not both the original parent subtilase and a possible variant of said parent subtilase, which are modified in any of said 18 conserved residues above, are a homologous subtilase within 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 a homologous subtilases and corresponding suitable residues to be modified according to the invention•

It is obvious that a similar or larger table covering other homologous subtilases may easily be produced by a person skilled in the art.
Wash performance
The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, wash-ability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property.
Isolated DNA sequence
The term "isolated", when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are

free of other genes with which they are ordinarily associated, but; may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of or-
dinaryi skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term "an isolated DNA sequence" may alternatively be termed "a cloned DNA sequence".
Isolated protein
When applied to a protein, the term "isolated" indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities" (see below)). It is preferred to provide the protein in a highly purified form, i.e., greater than 40% pure, greater than 60% pure, greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.
The term "isolated protein" may alternatively be termed "purified protein".
Homologous impurities
The term "homologous impurities" means any impurity (e.g. another polypeptide than the polypeptide of the invention) which originate from the homologous cell where the polypeptide of the invention is originally obtained from.
Obtained from The term "obtained from" as used herein in connection witah a specific microbial source, means that the polynucleotide and/or polypeptide produced by the specific source, or by a cell in which a gene from the source have been inserted.
Substrate

The term "Substrate" used in connection with a substrate for a protease is should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.
Product
The term "product" used in connection with a product derived from a protease enzymatic reaction should in the context of this invention be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.
•;
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows an alignment of a number of homologous subtilases, which are aligned to 18 highly conserved residues in subtilases.
DETAILED DESCRIPTION OF THE INVENTION
Subtilase variants with improved wash performance:
Numerous subtilase variants of the invention is tested herein and showing improved wash-performance in detergents (see working examples herein (vide infra)).
Accordingly, an embodiment of the invention relates to a subtilase enzyme variant according to the second aspect of the invention, wherein the modification is chosen 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
lft7F+170T
167F+170L
167L
17 OH
167S.
The present inventors have identified the improved wash
performance variants in BLS309 (SAVINASE®), which in its parent wild-type primary sequence comprise Y167 and R170 as the original wild-type amino acids (see figure 1).
Accordingly, a further embodiment of the invention relates to a subtilase enzyme variant according to the second aspect of the invention, wherein the modification is chosen 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}
Yi67{F,W or Y}+R170{E, or D}
Y167{F,W or Y}+R170{R,K, or H}
Yl67{F,W or Y}+R170{L,I, or V}
Y167{L,I, or V}
R170H
Y167{G,A,S, or T}; or more preferred

a subtilase enzyme variant according to the embodiment immediately above, wherein the modification is chosen from the group comprising (in BASBPN numbering):
Y167A+R170S
Y167A+R170L
Y167A+R170N
Y167P
Y167P+R170L
Y167V+R170T
Y167I+R170T
Yq67V+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 substitution of one amino acid to a similar conservative amino acid only give a minor change in the characteristic of the enzyme.
Table III below list groups of conservative amino acids.
Table III Conservative amino acid substitutions Basic: R = arginine
K = lysine

H = histidine Acidic: E = glutamic acid
D = aspartic acid Polar: Q = glutamine
N = asparagine Hydrophobic: L = leucine
I = isoleucine
V = valine
M = methionine
Aromatic: F = phenylalanine
W = tryptophan
Y = tyrosine
Small: G = glycine
A = alanine S = serine T = threonine
Accordingly, subtilase variants such as 167A4-170S, 167G+170S, 167S+170S, and 167T+170S, will have a similar wash-performance improvement•
Further, subtilase variants such as Y167G+R170S, Y167S+R170S, and Y167T+R170S, will have a similar wash-performance improvement as the variant Y167A+R170S. See e.g. working examples herein for a specific wash performance test of said Y167A+R170S variant.
Based on the disclosed and in particular the numerous exemplified subtilase variants herein, it is routine work, for a person skilled in the art, to identify further suitable conservative modification(s) , of in particular said exemplified variants, in order to obtain a subtilase variant with improved wash-performance, according to all aspects and embodiment of a subtilase variant of the invention.
In embodiments of the invention, the subtilases of interest are those belonging to the subgroups I-Sl and I-S2.
Relating to subgroup I-Sl preferred parent subtilase is chosen from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR or functional variants thereof having retained the characteristic of sub-group I-Sl.

Relating to subgroup I-S2 preferred parent subtilase is chosen from the group comprising BLS147, BLS309, BAPB92, TVTHER AND BYSYAB or functional variants thereof having retained the characteristic of sub-group I-S2.
In particular said parent subtilase is BLS309
(SAVINASE® NOVO NORDISK A/S) .
The present invention also comprises any one or more modifications in the above mentioned positions in combination with any other modification to the amino acid sequence of the parent enzyme. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged. The art describe a number of subtilase variants with differnt improved properties and a number of those are mentioiied in the "Background of the invention" section herein (vide supra). Those references are disclosed here as references to identify a subtilase variant, which advantageously can be combined with a subtilase variant of the invention.
Such combinations comprise the positions: 222 (improve oxidation stability), 218 (improves thermal stability), substitutions in the Ca-binding sites stabilizing the enzyme, e.gr. position 76, and many other apparent from the prior art.
In further embodiments a subtilase variant of the invention may advantageously be combined with one or more modification(s) 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 BLS309 and BAPB92 variants are considered appropriate for combination:
K27R, *36D, S57P, N76D, S87N, G97N, SIOIG, V104A, V104N, V104Y, H120D, N123S, Q206E, N218S, M222S, M222A, T224S, K235L and T274A.
Furthermore variants comprising any of the variants S101G+V104N, S87N4-S101G4-V104N, K27R+V104Y+N123S+T274A, or N7 6D+V104A or other combinations of these mutations (V104N, SIOIG, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with any one or more of the modification(s) mentioned above exhibit improved properties.

Even further subtilase variants of the main aspect(s) of the invention are preferably combined with one or more modification(s) in any of the positions 129, ,131, 133 and 194, preferably as 129K, 131H, 133P, 133D and 194P modifications, and most preferably as P129K, P131H, A133P, A133D and A194P modifications. Any of those modification(s) give a higher expression level of a subtilase variant of the invention. For further details reference is made to working examples herein (vide infna) .
Accordingly, an , even further embodiment of the invention relates to a variant according to the invention, wherein said modification is chosen from the group comprising:
Y167A+R170S+A194P
.Y167A+R170L+A194P
Y167A+R170N+A194P
Y167A+R170S+P129K
Y167A+R170L+P129K
Y167A+R170N+P129K
Y167A+R170S+P13IH
Y167A+R170L+P131H
Y167A+R170N+P131H
Y167A+R170S+A133P
Y167A+R170L+A133P
Y167A+R170N+A133P
Y167A+R170S+A133D
Y167A+R170L+A133D
Y167A+R170N+A133D
PRODUCING MUTATIONS IN SUBTILASE GENES
Many methods for cloning a subtilase of the invention and for introducing mutations into genes (e.g. subtilase genes) are well known in the art.
In general standard procedures for cloning of genes and introducing mutations (random and/or site directed) into said genes may be used in order to obtain a subtilase variant of the invention. For further description of suitable techniques reference is made to working examples herein {vide infra) 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; Harwood, 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 may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome(s) into which it has been integrated.
The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to 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, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the enzyme.
The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
Examples of suitable prompters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkalihe protease gen, or the Bacillus pumilus xylosidase gene,

or the phage Lambda PR or PL promoters or the E. coli Lac, trp or tac promoters.
I The DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.
The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides.
To direct an enzyme of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.).
HOST QELL
The DNA sequence encoding the present enzyme introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably

connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
TJae host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells.
Examples of bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram-pbsitive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. jbrevls, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circularise B. lautus, B. megatbBrium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or iS. murinu, or gram-negative bacteria such as Echerichia coli. The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
When expressing the enzyme in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.
When expressing the enzyme in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or may be directed to the

extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
METHOD OF PRODUCING SUBTILASE
The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting 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 enable heterologous recombinant production of the enzyme of the invention.
Thereby it is possible to make a highly purified subtilase composition, characterized in being free from homologous impurities.
In this context homologous impurities means any impurities (e.g. other polypeptides than the enzyme of the invention) which originate from the homologous cell where the enzyme pf the invention is originally obtained from.
The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells iin question. The expressed subtilase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
USE OF A SUBTILASE VARIANT OF THE INVENTION
A subtilase protease variant of the invention may be used for a number of industrial applications, in particular within the detergent industry.

Further the invention relates to an enzyme composition, which comprise a subtilase variant of the invention.
An summary of preferred industrial applications and corresponding preferred enzyme compositions are described below.
This summary is not in any way intended to be a complete list of suitable applications of a subtilase variant of the invention. A subtilase variants of the invention may be used in other industrial applications known in the art to include use of a protease, in particular a subtilase.
DETERGENT COMPOSITIONS COMPRISING THE MUTANT ENZYMES
The present invention comprises the use of the mutant enzymes of the invention in cleaning and detergent compositions and such compositions comprising the mutant subtilisin enzymes. Such cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions.
Further reference is made to workings example(s) herein showing wash performance improvements for a number of subtilase variants of the invention.
DETERGENT DISCLOSURE AND EXAMPLES Surfactant system
The detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant can be selected from nonibnic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants.
The surfactant is typically present at a level frqm 0.1% to 60% by weight.
The surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or
gel compositions the surfactant is most preferably formulated
in such a way that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.

Preferred systems to be used according to the present Lnvention comprise as a surfactant one or more of the nonionic and/or anionic surfactants described herein.
Polyethylene, polypropylene, and polybutylene oxide conden-sates of alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present inven-tion, with the polyethylene oxide condensates being preferred. These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight chain or branched-chain configuration' with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and Triton™ X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (e.g., alkyl phenol ethoxylates).
The condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the nonionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. Preferred are the condensation products of alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcohol. About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available nonionic surfactants of this type include TergitolTM 15-S-9 (The condensation product of C11-C15 linear alcohol with 9 moles ethylene oxide), TergitolTM 24-L-6 NMW (the condensation product of C12-C14 primary alcohol with 6

naoles ethylene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; WeodolTM 45-9 (the condensation product of C14-C15 linear
• TM
alcohol with 9 moles of ethylene oxide), Neodol 23-3 (the condensation product of C12-C13 linear alcohol with 3.0 moles of ethylene oxide) , NeodolTM 45-7 (the condensation product of C14-C15 linear alcohol with 7 moles of ethylene oxide), Neodol™ 45-5 (the condensation product of C14-C15 linear alcohol with 5 mol6s of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C13-C15 alcohol with 9 moles ethylene oxide), marketed by The Procter & Gamble Company, arid Genapol LA 050 (the condensation product of C12-C14 alcohol with 5 moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB in these products is from 8-11 and most preferred from 8-10.
Also useful as the nonionic surfactant of the surfactant systems of the present invention are alkylpolysaccharides disclosed in US 4,565,647, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e.g. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing saccharide containing 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside). The intersaccharide bonds can be, e.g., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6- positions on the preceding saccharide units.
The preferred alkylpolyglycosides have the formula
R20(CnH2nO)t(glycosyl)x
wherein R2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to

ibout 18, preferably from about 12 to about 14, carbon atoms; n Is 2 or 3, preferably 2; t is from 0 to about 10, pre-ferably 3; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the 1-position). The additional glycosyl units can then be attached between their 1-position and the preceding glycosyl units 2-, 3-, 4-, and/or 6-position, preferably predominantly the 2-position.
The ciondensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional nonionic surfactant systems of the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility. The addition of polyoxyethylene moieties 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 up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide. Examples of compounds of this type include certain of the commercially available PluronicTM surfactants, marketed by BASF.
Also suitable for use as the nonionic surfactant of the nonionic surfactant system of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene pxide and ethylenediamine. The hydrophobic moiety of these products consists of the reaction product of ethylenediamine and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000. This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product dontains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000

to abput 11,000. Examples of this type of nonionic surfactant include certain of the commercially available TetronicTM compounds, marketed by BASF.
Preferred for use as the nonionic surfactant of the surfactant systems of the present invention are polyethylene oxide condensates of alkyl phianols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylen6oxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C8-C14 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C8-C18 alcohol ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.
Highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the formula

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 hydroxyIs directly connected to the chain, or an alkoxylated derivative thereof. Preferably, R1 is methyl, R2 is straight C11-15 alkyl or C16-18 alkyl or alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction.
Highly preferred anionic surfactants include alkyl I alkoxylated sulfate surfactants. Examples hereof are water soluble salts or acids of the formula R0(A)mS03M wherein R is an unsubstituted C10-C-24 alkyl or hydroxyalkyl group having a C10-C24 alkyl component, preferably a C22-C20 alkyl or hydroxyalkyl, more preferably C12-C18 alkyl or hydroxyalkyl, A is an
ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about
0.5 and about 3, and M is H or a cation which can be, for
example, a metal cation (e.g., sodium, potassium, lithium.

calC1um, magnesium, etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein. SpeC1fic examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like. Exemplary surfactants are C12-C18 alkyl polyethoxylate (1.0) sulfate (C12-C18E(1.0)M), C12-C18 alkyl polyethoxylate (2-25) sulfate (C12-C18(2.25)M, and C12-C18 alkyl polyethoxylate (3.0) sulfate (C12-C18E(3.0)M) , and C12-C18 alkyl polyethoxylate (4.0) sulfate (C12~C18E(4.0)M) , wherein M is conveniently selected from sodium and potassium.
Suitable anionic surfactants to be used are alkyl ester sulfonate surfactants including linear esters of C8-C20 carboxylic acids (i.e., fatty acids) which are sulfonated with gaseous SO3 according to "The Journal of the American Oil Chemists SoC1ety", 52 (1975), pp. 323-329. Suitable starting materials would include natural fatty substances as derived from tallow, palm oil, etc.
The preferred alkyl ester sulfonate surfactant, espQc:.ally for laundry applications, comprise alkyl ester sulfonate surfactants of the structural formula:

wherein R3 is a C8-C20 hydrocarbyl, preferably an alkyl, or combination thereof, R is a C1-C5 hydrocarbyl, preferably an alkyl, or combination thereof, and M is a cation which forms a water soluble salt with the alkyl ester sulfonate. Suitable salt-forming cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethonolamine, and triethanolamine.

Preferably, R3 is C10-C16 alkyl, and R4 is methyl, ethyl or isopropyl. EspeC1ally preferred are the methyl ester sulfonates wherein R3 is C1Q-C1G alkyl.
Other suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO3M wherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C1Q-C20 alkyl component, more preferably a C12-C18 alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium), or ammonium or substituted ammonium (e.g. methyl-, dimethyl-, and trimethyl ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and quaternary ammonium cations derived from alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like). Typically, alkyl chains of C12-C16 are preferred for lower wash temperatures (e.g. below about 50oC) and C16-C18 alkyl chains are preferred for higher wash temperatures (e.g. above about 50oC) .
Other anionic surfactants useful for detersive purposes
can also be included in the laundry detergent compositions of
the present invention. Theses can include salts (including, for
example, sodium, potassium, ammonium, and substituted ammonium
salts such as mono- di- and triethanolamine salts) of soap, Ca-
C22 primary or secondary alkanesulfonates, C8-C24
olefinsulfonates, sulfonated polycarboxylic acids prepared by
sulfonation of the pyrolyzed product of alkaline earth metal
C1trates, e.g., as described in British patent speC1fication
No. 1,082,179, C8-C24 alkylpolyglycolethersulfates (containing
up to |0 moles of ethylene oxide); alkyl glycerol sulfonates,
fatty Acyl glycerol sulfonates, fatty oleyl glycerol sulfates,
alkyl phenol ethylene oxide ether sulfates, paraffin
sulfonates, alkyl phosphates, isethionates such as the acyl
' i isethibnates, N-acyl taurates, alkyl sucC1namates and
sulfosucC1nates, monoesters of sulfosucC1nates (espeC1ally
saturated and unsaturated C12-C18 monoesters) and diesters of
sulfosucC1nates (espeC1ally saturated and unsaturated C6-C12
diesters), acyl sarcosinates, sulfates of aIkyIpolysaccharides

such as the sulfates of alkylpolyglucoside (the nonionic noBsulfated compounds being described below), branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as those of the formula RO(CH2CH2O)k-CH2C00-M+ wherein R is a C8-C22 alkyl, k is an integer from 1 to 10, and M is a soluble salt forming cation. Resin acids and hydrogenated resin acids ar^ also suitable, such as rosin, hydrogenated rosin, and resin acids and hydrogenated iresin acids present in or derived from tall oil.A
Alkylbenzene sulfonates are highly preferred. EspeC1ally preferred are linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms.
Further examples are described in "Surface Active Agents and Detergents" (Vol. I and II by Schwartz, Perrry and Berch). A variety of such surfactants are also generally disclosed in US 3,9?9,678, (Column 23, line 58 through Column 29, line 23, herdinj incorporated by reference).
When included therein, the laundry detergent compositions of th^l present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants.
The laundry detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as well as the nonionic and/or anionic surfactants other than those already described herein.
Cationic detersive surfactants suitable for use in the laundry detergent compositions of the present invention are those having one long-chain hydrocarbyl group. Examples of such I cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides, and those surfactants having the formula:

wherein R^ is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R"^ is selected form the group consisting of -CH2CH2", -CH2CH(CH3)-, -CH2CH(CH20H)-, -CH2CH2CH2-/ and mixtures thereof; each R'* is selected from the group consisting of C1-C4 alkyl, C1-C4 hydroxyalkyl, benzyl ring structures formed by joining the two R"^ groups, -CH2CHOHCHOHCOR^CHOHCM20H, wherein R6 is any hexose or hexose polymer having a molecular weight less than about 1000, andfthydrogen when y is not 0; R5 is the same as R4 or is an alkyl chain,wherein the total number of carbon atoms or R plus R^ is not more than about 18; each y is from 0 to about 10,and the sum of the y values is from 0 to about 15; and X is any compatible anion.
Highly preferred cationic surfactants are the water soluble quaternary ammonium compounds useful in the present composition having the formula:
RlR2R3R4N+X- (i)
wherein R1 is Cg-C1g alkyl, each of R2, R3 and R4 is independently C1-C4 alkyl, C1-C4 hydroxy alkyl, benzyl, and -(C2H4o)xH where x has a value from 2 to 5, and X is an anion.
r
Not more than one of R2, R3 or R4 should be benzyl.
The preferred alkyl chain length for Ri is C12"C15, particularly where the alkyl group is a mixture of chain length, derived from coconut or palm kernel fat or is derived synthetically by olefin build up or 0X0 alcohols synthesis.
preferred groups for R2R3 and R4 are methyl and
hydroxyethyl groups and the anion X may be selected from
halide, methosulphate, acetate and phosphate ions.
Examples of suitable quaternary ammonium compounds of
formulae (i) for use herein are:
coconut trimethyl ammonium chloride or bromide;
coconut methyl dihydroxyethyl ammonium chloride or bromide;
decyl triethyl ammonium chloride;
decyl dimethyl hydroxyethyl ammonium chloride or bromide;
C12-15 dimethyl hydroxyethyl ammonium chloride or bromide;
coconut dimethyl hydroxyethyl ammonium chloride or bromide;

myrlstyd trlmethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy)4 ammonium chloride or bromide; choline esters (compounds of formula (i) wherein R1 is

di-alkyl imidazolines [compounds of formula (i)].
Other cationic surfactants useful herein are also described in US 4,228,044 and in EP 000 224.
When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 25%, preferably from about 1% to about 8% by weight of such cationic surfactants.
Ampholytic surfactants are also suitable for use in the laundry 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 from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g. carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants.
When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants.
Zwitterionic surfactants are also suitable for use in laundry detergent compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosplionium or tertiary sulfonium compounds. See US 3,929,678

(column 19, line 38 through column 22, line 48) for examples of zwitterionic surfactants.
When included therein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such zwitterionic surfactants.
Semi-polar nonionic surfactants are a speC1al category of nonionic surfactants which include water-soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; watersoluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms.
Semi-polar nonionic detergent surfactants include the amine oxide surfactants having the formula:

wherein H^ is an a],kyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms, R4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atom, or mixtures thereof; x is from 0 to about 3: and each R5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups. The R5 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C10-c18 alkyl dimethyl amine oxides and C8-C12 alkoxy ethyl dihydroxy ethyl amine oxides.
When included therein, the laundry detergent compositions of the present invention typically comprise from 0,2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar nonionic surfactants.
Builder system
The compositions according to the present invention may further comprise a builder system. Any conventional builder system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphosphonates, particularly ethylenediamine tetramethylene phosphonic acid and diethylene triamine pentamethylenephosphonic acid. Though less preferred for obvious environmental reasons, phosphate builders can also be used herein.
Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
Another suitable inorganic builder material is layered silicate, e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na2Si2O5) .
Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed in Belgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of sucC1nic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German Offenle-enschrift 2,446,686, and 2,446,487, US 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623. Polycarboxylates containing three carboxy groups include, in partiC1;ular, water-soluble C1trates, aconitrates and

C1traconates as well as sucC1nate derivatives such as the carboxymethyloxysucC1nates described in British Patent No. 1,379,241, lactoxysucC1nates described in Netherlands Application 7205873, and the oxypolycarboxylate materials such as 2-oxa-l,1,3-propane tricarboxylates described in British Patent No, 1,387,447.
Polycarboxylates containing four carboxy groups include oxydisucC1nates disclosed in British Patent No..1,261,829, 1,1,2,S,-ethane tetracarboxylates, 1,1,3,3-propane tetrackrboxylates containing sulfo substituents include the sulfosucC1nate derivatives disclosed in British Patent Nos. 1,398,421 and 1,398,422 and in US 3,936,448, and the sulfonated pyrolysed C1trates described in British Patent No. 1,082,179, while polycarboxylates containing phosphone substituents are disclosed in British Patent No. 1,439,000.
Alicyclic and heterocyclic polycarboxylates include cyclopentane-C1s,C1s-C1s-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydro-furan - C1s, C1s, C1s-tetrpicarboxylates, 2,5-tetrahydro-furan-C1s, discarboxylates, 2,2,^,5,-tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6-hexane - hexacarboxylates and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol. Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent No. 1,425,343.
Of the above, the preferred polycarboxylates are hydroxy-carboxylates containing up to three carboxy groups per molecule, more particularly C1trates.
Preferred builder systems for use in the present compositions include a mixture of a water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6), and a water-soluble carboxylate chelating agent such as C1tric acid.
A suitable chelant for inclusion in the detergent composi-ions in accordance with the invention is ethylenediamine-N,N'-disucC1nic acid (EDDS) or the alkali metal, alkaline earth metal, ammonium, or substituted ammonium salts thereof, or mixtures thereof. Preferred EDDS compounds

are the free acid form and the sodium or magnesium salt thereof. Examples of such preferred sodium salts of EDDS include Na2EDDS and Na4EDDS. Examples of such preferred magnesium salts of EDDS include MgEDDS and Mg2EDDS. The magnesium salts are the most preferred for inclusion in
it
compositions in accordance with the invention.
Preferred builder systems include a mixture of a water-insoluble aluminosilicate builder such as zeolite A, and a water soluble carboxylate chelating agent such as C1tric acid.
Other builder materials that can form part of the builder system for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates, amino polyalkylene phosphonates and amino polycarboxylates.
Other suitable water-soluble organic salts are the homo-or co-polymeric acids or their salts, in which the polyccarboxylic acid comprises at least two carboxyl radicals separated form each other by not more than two carbon atoms.
Polymers of this type are disclosed in GB-A-1,596,756. Examples of such salts are polyacrylates of MW 2000-5000 and their idiopolymers with maleic anhydride, such copolymers having a molecular weight of from 20,000 to 70,000, espeC1ally about 40,000.
Detergency builder salts are normally included in amounts of from 5% to 80% by v/eight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.
Enzymes
Preferred detergent compositions, in addition to the enzyme preparation of the invention, comprise other enzyme(s) which provides cleaning performance and/or fabric care benefits.
Such enzymes include other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g. laccases).
Proteases; Any other protease suitable for use in alkaline sol--utions can be used. Suitable proteases include those of animal,

vegetable or microbial origin. Microbial origin is 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 prpteases are subtilisins, espeC1ally those derived from BaC1llus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of pociline or bovine origin) and the Fusarium protease described in WO 89/06270.
Preferred commerC1ally available protease enzymes include those sold under the trade names Alcalase, Savinase, Primase, Durazym, and Esperase by Novo Nordisk A/S (Denmark), those sold under the tradename Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the tradename Opticlean and Optimase by Solvay Enzymes. Protease enzymes may be incorporated into the compositions in accordance with the invention at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to
0.2% of enzyme protein by weight of the composition.
Lipases; Any lipase suitable for use in alkaline solutions can be used. 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, e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcalioenes and P. pseudoalcaliqenes lipase, e.g., as described in EP 218 272, a P. cepaC1a lipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., as disclosed in GB 1,372,034, a P. fluorescens lipase, a BaC1l-

lus lipase, e.g., a B. subtilis lipase (Dartois et al., (1993), Biochemica et Biophysica acta 1131, 253-260), a B. stearo-thermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422).
Furthermore, a number of cloned lipases may be useful, including the PeniC1llium camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. et al., (1989), J. Biochem., 106, 383-388) and various Rhizopus lipases such as a R. delemar lipase (Hass, M.J et al., (1991), Gene 109, 117-113), a R niveus lipase (Kugimiya et al., (1992), BiosC1. Biotech. Biochem. 5fe, 716-719) and a R. oryzae lipase.
Other types of lipolytic enzymes such as cutinases may also be useful, e.g., a cutinase derived from Pseudomonas mendoC1na as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446) .
EspeC1ally suitable lipases are lipases such as Ml LipaseTM, Luma fastTM and LipomaxTM (Genencor) , LipolaseTM and Lipolase UltraTM (Novo Nordisk A/S) , and Lipase P "Amano" (Amano Pharmaceutical Co. Ltd.).
The lipases are normally incorporated in the detergent composition at a level of from 0-00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
Amylases: Any amylase (a and/or P) 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, a-amylases obtained from a speC1al strain of B. licheniformis described in more detail in GB 1,296,839. CommerC1ally available amylases are Duramyl™, Termamyl™, Fungamyl™ and BANTM (available from Novo Nordisk A/S) and RapidaseTM and Maxamyl PTM (available from Genencor).

The amylases are normally incorporated in the detergent composition at a level of from 0,00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0,2% of enzyme protein by weight of the composition.
Cellulafees; Any qellulase suitable for use in alkaline
solutions can be used. Suitable cellulases include those of
bacterikl or fungal origin. Chemically or genetically modified
mutants are inqluded. Suitable cellulases are disclosed in US 4,435,307, which discloses fungal cellulases produced from Humicola insolens. EspeC1ally suitable cellulases are the cellulases having colour care benefits. Examples of such cellulases are cellulases described in European patent application No. 0 495 257.
CommerC1ally available cellulases include CelluzymeTM produced by a strain of Humicola insolens, (Novo Nordisk A/S),
and KAC-500(B)™ (Kao Corporation).
Cellulases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
Peroxidases/Oxidases; Peroxidase enzymes are used in combination with hydrogen peroxide or a source thereof (e.g. a percarbonate, perborate or persulfate). Oxidase enzymes are used in combination with oxygen. Both types of enzymes are used for "solution bleaching", i.e. to prevent transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor, preferably together with an enhanC1ng agent as described in e.g. WO 94/12621 and WO

95/01426. Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included.
I Peroxidase and/or oxidase enzymes are normally incorporated in the detergent composition at a level of from 0.0000i% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
Mixtures of the above mentioned enzymes are encompassed herein, 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 normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01% to 0.2% of enzyme protein by weight of the composition.
Bleaching agents; Additional optional detergent ingredients that can be included in the detergent compositions of the present invention include bleaching agents such as PBl, PB4 and percarbonate with a particle size of 400-800 microns. These bleaching agent components can include one or more oxygen bleaching agents and, depending upon the bleaching agent chosen, one or more bleach activators. When present oxygen bleaching compounds will typically be present at levels of from about 1% to about 25%. In genqjral, bleaching compounds are optional added components in non-liquid formulations, e.g. granular detergents.
The bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygen bleaches as well as others known in the art.

The bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.
One category of oxygen bleaching agent that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chlqro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents are disclosed^in US 4,483,781, US 740,446, EP 0 133 354 and US 4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in US 4,634,551.
Another category of bleaching agents that can be used encompasses the halogen bleaching agents. Examples of hypohalite bleaching agents, for example, include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such materials are normally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.
The hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in US 4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG), which are perhydrolyzed to form a peracid as the active bleaching speC1es, leading to improved bleaching effect. In addition, very suitable are the bleach activators C8(6-octanamido-caproyl) oxybenzene-sulfonate, C9(6-nonanamido caproyl) oxybenzenesulfonate and C1O (6-decanamido caproyl) oxybenzenesulfonate or mixtures thereof. Also suitable activators are acylated C1trate esters such as disclosed in European Patent Application No. 91870207.7.
Useful bleaching agents, including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleaching compounds for use in cleaning compositions according to the invention are described in application USSN 08/136,626.
The hydrogen peroxide may also be present by adding an enzymatic system (i.e. an enzyme and a substrate therefore)

which is capable of generation of hydrogen peroxide at the beginning or during the washing and/or rinsing process. Such enzymatic systems are disclosed in European Patent Application EP 0 537 381.
Bleaching agents other than oxygen bleaching agents are also known in the art and can be utilized herein. One type of non-oxygen bleaching agent of particular interest includes photoactivated bleaching agents such as the sulfonated zinc and/or aluminium phthalocyanines. These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, in the presence of oxygen, such as by hanging cl6thes out to dry in the daylight, the sulfonated zinc phthalocyanine is activated and, consequently, the substrate is bleached. Preferred zinc phthalocyanine and a photoactivated bleaching process are described in US 4,033,718. Typically, detergent composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc phthalocyanine.
Bleaching agents may also comprise a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "EffiC1ent manganese catalysts for low-temperature bleaching". Nature 369. 1994, pp. 637-639.
Suds suppressors; Another optional ingredient is a suds
suppressor, exemplified by silicones, and silica-silicone
mixtures. Silicones can generally be represented by alkylated
polysiloxane materials, while silica is normally used in finely
divided forms exemplified by silica aerogels and xerogels and
hydrophobic silicas of various types. Theses materials can be
i incorporated as particulates, in which the suds suppressor is
advantageously releasably incorporated in a water-soluble or
waterdjispersible, substantially non surface-active detergent
imperm.eable carrier. Alternatively the suds suppressor can be
dissolved or dispersed in a liquid carrier and applied by
spraying on to one or more of the other components.
A preferred silicone suds controlling agent is disclosed
in US 3,933,672. Other particularly useful suds suppressors are
the self-emulsifying silicone suds suppressors, described in
German Patent Application DTOS 2,646,126. An example of such a

compound is DC-544, commerC1ally available form Dow Corning, which is a siloxane-glycol copolymer. EspeC1ally preferred suds controlling agent are the suds suppressor system comprising a mixture of silicone oils and 2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol which are commerC1ally available under the trade name Isofol 12 R.
Such suds suppressor system are described in European Patent Application EP 0 593 841.
Esecially preferred silicone suds controlling agents are described in European Patent Application No. 92201649.8. Said compositions can comprise a silicone/ silica mixture in combinatioh with fumed nonporous silica such as Aerosil.
The suds suppressors described above are normally employed at levels of from 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.
Other components; Other components used in detergent compositions may be employed such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bacteriC1des, tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes.
EspeC1ally suitable encapsulating materials are water soluble capsules which consist of a matrix of polysaccharide and polyhydroxy compounds such as described in GB 1,464,616.
Other suitable water soluble encapsulating materials comprise dextrirjs derived from ungelatinized starch acid esters of substituted c^icarboxylic acids such as described in US 3,455/838. These acid-ester dextrins are, preferably, prepared from ^uch starches as waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of said encapsulation materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material consists of a modified maize starch and glucose. The starch is modified by adding monofunctional substituted groups such as octenyl sucC1nic acid anhydride.
Antiredeposition and soil suspension agents suitable herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or co-polymeric polycarboxylic acids or their salts. Polymers of

particular preferred polymer in accordance with EP 0 272 033 has the formula:
(CH3(PEG)43)o,75(POH)o.25[T-PO)2.8(T-PEG)o.4]T(POH) 0.25 ( (PEG) 43CH3) 0.75
where PEG is -(OC2H4)0-, PO is (OC3H6O) and T is (POOC6H4CO) .
Also very useful are modified polyesters as random copolymers of dimethyl terephthalate, dimethyl
sulfoisophthalate, ethylene glycol and 1,2-propanediol, the end groups consisting primarily of sulphobenzoate and secondarily of mono esters of ethylene glycol and/or 1,2-propanediol. The target is to obtain a polymer capped at both end by sulphobenzoate groups, "primarily", in the present context most of said copolymers herein will be endcapped by sulphobenzoate groups. However, some copolymers will be less than fully capped, and therefore their end groups may consist of monoester of ethylene glycol and/or 1,2-propanediol, thereof consist "secondarily" of such speC1es.
The selected polyesters herein contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of 1,2-propanediol, about 10% by weight ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, and have a molecular weight of about 3.000. The polyesters and their method of preparation are described in detail in EP 311 342.
Softening agents: Fabric softening agents can also be incorporated into laundry detergent compositions in accordance with the present invention. These agents may be inorganic or organic in type. Inorganic softening agents are exemplified by the smectite clayg disclosed in GB-A-1 400898 and in US 5,019,292. Organic fabric softening agents include the water insoluble tertiary amines as disclosed in GB-Al 514 276 and EP 0 oil 340 and their combination with mono C12-C14 quaternary ammonium salts are disclosed in EP-B-0 026 528 and di-long-chain mides as disclosed in EP 0 242 919. Other useful organic

ingredients of fabric softening systems include high molecular weight polyethylene oxide materials as disclosed in EP 0 299
575 and 0 313 146.
Levels of smectite clay are normally in the range from 5% to 15%, more preferably from 8% to 12% by weight, with the material being added as a dry mixed component to the remainder of the formulation. Organic fabric softening agents such as the water-insoluble tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to 5% by weight, normally from 1% to 3% by weight whilst the high molecular weight polyethylene oxide materials and the water soluble cationic materials are added at levels of from 0.1% to 2%, normally from 0.15% to 1.5% by weight. These materials are normally added to the spray dried portion of the composition, although in some instances it may be more convenient to add them as a dry mixed particulate, or spray them as molten liquid on to other solid components of the composition.
Polymeric dye-transfer inhibiting agents: The detergent compositions according to the present invention may also comprise from 0*001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weight of polymeric dye-transfer inhibiting agents. Said polymeric dye-transfer inhibiting agents are normally incorporated into detergent compositions in order to inhibit the transfer of dyes from colored fabrics onto fabrics washed therewith. These polymers have the ability of complexing or adsorbing the fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become attached to other articles in the wash.
EspeC1ally suitable polymeric dye-transfer inhibiting agents are polyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone and N-vinylimidazole, polyvinylpyrrolidone poilymers, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
Addition of such polymers also enhances the performance op the enzymes according the invention.
The detergent composition according to the invention can be in liquid, paste, gels, bars or granular forms.

Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (poiyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 ta 80 ethylene oxide units; fatty alcohols; fatty acids;
and mono-- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. form 550 to 950 g/1; in such case, the granular detergent compositions according to the present invention will contain a lower amount of "Inorganic filler salt", compared to conventional granular detergents; typical filler salts are alkaline earth metal salts of sulphates and chlorides, typically sodium sulphate; "Compact" detergent typically comprise not more than 10% filler 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 lower amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergent is less than 30%, more preferably less than 20%, most preferably less than 10% by weight of the detergent compositions.
The compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations and dishwashing operations.

C1trate: Tri-sodium C1trate dihydrate
C1tric: C1tric acid
Perborate: Anhydrous sodium perborate monohydrate bleach,
empirical formula NaBO2.H2O2
PB4: Anhydrous sodium perborate tetrahydrate
Percarbonate: Anhydrous sodium percarbonate bleach of
empirical formula 2Na2O3.3H2O2
TAED: A Tetraacetyl ethylene diamine
CMC: Sodium carboxymethyl cellulose
DETPMP: .! Diethylene triamine penta (methylene phosphonic acid), marketed by Monsanto under the Tradename Dequest 2060
PVP: Polyvinylpyrrolidone polymer
EDDS: Ethylenediamine-N, N'-disucC1nic acid, [S,S] isomer
in the form of the sodium salt
Suds 25% paraffin wax Mpt 50°C, 17% hydrophobic silica,
58%
Suppressor: paraffin oil
Granular Suds 12% Silicone/silica, 18% stearyl alcohol, 70%
suppressor: starch in granular form
Sulphate: Anhydrous sodium sulphate
HMWPEO: High molecular weight polyethylene oxide
TAE 25^ fallow alcohol ethoxylate (25)
► Detergent Example I
jA granular fabric cleaning composition in accordance with
the iriVentioh may be prepared as follows:
Sodium linear C12 alkyl 6.5
benzene sulfonate
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 sulphonate 0.1
Minors Up to 100
Detergent Example II
A compact granular fabric cleaning composition (density 800 g/1) in accord with the invention may be prepared as follows:
45AS 8.0
25E3S 2.0
■i
25E5 3.0
25E3 3.0
TFAA 2.5
Zeolite A 17.0
NaSKS-6 12.0
C1tric acid 3.0
Carbonate 7.0
MA/AA 5.0
CMC 0.4
Enzyme of the invention 0.1
TAED i 6.0
Percarbonate 22.0
EDDS 0.3
Granular puds suppressor 3.5
water/minors Up to 100%
Detergent Example III
Granular fabric cleaning compositions in accordance with the invention which are espeC1ally useful in the laundering of coloured fabrics were prepared as follows:
LAS 10.7 -
TAS 2.4 -
TFAA - 4.0
45AS 3.1 10.0
45E7 4.0

25E3S - 3.0
68E11 1.8
25E5 - 8.0
C1trate 15.0 7.0
Carbonate - 10
C1tric 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 j of the invention 0.10 0. 05
Silicate 2.5
Sulphate 5.2 3.0
PVP I 0.5 -
Poly (4-vinylpyridine)-N- - 0.2
Oxide/copolymer of vinyl-
imidazole and vinyl-
pyrrol idone
Perborate 1.0
Phenol sulfonate 0.2 -
Water/Minors Up to 100%
Detergent Example IV
Granular fabric cleaning compositions in accordance with
the invention which provide "Softening through the wash"
capability may be prepared as follows:
45AS " 10.0
LAS 7.6
68AS 1.3
45E7 4.0
25E3 - 5.0
Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride
C1trate 5.0 3.0
Na--SKS-6 - 11.0
Zeolite A 15.0 15.0
MA/AA 4.0 4.0
DETPMP 0.4 0.4

Perborate 15.0
Percarbonate - 15.0
TAED 5.0 5.0
Smectite clay 10.0 10.0
HMWPEO - 0.1
Enzyme of the invention 0.10 0.05
Silicate 3.0 5.0
Carbonate 10.0 10.0
Granular suds suppressor 1.0 4.0
CMC 0.2 0.1
Water/Minors Up to 100%
■J
Detergent Example V
Heavy duty liquid fabric cleaning compositions in accordance with the invention may be prepared as follows:
I II
LAS acid form - 25.0
C1tric acid 5.0 2.0
2 5AS acid.form 8.0
25AE2S,acid form 3.0
25AE7 I 8.0
CFAA 5
DETPMP. 1.0 1.0
Fatty acid 8
Oleic acid - 1.0
Ethanql 4.0 6.0
Propanediol 2.0 6.0
Enzyme of the invention 0.10 0.05
Coco-alkyl dimethyl - 3.0
hydroxy ethyl ammonium
chloride
Smectite clay - 5.0
PVP 2.0
Water / Minors Up to 100%
LEATHER INDUSTRY APPLICATIONS

A subtilase of the invention may be used in the leather industry, in particular for use in depilation of sKins.
In said application a subtilase variant of the invention is preferably used in an enzyme composition which further comprise another protease.
For a more detailed description of suitable other proteases see section relating to suitable enzymes for use in a determent composition {vide supra).
WOOL INDUSTRY APPLICATIONS
A subtilase of the invention may be used in the wool industry, in particular for use in cleaning of clothes comprising wool.
In said application a subtilase variant of the invention is preferably used in an enzyme composition which further comprise another protease.
For a more detailed description of suitable other proteases see section relating to suitable enzymes for use in a detergent composition {vide supra).
The invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention as claimed.
MATERIALS AND METHODS
Strains;
B. subtilis DN1885 (Diderichsen et al., 1990).
B. lentus 309 and 147 are speC1fic strains of BaC1llus lentus, deposited with the NC1B and accorded the accession numbers NC1B 10309 and 10147, and described in US Patent No. 3,723,250 incorporated by reference herein.
E. coli MC 1000 (M.J. Casadaban and S.N. Cohen (1980); J. Mol. Biol. 138 179-207) , was made r- ,m+ by conventional methods and is also described in US Patent Application Serial No. 039,298.
Plasmids:

pJS3: E. call - B. snbtilis shuttle vector containing a synthetic gene encoding for subtilase 309. (Described by Jacob Schiodt et al. in Protein and Peptide letters 3:39-44 (1996)).
pSX222: B. subtilis expression vector (Described in WO
96/34946).
General molecular biology methods:
Unless otherwise mentioned the 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, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for BaC1llus", John Wiley and Sons, 1990).
Enzymes for DNA manipulations were used according to the speC1fications of the suppliers.
Enzymes for DMA manipulations
Unless otherwise mentioned all enzymes for DNA manipulations,
such las e.g. restiction endonucleases, ligases etc., are ob-
tained 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 relatively to an enzyme standard (SAVINASEO), and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50°C, pH 8.3, 9 min. reaction time, 3 min. measuring time. A folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
A GU is a GlyC1ne Unit, defined as the proteolytic enzyme activity which, under standard conditions, during a 15-minutes' incubation at 40 deg C, with N-acetyl casein as substrate, produces an amount of NH2-group equivalent to 1 mmole of glyC1ne.

Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate sucC1nyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in the Journal of American Oil Chemists SoC1ety, Rothgeb, T.M., Goodlander, B.D., Garrison, P.H., and Smith, L.A., (1988).
Fermentation:
Fermentation of subtilase enzymes were performed at 30°C on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days.
Consequently in order to make an e.g. 2 liter broth 20 Erlenmeyer flasks were fermented simultaneously.
Media:
BPX: Composition (per liter)
Potato starch 100g
Ground barley 50g
Soybean flour 20g
Na2HPO4 X 12 H2O 9g
Pluronic 0.1g
Sodium caseinate 10g
The starch in the medium is liquified with a-amylase and
i the medium is sterilized by heating at 120°C for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by addition 6f NaHCOa to 0.1 M.
EXAMPLES
EXAMPLE 1
Construction and Expression of Enzyme Variants;
Site-directed mutagenesis: Subtilase 309 site-directed variants was made by the "Unique site elimination (USE)" or the "UraCil-USE" technique described respectively by Deng et al. (Anal. Biochem. 200:81-88 (1992)) and Markvardsen et al. (BioTechniques 18(3):371-372 (1995)).

The template plasmid was pJS3, or a analogue of this containing a variant of Subtilase 309, e.g. USE mutagenesis was performed on pJS3 analogue containing a gene encoding the Y167A variant with a oligonucleotide directed to the construct R170L variant resulting in a final yi67A+R170L Subtilase 309 variant.
The in pJS3 constructed Subtilase 309 variants was then subcloned into the B.subtilis pSX222 expression plasmid, using the restriction enzymes Kpnl and Mlul.
LocmlizBd Random mutagenesis:
The overall strategy to used to perform localized random mutagenesis was:
a mutagenic primer (oligonucleotide) was synthesized which corresponds to the part of the DNA sequence to be mutagenized except for the nucleotide(s) corresponding to amino acid codon(s) to be mutagenized.
Subsequently, the resulting mutagenic primer was used in a PCR reaction with a suitable opposite primer. The resulting PCR fragment was purified and digested and cloned into a E.coli~B.subtil is shuttle vector.
Alternatively and if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a second suitable opposite primer so as to allow digestion and cloning of the mutagenized region into the shuttle vector. The PCR reactions are performed under normal conditions.
Following this strategy a localized random library was constructed in SAVINASE wherein both position Y167 and R170 was completely randomized.
One oligonucleotide was synthesized with 25% of each of the four bases (W) in the first and the second base at amino acid codons wanted to be mutagenized. The third nucleotide (the wobble base) in codons were synthesized with 50%G/50%C (S) to avoid two (TAA, TGA) of the three stop-codons .
The mutagenic primer (5'- GTT TGG ATC AGT AGC TCC GAC TGC CAT TGC GTT CGC ATA SNU CGC CGG SNN GCT GAT TGA GCC -3' (anti-sense)) were used In a PCR reaction with a suitable opposite primer (e.g. 5' GAA CTC GAT CCA GCG ATT TC 3' (sense)) and the plasmid pJS3 as template. This resulting PCR product was cloned

into the pJS3 shuttle vector by using the restriction enzymes
Asp 718 and Bel I.
The in pJS3 constructed localized random library was then subclored into the B.subtilis pSX222 expression plasmid, using the restriction enzymes KphI and Mlul,
The library prepared contained approximately 100,000 individual clones/library.
Ten randomly chosen colonies were sequenced to confirm the mutations Resigned.
In order to purify a subtilase variant of the invention the B. subtilis •'pSX222 expression plasmid comprising a variant of the invention was transformed into a competent B. subtilis strain and was fermented as described above in a medium
containing 10 |ig/ml Chloramphenicol (CAM) . EXAMPLE 2
Purification of Enzyme Variants;
This procedure relates to purification of a 2 litre scale fermentation of the Subtilisin 147 enzyme, the Subtilisin 309 enzyme or mutants thereof.
Approximately 1.6 litres of fermentation broth were centrifuged at 5000 rpm for 35 minutes in 1 litre beakers. The supernatants were adjusted to pH 6.5 using 10% acetic acid and filtered on Seitz Supra SlOO filter plates.
The filtrates were concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon SIYIO UF cartridge. The UF concentrate was centrifuged and filtered prior to absorption at rpom temperature on a BaC1traC1n affinity column at pH 7. The protease was eluted from the BaC1traC1n column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethyl-glutaric acid, 0.1 M boric acid and 0.002 M calC1um chloride adjusted to pH 7.
The fractions with protease activity from the BaC1traC1n purification step were combined and applied to a 750 ml Sephadex G25 column (5 cm dia.) equilibrated with a buffer

containing 0.01 dimethylglutaric acid, 0.2 M boric acid and 0.002 m calC1um chloride adjusted to pH 6.5.
Fractions with proteolytic activity from the Sephadex G25 column were combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm dia.) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid, and 0.002 M calC1um chloride adjusted to pH 6.5.
The protease was eluted using a linear gradient of 0-0.1 M sodium ,^^loride in 2 litres of the same buffer (0-0.2 M sodium chloride in case of Subtilisin 147).
In a final purification step protease containing fractions from the CM Sepharose column were combined and concentrated in an Amicon ultrafiltration cell equipped with a GR81PP membrane (from the Danish Sugar Factories Inc.)*
By using the techniques of Example 1 for the construction and the above isolation procedure the following subtilisin 309 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+R170A
Y167P+R170S
Y167I+R170L
Y167F+R170T
Y167F+R170E
Yr67F+R170H
Y167F+R170T
Y167F+R170L
Y167L

R170H
Y167S
EXAMPLE 3
Wash Performance of Detergent Compositions Comprising Enzyme Variants
The following examples provide results from a number of washing tests that were conducted under the conditions indicated

Experimental conditions
Table IV: Experimental conditions for evaluation of Subtilisin 309 variants.

Subsequent to washing the cloths were flushed in tap water and air-dried.
The above model detergent is a simple detergent formulation. The most characteristic features are that STP is used as builder and the content of anionic tenside (LAS) is quite high. Further the pH is adjusted to 10.4, which is within the normal range of a powder detergent.

Water hardness is adjusted by adding CaCl2 and MgCl2 to deionized water. pH of the detergent solution is modified to the desired value by addition of acid.
Measurement of remission (i?) on the test material has been done at 460 nm using an Elrepho 2000 photometer (without UV). Aperture: 10 mM.
The wash performance at XnM is calculated as:

Rx: is the wash effect of the enzyme at X nM (in remission units).
R90: is the wash effect of the enzyme at 0 nM (blank value)

Table VI: Variants and improvement factors for SAVINASE®,

I ■ ,| •
As it can be seen from Table VI SAVINASE® variants of
the invention exhibits an improvement in wash performance,
In a similar wash assay following SAVINASE® variants showed a wash performance valu6 in the range between the variant Y167I+R170L and Y167A+R170S:
Y167P
Y167P+R170L
Y167V+R170T
Y167H-R170T
Y167V+R170Q
Y167S+R170Q
Y167T+R170N
Y167A+R170A
Y167T+R170L
Y167T+R170A
Y167P+R170S
EXAMPLE 4
Wash Performance of Detergent Compositions Comprising Enzyme Variants
The following examples provide results from a number of wash ing
tests that were conducted under the conditions indicated
EXPERIMENTAL CONDITIONS

Table VII: Experimental conditions for evaluation of Subtilisin 309 variants.

The detergent used is a simple model formulation. pH is adjusted to 10,5 which is within the normal range for a powder detergent: The composition of model detergent 95 is as follows:
25% STP (NaaPaO10) 25% Na2SO4
10% Na2SO4 20% LAS (Nansa SOS)
5.0% Nonionic tenside (Dobanol 25-7)
5.0% Na2SO4
0.5% Carboxymethylcellulose (CMC)
9.5% Water
Water hardness was adjusted by adding CaCl2 and MgCl2 (Ca2 +:Mg2+ = 2:1) to deionized water (see also Surfactants in Consumer

Products - Theory, Technology and Application, springer 1986). pH of the detergent solution was adjusted to pH 10.5 by addition of HCl.
Measurement of reflectance (R) on the test material was done at 460 ,nm using a Macbeth ColorEye 7000 photometer (Macbeth, Division of Kollmorgen Instruments Corporation, Germany). The measurements were done according to the manufacturers protocol.
The wash performance of the Subtilisin 309 variants was evaluated by calculating a performance factor:

P: Performance factor
Rvariant: Reflectance of test material washed with variant
Rsavinase'- Reflectance of test material washed with Savinase®
Reiank: Reflectance of test material washed with no enzyme
The claimed Subtilisin 309 variants all have improved wash performance compared to Savinase® - i.e. P > 1.
The variants are divided into improvement classes designated with capital letters:
Class A: 1 Class B: 1.5 2
Table VIII: Subtilisin 309 variants and improvement classes.

The Savinase® variant Y167I+R170L+A194P was in a fermentation
experiment compared to a variant Y167I+R170L not having the A194P substitution.
Both variants were cloned in a pSX222 expression vector background and fermented as described above in a 100 ml BPX
medium containing 10 μg/ml CAM.
After 5 days fermentation 1.5 ml of the BPX fermentation medium was centrifuged and the supernatant was used to measure the ProtdiSlytic activity (KPNU) as described above.
The fermentation medium containing the Y167I+R170L+A194P variant had a significant higher level of proteolytic activity as compared to the fermentation medium containing the Y167I+R170L variant.
Both variants have the same speC1fic activity. Similar results were obtained with the variants Y167A+;R170S+A194P, Y167A+R170L+A194P, and Y167A+R170N+A194P compared with their corresponding variant without the A194P mutation.
Further, similar results were obtained with the variants A133P+Y167A+R170S, A133D+Y167A+R170S, P129K+Y167A+R170S, and P131H+Y167A+R170S compared with their corresponding variants without the A133P, A133D, P129K, and P131H mutations.

WE CLAIM:
1. A process for the preparation of 2-hydroxy-4-methylthiobutanoic acid or a salt
thereof comprising the steps of:
a) introducing an aqueous mineral acid and 2-hydroxy-4-methylthiobutanenitrile into a nitrile hydrolysis reactor comprising a first continuous stirred tank reactor;
b) continuously hydrolysing 2-hydroxy-4-methylthiobutanenitrile within said nitrile hydrolysis reactor to produce a nitrile hydrolysis reactor product stream containing 2-hydroxy-4-methylthiobutanamide; and
c) continuously hydrolysing 2-hydroxy-4-methylthiobutanamide in an amide hydrolysis flow reactor to produce a finished aqueous hydrolysate product containing 2-hydroxy-4-methylthiobutanoic acid.

2. The process as claimed in claim 1, further comprising, in between step (b) and step (c), continuously introducing water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid into said amide hydrolysis flow reactor; and optionally diluting said nitrile hydrolysis reactor product stream with said water before said nitrile hydrolysis reactor product stream and said water are introduced into said amide hydrolysis flow reactor.
3. The process as claimed in claim 2, wherein said water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid are mixed to form a finishing reaction stream such that the hydrolysis of 2-hydroxy-4-methylthiobutanamide is substantially completed as said finishing reaction stream flows through said amide hydrolysis flow reactor.

4. The process as claimed in claim 1, further comprising, in between step (b) and step (c), continuously introducing water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid into a continuous amide hydrolysis reactor comprising a second continuous stirred tank reactor such that a substantial portion of 2-hydroxy-4-methylthiobutanamide contained in said nitrile hydrolysis reactor product stream is hydrolysed in said second continuous stirred tank reactor to form a finishing reaction stream; optionally diluting said nitrile hydrolysis reactor product stream with said water before said nitrile hydrolysis reactor product stream and said water are introduced into said continuous amide hydrolysis reactor; and continuously introducing said finishing reaction stream into said amide hydrolysis flow reactor.
5. The process as claimed in claim 4, wherein said water, said nitrile hydrolysis reaction product stream and optionally said aqueous mineral acid are mixed to form an amide hydrolysis mixture that is introduced into the continuous amide hydrolysis reactor.
6. The process as claimed in claim 4 or claim 5, wherein said continuous amide hydrolysis reactor is operated at a temperature ranging from 70°C to 120^C.
7. The process as claimed in any preceding claim, wherein the aqueous mineral acid introduced into said nitrile hydrolysis reactor is sulfuric acid in an acid stream having a strength of between 50% by weight and 70% by weight sulfuric acid.
8. The process as claimed in anyone of claims 1 to 6, wherein the aqueous mineral acid introduced into said nitrile hydrolysis reactor is sulfuric acid in an acid stream having a strength of between 70% by weight and 99% by weight

sulfuric acid, and said acid stream is continuously introduced to said nitrile hydrolysis reactor concurrently with a water stream to form sulfuric acid having a strength of between 50% by weight and 70% by weight on an organic-free basis within said nitrile hydrolysis reactor.
9. The process as set further in claim 7 or 8, wherein the molar ratio of sulfuric acid to 2-hydroxy-4-methylthiobutanenitrile introduced into said nitrile hydrolysis reactor is between 1.0 and 2.0 during the period between start up of the process until steady state conditions are established in said amide hydrolysis flow reactor, and thereafter said molar ratio of sulfuric acid to 2-hydroxy-4-methylthiobutanenitrile is between 0.6 and 1.5.
10. The process as claimed in any preceding claim, wherein at least 90% of 2-hydroxy-4-methylthiobutanenitrile is converted to 2-hydroxy-4-methylthiobutanamide within said nitrile hydrolysis reactor.
11. The process as claimed in any preceding claim, wherein the ratio of the rate of mineral acid flow into said amide hydrolysis flow reactor to the rates of 2-hydroxy-4-methylthiobutanamide and 2-hydroxy-4-methylthiobutanenitrile flow into said amide hydrolysis flow reactor is controlled to provide an excess of at least 5% molar excess mineral acid than is stoichiometrically equivalent to 2-hydroxy-4-methylthiobutanamide and 2-hydroxy-4-methylthiobutanenitrile introduced into said amide hydrolysis flow reactor.
12. The process as claimed in any preceding claim, wherein said finished aqueous hydrolysate product produced upon start up of the process comprises up to 0.05 wt.% amide and up to 0.05 wt.% nitrile.

13. The process as claimed in anyone of claims 3 to 12, wherein said amide hydrolysis flow reactor is a plug flow reactor, and the flow of said finishing reaction stream through said plug flow reactor is turbulent.
14. The process as claimed in anyone of claims 3 to 12, wherein said amide hydrolysis flow reactor comprises a pipeline reactor and said finishing reaction stream moves through said pipeline reactor in turbulent flow.
15. The process as claimed in claim 14, wherein said amide hydrolysis flow reactor is operated at a Reynolds number greater than 3,000.
16. The process as claimed in anyone of claims 1 to 12, wherein said amide hydrolysis flow reactor is a plug flow reactor and is operated at a Peclet number of at least 50, a peak temperature of 90°C to 120°C and a residence time between 30 and 100 minutes.
17. The process as claimed in any preceding claim, wherein said amide hydrolysis flow reactor operates substantially adiabatically; or operates substantially isothermally; or operates adiabatically and autothermally.
18. The process as claimed in any preceding claim, wherein 2-hydroxy-4-methylthiobutanoic acid is recovered by extracting 2-hydroxy-4-methylthiobutanoic acid from said finished aqueous hydrolysate product.
19. The process as claimed in any preceding claim, wherein 2-hydroxy-4-methylthiobutanoic acid is recovered by neutralizing said finished aqueous hydrolysate product to form an organic phase containing 2-hydroxy-4-methylthiobutanoic acid and an aqueous phase, and separating said organic phase and said aqueous phase to recover 2-hydroxy-4-methylthiobutanoic acid.

20. The process as claimed in claim 1, wherein, in step (a), 2-hydroxy-4-methylthiobutanenitrile and aqueous mineral acid are introduced into an aqueous hydrolysis mixture comprising 2-hydroxy-4-methylthiobutanamide, mineral acid and unreacted 2-hydroxy-4-methylthiobutanenitrile; wherein the nitrile hydrolysis reactor comprises a back-mixed reaction zone and a circulation zone in fluid flow communication with said back-mixed reaction zone, said circulation zone comprising a circulating line; and wherein the aqueous hydrolysis mixture is continuously circulated in a circulating stream that is withdrawn from said back-mixed reaction zone, passed through said circulation zone and returned to said back-mixed reaction zone, said circulating stream as withdrawn from said back-mixed reaction zone containing unreacted 2-hydroxy-4-methylthiobutanenitrile.
21. The process as claimed in claim 20, further comprising, in between step (b) and step (c), removing a portion of said circulating aqueous hydrolysis mixture as said nitrile hydrolysis reactor product stream from a forward flow port in said circulation zone; transferring said nitrile hydrolysis reactor product stream, water and optionally aqueous mineral acid to said amide hydrolysis flow reactor; and diluting said nitrile hydrolysis reactor product stream with said water at a point downstream of said forward flow port; wherein the sum of the residence time of said circulating stream in said circulation zone upstream of said forward flow port and the residence time of said nitrile hydrolysis reactor product stream downstream of said forward flow port prior to dilution is sufficient to substantially extinguish residual 2-hydroxy-4-methylthiobutanenitrile prior to the dilution of said nitrile hydrolysis reactor product stream.

22. The process as claimed in claim 21, wherein said water, said nitrile hydrolysis reaction product stream and optionally said aqueous mineral acid are mixed to form a finishing reaction stream that is introduced into the amide hydrolysis flow reactor.
23. The process as claimed in claim 20, further comprising, in between step (b) and step (c), removing a portion of said circulating aqueous hydrolysis mixture as said nitrile hydrolysis reactor product stream from a forward flow port in said circulation zone; transferring said nitrile hydrolysis reactor product stream, water and optionally aqueous mineral acid to a continuous amide hydrolysis reactor; and diluting said nitrile hydrolysis reactor product stream with said water at a point downstream of said forward flow port to provide an amide hydrolysis mixture, a substantial portion of 2-hydroxy-4-methylthiobutanamide contained in said amide hydrolysis mixture being hydrolysed in said continuous amide hydrolysis reactor to form a finishing reaction stream; and transferring said finishing reaction stream to said amide hydrolysis flow reactor; wherein the sum of the residence time of said circulating stream in said circulation zone upstream of said forward flow port and the residence time of said nitrile hydrolysis reactor product stream downstream of said forward flow port prior to dilution is sufficient to substantially extinguish residual 2-hydroxy-4-methylthiobutanenitrile prior to the dilution of said nitrile hydrolysis reactor product stream.
24. The process as claimed in claim 23, wherein the flow regimes and residence times are such as to provide the equivalent of at least one sequential back-mixed reaction zone in a nitrile extinction reaction region comprising the portion of said circulation zone upstream of said forward flow port and a

forward flow reaction zone within which said nitrile reactor product stream flows between said forward flow port and said point of dilution or said continuous amide hydrolysis reactor.
25. The process as claimed in claim 23, wherein the flow regime in a forward flow reaction zone within which said nitrile reactor product stream flows between said forward flow port and said point of dilution or said continuous amide hydrolysis reactor is laminar, and the flow regime and residence time are such as to provide the equivalent of at least one sequential back-mixed reaction zone in said forward flow reaction zone.
26. The process as claimed in anyone of claims 21 to 25, wherein the flow regime in said circulating line is substantially turbulent, the temperature of said circulating stream is at least 50°C throughout said circulation zone, and the residence time of said circulating stream in said ciculation zone upstream of said forward flow port is at least 3 seconds.
27. The process as claimed in anyone of claims 21 to 26, wherein between said forward flow port and said point of dilution the temperature of said nitrile hydrolysis reactor product stream is at least 50°C and the residence time is at least 30 seconds.
28. The process as claimed in claim 23, wherein the sum of the residence time of said circulating stream in said circulation zone upstream of said forward flow port and the residence time of said nitrile hydrolysis reactor product stream downstream of said forward flow port prior to dilution is at least 20 seconds.

29. The process as claimed in anyone of claims 21 to 28, wherein said nitrile hydrolysis reactor product stream is transferred to said point of dilution via a vertical downcomer line.
30. The process as claimed in anyone of claims 20 to 29, wherein said finished aqueous hydrolysate product comprises up to 0.05 wt.% amide and up to 0.05 wt.% nitrile.
31. The process as claimed in claim 1, wherein at least 90% of 2-hydroxy-4-methylthiobutanenitrile is converted to 2-hydroxy-4-methylthiobutanamide within said nitrile hydrolysis reactor and the process further comprises continuously removing heat of nitrile hydrolysis from the nitrile hydrolysis reactor during step (b); and, in between step (b) and step (c), continuously introducing water, said nitrile hydrolysis reactor product stream and optionally aqueous mineral acid into said amide hydrolysis flow reactor.
32. The process as claimed in claim 4, wherein at least 90% of 2-hydroxy-4-methylthiobutanenitrile is converted to 2-hydroxy-4-methylthiobutanamide within said nitrile hydrolysis reactor and the process further comprises continuously removing heat of nitrile hydrolysis from the nitrile hydrolysis reactor during step (b); and diluting the nitrile hydrolysis reactor product stream with said water before said nitrile hydrolysis reactor product stream and said water are introduced into said amide hydrolysis flow reactor for preventing liquid phase separation.
33. An apparatus for use in a process for the preparation of 2-hydroxy-4-methylthiobutanoic acid, comprising:
a nitrile hydrolysis reactor (10) comprising a first continuous stirred tank

reactor for the continuous hydrolysis of 2-hydroxy-4-methylthiobutanenitrile to
produce an aqueous hydrolysis mixture containing 2-hydroxy-4-
methylthiobutanamide, said nitrile hydrolysis reactor being adapted to receive
aqueous mineral acid and 2-hydroxy-4-methylthiobutanenitrile feed streams
and having an exit for the aqueous hydrolysis mixture;
a circulating line (14) for circulating the aqueous hydrolysis mixture from said
exit of said nitrile hydrolysis reactor through said circulating line and back to
said nitrile hydrolysis reactor;
a forward flow port (26) in said circulating line for removing a portion of the
aqueous hydrolysis mixture to form a nitrile hydrolysis reactor product stream;
and
an amide hydrolysis flow reactor (16) for the continuous hydrolysis of 2-
hydroxy-4-methylthiobutanamide in the nitrile hydrolysis reactor product
stream to produce a finished aqueous hydrolysate product containing 2-
hydroxy-4-methylthiobutanoic acid.
34. The apparatus as claimed in claim 33, wherein said amide hydrolysis flow reactor is insulated for adiabatic operation.
35. The apparatus as claimed in claim 33 or claim 34, further comprising a transfer line (28) for transporting the nitrile hydrolysis reactor product stream to a point of dilution (30), said circulating line and said transfer line providing additional residence time for substantially extinguishing residual 2-hydroxy-4-methylthiobutanenitrile prior to dilution of the nitrile hydrolysis reactor product stream.
36. The apparatus as claimed in any one of claims 33 to 35, further comprising a second continuous stirred tank reactor (36) which is located upstream of said

amide hydrolysis flow reactor and is adapted to receive water and the aqueous hydrolysis mixture exiting said nitrile hydrolysis reactor and to transfer a finishing reaction stream exiting from said second continuous stirred tank reactor to said amide hydrolysis flow reactor.
37. The apparatus as claimed in any one of claims 33 to 36, further comprising means for removing heat generated by dilution of mineral acid and reaction of 2-hydroxy-4-methylthiobutanenitrile and water in order to maintain a reaction temperature for hydrolysis of 2-hydroxy-4-mehtylthiobutanenitrile.
38. A process for the preparation of 2-hydroxy-4-methylthiobutanoic acid or a salt thereof substantially as herein described with reference to the accompanying drawings.
39. An apparatus for use in a process for the preparation of 2-hydroxy-4-
methylthiobutanoic acid substantially as herein described with reference to the
accompanying drawings.
Dated this 3 day of June 1996

Documents:

2509-mas-1997- assignment.pdf

2509-mas-1997- claims duplicate.pdf

2509-mas-1997- claims original.pdf

2509-mas-1997- correspondence others.pdf

2509-mas-1997- correspondence po.pdf

2509-mas-1997- description complete duplicate.pdf

2509-mas-1997- description complete original.pdf

2509-mas-1997- drawings.pdf

2509-mas-1997- form 1.pdf

2509-mas-1997- form 26.pdf

2509-mas-1997- form 3.pdf

2509-mas-1997- form 4.pdf

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Patent Number

208018

Indian Patent Application Number

2509/MAS/1997

PG Journal Number

26/2007

Publication Date

29-Jun-2007

Grant Date

06-Jul-2007

Date of Filing

04-Nov-1997

Name of Patentee

NOVOZYMES A/S

Applicant Address

KROGSHOEJVEJ 36,DK 2880 BAGSVAERD .

Inventors:

#	Inventor's Name	Inventor's Address
1	HANSAN PETER KAMP	KROGSHOEJVEJ 36,DK 2880 BAGSVAERD .

PCT International Classification Number

C11D3/37

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	1236/96	1996-11-04	Denmark