MXPA01006047A - Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region - Google Patents

Abstract

Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in position 103 of the active site loop (b) region from position 95 to 103. Variant subtilases exhibit improved wash performance in a detergent in comparison to its parent enzyme.

Description

SUBSTITUTE ENZYMES OF SUB-GROUPS IS-1 AND IS-2 THAT HAVE ADDITIONAL AMINO-ACID RESIDUE IN AN ACTIVE SITE CIRCUIT REGION. Field of Invention This invention relates to novel subtylase enzymes of subgroups IS-1 and IS-2 having at least one additional amino acid residue at position 103 of the active site circuit region (b) from position 95 to 103 These proteases are useful in showing excellent or improved performance for washing when used in detergents; detergent and cleaning compositions. The invention further relates to gene coding for the expression of said enzymes when inserted into an appropriate host cell or organism; and such host cells are transformed with them and are capable of expressing variants of enzymes, and to methods for producing novel enzymes. Background of the Invention In the detergent industry, enzymes have been implemented for more than 30 years in washing formulations. "The enzymes used in such formulations comprise proteases, lipases, amylases, cellulases as well as other enzymes or Ref: 130427 mixtures thereof. The most important commercial enzymes are proteases. An increasing number of commercially used proteases are variants of proteins naturally prepared by naturally occurring proteases, for example DURAZYM® (Novo Nordisk A / S), RELASE® (Novo Nordisk A / S), MAXAPEM® / Gist- Brocades NV), PURAFECT® (Genencor International, Inc.). In addition, various protease variants are described in the art such as EP 130756 (GENENTECH) (corresponding to US Reissue Patent No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP (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 (A gene); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER &GAMBLE COMPANY); WO 95/30010 (PROCTER &GAMBLE COMPANY); WO 95/29979 (PROCTER &GAMBLE COMPANY); 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, although a variety of useful protease variants have been described, there is still a need for improved new proteases or protease variants for a variety of industrial uses. Therefore, an object of the present invention is to provide improved proteases or protease variants prepared by protein engineering, especially for use in the detergent industry. Brief description of the invention. Current inventors have found that subtilisins, wherein at least one of the active site circuits are larger than those currently known, show improved wash performance in detergent compositions. The identification of these was done when constructing subtilisin variants, especially subtilisin. 309 (BLSAVI or Savinase®), which showed improved performance in washing performance in detergent compositions relative to the original wild-type enzyme. This has been described in our previous application DK1332 / 97. It has now been found that certain subtilases or variants thereof of the subgroups IS-1 (true subtypes) and IS-2 (subtypes of high alkaline content) having at least one additional amino acid residue at position 103 (or rather between positions 103 and 104) of the region of the active site circuit (b) from position 95 to 103, surprisingly show an improved wash performance compared to those currently known and those described in said application. The improved proteases according to the invention can be obtained by isolation of natural resources or by the introduction of at least one additional residue, of amino acid (an insert) in the active site circuit (b) between positions 103 and 104 in a wild type subtyla (for a definition of the active site circuits and the numbering of the positions see below). Although this finding was made in subtilisin 309, it is predictable that it will be possible to produce or isolate similar advantageous subtilases or variants of subtilases. In addition, it will be possible to specifically screen the natural isolates to identify novel wild-type subtilases comprising an active site circuit (b) that is longer than the corresponding active site circuit in known wild type subtilases, such as subtilisin 309 , which subtilases can be considered to have an inserted amino acid residue between positions 103 and 104, and exhibit excellent washing performance in a detergent, as compared to the closest known known subtilisin, such as subtilisin 309. the alignment and numbering, reference is made to figures 1, 2, and 2a below, the alignments are shown between the subtilisin BPN '(BASBPN) (a) and subtilisin 309 (BLSAVI) (b), and the alignments between the subtilisin BPN '(a) and Carlsberg subtilisin (g) In Figures 1 and 2, the alignments were established by using the GAP routine of the GCG package as indicated below, while the alignments of the figures la and 2a are the same as shown in WO 91/00345. These alignments are in this patent application used as a reference to number the waste. The seven active site circuits (a) through (g) (including the terminal amino acid residues indicated) are defined herein to group the amino acid residues in the segments given below or (a) the region between amino acid residues 33 and 43; (b) the region between amino acid residue 95 and 103; (c) the region between amino acid residue 125 and 132; (d) the region between amino acid residue 153 and 173; (e) the region between amino acid residue 181 and 195; (f) the region between amino acid residue 202 and 204; (g) the region between amino acid residue 218 and 219. Thus, in a first aspect the invention relates to isolated subtylase enzymes (ie, more than 10% pure), subgroups IS-1 and IS -2 having at least one additional amino acid residue at position 103 of the active site circuit region (b) from position 95 to 103, whereby the additional amino acid residue (s) corresponds to the insertion of at least one amino acid residue between positions 103 and 104. In a second aspect the invention relates to an isolated DNA sequence encoding a subtyla variant of the invention. In a third aspect, the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtyla variant of the invention. In a fourth 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 subtilisin enzymes of the invention. The enzymes of the invention can be produced generally by culturing a microbial strain from which the enzyme is isolated and recovering the enzyme in a substantially pure form; or by inserting an expression vector according to the fourth aspect of the invention into an appropriate microbial host, culturing the host to express the desired subtylase enzyme and recovering the enzyme product. Furthermore, the invention relates to a composition comprising a subtilase or a subtyla variant of the invention. Still further, the invention relates to the use of the enzymes of the invention for a variety of relevant industrial uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant enzymes. of sub ilisina. Definitions Prior to discussing this invention in greater detail, the following terms and conversions will be defined first. 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 = Gln = 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 AND CONVENTIONS FOR THE DESIGNATION OF VARIANTS In Description of the various enzyme variants produced or contemplated according to the invention, the following nomenclatures and conventions have been adapted for ease of reference: A reference frame is first defined by aligning the original or isolated wild-type enzyme with subtilisin BPN '(BASBPN). The alignment can be obtained by the GAP routine of the GCG package version 9.1 to number the variants using the following parameters: penalty for the creation of space = 8 and penalty for the extension of space = 8 and all other parameters are kept at their values predefined Another method is to use known known alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance. Such alignments between the subtilisin BPN ' (BLSCAR), and subtilisin 309 (BLSAVI) and Carlsberg subtilisin (BLSCAR), respectively are indicated in figures 1, la, 2 and 2a. By this, various eliminations and insertions will be defined in relation to BASBPN. In Figure 1, subtilisin 309 has 6 deletions at positions 36, 58, 158, 162, 163, and 164 compared to BASBPN, whereas in Figure la, subtilisin 309 has the same deletions at positions 36, 56, 159, 164, 165, and 166 compared to BASBPN. In Figure 2 the Carlsberg subtilisin has a deletion at position 58 compared to BASBPN, whereas in Figure 2a the Carlsberg substilisin has a deletion at position 56 compared to BASBPN. These eliminations are in figures 1, 2, and 2a indicated by asterisks (*). The various modifications carried out on a wild-type enzyme are generally indicated using three elements as follows: Amino acid substituted at the original position of the amino acid The notation G195E thus means a substitution of a glycine at position 195 with a glutamic acid. In the case when the original amino acid residue can be any amino acid residue, a short notation can sometimes be used only indicating the position and the substituted amino acid Substituted amino acid in position Such notation is particularly relevant in connection with the (s) modification (en) in homologous subtilases (vide infra), similarly when the identity of the substituent amino acid residue is non-material Original position of amino acid When both the original amino acid and the substituted amino acid can comprise any amino acid, then only the position is indicated, example: 170. When the original amino acid (s) and / or substituted amino acid can comprise more than one, but not all the amino acids (s) then the selected amino acids are indicated within parentheses {,.}.,. {amino acid substituted lr ... an amino acid substratum.} For specific variants, we use an the specific codes of three or one letter, including the Xaa and X codes to indicate any amino acid residue.

SUBSTITUTIONS: The replacement of glutamic acid by glycine at position 195 is designated as: Glyl95Glu or G195E or the substitution of any amino acid residue by glycine at position 195 is designated as: Glyl95Xaa or G195X Gly or gl95 The substitution of serine by any amino acid residue at position 170 will then be designated Xaal70Ser or X170S. 170Ser or 170S Such notation is particularly relevant in connection with the modification (s) of the homologous subtilases (vi de infra). 170Ser means then that it comprises, for example, a Lysl70Ser modification in BASBPN and an Argl70Ser modification in BLSAVI (cf Figure 1). For a modification 'wherein the original amino acid and / or substituted amino acid may comprise more than one, but not all amino acids, the substitution of glycine, alanine, serine, or threonine for arginine at position 170 shall be indicated by Argl70. { Gly, Ala, Ser, Thr} or R170. { GAS , } to indicate the variants R170G, R170A, R170S, and R170T. ELIMINATIONS A glycine removal at position 195 will be indicated by: Glyl95 * or G195 * Correspondingly, the removal of more than one amino acid residue such as the removal of glycine and leucine at positions 195 and 196, will be designated Glyl95 * + Leul96 * or G195 * + L196 + INSERTIONS: The insertion of an additional amino acid residue such as for example a lysine after G195 is: Glyl95GlyLys or G195GK; or when more than one amino acid residue is inserted, such as for example a Lys, Ala and Ser after Gl 95 this is: Glyl95GlyLysAlaSer or G195GKAS In such cases, the inserted amino acid residue (s) is numbered by the addition of letters lower case in the position number of the amino acid residue preceding the inserted amino acid residue. In the previous example, sequences 194 to 196 would be as follows: 194 195 196 BLSAVI A - G - L 194 195 195a 195b 195c 196 Variant A - G - K - A - S - L In cases where an amino acid residue identical to the amino acid residue is inserted existing amino acid residue, it is clear that there is a degeneration in the nomenclature. If for example a glycine is inserted after the glycine in the previous example this will be indicated by G195GG. The same current change could also be indicated, A194AG for the change of 194 195 196 BLSAVI A - G - L 194 195 195.a 196 Variant A - G - G - L 194 194A 195 196 Such examples will be apparent to the person authorized and the indication G195GG and the corresponding indications for this type of insertion thus mean that they include such degenerate equivalent indications. FILLING A SPACE: When there is a deletion in an enzyme in the reference comparison, with the subtilisin sequence BPN 'used for the numbering, an insert in such position is indicated as: * 36Asp or * 36D for the insertion of an acid aspartic MULTIPLE MODIFICATIONS Variants comprising multiple modifications are separated by further signs, for example: Argl70Tyr-Glyl95Glu or R170Y + G195E Representing modifications at positions 170 and 195 replacing tyrosine and glutamic acid with arginine and glycine, respectively. or for example, Tyrl67. { Gly, Ala, Ser. Thr} + Argl70. { Gly, Ala, Ser, Thr} designates the variants Tyrl67Gly + Argl70Gly, Tyr 167Gly + Argl7 OAla, Tyrl67Gly + Argl70Ser, Tyr167Gly + Argl7 OThr, Tyrl67Ala + Argl70Gly, Tyrl 67Ala + Argl7 OAla, Tyrl67Ala + Argl70Ser, Tyr167Ala + Argl7 OThr, Tyrl67Ser + Argl70Gly, Tyrl67Ser + Argl70Ala, Tyrl67Ser + Argl70Ser, Tyr 167Ser + Argl7 OThr, Tyrl67Thr + Argl70Gly, Tyr167Thr + Argl7 OAla, Tyrl67Thr + Argl70Ser, and Tyr167Thr + Argl7 OThr. This nomenclature is particularly relevant in relation to the modifications considered to substitute, replace, insert or eliminate amino acid residues that have specific common properties such as positive charge residues (K, R, H), negative charge (D, E), or conservative modifications of amino acids, for example, Tyrl67. { Gly, Ala, Ser, Thr} + Argl70. { Gly, Ala, Ser, Thr} , which means replacing a small amino acid with another small amino acid. See the section "Detailed description of the invention" for more details. Proteases Enzymes that split amide ligatures into protein substrates are classified as proteases or peptides (interchangeably) (see Walsh, 1979, Enzymatic Reaction Mechanisms, W.H. Freeman and Company, San Francisco, Chapter 3).

Numbering of amino acid positions / residues. If nothing else is mentioned, the numeration of the amino acid used here corresponds to that of the subtylase BPN sequence (BASBPN). For an additional description of the sequence BPN 'see figures 1 and 2, or Siezen et al., Protein Engng. 4 (1991) 719-737. Serine Proteases A serine protease is an enzyme that catalyzes the hydrolysis of peptide bonds, and in which there is an essential residue of serine in the active site (White, Handler and Smith, 1973"Principles of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pages 271-272). Bacterial serine proteases have molecular weights in the range of 20,000 to 45,000 daltons. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A narrower term, alkaline protease, which covers a subgroup reflects the high optimum pH of some of the serine proteases, from pH 9.0 to 11.0 (for review see Priest (1977) Bacteriological! Rev. 41 711-753). SUBTILAAS A sub-group of serine proteases tentatively designated as subtilases has been proposed by Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of the serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Ziezen et al., It is now a subgroup of subtylase. A wide variety of subtilases have been identified, and the amino acid sequence of a variety of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences, reference is made in Siezen et al. (1997). A sub-group of subtilases, IS-1 or "true subtilisins" comprise the "classical" subtilisins such as subtilisin 168 (BSS168), subtilisin BPN ', subtilisin Carlsberg (ALCALASE®, NOVO NORDISK A / S), and Subtilisin DY (BSSDY). An additional sub-group of subtilases, I-S2 or subtilisins of high alkaline content, are recognized by Siezen et al., (Supra). The proteases of subgroup I-S2 are described as highly alkaline subtilisins and comprise enzymes such as subtilin PB92 (BAALKP) (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A / S), Subtilisin 147 (BLS147 ) (ESPERASE®, NOVO NORDISK A / S), alkaline elastase YaB (BSEYAB). List of acronyms for subtylases I-SI Subtilisin 168 BSS168 (BSSAS (subtilisin amylosacchariticus), BSAPRJ (subtilisin J), BSAPRN (subtilisin NAT), BMSAMP (Mesentericopept idasa), Subtilisin BPN ', BASBPN, Subtilisin DY, BSSDY, Subtilisin Carlsberg, BLSCAR (BLKERA (Keratinase), BLSCA1, BLSCA2, BLSCA3), BSSPRC, serine protease C BSSPRD, serine protease D I-S2 Subtilisin Sendai, BSAPRS Subtilisin ALP 1, BSAPRQ, Subtilisin 147, Esperase® BLS147 (BSAPRM (SubtilisinAprM), BAH101) , Subtilisin 309, Savinase®, BLS309 / BLSAVI (BSKSMK (M-protease, BAALKP (Subtisolysin PB92, Bacillus alkalophilic alkaline protease), BLSUBL (subtilisin BL)), Alkaline Elastase YaB, BYSYAB, "SAVINASE®" The SAVINASE® is marketed by NOVO NORDISK ACE. It is subtilisin 309 of B. Lentus and differs from BAALKP only in one position (N87S, see figure 1 here). SAVINASE® has the amino acid sequence designated b) in figure 1. Original subtyla The term "original subtylase" describes a subtyla defined according to Siezen et al. (1991 and 1997). For more detail see the description of "SUBTILASAS" immediately above.

An original subtilase can also be an isolated subtyla from a natural source, where subsequent modification has been made while retaining the characteristics of a subtyla. Alternatively, the term "original subtylase" can be termed "wild-type subtylase". Modification (s) of a subtilase variant The term "modification (s)" as used herein is defined to include the chemical modification of a subtyla as well as the genetic manipulation of the DNA encoding a subtyla. The modification (s) may be the replacement of the amino acid side chain, substitutions, deletions and / or insertions in the amino acid of interest. Subtylase variant In the context of this invention, the term variant subtylase or mutated subtilase, means a subtilase that has been produced by an organism that is expressing a mutant gel of an original microorganism that has an original gene and which produces an enzyme Originally, the original gene has been mutated in order to produce the mutant gene from which the mutated subtylase protease is produced when expressed in an appropriate host. Subtylase homologous sequences The specific regions of the active site circuit, and the amino acid insertions in such circuits of the SAVINASE® subtyla, are identified herein for modification to obtain a subtyla variant of the invention. However, the invention is not limited to modifications of this subtyla in particular, but extends to other original subtilases (wild type) that have a primary homologous structure to that of SAVINASE®. The homology between two amino acid sequences is in this context described by the parameter "identity". In order to determine the degree of identity between two subtilases, the GAP routine of the GCG package version 9.1 can be applied (in brief) using the same parameters. The output of the routine is to one side of the alignment of the amino acids, the calculation of the "percent identity" between the two sequences. Based on this description, it will be routine for a person skilled in the art to identify the appropriate homologous subtilases and the corresponding active site circuit homolog regions, which can be modified in accordance with the invention.

Washing performance The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present in objects to be cleaned during, for example, hard surface cleaning or washing, is often referred to as the washing, detergency, or washing performance, through this application, the term washing performance will be used to cover this property. Isolated sequence of DNA The term "isolated", when applied to a DNA sequence molecule, denotes that the DNA sequence has separated from its natural genetic site and is thus free of other undesirable or foreign coding sequences., and is in a form suitable for use within production systems of proteins prepared by genetic engineering. Such isolated molecules are those that are separated from their natural environment and include genomic and cDNA clones. The isolated molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 'and 3' untranslated regions such as promoters and terminators. The identification of the associated regions will be apparent to someone with ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). The term "an isolated DNA sequence" can alternatively be referred to as "a cloned DNA sequence". Isolated protein When a protein is applied, the term "isolated" indicates that the protein has been separated from its natural environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (ie, "homologous impurities" (see below)). An isolated protein is greater than 10% pure, preferably greater than 20% pure, more preferably greater than 30% pure, as determined by SDS-PAGE. It is further preferred to supply the protein in a highly purified form, ie, more than 40% pure, more than 60% pure, more than 80% pure, more preferably more than 95% pure and even more preferably more than 99% pure as it is determined by SDS-PAGE. The term "isolated protein" can alternatively be referred to as "purified protein". Homologous impurities The term "homologous impurities" means any impurity (eg, another polypeptide than the polypeptide of the invention) that originates from the homologous cell in which the polypeptide of the invention is originally obtained. Obtained from The term "obtained from" as used herein in conjunction with a specific microbial source, means that the polynucleotide and / or polypeptide is produced by the specific source, or by a cell into which a gene from the source has been inserted. . Substrate The term "substrate" used in conjunction with a substrate for a protease, should be construed in its broadest form comprising a compound that contains at least one peptide bond susceptible to hydrolysis by a subtilisin protease.

Product The term "product" used in conjunction with a product derived from an enzymatic reaction of proteases must, in the context of this invention, be construed as including the products of a hydrolysis reaction involving a subtylase protease. A product can be the substrate in a subsequent hydrolysis reaction. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an alignment between subtilisin BPN '(a) and Savinase® (b) using the GAP routine mentioned above. The figure shows the alignment between subtilisin BPN 'and Savinase® as taken from WO 91/00345. Figure 2 shows an alignment between the subtilisin BPN 'and the subtilisin Carlsberg using the GAP routine mentioned above. Figure 2a shows the alignment between the subtilisin BPN 'and the Carlsberg subtilisin as taken from WO 91/00345. Figure 3 shows the three-dimensional structure of the Savinase® (protein data bank (PDB) input 1SVN). The active site circuit (b) is indicated in the figure.

Detailed Description of the Invention The subtilases of the invention in a first aspect, relate to an isolated subtylase enzyme (ie, more than 10% pure) of sub-groups I-SI and I-S2 having at least one additional amino acid residue at position 103 of the active site circuit region (b) from position 95 to 103, whereby the additional amino acid residue corresponds to the insertion of at least one amino acid residue between positions 103 and 104. In other words, the subtilases of the invention are characterized by comprising an active site circuit region (b) of more than 9 amino acid residues and wherein the additional amino acid residue is or can be considered to be inserted between positions 103 and 104 in comparison to the known subtyla of wild type or original. A subtilase of the first aspect of the invention may be an original or wild type subtyla identified and isolated from nature.

Such an original wild-type subtylase can be specifically screened by standard techniques known in the art. A preferred way of doing this may be by specifically amplifying in PCR the known DNA regions encoding the active site circuits in subtyla from several different microorganisms, preferably different Bacillus strains. Subtilases are a group of conserved enzymes, in the sense that their DNA and amino acid sequences are homologous. In this way, it is possible to construct relatively specific primers that surround the active site circuits. One way of doing this is by investigating the alignment of different subtilases (see, for example, Siezen and collaborators Protein Science 6 (1997) 501-523). It is from this routine work for a person skilled in the art, the construction of the PCR primers that surround the active site circuit that corresponds to the active site circuit (b) between amino acid residue 95 to 103 in any of the group I-SI or group I-S2 such as BLSAVI.

Using such PCR primers to amplify the DNA from a variety of different microorganisms, preferably different strains of Bacillus, followed by the formation of DNA sequences from said amplified PCR fragments, it will be possible to identify the strains that produce the subtilase of these groups comprising a longer active site region, as compared to for example, BLSAVI corresponding to the active site circuit region from positions 95 to 103, and where an insertion between positions 103 may be considered. and 104. Once the strain 'and a partial DNA sequence of such subtylae of interest have been identified, it is routine work for a person skilled in the art to complete the cloning, expression and purification of said subtyla of the invention. However, it is envisioned that a subtylase enzyme of the invention is predominantly a variant of an original subtylase. In this way, in one embodiment of the invention relates to a subtylase-isolated enzyme according to a first aspect of the invention, wherein said subtylase enzyme is a constructed variant having a longer active site circuit (b) than its enzyme original having at least one amino acid insertion between amino acid residues 103 and 104. The subtilases of the invention show excellent washing performance in a detergent, and if the enzyme is a constructed variant, it has an improved wash performance in a detergent in comparison with the more closely related subtylase, such as subtilisin 309. Different subtilase products will show different wash performance in different types of detergent compositions. The subtyla of the invention has improved wash performance, as compared to its closest similar in a majority of different types of detergent compositions. Preferably, a subtylase enzyme of the invention has improved wash performance, as compared to its closest similar in the detergent composition shown in Example 3 herein (vide infra). In order to determine whether a given subtyla amino acid sequence (irrelevant whether the subtylase sequence is an original wild-type subtyla sequence or a subtyla variant sequence produced by any other method than by site-directed mutagenesis) is within the scope of the invention, the following procedure can be used: i) aligning the subtylase sequence to the amino acid sequence of the subtilisin BPN '"Definitions" here (vi of s upra); ii) based on the alignment performed in step i) identified in the active site circuit (b), in the subtylase sequence corresponding to the active site circuit region (b) of the subtilisin BPN 'comprising the region (both of the terminal amino acids included) between the amino acid residue from 95 to 103. iii) determine if the active site circuit (b) in the subtylase sequence, identified in step ii), is longer than the corresponding active site circuit in BLSAVI and if said prolongation corresponds to the insertion of at least one amino acid residue between positions 103 and 104. If this is the case, the subtilase investigated is a subtilase within the scope of the present invention. The alignment carried out in step i) above is carried out as described above using the GAP routine. Based on this description, it is routine for a person skilled in the art to identify the active site circuit (b) in a subtilase and determine whether the subtyla in question is within the scope of the invention. If a variant is constructed by site-directed mutagenesis, it is of course known in advance whether the subtyla variant is within the scope of the invention.

A subtylase variant of the invention can be constructed by standard techniques known in the art such as by site-directed random mutagenesis or by DNA mixing of different subtylase sequences. See the section "PRODUCE A SUBSTITUTE VARIANT" and the methods and materials here (vi de in fra) for more details.

In further embodiments, the invention relates to: 1. An enzyme subtylase isolated according to the invention, wherein at least one inserted amino acid residue is selected from the group comprising: T, G, A and S; 2. An isolated subtylase enzyme according to the invention, wherein at least one inserted amino acid residue is selected from the group of amino acid-laden residues comprising: D, E, H, K, and R, more preferably D, E , K and R,; . An enzyme subtyla isolate according to the invention, wherein at least one inserted amino acid residue is chosen from the group of hydrophilic amino acid residues comprising: C, N, Q, S and T., more preferably N, Q , S and T; . An isolated subtylase enzyme according to the invention, wherein at least one inserted amino acid residue is selected from the group of small hydrophobic amino acid residues comprising: A, G and V; or An isolated subtylase enzyme according to the invention, wherein at least one inserted amino acid residue is selected from the group of large hydrophilic amino acid residues comprising: F, I, L, M, P, W and Y, more preferably F , I, L, M, and Y. In a further embodiment, the invention relates to an isolated subtylase enzyme according to the invention, wherein said insertion between positions 103 and 104 comprise at least two amino acids, as compared to the corresponding circuit of active site in BLSAVI. In further embodiments, the invention relates to a subtylase-isolated enzyme comprising at least one insert, chosen from the group comprising (in BASBPN numbering): X103X. { T, G, A, S.}. X103X { D, E, K, R} X103X { H, V, C, N, Q } X103X { F, I, L, M, P, W, Y.}. or more specific for subtilisin 309 and the closely similar subtilases, such as BAALKP, BLSUBL, and BSKSMK S103SA S103ST S103SG S103SS S03 SD S103SE S103SK S103SR S103SH S103SV S103SC S103SN S103SQ S103SF S103SI S103SL S103SM S103 S103SW S103SY Additionally, the invention relates to subtilases comprising multiple inserts at position 103, or Any of the following combinations S103ST + Y167A It is well known in the art that the so-called conservative substitution of an amino acid residue to a similar amino acid residue is expected to produce only a minor change in the characteristic of the enzyme. Table III below lists groups of conservative amino acid substitutions Table III Conservative amino acid substitutions Common property Basic amino acid (positive charge. 'K = lysine H = histidine Acid (negative charge) E = glutamic acid ID = aspartic acid Polar Q = glutamine N = aspargin Hydrophobic L = leucine 1 = isoleucine V = valine M = methionine Aromatic F = phenylalanine W = tryptophan Y = tyrosine Small G = glycine A = alanine S = serine T = threonine In accordance with this principle, 'subtylase variants comprising conservative substitutions, such as G97A + A98AS + S99G, G97 + A98AT + S99A, are expected to show characteristics that are not drastically different from one another. Based on the subtilase variants described and / or exemplified herein, it is a routine work for a person skilled in the art to identify appropriate conservative modifications of these variants in order to obtain other subtyla variants that show a washing performance similarly improved. According to the invention, the subtilases of the invention belong to the subgroups I-SI and I-S2, especially the subgroup I-S2, both to isolate novel enzymes of the invention from nature, as well as for the artificial creation of diversity, and for the design and production of variants from an original subtilasa. Regarding variants of subgroup I-Sl, it is preferred to choose an original subtyla from the group comprising BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA, BLSCAl, BLSCA2, BLSCA3), BSSPRC, and BSSPRD, or functional variants thereof which have retained the characteristic of sub-group I-SI. With regard to the sub-group I-S2 variants, it is preferred to choose an original subtyla from the group comprising BSAPRQ, BLS147 (BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYVA, and BSAPRS, or functional variants of the they have retained the characteristic of subgroup I-S2. In particular, the original subtyla is BLSAVI (SAVINASE® NOVO NORDISK A / S), and a preferred variant of the subtilase of the invention is, in this manner, a variant of SAVINASE®. The present invention also comprises any of the aforementioned subtilases of the invention, in combination with any other modification to the amino acid sequence thereof. Especially, combinations with other modifications known in the art to provide improved properties to the enzyme, are glimpsed. The art describes a variety of subtilase variants with different improved properties, and a variety of those are mentioned in the "Background of the Invention" section here (vi de s upra). These references are described herein as references to identify a subtyla variant, which may be advantageously combined with a subtyla variant of the invention. Such combinations comprise the positions: 222 (improves oxidation stability), 218 (improves thermal stability), substitutions at Ca binding sites that stabilize the enzyme, e.g., position 76, and many other apparent prior art . In additional embodiments, a subtyla variant of the invention can be advantageously combined with one or more modifications at any of the positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206 , 218, 222, 224, 235 and 274. Specifically, the following variants BLSAVI, BLSUBL, BSKSMK, and BAALKP are considered appropriate for combination: N27R, * 36D, S57P, N76D, S87N, G97N, S101G, S103A, V104A, V104I , V104N, V104Y, H120D, N123S, Y167, R170, Q206E, N218S, M222S, M222A, T224S, K235L and T274A. In addition, the variants comprise any of the variants S101G + V104N, S87N + S101G + V104N, K27R + V1O4Y + N123S + T274A, N76D + S103A + V104I or N76D + V104A or other combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A) in combination with any one or more of the aforementioned modifications that show enhanced properties. Still further, the subtilase variants of the main aspect of the invention are preferably combined with one or more modifications at any of positions 129, 131, 133 and 194, preferably as modifications 129K, 131H, 133P, 133D and 194P, and more preferably as modifications P129K, P131H, A133P, A133D and A194P. any of those modifications are expected to provide a higher level of expression of a subtyla variant of the invention in the production thereof. In this way, a still further embodiment of the invention, refers to a variant according to the invention, wherein the modification is chosen from the group comprising: Production of a Subtylase Variant. Many methods for the cloning of the subtyla of the invention and for the introduction of insertions into the genes (for example, subtyla genes), cf., references cited in the section "Background of the Invention" are well known in the art. . In general, standard procedures for gene cloning and the insertion of inserts (random and / or site-directed) into the genes can be used in order to obtain a subtyla variant of the invention. For further description of the appropriate techniques, reference is made to the Examples herein (vi of infra) and (Sambrook et al., (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F.M. and collaborators, (eds) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C.R., and Curring, S.M. (eds) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990); and WO 96/34946. In addition, a subtyla variant of the invention can be constructed by standard techniques for the artificial creation of diversity, such as by mixing DNA from different subtylase genes (WO 95/22625; Stemmer WPC; Nature 370: 389-91 (1994)). The mixing of DNA of, for example, the gene encoding Savinase®, with one or more partial sequences of aubthylase identified in nature, to comprise active site circuit regions (b) longer than the active site circuits ( b) of the Savinase®, will provide after a subsequent screening for the improved washing performance variants, subtilase variants according to the invention. Expression Vectors. A recombinant expression vector comprises a DNA construct encoding the enzyme of the invention, can be any vector that can be conveniently attached to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example a plasmid. Alternatively, the vector can be one that, with introduction into a host cell, is integrated into the genome of the host cell in part or in its entirety and replicates 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 the additional segments required for transcription of the DNA. In general, the expression vector is derived from the plasmid or viral DNA, or may contain elements of both. The term "operably linked" indicates that the segments are positioned to function in harmony for the intended purposes, for example, transcription starts at a promoter and continues through the DNA sequence encoding the enzyme. The promoter can be any DNA sequence that exhibits transcriptional activity in the host cell of choice and can be derived from genes encoding proteins homologous or heterologous to the host cell. Examples of suitable promoters for use in bacterial host cells include the promoter of the maltogenic amylase gene Bacillus stearothermophilus, the alpha amylase gene Bacillus licheniformis, the alpha amylase gene Bacillus amyloliquefaciens, the alkaline protease gene Bacillus subtilis, or the gene of xylosidase Bacillus pumilus, or the promoters of phage Lambda PR or PL or the promoters E. coli lac, trp or tac. The DNA sequence encoding the enzyme of the invention can also, if necessary, be operatively connected to an appropriate terminator. The recombinant vector of the invention can further comprise a DNA sequence that allows the vector to replicate in the host cell in question. The vector may also comprise a selectable marker, eg, a gene, the product of which complements a defect in the host cell, or a gene encoding resistance for, for example, antibiotics such as kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycin. , or similar, or resistance to heavy metals or herbicides. To direct an enzyme of the present invention within the secretory path of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) can be provided in the recombinant vector. The secretory signal sequence binds to the DNA signal sequence encoding the enzyme in the correct reading structure. The secretory signal sequences are commonly positioned 5 'to the DNA sequence encoding the enzyme. The secretory signal sequence may be that it is normally associated with the enzyme or it may be from a gene encoding another secreted protein. The methods used to ligate the DNA sequences encoding the current enzyme, the promoter and optionally the terminator and / or the secretory signal sequence, respectively, or to assemble these sequences by appropriate PCR amplification schemes, and insert them into appropriate vectors which contain the information necessary for replication or integration, are well known to those skilled in the art (see, for example, Sambrook et al., op.cit.).

Host Cell. The DNA sequence encoding the current enzyme introduced into the host cell can be homologous or heterologous to the host in question. If it is homologous to the host cell, this is produced by the host cell in nature, it will be operatively connected in a manner typical of another promoter sequence or, if applicable, another segregating signal sequence and / or terminator sequence which in its natural environment The term "homologous" is intended to include a DNA sequence that encodes a natural enzyme 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 can be from another organism or it can be synthetic.

The host cell into which the DNA construct or the recombinant vector of the invention is introduced can be a cell that is capable of producing the current enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells including plants. Examples of bacterial host cells which, under culture, can produce the enzyme of the invention, are Gram-positive bacteria such as Bacillus strains, such as strains of B. Subtilus, B. Licheniformis, B. Lentus, B. Brevis, B. Stearothermophilus, B. Alcalophilus, B. Amyloliqu faciens, B. coagulans, B. Circulans, B. Lautus, B-megatherium or B. thruringiensis, or strains of Streptomyces, such as S. lividans or S. mu inus, or Gram-negative bacteria such as Escherichia coli. The transformation of the bacteria can be effected by transformation of protoplasts, electrophoration, conjugation, or by the use of competent cells in a manner known per se (cf Sambrook et al., Supra). When the enzyme is expressed 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 above case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded upon diluting the denaturing agent. In the latter case, the enzyme can be recovered from the periplasmic space by breaking the cells, for example by sonication or osmotic shock, to release the contents of the periplasmic space and recover the enzyme. When the enzyme is expressed in a Gram-positive bacterium, such as strains of Bacillus or Streptomyces, the enzyme can be retained in the cytoplasm, or it can be directed to the extracellular medium by a bacterial sequence of secretion. In the latter case, the enzyme can be recovered from the medium as described below. SUBTILAE PRODUCTION METHOD The present invention provides a method of producing an isolated enzyme according to the invention, wherein an appropriate host cell that has been transformed with a DNA sequence encoding the enzyme is cultured under conditions that allow the production of the enzyme, and the enzyme resulting from the culture is recovered. When an expression vector comprising a DNA sequence encoding the enzyme is transformed within a heterologous host cell, it is possible to allow heterologous recombinant production of the enzyme of the invention.

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

Use of a subtyla variant of the invention A variant of the subtyla protease of the invention can be used for various industrial applications, in particular within the detergent industry. Furthermore, the invention relates to an enzyme composition, which comprises a subtyla variant of the invention. A summary of the preferred industrial applications and the corresponding preferred compositions of enzymes are described below. This summary is in no way intended to be a complete listing of the appropriate applications of a subtyla variant of the invention. The subtilase variants of the invention can be used in other industrial applications known in the art, including the use of a protease, in particular a subtylase. DETERGENT COMPOSITIONS COMPRISING THE MUTANT ENZYMES The present invention comprises the use of mutant enzymes of the invention in detergent and cleaning compositions and such compositions comprise the mutant enzymes of subtilisin.

Such detergent and cleaning 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 the appropriate detergent and cleaning compositions. Additionally, the examples below demonstrate improvements in washing performance for a variety of subtyla variants of the invention. Detergent compositions The enzyme of the invention can be added and thus become a component of the detergent composition. The detergent composition of the invention can be formulated, for example, as a detergent composition for machine or hand washing, including an additive laundry composition suitable for pretreatment of stained fibers and a rinse composition added to fabric softeners, or formulated as a detergent composition for use in general domestic cleaning cooperations on hard surfaces, or formulated for machine or hand dishwashing operations.

In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, a arabinase, a galactanase, a xylanase, an oxidase, for example a laccase, and / or a peroxidase. In general, the properties of the chosen enzyme must be compatible with the selected detergent (ie, optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.) and the enzyme must be present in effective amounts. Proteases: appropriate proteases include those of animal, plant or microbial origin. The microbial origin is preferred. Chemically modified mutants or engineered proteins are included. The protease can be a serine protease or a metallo protease, preferably an alkaline microbial protease or a protease such as trypsin. Examples of the alkaline proteases are the subtilisins, especially those derived from Bacillus, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (for example of porcine or bovine origin), and Fusarium protease described in WO 89/06270 and WO 94/25583. Examples of the useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57 , 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274. Preferred commercially available protease enzymes include Alease ™, Savinase ™, Primase TM Duralase TM Wait, T'M TM and Kannase " Novo Nordis k A / S; Maxatase TM Maxacal TM Maxapem, Properase, Purafect PxP, FN2, and FN3 ™ (Genencor International Inc.). Lipases: Appropriate lipases include those of bacterial or fungal origin. Chemically modified mutants or engineered proteins are included. Examples of useful lipases include Humicola lipases (synonymous with Thermomyces), for example from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580 , a Pseudomonas lipase, for example, from P. "alcaligenes or P. seudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, strains of Pseudomonas sp. SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, for example from B. subtilis (Dartois et al., (1993), Biochemica et Biophysica Acta, 1131 , (253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96700292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202. Preferred lipase enzymes Commercially available include Lipolase ® and Lipolasa Ultra® (Novo Nordisk A / S). Amylases: The appropriate amylases (a and / or ß) include those of bacterial or fungal origin. Chemically modified mutants or prepared by protein engineering are included. Amylases include, for example, α-amylases obtained from Bacillus, for example a special strain of B. licheniformis, described in more detail in GB 1,296,839. Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444. The commercially available amylases are Duramyl® , Termamyl®, Fungamyl® and BAN® (Novo Nordisk A / S), Rapidase® and Purastar® (from Genencor International Inc.). Cellulases: Appropriate cellulases include those of bacterial or fungal origin. Chemically modified or engineered mutants are included. Suitable cellulases include cellulases of the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example fungal cellulases produced from Humicola insolens, Mycel and oph thora thermophi la and Fusa ri um oxysporum described in US 4,435,307, US 5,648,263 , US 5,691,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are alkaline or neutral cellulases that have color care benefits. Examples of such cellulases are the cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT / DK98 / 00299. Commercially available cellulases include Celluzyme®, and Carezyme® (Novo Nordisk A / S), Clazinase®, and Puradax HA® (Genencor International Inc.), and KAC-500 (B) ® (Kao Corporation). Peroxidases / Oxidases: Appropriate peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified mutants or prepared by protein engineering are included. Examples of useful peroxidases include Coprin peroxidases, for example from C. cin erus, and variants thereof as described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme® (Novo Nordisk A / S). Detergent enzymes may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, this is a separate additive or a combined additive, it can be formulated, for example, as a granulate, a liquid, a thick liquor, etc. Preferred formulations of detergent additives are granules, in particular granules which do not produce dust, liquids, in particular stabilized liquids, or thick liquors. Granules that do not emit dust can be produced, for example, as described in US Patents 4,106,991 and 4,661,452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are the products of poly (ethylene oxide), (polyethylene glycol, PEG) with average molar weights of 1000 to 20,000; 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 from 15 to 80 units of ethylene oxide; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of suitable film-forming coating materials for application by fluidized bed techniques are given in GB 1483591. Liquid enzyme preparations can, for example, be stabilized by adding a polyol such as propylene glycol, a sugar or alcohol of sugar, lactic acid or boric acid in accordance with established methods. Protected enzymes can be prepared according to the method described in EP 238, 216. The detergent composition of the invention can be in any convenient form, for example, a stick, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent can be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous solvent.

The detergent composition comprises one or more surfactants, which may be nonionic including semi-polar and / or anionic and / or cationic and / or zwitterionic. Surfactants are typically present at a level from 0.1% to 60% by weight. When included with the detergent, they will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzene sulfonate, alpha-olefinsul fonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkan sulfonate, methyl ester of fatty alpha-sulfo acid, alkyl or alkenyl succinic acid or soap. When included with the detergent, they will usually contain from about 0.2% to about 40% of a nonionic surfactant such as an alcohol ethoxylate, ethoxylated nonylphenol, alkyl polyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, acid monoethanolamide. fatty acid, polyhydroxy alkylamide of fatty acid, or N-acyl derivatives and N-alkyl of glucosamine ("glucamides").

The detergent may contain from 0 to 65% of a detergent additive or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediamine et racerate acid, diethylene triamine pentacetic acid, alkyl acid or alkenylsuccinic, soluble silicates or layered silicates (for example, SKS-6 from Hoechst). The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly (vinylpyrrolidone), poly (ethylene glycol), polyvinyl alcohol, polyvinylpyridine N-oxide, poly (vinylimidazole), polycarboxylates such as polyacrylates, acrylic / maleic acid copolymers and acrylic acid / lauryl methacrylate copolymers. The detergent may contain a bleach system which may comprise a source of H202 such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetracetylethylenediamine or nonaoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type.

Enzymes of the detergent composition of the invention can be stabilized using conventional stabilizing agents, for example, a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or boric acid derivatives , for example, an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition can be formulated as described in, for example, WO 92/19709 and WO 92/19708. The detergent may also contain other conventional detergent ingredients such as, for example, fabric conditioners including clays, foam-enhancing agentssuds suppressors, anti-corrosion agents, soil suspending agents, anti-dirt re-deposition agents, dyes, bactericides, optical brighteners, hydrotropes, stain inhibitors, or perfumes. It is currently contemplated that in detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg of protein. of enzyme per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor. The enzyme of the invention can, additionally, be incorporated into the detergent formulations described in WO 97/07202 which is incorporated herein by reference. Applications in the Leather Industry. The subtyla of the invention can be used in the skin industry, in particular for use in skin depilation. In said application a subtylase variant of the invention is preferably used in an enzyme composition which further comprises another protease. For a more detailed description of other appropriate proteases, see the section on enzymes for use in detergent compositions (vi de s upra). Applications in the Wool Industry. The subtyla of the invention can be used in the wool industry, in particular for use in cleaning garments comprising wool.

In such an application, a subtyla variant of the invention is preferably used in an enzyme composition which also comprises another protease. For a more detailed description of other appropriate proteases, see the section related to the appropriate enzymes for use in a detergent composition (vide supra). The invention is described in greater detail in the following examples, which are in no 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. specific strains of Bacil lus lentus, deposited with the NCIB and with accession number agreements NCIB 10309 and 10147, and described in US Patent No. 3,723,250 which is incorporated herein by reference. E. coli MC 1000 (MJ Casadaban and SN Cohen (1980); J. Mol. Biol. 138 179-207), was made r-, m + by conventional methods and is also described in the US Patent Application No. Series 039,298. Plasmids: pJS3: The transfer vector E. col i - B. s ub ti l i s that contains a synthetic gene that encodes subtyla 309. (Described by Jacob Schi0dt et al., In Protein and Peptide letters 3: 39-44 (1996)). pSX222: The expression vector B. s ub t i l i s (described in WO 96/34946). , General Methods of Molecular Biology: Unless otherwise mentioned, manipulations and transformations of DNA were carried out 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. and collaborators (eds) "Current protocols in Molecular Biology". John Wiley and -Sons, 1995; Harwood, C. R., and Cutting S.M. (eds) "Molecular Biologicals Methods for Bacillus. "John Wiley and Sons, 1990.) Enzymes for DNA manipulations were used according to the specifications of the suppliers.

Enzymes for DNA manipulations. Unless otherwise mentioned, all enzymes for DNA manipulations, such as, for example, restriction endonucleases, ligases, etc., are obtained from New England Biolabs, Inc Proteolytic activity. In the context of this invention, the proteolytic activity is expressed in kilo NOVO (KNUP) protease units. The activity is determined relatively to an enzyme standard (SAVINASE®), and the determination is based on the digestion of a solution of dimethyl casein (DMC) by the proteolytic enzyme at standard conditions, that is, 50 ° C, pH 8.3, reaction time of 9 minutes, measurement time 3 minutes. An AF 220/1 folder is available upon request at Novo Nordisk A / S, Denmark, the folder is included here for reference. A GU is a glycine unit, defined as the? proteolytic enzyme activity which, under standard conditions, during a 15 minute incubation at 40 ° C, with N-acetyl casein as substrate, yields an amount of an NH 2 group equivalent to 1 mmol glycine.

Enzyme activity can also be measured using a PNA assay, according to the reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitro-phenol, which is described in the Journal of the American Oil Chemists Society, Rothged , TM, Goodlander, BD, Garrison, PH, and Smith, LA, (1988). Fermentation The fermentations for the production of subtylase enzymes were carried out at 30 ° C on a rotary shaking table (300 r.p.m.) in Erlenmeyer flasks with 500 ml screens containing 100 ml of a BPX medium for 5 days. Consequently, in order to make, for example, a 2-liter broth, 20 Erlenmeyer flasks were fermented simultaneously. Means: Composition of BPX Medium (per liter) Potato starch 100 g Ground barley 50 g Soy flour 20 g Na2HP04 x 12 H20 9 g Pluronic 0.1 g Sodium caseinate 10 g The starch in the medium is liquefied with α-amylase and the medium is sterilized by heating at 120 ° C for 45 minutes. After sterilization, the pH of the medium is adjusted to 9 by the addition of 0.1 M NaHC03. Example 1 Construction and Expression of the Variants of Enzymes: Site Directed Mutagenesis: The variants directed to the site of subtylase 309 of the invention, comprise specific insertions in the active site circuit (b) between positions 103 and 104, were made by traditional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced by PCR of the oligos containing the desired inserts (see below). The template plasmid DNA was pJS3, or an analog thereof containing a variant of subtilas 309. Inserts were introduced by mutagenesis directed to the oligos for the construction of the insertion variants S103SX (X = to any amino acid residue inserted between positions 103 and 104) resulting in variants of subtilasa 309 S103SX. The subtylase 309 variants were transformed into E. col i. The purified DNA of an overnight culture of these transformants was transformed into B. s ubtil i s by digestion of restriction endonucleases, purification of DNA fragments, ligation, transformation of B. subtili s. The transformation of B. s ubti l i s was carried out as described by Dubnau et al., 1971, J. Mol. Biol. 56, pages 209-221. Localized Random Mutagenesis to Insert Random Inserts in a Localized Region: The global strategy to be used to carry out the localized random mutagenesis was: A mutagenic primer was synthesized (oligonucleotide), which corresponds to the DNA sequence that limits the insertion site, separated by the base pairs of DNA that define the insertion. Subsequently, the resulting mutagenic primer was used in a PCR reaction with an appropriate opposite primer. The resulting PCR fragment was purified and extended in a second PCR reaction, before being digested by the endonucleases and cloned into the E transfer vector. col i - B. s ub t i l i s (see below). Alternatively, and if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a proper second opposite primer to allow digestion and cloning of the mutagenized region within the transfer vector. PCR reactions are carried out under normal conditions. Following this strategy, a randomized collection located in SAVINASE was constructed, where the insertions were introduced in the active site circuit region between positions 103 and 104. Mutations were introduced by mutagenic primers (see below), so that represent all 20 amino acids (N = 25% of A, T, C, and G, while S = 50% of C and G. The PCR produced fragment extends to the N-terminus of Savinase by another round of PCR by the combination of a sequence of overlap with a PCR fragment produced by the PCR amplification with primers; 5 'CTA AAT ATT CGT GGTGGC GC 3' (sense) and 5 'GAC TTT AAC AGC GTA TAG CTC AGC 3' (antisense). The extended DNA fragments were cloned into the Hind III- and Mlu I sites of the modified plasmid pJS3 (see above), and ten colonies randomly selected from E. coli were sequenced to confirm the designated mutations.The mutagenic primer (5 ') CTA GGG GCG AGC GG T TCA GGT TCG NNS GTC AGC TCG ATT GCC CAGA GGA TTG 3 '(sense)) was used in a PCR reaction with an appropriate anti-sense opposite primer, located in the 3' direction of the Mlu I site in pJS3 (e.g. 5 'CCC TTT AAC CGC ACA GCG TTT -3' (antisense)) and the plasmid pJS3 as template. This resulting PCR product was cloned into the pJS3 transfer vector by using the restriction enzymes Hind III and Mlu I. The random collection was transformed into E. coli by well-known techniques. The prepared collection contained approximately 100,000 individual clones / collection. Ten randomly chosen colonies were sequenced to confirm the designated mutations.

In order to purify a subtyla variant of the invention, the expression plasmid B. sub ti l i s pJS3 comprising a variant of the invention, was transformed into a competent strain B. s ub t i l i s and fermented as described above in a medium containing 10 μg / ml chloramphenicol (CAM). Example 2. Purification of Enzyme Variants: This procedure relates to the purification of a 2-liter fermentation for the production of the subtyla of the invention in a Ba ci lus host cell. About 1.6 liters of fermentation broth was centrifuged at 5000 rpm for 35 minutes in 1 liter agitators. The supernatants were adjusted to a pH of 6.5 using 10% acetic acid and filtering on Seitz Supra S100 plate filters. The filtrates were concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate was centrifuged and filtered before absorption at room temperature in a Bacitracin affinity column at a pH of 7.

Protease was eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1M sodium chloride in a buffer solution with 0.01 dimethylglutaric acid, 0.1M boric acid and 0.002M calcium chloride adjusted to a pH of 7. combined fractions with protease activity from the purification step in Bacitracin and were applied to a 750 ml G25 Sephadex column (5 cm diameter) equilibrated with a buffer solution containing 0.01 dimethylglutaric acid, 0.2 M boric acid and calcium chloride 0.002 adjusted to a pH of 6.5. Fractions with proteolytic activity from the "Sephadex G25 column were combined and applied to a CM Sepharose CL 6 B 150 ml cation exchange column (5 cm diameter) equilibrated with a buffer solution containing 0.01 M dimethylglutaric acid, boric acid 0.2 M, and 0.002 M calcium chloride adjusted to a pH of 6.5 The protease was eluted using a linear gradient of 0-0.1 M sodium chloride in 2 liters of the same buffer (0-0.2 M sodium chloride) of Subtilisin 147).

In a final purification step, the fractions containing protease from the CM column Sepharose, were combined and concentrated in an ultrafiltration cell equipped with a GR81PP membrane (from Danis Sugar Factories Inc.). Using the techniques of Example 1 for the construction and fermentation, and the above isolation procedure, the following variants of subtilisin 309 were produced and isolated: S103ST S103SA S103SS S103SD S103SE S103SP S103SG S103SH S103SI S103ST + Y167A These variants showed better performance of washing that Savinase in a preliminary trial. Example 3. Washing Performance of Detergent Compositions Comprising Enzyme Variants.

The following examples provide results of a variety of washing tests that were carried out under the indicated conditions. Mini Wash. Washing Conditions: Detergents: The detergents used are obtained from supermarkets in Denmark (OMO, catalog ED-9745105) and in the USA (Wisk, catalog ED-9711893), respectively. Before using all enzymatic activity in the detergents, they were inactivated by microwave treatment. Fabric Samples: The fabric samples used were EMPA116 and EMPA117, obtained from EMPA Tes tmaterialen, Movenstrasse 12, CH-9015 St. Gail, Switzerland. Reflectance The measurement of the reflectance (R) in the test material was made at 460 nm using a Macbeth ColorEye 7000 photometer. The measurements were made according to the manufacturer's protocol. Evaluation .

The evaluation of the wash performance of a subtyla is determined by the improvement factor or the performance factor for the subtilase investigated. The improvement factor, I FDosis / response. it is defined as the ratio between the slopes of the washing performance curves for a detergent containing the subtilase investigated and the same detergent containing a reference subtyla at an asymptotic concentration of the subtilase going to zero. -1- - 'dose / espues t a = reí The washing performance is calculated according to formula I: a-? R R = R max 0? R (I) max + a-c where R is the washing performance in reflectance units; R0 is the intersection of the curve fitted with the Y axis (blind); a is the slope of the curve set as c? 0; c is the enzyme concentration; and? Rmax is the maximum theoretical wash effect as c? oo The performance factor, P, is calculated according to formula II: ^ Variant ~ White P - (II) ^ Savinase "" "^ White where Rvariant is the reflectance of the washed test material with the lOnM variant; Rsavinase is reflectance of the washed test material with Savinase 10 nM; Rbianco is the reflectance of the test material washed without enzyme. USA (detergent: USA Wisk, Fabric sample: EMPA117) The subtilases of the invention are thus seen to show improved wash performance compared to Savinase®. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (34)

1. Claims Having described the invention as above, the content of the following claims * is claimed as property. 1. A subtylase enzyme of sub-groups I-SI and I-S2 characterized by having at least one additional amino acid residue at position 103 of the active site circuit region (b) from position 95 to position 103 , whereby the additional amino acid residue corresponds to the insertion of at least one amino acid residue between positions 103 and 104.
2. 2. The isolated subtylase enzyme according to claim 1, characterized in that the subtylase enzyme is a variant constructed having at least one amino acid residue inserted between positions 103 and 104 of a precursor subtyla.
3. 3. The subtylase enzyme isolated according to claim 1 or 2, characterized in that it is selected from the group comprising X103X. { A, T, G, S.}. , X103X. { D, E, K, R.}. , X103X. { H, V, C, N, Q.}. , and X103X. { F, I, L, M, P, W, Y.}.
4. 4. The subtylase enzyme isolated according to claim 3, characterized in that at least one additional or inserted amino acid residue is chosen from the group comprising: T, G, A, Y S.
5. 5. The subtylase enzyme isolated in accordance with with claim 3, characterized in that at least one additional or inserted amino acid residue is chosen from the group of amino acid-laden residues comprising: D, E, H, K, and R, more preferably D, E, K and R.
6. The subtylase enzyme isolated according to claim 3, characterized in that at least one additional or inserted amino acid residue is chosen from the group of hydrophilic amino acid residues comprising: C, N, Q, S and T, more preferably N, Q, S and T.
7. 7. The subtylase enzyme isolated according to claim 3, characterized in that at least one additional or inserted amino acid residue is chosen from the group of small hydrophobic amino acid residues comprising: A, G and V.
8. 8. The subtylase enzyme isolated according to claim 3, characterized in that at least one additional or inserted amino acid residue is chosen from the group of large hydrophobic residues of amino acids comprising: F, I, L, M, P, W and Y, more preferably F, I, L, M, and Y.
9. 9. The isolated subtylase enzyme according to any of the preceding claims, characterized in that at least one additional or inserted amino acid residue comprises more than one additional or inserted amino acid residue. in the active site circuit (b).
10. 10. The subtilase variant according to any of the preceding claims, characterized in that the inserts between the positions 103 and 104 are combined with one or more additional modifications in any other position.
11. 11. The subtilase variant according to claim 15, characterized in that the additional modifications are in one or more of the positions 27, 26, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224, 235 and 274.
12. 12. The subtilase variant according to any of the preceding claims, characterized in that the modifications are combined with modifications in one or more of the positions 129, 131, 133 and 194.
13. 13. The subtilase according to any of the preceding claims, characterized because the subtilase, or if the subtyla is a variant of the original subtyla, belong to the sub-group I-SI.
14. 14. The subtilase according to claim 13, characterized in that the original subtilase is chosen from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR, or functional variants thereof which retain the characteristic of the sub-group I-SI.
15. 15. The subtilase according to any of claims 1-14, characterized in that the subtilase, or if the subtyla is a variant, the original subtyla belongs to the subgroup I-S2.
16. 16. The subtilase according to claim 15, characterized in that the original subtylase is chosen from the group comprising BLS147, BLS309, BAPB92, TVTHER and BYSYAB, or functional variations thereof which have retained the characteristic of sub-group I-S2.
17. 17. The subtylase enzyme isolated according to claims 3, 15 or 16, characterized in that it is selected from the group comprising S103SA, S103ST, S103SG, S103SS, S103SD, S103SE, S103SK, S103SR, S103SH, S103SV, S103SC, S103SN, S103SQ, S103SF, S103SI, S103SL, S103SM, S103SP, S103SW, and S103SY.
18. 18. The subtilase variant according to any of claims 15 to 17, characterized in that the further modifications are selected from the group comprising K27R, * 36D, S57P, N76D, S87N, G97N, S101G, V104A, V104N, V104Y, H120D , N123S, Y167X, R170X, Q206E, N218S, M222S, M222A, T224S, K235L, and T274A.
19. 19. The subtilase variant according to any of claims 15 to 17, characterized in that the additional modifications are chosen from the group comprising S101G + V104N, S87N + S101G + V104N, " K27R + V104Y + N123S + T274A, N76D + S 103A + V104 I or N76D + V104A, or other combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with one or more of the substitutions, deletions and / or insertions mentioned in any of claims 1 to 14.
20. 20. The subtilase variant according to any of claims 15 to 17, characterized in that the modifications are chosen from the group further comprising P129K, P131H , A133P, A133D and A194P.
21. 21. The variant according to any of the preceding claims, characterized in that it comprises the chosen modification of the group comprising: S103ST + Y167A
22. 22. A subtyla belonging to the sub-group I-SI, characterized in that it has the amino acid sequence: 1 10 20 30 A-Q-T-V-P-Y-G-I-P-L-I-K-A-D-K-V-Q-A-Q-G-F-K-G-A-N-V-K-V-A-V 40 50 60 L-D-T-G-I-Q-A-S-H-P-D-L-N-V-V-G-G-A-S-F-V-A-G-E-A - * - Y-N-T-D 70 80 90 G-N-G-K-G-T-H-V-A-G-T-V-A-A-L-D-N-T-T-G-V-L-G-V-A-P-S-V-S-L 103a 110 120 Y-A-V-K-V-L-N-S-S-G-S-G-T-X-Y-S-G-1-V-S-G-1-E-W-A-T-T-N-G-M-D 130 140 150 V-I-N-M -? - L-G-G-P-S-G-S-T-A-M-K-Q-A-V-D-N-A-Y-A-R-G-V-V-V-V 160 170 180 A-A-G-N-S-G-S-S-G-N-T-N-T-I-G-Y-P-A-K-Y-D-S-V-I-A-V-G-A-V 190 200 210 D-S-N-S-N-R-A-S-F-S-S-V-G-A-E-L-E-V-M-A-P-G-A-G-V-Y-S-T-Y-P 220 230 240 T-S-T-Y-A-T-L-N-G-T-S-M-A-S-P-H-V-A-G-A-A-A-L-I-L-S-K-H-P-N 250 260 270 L-S-A-S-Q-V-R-N-R-L-S-S-T-A-T-Y-L-G-S-S-F-Y-Y-G-K-G-L-I-N-V 275 E-A-A-A-Q or a homologous subtylase having a sequence - of amino acid comprising a position of amino acid residue 103a and showing an identity of more than 70%, 75%, 80%, 85%, 90%, or 95% therewith.
23. 23. A subtilasa that belongs to the sub-group I-S2, characterized because it has the amino acid sequence: .1 10 20 30 .A-Q-S-V-P-W-G-I-S-R-V-Q-A-P-A-A-H-N-R-G-L-T-G-S-G-V-K-V-A-V- 40 50 60 .L-D-T-G -? - * - S-T-H-P-D-L-N-I-R-G-G-A-S-F-V-P-G-E-P - * - S-T-Q-D ~ 70 80 90 . G-N-G-H-G-T-K-V-A-G-T-1 -A-A-L-N-N-S- 1 -G-V-L-G-V-A- P-S-A-E-L-103a 110 120 .Y-A-V-K-V-L-G-A-S-G-S-G-S-X-V-S-S -? - A-Q-G-L-E-W-A-G-N-N-G-M-H-130 140 150 .V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S-R-G-V-L-V-V- 160 170 180 .A-A-S-G-N-S-G-A - * - G-S-I-S - * - * - * - Y-P-A-R-Y-A-N-A-M-A-V-G-A-T-190 200 210 .D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A-P-G-V-N-V-Q-S-T-Y-P-220 230 240 .G-S-T-Y-A-S-L-N-G-T-S-K-A-T-P-H-V-A-G-A-A-A-L-V-K-Q-K-N-P-S-250 260 270 .WSNVQIRNHLKNTATSLGSTN-LYGSGLVNA- 275 .EAATR or a homologous subtylase having an amino acid sequence comprising a position of amino acid residue 103a and showing an identity of more than 70%, 75%, 80%, 85%, 90%, or 95% with it.
24. 24. The subtilase variant according to claim 22 or 23, characterized in that X at position 103a is selected from the group comprising T, A, G, S, and P.
25. 25. An isolated DNA sequence encoding a subtyla or a subtyla variant according to any one of claims 1 to 24.
26. 26. An expression vector, characterized in that it comprises an isolated DNA sequence according to claim 25.
27. 27. A microbial host cell transformed with an expression vector according to claim 26.
28. 28. The microbial host according to claim 27, characterized in that it is a bacterium, preferably a Bacillus, especially B. l in t us.
29. 29. The microbial host according to claim 27, characterized in that it is a fungus or yeast, preferably a filamentous fungus, especially an Aspergillus.
30. 30. A method for the production of a subtilase or subtyla variant, according to any of claims 1 to 24, characterized in that a host of any of claims 27 to 29 is cultured under conditions that lead to expression and secretion. of the variant, and the variant is recovered.
31. 31. A composition, characterized in that it comprises a subtilase or subtilase variant according to any of claims 1 to 24.
32. 32. The composition according to claim 31, characterized in that it additionally comprises a cellulase, lipase, cutinase, oxidoreductose, another protease, or an amylase.
33. 33. The composition according to claim 31 or 32, characterized in that the composition is a detergent composition.
34. 34. The use of a subtilase or subtyla variant according to any one of claims 1 to 24, or an enzyme composition according to claim 31 or 32, which is in a dishwashing detergent and / or laundry .