MXPA01006219A - 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 101 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

SUBTILLAS ENZYMES OF SUB-GROUPS I-SI AND I-S2 WHICH HAVE AN ADDITIONAL AMINO ACID RESIDUE IN A RIZO REGION OF THE ACTIVE SITE TECHNICAL FIELD This invention relates to the novel subtylase enzymes of subgroups I-SI and I-S2 having at least one additional amino acid residue at position 101 of region (b) of the active site loop at position 95 to 103. These proteases are useful showing excellent or improved wash performance when used in detergents; cleaning compositions and detergents. The invention further relates to genes that code 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 and methods for producing the novel enzymes.

BACKGROUND OF THE INVENTION In the detergent industry, enzymes have been implemented in washing formulations for more than 30 years. The enzymes used in such REF: 130242 formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. The most important commercial enzymes are proteases. An increasing number of commercially used proteases are variants of genetically engineered proteins, wild type proteases of natural origin, for example DURAZYM (Novo Nordisk A / S), RELASE® (Novo Nordisk A / S), MAXAPEM ® (Gist-Brocades NV), PURAFECT * (Genencor International, Inc.). In addition a number of protease variants are described in the art, such as in European Patent EP 130756 (GENENTECH) (corresponding to US Reissue Patent No. 34,606 (GENENCOR)); European Patent EP 214435 (HENKEL); and 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) "Mol. Biol. 193 803-813; Russel and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95 / 27049 (SOLVAY SA), WO 95/30011 (PROCTER &GAMBLE COMPANY), WO 95/30010 (PROTECTER &GAMBLE COMPANY), WO 95/29979 (PROTER &GAMBLE COMPANY) '; US 5,543,302 (SOLVAY SA) EP 251 446 (GENENCOR), WO 89/06279 (NOVO NORDISK A / S), WO 91/00345 (NOVO • NORDISK A / S), EP 525 610 A1 (SOLVAY) and WO 94/02618 (GIST-BROCADES N.V.). However, even when a number of useful protease variants have been described, there is still a need for new improved proteases and protease variants for various industrial uses. Therefore, an object of the present invention is to provide improved proteases for protein-engineered protein protease variants, especially for use in the detergent industry.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have found that subtilisins in which at least one of the active site loops are longer than those currently known, show improved washing performance properties in detergent compositions. The identification thereof was carried out in the construction of the subtilisin variants, especially subtilisin 309 (BLSAVI or Savinase), showing improved washing performance properties in detergent compositions in relation to the wild-type proger enzyme. This has been described in our first application DK1332 / 97.

It has now been found that certain subtilases or variants thereof of the I-SI subgroups ("true subtilisins") and I-S2 (highly alkaline subtilisins) having at least one additional amino acid residue at position 101 (or rather between positions 101 and 102) of region (b) of the active site curl from position 95 to 103, show surprisingly improved wash performance compared to those currently known and those described in this application. The improved proteases according to the invention can be obtained by isolation from natural resources or by introducing at least one additional amino acid residue (one insert) into the loop (b) of the active site between positions 101 and 102 in a subtyla of wild type (for a defion of the curls of active type and the numbering of the positions, See later) . Although this finding was made in subtilisin 309, it is predicted that it will be possible to produce or isolate similar advantageous subtilases or subtylase variants. In addition, it will be possible to specifically select the natural isolates to identify the novel wild-type subtilases comprising an active site loop (b) which is longer than the corresponding active site loop in the known wild type subtilases, such as subtilisin 309, which subtilases can be considered as possessing an amino acid residue inserted between positions 101 and 102, and which exhibit excellent washing performance in a detergent as compared to its more closely related known subtilisin, such as subtilisin 309. to alignment and numbering, reference is made to figures 1, la, 2 and 2a below, which show the alignments between the subtilisin BPN '(BASBPN) (a) and subtilisin 309 (BLSAVI) (b), and the alignments between subtilisin BPN '(a) (BASPN) and subtilisin Carlsberg (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 for the numbering of the waste. The seven active site loops (a) through (g) (including both indicated extreme amino acid residues) are defined herein to encompass the amino acid residues in the segments given below (a) the region between amino acid residue 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; Accordingly, in a first aspect the invention relates to an isolated subtylase enzyme (for example more than 10% pure) of subgroups I-SI and I-S2 having at least one additional amino acid residue at position 101 of the region (b) of the curl of the active site 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 101 and 102. 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 by 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. The enzymes of the invention can be generally produced either by culturing a microbial strain from which the enzyme was isolated, and recovering the enzyme in substantially pure form; or by inserting an expression vector according to the fourth aspect of the invention into a suitable microbial host, culturing the host to express the desired subtylase enzyme, and recovering the enzyme product. In addition, the invention relates to a composition comprising a subtilase or subtyla variant of the invention. Additionally the invention relates to the use of the enzymes of the invention for a number of industrially relevant uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes.

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

NOMENCLATURE OF AMINO ACIDS A Ala Alanine V Val Valine L Leu Leucine I He Isoleucine P Pro Proline F Phe Phenylalanine W Trp Tryptophan M Met Methionine G Gly Glycine S Serine T Thr Threonine C Cys Cysteine And 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 describing the various enzymatic variants produced or contemplated according to the invention, the following nomenclatures and conventions have been adapted for ease of reference: A reference structure is first defined by alignment of the isolated wild type enzyme or progenitor with subtilisin BPN '(BASBPN). The alignment can be obtained through the GAP routine of the GCG package, version 9.1, to number the variants using the following parameters: penalty for creation = 8 and penalty for extension of empty space = 8 and all other parameters maintained in their default values. Another method is to use recognized, 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 subtilisin BPN '(BASBPN) and subtilisin 309 (BLSAVI) and subtilisin Carlsberg (BLSCAR), respectively, are indicated in figures 1, 2, and 2a. Through this, a number of deletions 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 the figure subtilisin 309 has the same deletions at positions 36, 56 , 159, 164, 165 and 166 compared to BASBPN. In Fig. 2 the Carlsberg subtilisin has a deletion at position 58 compared to BASBPN, while in Fig. 2a the Carlsberg subtilisin has a deletion at position 56 compared to BASBPN. These deletions are in figures 1, 2, and 2a indicated by asterisks (*).

The various modifications made to a wild-type enzyme are generally indicated using three elements as follows: Amino acid substituted at the position of the original 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 manual notation can sometimes be used indicating only the position and the substituted amino acid.

Amino acid substituted in position Such notation is particularly relevant in connection with the modification (s) in the homologous subtilases (see below). Similarly when the identity of the resulting amino acid residues is not important.

Original amino acid position When the original amino acid (s) and the substituted amino acid (s) can comprise any amino acid, then only the position is indicated, for example: 170. When the original amino acid (s) and / or the substituted amino acid (s) may comprise more than one, but Not all amino acids, then the selected amino acids are indicated inside braces. { } .

Original amino acid position (substituted amino acid, ..., substituted amino acid For the specific variants, the specific codes of three letters or one letter are used, including Xaa and X codes to indicate any amino acid residue.

SUBSTITUTIONS: The replacement of glutamic acid by glycine at position 195 is indicated as: Glyl95Glu or G195E or the substitution of any amino acid residue by glycine at position 195 is designated as: Glyl95Xaa G195X Glyl95 or G195 Substitution of serine by any amino acid residue at position 170 could be designated in this way: Xaal70Ser or X170S 170Ser 170S Such notation is particularly relevant in connection with the modification (s) in the homologous subtilas (see below). 170Ser is understood in this way as comprising, for example, a modification Lysl70Ser in BASBPN and the modification Argl70Ser in BLSAVI (see figure 1). For a modification where the original amino acids and / or the substituted amino acid (s) may comprise more than one, but not all of the amino acids, the substitution of glycine, alanine, serine or threonine by arginine at position 170 could be indicated by Argl70. { Gly, Ala, Ser, Thr} or R170. { G, A, S, T.}. to indicate the variants R170G, R170A, R170S, and R170T.

SUPPRESSIONS: A deletion of glycine at position 195 will be indicated by: Glyl95 * or G195 * Correspondingly, the deletion of more than one amino acid residue, such as deletion 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 Lys, Ala and Ser after G195 this is: Glyl95GlyLysAlaSer or G195GKAS In such cases the inserted amino acid residue (s) is numbered by the insertion of lowercase letters to the number of position of the amino acid residue that precedes the inserted amino acid residue (s). In the previous example, sequences 194 to 196 could 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 a waste of amino acid identical to the existing amino acid residue is inserted, it is clear that a degeneration arises in the nomenclature. If for example a glycine is inserted after the glycine in the previous example, this could be indicated by G195GG. The same effective change could also be indicated just as A194AG by the change from: 194 195 196 BLSAVI A - G - L 194 195 195a 196 Variant A - G - G - L 194 194a 195 196 Such cases will be apparent to the person skilled in the art, and the indication G195GG and the corresponding indications for this type of insertions are understood in this way to include such indications equivalent degenerates.

FILLING AN EMPTY SPACE: When there is a deletion in an enzyme in the reference comparison with the subtilisin sequence BPN 'used for numbering, an insert in such position is indicated as: * 36Asp O * 36D for the insertion of an aspartic acid in position 36.

MULTIPLE MODIFICATIONS Variants comprising multiple modifications are separated by plus signs, for example: Argl70Tyr + Glyl95Glu or R170Y + G195E representing modifications at positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively, or for example Tyrl67. { Gly, Ala, Ser, Thr} + Argl70. { Gly, Ala, Ser, Thr} designates Tyrl67Gly + Argl70Gly variants, Tyrl67Gly + Argl70Ala, Tyrl67Gly + Argl70Ser, Tyrl67Gly + Argl70Thr, Tyrl67Ala + Argl70Gly, Tyrl67Ala + Argl70Ala, Tyrl67Ala + Argl70Ser, Tyrl67Ala + Argl70Thr, Tyrl67Ser + Argl70Gly, Tyrl67Ser + Argl70Ala, Tyrl67Ser + Argl70Ser, Tyrl67Ser + Argl70Thr , Tyrl67Thr + Argl70Gly, Tyrl67Thr + Argl70Ala, Tyrl67Thr + Argl70Ser, Tyr167Thr + Argl7OThr, This nomenclature is particularly relevant in relation to modifications directed to the rcement, rcement, insertion or deletion of amino acid residues that have specific common properties, such as positive charge residues (K, R, H), negatively charged (D, E), or one or more conservative amino acid modifications for example from Tyrl67. { Gly, Ala, Ser, Thr) + Argl70. { Gly, Ala, Ser, Thr} , which means the substitution of a small amino acid for another small amino acid. See section "Detailed description of the invention" for additional details.

Proteases Enzymes that break amide bonds in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzyme tic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3).

Numbering of amino acid positions / residues If nothing is mentioned, the amino acid numbering used herein corresponds to that of the BPN subtylase sequence (BASBPN). For an additional description of the BPN 'sequence, 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 serine residue in the active site (White, Handler and Smith, 1973"Principies 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. These are inhibited by diisopropyl fluorophosphate. They hydrolyse the 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 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 subgroup of serine proteases tentatively designated as subtilases has been proposed by Siezen et al., Protein Engng. (1991) 719-737 and Siezen et al., Protein Science 6 (1997) 501-523. These 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 previously defined as a serine protease produced by Gram-positive bacteria or fungi, and in accordance a Siezen et al. is now a group of subtylases A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined.For a more detailed description of such subtilases and their amino acid sequences is refers to Siezen et al (1997) .A subgroup of the subtilases, I-SI 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 subgroup of subtilases, I-S2 or highly alkaline subtilisins, is recognized by Siezen et al. (supra) The proteases of subgroup I-S2 are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (BAALKP) (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A / S), subtilisin 147 (BLS147) (WAIT *, NOVO NORDISK A / S), and alkaline elastase YaB (BSEYAB).

List of acronyms for subtilasas: I-SI Subtilisin 168, BSS168 (Subtilisin amylosacchariticus), BSAPRJ (Subtilisin J), BSAPRN (Subtilisin NAT), BMSAMP (Mesentericopeptidase), 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,? Ubtilisin 147, Esperase® BLS147 (BSAPRM (SubtilisinAprM), (BAH101), Subtilisin 309, Savinase®, BLS3s9 / BLSAVI (BSKSMK (M-protease), BAALKP (Subtilisin PB92, Alkalophilic alkaline protease of Bacillus), BLSUBL (Subtilisin BL), Alkaline elastase YaB, BYSYAB, "SAVINASE" "SAVINASE®" is marketed by 'NOVO NORDISK A / S. This is subtilisin 309 of B. Lentus and differs from BAALKP only in one position (N87S, see figure 1 herein). "SAVINASE®" has the amino acid sequence designated b) in Figure 1.

Progenitor Subtilasa The term "Subtilasa progenitora" describes a subtilase defined according to Siezen et al. (1991 and 1997). For additional details see the description of "Subtilasas" immediately above. A progenitor subtilasa can also be an isolated subtyla from a natural source, where subsequent modification has been made while retaining the characteristic of a subtilase. Alternatively, the term "progenitor subtylase" can be termed "wild-type subtylase".

Modification (s) of a subtyla variant The term "modification (s)" 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 or modifications may be one or more replacements of the amino acid side chain (s), substitution (s), deletion (s) and / or insertions within or in the amino acid or amino acids of interest.

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

Subtylase Homologous Sequences The curl regions of the specific active site, and the amino acid insertions in the curls of the subtilase SAVINASE are identified for modification herein, to obtain a subtyla variant of the invention. However, the invention is not limited to the modifications of this particular subtilase, but it extends to other progenitor subtilases (wild type), which have a primary structure homologous 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 version 9.1 of the GCG package can be applied (infra) using the same settings. The result of the routine is in addition to the alignment of the amino acids the calculation of the "Percentage Identity" between the two sequences. Based on this description, it is routine for a person skilled in the art to identify the appropriate homologous subtilases and the corresponding homologous active site loop regions, which can be modified according to the invention.

Washing performance The ability of an enzyme to catalyze the degradation of various substrates of natural origin present on objects that are to be cleaned, for example during washing or cleaning hard surfaces, is frequently referred to as its washing ability, washability, Detergency, or washing operation. Throughout this specification, the term wash operation will be used to encompass this property.

SEQUENCE OF ISOLATED DNA The term "isolated or isolated", when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic environment and is thus free of other foreign or undesired coding sequences, and is in a form suitable for use within protein production systems, engineered by genetic engineering. Such isolated molecules are those that are separated from their natural environment and include cDNAs and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include the 5 'and 3' non-translated regions of natural origin such as promoters and terminators. The identification of the associated regions will be apparent to a person of 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 called "a cloned DNA sequence".

Isolated protein When applied to a protein, the term "isolated or isolated" indicates that the protein has been removed from its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (eg, "homologous impurities" (see below)). An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. In addition, it is preferred to provide the protein in a highly purified form, for example, 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 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 (for example another polypeptide than the polypeptide of the invention) that originates from the homologous cell from which the polypeptide of the invention was originally obtained.

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

Substrate The term "substrate" used in connection with a substrate for a protease should be interpreted in its broadest form as that 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 an enzymatic protease reaction should be interpreted in the context of this invention to include 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 using the aforementioned GAP routine. The figure shows the alignment between subtilisin BPN 'and Savinase as taken from WO 91/00345. Figure 2 shows an alignment between subtilisin BPN 'and subtilisin Carlsberg using the aforementioned GAP routine. Figure 2a shows an alignment between the subtilisin BPN 'and the subtilisin Carlsberg as taken from WO 91/00345. Figure 3 shows the three-dimensional structure of Savinase (Protein Data Bank (PDB) entry 1SVN). In the figure, the curl of the active site b) is indicated.

DETAILED DESCRIPTION OF THE INVENTION The subtilases of the invention in a first aspect refer to an isolated subtylase enzyme (for example more than 10% pure) of subgroups I-SI and I-S2 having at least one additional amino acid residue at position 101 of the region (b) of the curl of the active site 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 101 and 102. In other words, the. subtylases of the invention are characterized by comprising a region (b) of active site curl of more than 9 amino acid residues and wherein the additional amino acid residue is or can be considered to be inserted between positions 101 and 102 in relation to to the parent, or to a known wild type subtyla. A subtylase of the first aspect of the invention may be a progenitor or wild type subtylase identified and isolated from nature. Such subtylase of wild type progenitor can be specifically selected by standard techniques known in the art. A preferred way to do this can be by PCR amplification specifically, of DNA regions that are known to encode the active site loops in the subtilases from a number of different microorganisms, preferably different Bacillus strains. Subtilases are a group of conserved enzymes, in the sense of their DNA and amino acid sequences that are homologous. Consequently, it is possible to construct relatively specific primers flanking the active site loops. One way to do this is by investigating an alignment of different subtilases (see for example Siezen et al., Protein Science 6 (1997) 501-523). It is from this routine work for a person skilled in the art that PCR primers flanking the active site loop corresponding to the active site loop (b) between amino acid residue 95 to 103 can be constructed in any of the groups I-SI or I-S2 such as from BLSAVI. Using such PCR primers to amplify the DNA from a number of different microorganisms, preferably different strains of Bacillus, followed by sequencing the DNA of the amplified PCR fragments, it will be possible to identify the strains that produce the subtilases of these groups, which they comprise a longer active site region, compared for example to BLSAVI, corresponding to the active site curl region of positions 95 to 103, and where an insertion can be considered to exist between positions 101 and 102. Having identified the strain and a partial DNA sequence of such subtylae of interest, it is routine work for a person skilled in the art to complete the cloning, expression and purification of such subtyla of the invention. However, it is considered that a subtylase enzyme of the invention is predominantly a variant of a progenitor subtylase. Accordingly, in one embodiment the invention relates to a subtylase enzyme isolated according to the first aspect of the invention, wherein the subtylase enzyme is a constructed variant that has a longer active site loop (b), than its parent enzyme. , having at least one amino acid insert between the amino acid residues 101 and 102. The subtilases of the invention show excellent washing performance in a detergent, and if the enzyme is a constructed variant an improved washing operation in a detergent is obtained. in comparison to its closest related subtilase, such as subtilisin 309. Different subtilase products will show a different washing performance in different types of detergent compositions. A subtyla of the invention has improved wash performance, as compared to its closest relative in a majority of such different types of detergent compositions.

Preferably, a subtylase enzyme of the invention has improved wash performance, as compared to its closest relative in the detergent composition, shown in Example 3 herein (see below). In order to determine whether a given subtylase amino acid sequence (irrelevant if said subtylase sequence is a subtylase wild-type progenitor sequence or a subtylase variant sequence produced by any other method other than site-directed mutagenesis), it 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 '(see section "Definitions" herein (see above); ii) based on the alignment performed in step i) identifying the active site curl (b), in the subtylase sequence corresponding to the region (b) of the active site curl of the subtilisin BPN 'comprising the region (both extreme amino acids included) between the amino acid residue of 95 to 103; iii) determining whether the curl (b) of the active site in the subtilase sequence, identified in step ii) is longer than the curl of the corresponding active site in BLSAVI and if said prolongation corresponds to the insertion of at least one residue of amino acid between positions 101 and 102. If this is the case, the subtilase investigated is a subtyla within the scope of the present invention. The alignment performed in step i) above is carried out as described above by the use of the GAP routine. Based on this description, it is routine for a person skilled in the art to identify the curl (b) of the active site in a subtyla 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 random / site-directed mutagenesis or by DNA shuffling in different subtylase sequences. See section "PRODUCTION OF A SUBSTITUTE VARIANT" and Material and methods herein (see below) for additional details. In the further embodiments, the invention relates to: An isolated subtylase enzyme according to the invention, wherein at least one inserted amino acid residue is selected from the group comprising: T, G, A, and S; An isolated subtylase enzyme according to the invention, wherein at least one inserted amino acid residue is selected from the group of charged amino acid residues comprising: D, E, H, K, and R, more preferably D, E, K, and R; An isolated subtylase enzyme according to the invention, wherein at least one inserted amino acid residue is selected 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, most 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 the positions 101 and 102 comprises at least two amino acids, in comparison to the site curl, corresponding active in BLSAVI. In further embodiments, the invention relates to an isolated subtylase enzyme comprising at least one insert, chosen from the group comprising (in the BASBPN numbering): X101XT. { T, G, A, S} X101X { D, E, K, R} X101X { H, V, C, N, Q } X101X { F, I, L, M, P, W, Y} or more specific for subtilisin 309 and the closely related subtilases, such as BAALKP, BLSUBL, and BSKSMK S101A S101ST S101SG S101SS S101SD S101SE S101SK S101SR S101SH S101SV S101SC S101SN S101SQ S101SF S101SI S101SL S101SM S101SP S101SW S101SY In addition, the invention relates to subtilases comprising the following multiple inserts in the position 101 S101SGAA or any of the following combinations A98G + S101ST, A98G + S101SG + S103T, A98G + S99A + S101ST It is known in the art that a so-called substitution Conservation 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 the groups of conservative amino acid substitutions.

TABLE III Conservative amino acid substitutions Common property Amino acid Basic (positive charge) K = Lysine H = histidine Acids (negative charge) E = Glutamic acid D = Aspartic acid Polar Q = Glutamine N Hydrophobic Asparagine L Leucine I Isoleucine V Valine M Methionine Aromatics F Phenylalanine W Tryptophan and Tyrosine Small G Glycine A Alanine S = Serine T = Threonine According to this principle, subtylase variants that comprise conservative substitutions, such as G97A + A98AS + S99G, G97S + 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 routine work for a person skilled in the art to identify one or several conservative modifications suitable for these variants, in order to obtain other subtyla variants that show functioning similarly improved wash. According to the invention, the subtilases of the invention belong to the subgroups I-SI and 1-52, especially to the subgroup 1-S2, to isolate the novel enzymes of the invention from nature or from the artificial creation of diversity ", and to design and produce variants from a parent subtylase In relation to the variants of subgroup I-SI, it is preferred to choose a parent subtyla from the group comprising BSS168 (BSSAS, BSAPRJ, BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA, BLSCA1, BLSCA2, BLSCA3), BSSPRC, and BSSPRD, or the functional variants thereof that have retained the characteristic of the subgroup I-SI In relation to the subgroup I-S2 variants, it is preferred to choose a progestin subtyla group comprising BSAPRRQ, BLS147 (BSAPRM, BAH101), BLSAVI (BSKSMK, BAALKP, BLSUBL), BISYAB, and BSAPRS, or the functional variants thereof which have the characteristic of subgroup I-S2. In particular, the progenitor subtilase is BLSAVI (SAVINASE® NOVO NORDISK A / S), and a preferred subtylase variant of the invention is consequently 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, they are considered combinations with other modifications known in the art, to provide improved properties to the enzyme. The technique describes a number of subtilase variants with different improved properties and a number of those are mentioned in the "Background of the invention" section herein (watch up) . These references are described herein as references for identifying a subtyla variant, which can be advantageously combined with a subtyla variant of the invention. Such combinations comprise the positions: 222 (improve oxidation stability), 218 (improve thermal stability), substitutions at Ca binding sites that stabilize the enzyme, for example, position 76, and many others apparent to from the prior art. In additional embodiments, a subtilase 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 are considered suitable for combination: BLSAVI, BLSUBL, BSKSMK, and BAALKP: K27R, * 36D, S57P, N76D, S87N, G97N, S101G, S103A, V104A, V104I, V104N, V104Y, H120D, H123S, Y167, R170, Q206E, N218S, M222S, M222A, T224S, K235L and T274A. Additional variants comprising any of the variants S101G + V104N, S87N + S101G + V104N, K27R + V104Y + 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 one or more of the modifications mentioned above, show improved properties. Still further subtylase variants of the main aspects of the invention are preferably combined with one or more modifications in any of positions 129, 131, 133 and 194, preferably as modifications 129K, 131H, 133P, 133D and 194P, and more preferably as modifications P129K, P131H, A133P, A133D and A194P. Any of these modifications are expected to provide a higher level of expression of a subtyla variant of the invention in the production thereof. Accordingly, a further embodiment of the invention relates to a variant according to the invention, wherein the modification is chosen from the group comprising: THE PRODUCTION OF A SUBTYASE VARIANT Many methods for the cloning of a subtyla of the invention and for the insertion of inserts into genes (for example subtyla genes) are well known in the art, see references cited in the section of "BACKGROUND OF THE INVENTION".

In general, standard procedures for the cloning of genes and the introduction of inserts (random and / or site-directed) into said genes can be used in order to obtain a subtyla variant of the invention. For further description of suitable techniques, reference is made to the Examples herein (see below) and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (Eds.) "Current protocols in Molecular Biology" John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus." John Wiley and Sons, 1990); and WO 96/34946. In addition, a subtyla variant of the invention can be constructed by standard techniques for the artificial creation of diversity, such as by DNA shuffling of different subtylase genes (WO 95/22625; Stemmer WPC, Nature 370: 389-91 ( 1994)). The DNA blotting, for example of the "gene encoding Savinase®, with one or more partial subtylase sequences identified in nature, comprising an active site (b), the curl regions longer than the active site (b) ) of the Savinase® curl, after the subsequent selection for variants with improved wash performance, will provide subtylase variants according to the invention.

VECTORS OF EXPRESSION A recombinant expression vector comprising a DNA construct encoding the enzyme of the invention can be any vector that can be conveniently subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it will be introduced. In this way, the vector can be a vector of autonomous replication, for example a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector may be one that after introduction into a host cell is integrated into the genome of the host cell 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 the additional segments required for DNA transcription. In general, the expression vector is derived from plasmid or viral DNA, or it may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in unison for their intended purposes, for example, transcription starts in a promoter and proceeds through the DNA sequence encoding the enzyme. The promoter can be any DNA sequence that shows the transcriptional activity in the host cell of choice and can be derived from the genes that code for the proteins either 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 of Bacillus stearo thermophilus, the alpha-amylase gene of Bacillus licheniformis, the alpha-amylase gene of Bacillus amyloliquefaciens, the gene of the Bacillus subtilis alkaline protease, or the Bacillus pumilus xylosidase gene, or the "PR or PL promoters of the Lambda phage or the lac, trp or tac promoters of E. coli." The DNA sequence encoding the enzyme of the invention can also, if necessary, be operably connected to a suitable terminator.

The recombinant vector of the invention can further comprise a DNA sequence that makes it possible for the vector to replicate in the host cell in question. The vector may also comprise a selectable marker, for example, a gene, the product of which complements a defect in the host cell, or a gene encoding for resistance, for example for 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 towards the secretory pathway of 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 is linked to the DNA sequence encoding the enzyme in the correct reading structure. Secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or it may be from a gene encoding another secreted protein. The methods used to ligate the DNA sequences encoding the present enzyme, the promoter and optionally the terminator and / or 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 those of ordinary skill in the art (see, for example, Sambrook et al., cited reference).

HOST CELL The DNA sequence encoding the present enzyme introduced into the host cell can be either homologous or heterologous to the host in question. If it is homologous to the host cell, for example, produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, to another secretory signal sequence and / or terminator sequence, which in its natural environment. The term "homologue" is intended to include a DNA sequence that encodes a native enzyme for the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. In this way, the DNA sequence can be from another organism, or it can be a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector of the invention is introduced can be any cell that is capable of producing the present enzyme and includes bacteria, yeasts, fungi and higher eukaryotic cells including plants. Examples of bacterial host cells which, with the culture, are capable of producing the enzyme of the invention are gram-positive bacteria such as Bacillus strains, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. Lividans or S. Murinus, or gram-negative bacteria such as Escherichia coli. The transformation of the bacterium can be effected by protoplast transformation, electroporation, conjugation, or by the use of competent cells in a manner known per se (see Sambrook et al., Supra). When the enzyme is expressed in bacteria such as E. coli, the enzyme can be retained in the cytoplasm, typically as insoluble grains (known as the inclusion body), or it can be directed into the periplasmic space by a sequence of bacterial secretion. In the first 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 can be recovered from the periplasmic space by disintegrating the cells, for example by sonication or osmotic shock, to release the contents of the periplasmic space and recover the enzyme. When the enzyme is expressed in gram-positive bacteria such as strains of Bacillus or Streptoinyces, the enzyme can be retained in the cytoplasm, or it can be directed into the extracellular medium by a sequence of bacterial secretion. In the latter case, the enzyme can be recovered from the medium as described below.

METHOD TO PRODUCE SUBTILASA The present invention provides a method for producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions that allow the production of the enzyme , and the resulting enzyme is recovered from the culture.

When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell, it is possible to carry out the heterologous recombinant production of the enzyme of the invention. With this it is possible to produce a highly purified subtyla composition, characterized in that it is free of homologous impurities. In this context, homologous impurities means any impurities (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 for the culture of the transformed host cells can be any conventional means suitable for the development of the host cells in question. The expressed subtylase can be conveniently secreted into the culture medium and can be recovered from it by well-known methods which include the separation of the cells from the medium by centrifugation or filtration, precipitation of the protein components of the medium, by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

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

DETERGENT COMPOSITIONS THAT COMPRISE MUTATING ENZYMES The present invention comprises the use of the mutant enzymes of the invention in cleaning compositions and detergents and compositions such as the mutant subtilisin enzymes. Such cleaning compositions and detergents are also described in the art and reference is made to WO 96/24946; WO 97/07202; WO 95/30011 for further description of suitable cleaning compositions and detergents. In addition, the following example (s) demonstrate improvements in the operation of the wash for a number of subtilase variants of the invention.

Compositions Detergents The enzyme of the invention can be added to and thus become a component of a detergent composition. The detergent composition of the invention can for example be formulated as a detergent composition for hand or machine washing, including an additive laundry composition, suitable for pretreatment of the dyed fabrics and a fabric softening composition, added to the rinse, or be formulated as a detergent composition for use in general domestic hard surface cleaning operations, or be formulated for manual or machine 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, an arabinase, a galactanase, a xylanase, an oxidase, for example, a laccase and / or a peroxidase. In general, the properties of the enzyme (s) chosen must be compatible with the selected detergent (eg, the optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme (s) must be present in effective amounts .

Proteases: Suitable proteases include those of animal, plant or microbial origin. The microbial origin is preferred. Mutants engineered by proteins or chemically modified are also included. The protease can also be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are 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 useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially 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 Alcalase ™, Savinase ™, Primase ™, Duralase ™, Esperase ™, and KannaseMR ~ (Novo Nordisk A / S), Maxatase ™, Maxacal ™, Maxapem ™, Properase ™, Purafect ™, Purafect OxPMR, FN2 ™, and FN3 ™ (Genencor International Inc.) Lipases: Suitable lipases include those of bacterial or fungal origin. Mutants engineered by proteins or chemically modified are also included. Examples of such lipases include lipases from Humicola (synonym Thermomyces), for example from H. lanuginosa (T. lanuginosus) as described in European Patent EP 258 068 and EP 305 216 or from H. Insolens as described in WO 96 / 13580, a lipase from Pseudomonas, for example from P. Alcaligenes or P. pseudoalcaligenes (European Patent EP 218 272), P. cepacia (European Patent EP 331 376), P. stutzeri (GB 1,372,034), P. Fluorescens, Pseudomonas sp. strain 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 variants of lipases, such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94 / 25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202. Preferred commercially available lipase enzymes include Lipolase "1 * u Lipolase Ultra ™ (Novo Nordisk A / S).

Amylases: Suitable amylases (a and / or ß) include those of bacterial or fungal origin. Mutants engineered by proteins or chemically modified are also included. Amylases include, for example, α-amylases obtained from Bacillus, for example, a special strain of B. lichenifor is, described in more detail in British patent 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 BANTM (Novo Nordisk A / S), Rapidase "11 and Purastar ™ (from Genencor International Inc.) Cellulases: Suitable cellulases include those of bacterial or fungal origin. Mutants engineered by protein engineering or chemically modified are also included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example the fungal cellulases produced by Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum described in US 4,435,307, US 5,648,263, US 5,691,178, US Pat. 5, 776, 151 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 the documents 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 HAMR (Genencor International Inc.) and KAC-500 (B) MR (Kao Corporation).

Peroxidases / Oxidases: Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Also included are mutants engineered by proteins, or chemically modified. Examples of useful peroxidases include peroxidases from Coprinus, for example from C. cinereus, and variants thereof such as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme "11 (Novo Nordisk A / S).

The detergent enzyme (s) can be included in a detergent composition by the addition of separate additives containing one or more enzymes, or by the addition of a combined additive comprising all of these enzymes. A detergent additive of the invention, for example, a separate additive or a combined additive, can be formulated, for example, as a granulate, a liquid, a suspension, etc. Preferred additive formulations for detergents are granules, in particular granules which do not release dust, liquids, in particular stabilized liquids, or suspensions. Non-dusting granulates can be produced, for example, as described in US Pat. Nos. 4,106,991 and 4,661,452 and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethylene glycol, PEG) with average molar weights of 1,000 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 ethylene oxide units; fatty alcohols; fatty acids; and mono-, and di-, and tri-glycerides of fatty acids. Examples of film-forming coating materials, suitable for application by fluidized bed techniques, are given in British patent GB 1483591. Liquid enzyme preparations can, for example, be stabilized by the addition of a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes can be prepared according to the method described in European Patent EP 238,216. The detergent composition of the invention may 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 amphoteric. Surfactants are typically present at a level of 0.1% to 60% by weight. When included herein, the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkan sulfonate, ester methyl of alpha-sulfograso acid, alkyl- or alkenyl-succinic acid or soap. When it is included in. present, the detergent will usually contain from about 0.2% to about 40% of a nonionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkyl polyglycoside, alkyldimethylaminoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxyalkyl acid amide, or N-acyl or N-alkyl glucosamine derivatives ("glucamides"). The detergent may contain 0-65% of a detergent additive or complexing agent, such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminpentaacetic acid, alkyl- or alkenylsuccinic acid, 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), poly (vinyl alcohol), poly (vinylpyridine-N-oxide), poly (vinylimidazole), polycarboxylates such as polyacrylates, maleic / acrylic acid copolymers, and copolymers of lauryl methacrylate / acrylic acid. 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 tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleach system may comprise peroxyacids for example of the amide, imide, or sulfone type. The enzyme (s) 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 a boric acid derivative, for example, an aromatic borate ester, or a phenylboronic acid derivative, such as 4-formylphenylboronic acid, and the composition can be formulated as described for example in "WO 92/19709 and WO 92/19708. detergent may also contain other conventional detergent ingredients such as for example fabric conditioners including clays, foam triggers, soapy water suppressors, anti-corrosion agents, dirt suspending agents, anti-grime redeposition agents, colorants, bactericides, optical brighteners , hydrotropes, impingement inhibitors, or perfumes.It is contemplated to date that in compositions Any enzyme, in particular the enzyme of the invention, can 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 enzyme protein 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 be further incorporated into the detergent formulations described in WO 97/07202 which is incorporated by reference herein.

APPLICATIONS IN THE LEATHER INDUSTRY A subtyla of the invention can be used in the leather industry, in particular for use in hair removal. In such an application, a subtylase variant of the invention is preferably used in an enzymatic composition further comprising another protease.

For a more detailed description of other suitable proteases, see the section related to enzymes suitable for use in a detergent composition (see above).

APPLICATIONS IN THE USE OF WOOL A subtyla of the invention can be used in the wool industry, in particular for use in cleaning garments containing wool. In such an application, a subtylase variant of the invention is preferably used in an enzymatic composition further comprising another protease. For a more detailed description of other suitable proteases, see the section related to enzymes suitable for use in the detergent composition (see above). The invention is described in greater detail in the following examples, which are not in any way limiting the scope of the invention, as claimed.

MATERIALS AND METHODS CEPAS B. sutilis DN1885 (Diderichsen et al., 1990). B. lentus 309 and 147 are specific strains of Bacillus lentus, deposited with the NCIB and assigned with accession numbers NCIB 10309 and 10147, and described in U.S. Patent No. 3,723,250 incorporated by reference herein. E. coli MC 1000 (C. Casadaban and S.N. Cohen) (1980); J. Mol. Biol. 138 179-207), was made r ", m + by conventional methods, and is also described in U.S. Patent Application Serial No. 039,298.

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

GENERAL METHODS OF MOLECULAR BIOLOGY: Unless otherwise mentioned, 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, FM et al. (Eds.) "Current protocols in Molecular Biology." John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus." John Wiley and Sons, 1990). Enzymes for DNA manipulations were used according to the specifications of the suppliers.

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

PROTEOLITIC ACTIVITY In the context of this invention, the proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU). The activity is determined relatively to an enzyme standard (SAVINASE®), and the determination is based on the digestion of a dimethylcasein (DMC) in solution by the proteolytic enzyme under standard conditions, for example 50 ° C, pH 8.3, 9 minutes of reaction time, 3 minutes of measurement time. An AF 220/1 folder is available at the request of Novo Nordisk A / S, Denmark, a folder that is incorporated by reference herein. A GU is a Glycine Unit, defined as the activity of proteolytic enzyme which, under standard conditions, during a 15 minute incubation at 40 ° C, with N-acetylcasein as a substrate, produces an amount of NH2 group equivalent to 1 mmol of glycine. Enzymatic activity can also be measured using the PNA assay, according to the reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in the Journal of the American Oil Chemists Society, Rothgeb, 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 buffered 500 ml Erlenmeyer flasks, containing 100 ml of BPX medium for 5 days. Consequently, in order to make, for example, a 2-liter broth, 20 Erlenmeyer flasks were fermented simultaneously.

Media Composition of BPX Medium (per liter) Potato starch 100 g Milled 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 an 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 NaHCO3.

EXAMPLE 1 CONSTRUCTION AND EXPRESSION OF ENZYMATIC VARIANTS: MUTAGENESIS DIRECTED TO THE SITE: The variants directed to the subtylase site 309 of the invention, which comprise specific insertions in the active site loop (b) between positions 101 and 102 were made by traditional cloning of the DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 *. Ed., Cold Spring Harbor, 1989) produced by PCR of the oligos containing the desired inserts (see below). The plasmid DNA template was pJS3, or an analogue thereof containing a variant of subtylase 309. The insertions were introduced by oligo-directed mutagenesis to the construction of the insertion variants S101SK (X = any amino acid residue inserted between the positions 101 and 102) that result in the S101SX variants of subtylase 309. Subtylase variants 309 were transformed into E. coli. The purified DNA of an overnight culture of these transformants was transformed into B. subtilis by restriction endonuclease digestion, purification of DNA fragments, ligation, transformation of B. subtilis. The transformation of B. subtilis was performed as described by Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.

RANDOM MUTAGENESIS LOCATED IN ORDER TO INSERT RANDOM INSERTS IN THE LOCALIZED REGION: The complete strategy used to perform the localized random mutagenesis was: A mutagenic primer (oligonucleotide) was synthesized, which corresponds to the DNA sequence flanking 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 a suitable opposite primer. The resulting PCR fragment was purified and spread in a second PCR reaction, before being digested with the endonucleases and cloned into the shuttle vector of E. coli-B. subtilis (see below). Alternatively, and if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a second opposing primer suitable to allow digestion and cloning of the mutagenized region within the shuttle vector. PCR reactions are performed under normal conditions. Following this strategy a random library located in SAVINASE was built, where insertions were introduced in the region of active site curl between positions 101 and 102. Mutations were introduced by mutagenic primers (see below), so that the 20 amino acids are represented (N = 25% of A, T, C, and G, while S = 50% of C and G. The PCR fragment produced was extended towards the N-terminus of Savinase by another round of PCR, by combining a sequence of overlap with a PCR fragment produced by the PCR amplification with the 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 randomly chosen E. coli colonies were sequenced, to confirm the designated mutations. The mutagenic primer (5 'GTC CTA GGG GCG AGC GGT TCA NNS GGT TCG GTC AGC TCG ATT GCC 3' (sense) was used in a PCR reaction with a suitable anti-sense opposite primer, located downstream or 3 'from the site Mlu I in pJS3. {For example, 5 '- CCC TTT AAC CGC ACA GCG TTT-3' (anti-sense)) and plasmid pJS3 as template. This resulting PCR product was cloned into the shuttle vector pJS3 by the use of the restriction enzymes Hind III and Mlu I. The random library was transformed into E. coli by well-known techniques. The prepared library contained approximately 100,000 individual clones / library. Ten randomly chosen colonies were sequenced to confirm the designated mutations. In order to purify a subtyla variant of the invention, the expression plasmid pJS3 of B. subtilis comprising a variant of the invention was transformed into a competent strain of B. subtilis and fermented as described above in a medium containing 10 μg / ml chloramphenicol (CAM).

EXAMPLE 2 PURIFICATION OF ENZYMATIC VARIANTS: This process relates to the purification of a 2-liter fermentation for the production of the subtilases of the invention in a Bacillus host cell.

Approximately 1.6 liters of the fermentation broth were centrifuged at 5000 rpm for 35 minutes in 1 liter containers. The supernatants were adjusted to pH 6.5 using 10% acetic acid and filtered on Seitz Supra S100 filter plates. The filtrates were concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate was centrifuged and filtered before absorption at room temperature on a Bacitracin affinity column at pH 7. The protease was eluted from the Bacitracin column at room temperature using 25% 2-propanol and sodium chloride 1 M in a buffer solution with 0.01 M dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7. The protease activity fractions from the Bacitracin purification step were combined and applied to a Sephadex G25 column of 750 ml (5 cm in diameter) balanced with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chloride adjusted to pH 6.5. The fractions with proteolytic activity of the Sephadex G25 column were combined and applied to a cation exchange column of 150 ml CM Sepharose CL 6B (5 cm diameter), equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid , and 0.002 M calcium chloride adjusted to pH 6.5. The protease was eluted using a linear gradient of 0 - 0.1 M sodium chloride in 2 liters of the same buffer (sodium chloride 0 - 0.2 M in the case of Subtilisin 147). In a . final purification step, the fractions containing a protease 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 fermentation, and the above isolation procedure, the following subtilisin 309 variants were produced and isolated: S101ST S101SS S101SA S101SD S101SE S101SP S101SG S101SH S101SI S101SGAA S101ST + Y167A A98G + S101ST A98G + S101SG + S103T A98G + S99A + S101ST These variants showed better washing performance than Savinase in a preliminary trial.

EXAMPLE 3 OPERATION OF WASHING OF DETERGENT COMPOSITIONS COMPRISING ENZYMATIC VARIANTS The following examples provide the results of a number of wash tests that were conducted under the indicated conditions.

MINI WASHING WASHING CONDITIONS: DETERGENTS: The detergents used were either a detergent model, called Detergent 95 or obtained from supermarkets in Denmark (OMO, data sheet ED-9745105) and the United States (Wisk, data sheet ED-9711893), respectively. Before use, all enzymatic activity in the detergents was inactivated by the microwave treatment. The detergent 95 is a simple model formulation. The pH is adjusted to 10.5 which is within the normal range for a powder detergent. The composition of the model detergent 95 is as follows: % STP (Na5P3O10) 25% Na2S04 10% Na2C03 20% LAS (Nansa 80S) 5. 0% non-ionic tenside (Dobanol 25 - 7) 5. 0% Na2Si205 0.5% Carboxymethylcellulose (CMC) 9.5% Water SAMPLES: The samples used were EMPA116 and EMPA117, obtained from EMPA Testmaterialen, Movenstrasse 12, CH-9015 St. Gall, Switzerland.

REFLEGTANCE The measurement of the reflectance "(R) on the test material was made at 460 nm using a Macbeth ColorEye 7000 photometer. The measurements are made according to the manufacturer's protocol.

EVALUATION The evaluation of the washing performance of a subtyla is determined either by the improvement factor or the operating factor for the subtilase investigated. The improvement factor, IFDosls / Response / is identified as the ratio between the slopes of the washing performance curves for a detergent containing the subtilase investigated and the same detergent containing a subtyla of reference to the asymptotic concentration of the subtilas goes to zero. ll 'Dosage / Answer = 9- / aref The washing operation is calculated according to the formula I: af? Rmax.C R = Ro +? Rmax + a «c (I) where R is the washing operation in reflectance units; R0 is the intercept 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 theoretical maximum wash effect as c - > 8. The operating factor, P, is calculated according to formula II Rvari before "R-Bla R¡ Savinase - R - ^ - Belíaa nco (II) where Rvariant is the reflectance of the washed test material with the 10 nM variant; Rsav? Nase is the reflectance of the test material washed with Savinase -iO nM; Rbianco is the reflectance of the test material washed without enzyme.

US (detergent: Us Wisk, Test: EMPA117; * P calculated at [E] = 5 nM It is thus observed that the subtilases of the invention show improved wash performance in comparison to Savinase.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (34)

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

1. A subtylase enzyme of subgroups I-SI and I-S2, characterized in that it has at least one additional amino acid residue at position 101 of the active site curl region (b) from position 95 to 103, whereby the or the additional amino acid residues correspond to the insertion of at least one amino acid residue between positions 101 and 102.

2. The subtylase enzyme isolated according to claim 1, characterized in that the subtylase enzyme is a constructed variant that has at least an amino acid residue inserted between positions 101 and 102 of a precursor subtylase.

3. The isolated subtylase enzyme according to claim 1 or 2, characterized in that it is selected from the group comprising X101X. { A, T, G, S} , X101X. { D, E, K, R} , X101X. { H, V, C, N, Q } , and X101X. { F, I, L, M, P, W, Y}

4. The isolated subtylase enzyme 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, and S.

5. The subtylase enzyme isolated in accordance with claim 3, characterized in that at least one additional or inserted amino acid residue is chosen from the group of charged amino acid residues comprising: D, E, H, K, and R, more preferably D, E, K and R.

6. The isolated subtylase enzyme 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. The isolated subtylase enzyme 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.

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 amino acid residues comprising: F, I, L, M, P, W and Y, more preferably F, I, L, M, and Y.

9. The subtylase enzyme isolated according to any of the preceding claims, characterized in that at least one additional or inserted amino acid residue comprises more than one residue of additional amino acid or inserted into the active site loop (b).

10. The subtilase variant according to any of the preceding claims, characterized in that the insert (s) between the positions 101 and 102 are combined with one or more additional modifications in any other or other positions.

11. The subtilase variant according to claim 15, characterized in that the one or more modifications are in one or more of the positions 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224, 235 and 274. ~

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

13. The subtilase according to any of the preceding claims, characterized in that the subtilase, or if the subtyla is a variant, the progenitor subtilasa belongs to the subgroup I-SI.

14. The subtilase according to claim 13, characterized in that the parent subtylase is chosen from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR, or functional variants thereof which have retained the characteristic of subgroup I-SI.

15. The subtilase according to any of claims 1-14, characterized in that the subtilase, or if the subtilases is a variant, the progenitor subtilasa belongs to the subgroup I-S2.

16. The subtilase according to claim 15, characterized in that the subtilasa progenitura is chosen from the group comprising BLS147, BLS309, BAPB92, TVTHER and BYSYAB, or functional variants thereof which have retained the characteristic of subgroup I-S2.

17. The isolated subtylase enzyme according to any of claims 3, 15 or 16, characterized in that it is selected from the group comprising S101A, S101ST, S101SG, S101SS, S101SD, S101SE, S101SK, S101SR, S101SH, S101V, S101SC, S101SN , S101SQ, S101SF, S101SI, S101SL, S101SM, S101SP, S101SW, and S101SY,

18. The subtilase variant according to any of claims 15 to 17, characterized in that the one or more modifications are chosen 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. The subtilase variant according to any of claims 15 to 17, characterized in that the one or more modifications are chosen from the group comprising S101G + V104N, S87N + S101G + V104N, K27R + V104Y + N123S + T274A, N76D + S103A + V104I OR N76D + V104A; Or combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with any one or more of the substitutions, deletions and / or insertions mentioned in any of claims 1 to 14.

20. The subtilase variant according to any of claims 15 to 17, characterized in that the one or more modifications are chosen from the group further comprising P129K, P131H, A133P, A133D and A194P.

21. The variant according to any of the preceding claims, characterized in that it comprises the chosen modification of the group comprising: A98G + S101ST, S101SGAA, A9ffG + S101SG + S103T, and A98G + S99A + S101ST.

22. A subtyla belonging to subgroup I-Sl, characterized by having the amino acid sequence: i 10 20 30 AQT- VPYGI -PLI- KADKVQAQ -GF- -ANVKVAV KG 40 50 60 LDTGIQASHPDLNVVGGASFV-AGEA - * - 70 80 90 GNGHGTHVAGTVAALDNTTGV- YNTD -GVAPSVSL YAVKVLNSSGSXGTYSGIVSG-101a 110 120 130 140 150 IEWATTNGMD VINMSLGGPSGSTAMKQAVDN-AYARGVVVV 160,170,180 AAAGNSGSSGNTNTIGYPAKY -DSVIAVGAV 190 200 210 DSNSNRASFSSVGAE- -EVMAPGAGVYSTYP 220 230 240 TSTYATLNGTSMASPHVAGAA-A- -ILSKHPN 250 260 270 LSASQVRNRLSSTATYLGSSF-YYGKGLINV 275 EAAAQ or a homologous subtylase having an amino acid sequence comprising an amino acid residue at position 101a and showing a identity of more than 70%, 75%, 80%, 85%, 90% or 95% with this one.

23. A subtyla belonging to the subgroup I-S2, characterized because it has the amino acid sequence: January 10 20 30 40 50 60 AQSVPWGISRVQAPAAHNRG- -TGSGVKVAV- -DTGI - * - STHPDLNIRGG ASFVPGEP ~ - * - 70 80 90 GNGHGTHVAGTIAALNNSIGV- STQD -GVAPSAEL- YAVKVLGASGSXGSVSSIAQG-101a 110 120 130 140 150 LEWAGNNGMH- -VANLSLGSPSPSATLEQAVNS-ATSRGV- -VV- 160 170 180 .AASGNSGA - * - GSIS - * - * - * - YPARYANAMAVGAT- 190 200 210 DQNNNRASFSQYGAGLDIVAP-GVNVQSTYP- 220 230 240 GSTYASLNGTSMATPHVAGAA-ALVKQKNPS- 250 260 270.WSNVQIRNHLKNTATSLGSTN- -YGSGLVNA- 275 EAATR or a homologous subtilase having a amino acid sequence comprising an amino acid residue at position 101a, and showing an identity of more than 70%, 75%, 80%, 85%, 90% or 95% with it.

24. The subtilase variant according to claim 22 or 23, characterized in that X in position 101a is chosen from the group comprising T, A, G, S and P.

25. An isolated DNA sequence, characterized in that it encodes for a subtyla or subtyla variant according to any one of claims 1 to 24.

26. An expression vector, characterized in that it comprises an isolated DNA sequence according to claim 25.

27. A microbial host cell, characterized in that it is transformed with an expression vector according to claim 26.

28. The microbial host according to claim 27, characterized in that it is a bacterium, preferably a Bacillus, especially B. lentus

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. A method for producing a subtyla or subtyla variant according to any of claims 1 to 24, characterized in that a host according to any of claims 27 to 29 is cultured under the conditions that lead to the expression and secretion of said variant, and the variant is recovered.

31. A composition, characterized in that it comprises a subtilase or subtyla variant according to any of claims 1 to 24.

32. The composition according to claim 31, characterized in that it also comprises a cellulase, lipase, cutinase, oxidoreductase, another protease, or an amylase.

33. The composition according to claim 31 or 32, characterized in that the composition is a detergent composition.

34. The use of a subtilase or subtyla variant according to any one of claims 1 to 24, or an enzymatic composition according to claims 31 or 32 in a laundry detergent and / or for dish washing.