MXPA00001889A - Protease variants and compositions - Google Patents

Abstract

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

Description

VARIANTS AND COMPOSITIONS OF PROTEASE FIELD OF THE INVENTION This invention relates to new enzymes of mutant proteases or variant enzymes used in the formulation of detergent compounds and which exhibit improved washing performance in detergents; detergent and cleaning compositions containing said enzymes; imitated genes that code for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed immediately and capable of expressing said variant enzymes.

BACKGROUND OF THE INVENTION In the detergent industry, enzymes are more than 30 years old in the wash formulations. The enzymes used in these formulations contain proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially the most important enzymes are REF: 32787 proteases. An increase in the number of commercially used proteases are variants of naturally occurring wild type protease protein designs, for example DURAZYM® (Novo Nordisk A / S), RELASE® (Novo Nordisk A / S). ), MAXAPEN® (Gist-Brocades NV), PURAFECT® (Genencor International, Inc.). In addition, a number of protease variants are described in the art, such as in EP 130756 (GENENTECH) (corresponding to US Patent Reprinted No.34, 606 (GENENCOR), EP 214435 (HENKEL), WO 87/04461 ( AMGEN), WO 87/05050 (GENEX), EP 260105 (GENENCOR), Thomas, Russell, and Fersht (1985) Na ture 318 375-376, Thomas, Russell, and Fersht (1987) J. Mol. Biol. 193 803 -813, Russell and Fersht Na ture 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 (PROCTER &GAMBLE COMPANY), WO 95/29979 (PROCTER &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), WO 94/02618 (GIST-BROCADES NV), and WO 96/34946 (NOVO NORDISK A / S). However, even though a number of uses of the protease variants have been described, there is still a need for new improved protease variants for a number of industrial uses. Therefore, an object of the present invention is to provide improved protein protease variants, especially for use in the detergent industry.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have intensively studied numerous of the possible combinations of residues T134 and Q137 of the SAVINASE®, and identified a number of variants with an increase in the improved washing behavior. For further details reference is made to work examples here (see below). Accordingly, the present invention relates in its first aspect to a subtylase protease variant having an improved washing behavior in detergents, comprising modification (s) in the position (s) 134 and / or 137. Preferably a subtyla variant according to the invention contains modifications at position 137, and most preferably contains modifications in both positions 134 and 137.

In a second aspect, the invention relates to a variant of the subtylase enzyme having an improved washing performance in detergents, comprising at least one modification selected from the group comprising: 134A + 137L 134S + 137L 134A + 137E 137F 137L 134V + 137T 134C + 137S 134A + 137C 137C 137D; or a variant comprising one more conservative modifications in any of the above-mentioned variants (eg, a conservative modification of a variant 134A (small aa) + 137L including variants such as 134G (small aa) + 137L, 134S (small aa) ) + 137L, 134T (small aa) + 137L, and 134M (small aa) + 137L In a third aspect, the invention relates to an isolated DNA sequence encoding a variant of the subtyla of the invention.

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

DEFINITIONS Prior to discussing this invention in further detail, the following term will be first defined.

Amino acid nomenclature 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 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) or Uracil (only in RNA) Nomenclature of variants In describing the various variants of enzymes produced or contemplated according to the invention, the following nomenclatures have been adapted for ease of reference: The amino acid or amino acids are substituted for the original amino acid or amino acid position or positions. Accordingly, the substitution of glutamic acid by glycine at position 195 is designated as: Gly 195 Glu or G195E a deletion of glycine in the same position is: Gly 195 * or G195 * and the insertion of an additional amino acid residue such as the lysine is: Gly 195 GlyLys or G195GK Where a suppression is indicated compared to the sequence used for the numbering, an insert in such position is indicated as: * 36 Asp 36D for the insertion of an aspartic acid in the position 36 Multiple mutations are separated by positive signs, ie: Arg 170 Tyr + Gly 195 Glu or R170y + G195E representing mutations at positions 170 and 195, which substitute tyrosine and glutamic acid for arginine and glycine, respectively .

Proteases Enzymes that split amide bonds in protein substrates are classified as proteases, or (reciprocally) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms, W.H. Freeman and Company, San Francisco, Chapter 3) Numbering of amino acid positions / residues If other numerations of the amino acids used here are not mentioned, they correspond to the subtyle BPN sequence (BASBPN). For additional description of the BPNX sequence see Siezen et al., Protein Engineering 4 (1991) 719-737 and Figure 1.

Serine proteases A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue in the active site (White, Handler and Smith, 1973"Principles of Biochemistry", Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).

Serine bacterial proteases have molecular weights in the range of 20,000 to 45,000 Daltons. These are inhibited by the diisopropylfluorophosphates. These hydrolyse the simple terminal esters and are similar in activity to the eukariotic chymotrypsin, also to a serine protease. In narrower terms, the alkaline protease, which covers a subgroup, reflects the high optimum pH of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological! Rev. 41 711-753 ).

Subtilasas _ A subgroup of subtilases tentatively designated as serine proteases have been proposed by Siezen et al., Protein Engineering A (1991) 719-737. These are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by Gra -posit bacteria or fungi, and in accordance with Siezen et al., Is now a subgroup of subtilases. 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 said subtilases and their amino acid sequences reference is made to Siezen et al. and to Figure 1 here.

A subgroup of the subtilases, I-SI, comprises the "classical" subtilisins, such as subtilisin 168, subtilisin BPN ', subtilisin Carlsberg (ALCALASE®, NOVO NORDISK A / S), and subtilisin DY. An additional subgroup of Subtilases I-S2 is recognized by Siezen et al. (supra) The proteases of Subgroup I-S2 are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A / S), subtilisin 147 (ESPERASE®, NOVO NORDISK A / S), and alkaline elastase YaB.

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

Original Subtilasa The term "original subtylase" is a subtyla defined in accordance with Siezen et al. (Engineering of Protein 4: 719-737 (1991)). For more details see the description of "SUBTILASAS" immediately above. An original subtylase may also be an isolated subtyla from a natural source, wherein the subsequent modification has been made while retaining the characteristic of a subtylase. Alternatively, the term "original subtylase" can be termed "subtylase of native type".

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

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

Homologous subtylase sequences The amino acid specific residues of the SAVINASE® subtyla are identified by the modification herein, to obtain a variant subtyla of the invention. However, the invention is not limited to the modifications of this particular subtilase, but it extends to other original subtilases (of native type), which have a primary structure homologous to that of SAVI ASE®. To identify other homologous subtilases, within the scope of this invention, an alignment of said subtylase (s) for a group of previously aligned subtilases is performed maintaining the previous alignment constant. A comparison is made for 18 highly conserved residues in subtilases. The 18 highly conserved residues are shown in Table I (see Siezen et al., For additional details related to such conserved residues).

Table I residues highly conserved in the Subtilasas Position: Residue conserved 23 G 32 D 34 G 39 H 64 H 65 G 66 T 70 G 83 G 125 S 127 G 146 G 154 G 155 N 219 G 220 T 221 S 225 P The permitted alignments for the necessary insertions and deletions are then identified to maintain the proper alignment of the homologous residues. Said homologous residues can then be modified according to the invention. Using the computer alignment program CLUTSTALW (version 1.5, April 1995) (Thompson, JD, Higgins, DG and Gibson, TJ (1994) Nucleic Acids Research, 22: 4673-4680.), With the disadvantage of open GAP of 10.0 and disadvantage of GAP extension of 0.1, the weight matrix of prbtein BLOSUM30 is used, the alignment of the subtyla given for a group of previously aligned subtilases was achieved using the option of alignment profiles in the program. For a subtyla given to be within the scope of the invention, preferably 100% of the 18 highly conserved residues should be conserved. However, the alignment of greater than or equal to 17 of the 18 residues, or at least 16 of said conserved residues is also adequate for the identification of the homologous residues. The preservation of, in the subtilases, the catalytic trivalent Asp32 / His64 / Ser221 should be maintained.

The previously defined alignment is shown in Figure 1, where the percent identity of the individual Subtilases in this alignment is also shown for the 18 highly conserved residues.

Based on this description, it is routine for a person skilled in the art to identify the appropriate homologous subtilases and corresponding homologous residues, which can be modified according to the invention. To illustrate this, Table II below shows a limited list of a corresponding subtylase and corresponding suitable residues, to be modified in accordance with the invention.

Table II Homologous subtilases and their corresponding homologous residues, suitable to be modified according to the invention.

Pos \ Enz. BASBPN BYSYAB BLS309 BLS147 TVTHER 134 + 137 A134A + T134A + Q13 T134A + T134A + G134A + A137L 7L Q137L L137L Q137L 134 + 137 A134S + T134S + T134S + T134S + G134S + A137L Q137L Q137L L137L Q137L 137 A137C Q137C Q137C L137C Q137C It is obvious that a similar or larger table, covering other homologous subtilases can easily be produced by a person skilled in the art.

Washing performance The ability of an enzyme to catalyze the degradation of several naturally occurring substrates, present in the objects that will be cleaned during, for example, washing, is often referred to as washing ability, washability, detergency, or wash performance. Through this application, the term wash performance will be used to encompass this property.

DNA isolated sequences The term "isolated", when applied to a molecule of a DNA sequence, indicates that the DNA sequence has been removed from its natural genetic environment and is thus free of other foreign or unwanted sequences, and is in a suitable form to be used within the systems of production of the protein, designed genetically. Such isolated molecules are those separated from their natural environment and include cDNAs and genomic clones. The isolated DNA molecules of the present invention are free of others of other genes with which they are ordinarily associated, but may naturally originate 5 'and 3' untranslated regions such as promoters and terminators. The identification of the associated regions will be apparent to one skilled in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). The term "an isolated DNA sequence" can alternatively be referred to as "a cloned DNA sequence".

Isolated protein When applied to a protein, the term "isolated" indicates that the protein is in a condition different 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)). It is preferred to provide the protein in a highly purified form, ie, greater than 40% pure, greater than 60% pure, greater than 80% pure, more preferably greater than 95% pure, and even more preferably higher 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 of the polypeptide of the invention) which originates from the homologous cell, wherein the polypeptide of the invention is originally obtained therefrom.

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

Substrate The term "substrate" used in conjunction with a substrate for a protease should be interpreted in its most extensive form to comprise 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 protease reaction in the context of this invention should be interpreted 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 FIGURES Figure 1 shows an alignment of a number of homologous subtilases, which are aligned to the 18 highly conserved residues in the subtilases. The 18 highly conserved residues are highlighted in bold. All subtilasas show, except JP170, have 100% identity in said conserved residues. The JP170 that has an "N" instead of "G" in the conserved residues G146.

DETAILED DESCRIPTION OF THE INVENTION Variants of subtyla with improved wash performance: The present inventors have identified variants in the performance of improved washing in BLS309 (SAVINASE®). Accordingly, one embodiment of the invention relates to a variant of the subtylase enzyme, wherein the modification is selected from the group comprising: T134A + Q137L T134S + Q137L T134A + Q137E Q137F Q137L T134V + Q137T T134V + Q137L T134C + Q137S T134A + Q137C Q137C Q137D; or a variant comprising one or more conservative modification (s) in any of the above-mentioned variants (for example a conservative modification of a variant T134A (small aa) + Q137L including variants such as, T134G (small aa + Q137L, T134T (small aa) + Q137L, and T134M (small aa) + Q137L).

Numerous variants of the subtyla of the invention are tested here and show improved washing performance in detergents (see working examples here (see below)). It is well known in the art that the substitution of an amino acid to a similar conservative amino acid gives only a minor change in the characteristics of the enzyme. Table II below lists the groups of Amino acid preservatives Table III Substitutions of preservative amino acids Basic: Arginine Lysine Histidine Acid: Glutamic Acid Aspartic Acid Polar: Glutamine Hydrophobic Asparagine: Leucine Isoleucine Valine Aromatic: Phenylalanine Tryptophan Tyrosine Small: Glycine Alani to Serine Threonine Methionine Consequently, subtyla variants such as 134A + 137L, 134G + 137L, 134S + 1371, 134T + 137L, and 13M + 137L will have an improvement in similar wash performance. In addition, variants of subtylase such as T134A + Q137L, T134G + Q137L, T134S + Q137L, T134T + Q137L, and T134M + Q137L will have an improvement in wash performance also similar. Based on the descriptions of subtylase variants herein, it is routine work, for a person skilled in the art, to further identify suitable conservative substitutions in order to obtain a variant of subtylase with improved wash performance. In the embodiments of the invention, the subtilases of interest are those belonging to the subgroups I-SI and I-S2. In relation to subgroup I-SI the preferred original subtylase is chosen from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR or the functional variants thereof which have retained the characteristics of subgroup I-SI. In relation to the subgroup I-S2, the subtilase of preferred origin is chosen from the group comprising BLS147, BLS309., BAPB92, TVTHER and BYSYAB or the functional variants thereof that have retained the characteristics of sub-group I-S2.

The present invention also comprises any one or more modifications in the above-mentioned positions in combination with any other modification of the amino acid sequence of the original enzyme. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are considered. The technique describes a number of subtyla variants with different improved properties and a number of these are mentioned in the "Background of the invention" section herein (see above). These references are published herein as references to identify the subtilase variant, which advantageously can be combined with a variant of the subtilase of the invention. Such combinations include the positions: 222 (improving the oxidation stability), 218 (improving thermal stability), substitutions at the Ca bond sites that the enzyme establishes, for example, position 76, and many other apparent ones of the prior art. In further embodiments, a variant of the subtilase of the invention can be advantageously combined with one or more modifications in any of the positions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 167, 170, 206 , 218, 222, 224, 235 and 274. Specifically, the following variants BLS309 and BAPN92 are considered appropriate for the combination: K27R, * 36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Y167A, Y167I, R170S, R170L, R170N, Q206E, N218S, M222S, M222A, T224S, K235L and T274A. In addition, variants comprising any of the variants V104N + S101G, K27R + V104Y + N123S + T274A, or N76D + V104A or other combinations of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with one or more of any of the modifications mentioned above, exhibit improved properties. Still, additional subtyla 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 these modifications can give a high level of expression of a variant of the subtilase of the invention.

PRODUCTION OF MUTATIONS IN THE GENES OF THE SUBTILASA Many methods for the cloning of a subtyla of the invention and for introducing mutations within the genes (for example the subtyla genes) are well known in the art. In general, standard procedures for gene cloning and introduction of mutations (randomized and / or targeted sites) into said genes can be used to obtain a subtyla variant of the invention. For further description of suitable techniques, reference is made to the examples elaborated here (see below) and (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Ausubel , F: M: et al. (Editions) "Current Protocols in Molecular Biology." John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (edits) "Methods of Molecular Biology for Bacilli." John Wiley and Sons , 1990); and WO 96/34946.

VECTORS OF EXPRESSION A recombinant expression vector comprising a DNA construct encoding the enzyme of the invention, can be any vector which can be conveniently subjected to DNA recombination procedures, and the choice of vector will often depend on the host cell within from which it will be introduced. Thus, the vector can be a vector of autonomous replication, for example a vector which exists as an extrachromosomonal entity, the replication of which is independent of chromosomal replication, for example a plasmid. Alternatively, the vector can be one which, when introduced into a host cell, it is integrated into the genome of the host cell in part, or in its entirety and is replicated together with the chromosomes within which it has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for DNA transcription. In general, the expression vector is derived from the viral plasmid or DNA, or it may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that their functions are in function with their proposed purposes, for example the transcription is initiated in a promoter and proceeds through the DNA sequence that encodes the enzyme .

The promoter can be any DNA sequence which shows transcriptional activity in the host cell of choice and can be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for use in bacterial host cells include the maltogenic amylase gene promoter Bacillus stearothermophilus, the alpha-amylase gene Bacillus licheni ormis, the alpha-amylase gene Bacillus amyloliquefaciens, the alkaline protease gene Bacillus subtilis, or the xylosidase gene Bacillus pumilus , either the Lambda PR or PL phage promoters or the E. coli lac, trp or tac promoters. The encoded DNA sequence of the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator. The recombinant vector of the invention may also contain a DNA sequence that allows the vector to replicate in the host cell in question. The vector may also contain a selectable marker, for example a gene from the product of which a defect is complemented in the host cell, or a gene encoding resistance for example antibiotics such as kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycin, or the same, or resistance to heavy metals or herbicides. To direct an enzyme of the present invention within the secretory path 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 that encodes the enzyme in the correct reading structure. The secretory signal sequences are commonly those of 5 'positions for the DNA sequence encoding the enzyme. The secretory signal sequence may be normally associated with the enzyme or may be from a gene encoding another secreted protein. The methods used to ligate the DNA sequences encoding the present enzyme, the promoter and optionally the terminator and / or the 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 skilled in the art (see, eg, Sambrook et al., op.cit.).

HOST CELL The DNA sequence encoding the present enzyme that is introduced into the host cell can be either homologous or heterologous to the host in question. If it is homologous to the host cell, i.e., produced by the wild-type host cell, it will typically be operably linked to another promoter sequence or, if applicable, to another secretory signal sequence and / or to a terminal sequence that within its natural environment The term "homologous" is proposed to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is proposed to include a DNA sequence not expressed by the host cell in nature. In this way, the DNA sequence can be from another organism, or it can be a synthetic sequence. The host cell into which the DNA construct or the recombinant vector of the invention is introduced can be any cell which is capable of producing the present enzyme and includes bacteria, yeast, fungi and highly eukaryotic cells. Examples of bacterial host cells which, in culture, are capable of producing the enzyme of the invention are gram-positive bacteria such as Bacillus strains, such as strains of B. subtilis, B. lichenif ormis, B. lent? S , B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. mega therium or B. thuringiensis, or Streptomycin strains, such as S. lividans or S. murinus, or large negative bacteria such as Echerichia coli. 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 (cotéjese, Sambrook et al., Supra). When the enzyme is expressed in bacteria such as E. coli, the enzyme can be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it can be directed to the periplasmic space by a bacterial secretion sequence. In the first case, the cells are dissolved or used and the granules are recovered and denatured after which the enzyme is partitioned by the dilution of the denaturing agent. In the latter case, the enzyme can be recovered from the periplasmic space by breaking the cells, for example by acoustic or osmotic shock, to release the contents of the periplasmic space and the enzyme is recovered. When the expression of the enzyme in grampositive bacteria such as Bacillus or Streptomycin strains, the enzyme can be retained in the cytoplasm, or it can be directed to the extracellular medium by a sequence of bacterial secretion. In the latter case, the enzyme can be recovered from the medium as described below.

METHOD OF PRODUCTION OF THE SUBTILASE The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions that allow the production of the enzyme, and the resulting enzyme is recovered from the culture. When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell, it is possible to facilitate the heterologous recombinant production of the enzyme of the invention. In this way it is possible to make a highly purified subtyla composition, characterized by being free of homologous impurities. In this context the homologous impurities mean any impurity (for example other polypeptides of the enzyme of the invention) which originate from the cell where the enzyme of the invention is obtained in an original manner. The medium used to cultivate the transformed host cells can be any conventional means suitable for the growth of the host cells in question. The subtyla of expression can be conveniently secreted into the culture medium and can be recovered therefrom by well known procedures including the separation of the cells from the medium by centrifugation or filtration, the precipitation of the proteinaceous 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 VARIANT OF THE SUBTILASE 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 enzyme composition, which comprises a variant of the subtilase of the invention. A summary of the preferred industrial applications and corresponding to preferred enzyme compositions are described below. This summary is not intended in any way to be a complete list of suitable applications of a variant of the subtilase of the invention. Variants of the subtyla of the invention can be used in other industrial applications known in the art to include the use of a protease, in particular a subtyla.

DETERGENT COMPOSITIONS THAT COMPRISE IAS ENZIMAS MIJTANTF.S The present invention includes the use of the mutant enzymes of the invention in cleaning compositions and detergents and such compositions that include the enzymes of the mutant subtilisin. Such cleaning compositions and detergents are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further descriptions of suitable cleaning compositions and detergents. The additional reference is made to the examples elaborated herein, which show improvements in washing performance for a number of variants of the subtilase of the invention.

DESCRIPTION AND EXAMPLES OF THE DETERGENT Surfactant system The detergent compositions according to the present invention include a surfactant system, wherein the surfactant may be selected from nonionic and / or anionic and / or cationic and / or ampholytic and / or zwitterionic and / or semi-polar surfactants. The surfactant is typically present at a level from 0.1% to 60% by weight. The surfactant is preferably formulated to be compatible with the components of the enzyme present in the composition. In liquid or gel compositions, the surfactant is formulated more preferably in such a way that it promotes, or at least does not degrade, the stability of any enzyme in these compositions. Preferred systems to be used in accordance with the present invention include as one surfactant one or more of the nonionic and / or anionic surfactants described herein. Polyethylene oxide, the polypropylene oxide and the condensed polybutylene oxide of the alkyl phenols are suitable for use as nonionic surfactants of the surfactant system of the present invention, with condensate of the polyethylene oxide being preferred. This compound includes the condensation products of the alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight or straight chain or in a branched chain configuration with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include the Igepal ™ co-630, marketed by the Gaf Corporation; and the Triton ™ X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas. These surfactants are commonly referred to as alkyl phenol alkoxylates (eg, alkyl phenol ethoxylate). The condensation products of the primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide are suitable for use as nonionic surfactants of the nonionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can also be straight or branched, primary or secondary, and generally comprises from about 8 to about 22 carbon atoms. Preferably they are the products of the condensation of the alcohols having an alkyl group including from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcohol. Approximately 2 to about 7 moles of ethylene oxide and more preferably 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensed products. Examples of commercially available nonionic surfactants of this type include Teritol ™ 15-S-9 (the product of the condensation of the linear alcohol Cn-Cis with 9 moles of ethylene oxide), the Tergitol ™ 24-L6 NMW (the product of the condensation of the primary alcohol Ci2-C? with 6 moles of ethylene oxide with a distribution reducing the molecular weight), both marketed by the Union Carbide Corporation; Neodol ™ 45-9 (the product of the condensation of the linear alcohol C1-Ci5 with 9 moles of ethylene oxide), Neodol ™ 23-3 (the product of the condensation of the linear alcohol C_2- _3 with 3.0 moles of ethylene oxide ), Neodol ™ 45-1 (the product of the condensation of the linear alcohol C14-C15 with 7 moles of ethylene oxide), Neodol ™ 45-5 (the product of the condensation of the linear alcohol C14-C15 with 5 moles of oxide of ethylene) marketed by the Shell Chemical Company, Kyro ™ EOB (the product of the condensation of alcohol C13-C15 with 9 moles of ethylene oxide), marketed by the Company Procter & Gamble, and Genapol ™ LA 050 (the product of the condensation of C12-C14 alcohol with 5 moles of ethylene oxide) marketed by Hoechst. The preferred range of HLB in these products is 8-11 and more preferably 8-10. Also used as a non-ionic surfactant of the surfactant systems of the present invention are the alkyl polysaccharides shown in US 4,565,647, which have a hydrophobic group containing about 16 carbon atoms and a polysaccharide, for example a polyglucoside, a hydrophilic group containing about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 units of saccharide. Any reduced saccharide containing 5 or 6 carbon atoms can be used, for example, glucose, galactose and parts of the galactoside can be replaced by the parts of the glucoside (optionally the hydrophobic group is attached to positions 2-3- , 4-, etc. Thus giving a glucose or a galactose as opposed to a glycoside or galactoside). The intersaccharide linkages may be, for example, between a position of the additional saccharide units and positions 2-, 3-, 4-, and / or 6- in the preceding units of the saccharide. The alkyl polyglucosyl has the formula R20 (CnH2nO) t (glucosyl) * wherein R2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl group comprises from about 10 to about 18 carbon atoms, preferably from about 12 to about 14 carbon atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7. the glycoside is preferably derived from glucose. To prepare these compositions, the alcohol or alkylpolyethoxy alcohol is first formed and then reacted with glucose, or a source of glucose, to form the glucoside (annexed to position 1-). The additional glycoside units can then be linked between their 1- positions and the preceding glycoside units in positions 2-, 3-, 4-, and / or 6-, preferably with the 2- position predominating. The products of the condensation of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional nonionic surfactant system of the present invention. The hydrophobic part of these compositions will preferably have a molecular weight of from about 1500 to about 1800 and will exhibit insolubility in water. The addition of the polyoxyethylene moiety to this hydrophobic part will tend to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide. Examples of compositions of this type include certain of the commercially available Pluronic ™ surfactants marketed by BASF. Also suitable for use as the non-ionic surfactant of the nonionic surfactant system of the present invention are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamine. The hydrophobic moiety of these products consists of the reaction product of ethylenediamine and the excess of propylene oxide, and generally has a molecular weight of about 2500 to about 3000. This hydrophobic moiety is condensed with ethylene oxide to the extent that the product of the condensation comprises about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of about 5., 000 to approximately 11,000. Examples of this type of nonionic surfactants certainly include the commercially available Tetronic ™ compositions commercialized by BASF. Preferred to be used as the non-ionic surfactant of the surfactant systems of the invention are the condensed polyethylene oxide of the alkyl phenols, the condensation products of the primary and secondary aliphatic alcohols with about 25 moles of ethylene oxide, alkyl polysaccharides, and the mixtures of these. More preferred are the ethoxylates of the alkyl phenol -8 ~ Ci4 having from 3 to 15 ethoxy groups and the ethoxylated alcohols C8-Ciñ (preferably with average) having from 2 to 10 ethoxy groups, and mixtures thereof. The highly preferred non-ionic surfactants are the polyhydroxy amide fatty acid surfactants of the formula ? C - C - N - Z, wherein R1 is H, or R1 is C? _4 hydrocarbyl, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R2 cs C5-31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl sequence with at least 3 hydroxyls directly connected to the sequence, or an alkoxylate derived from them, preferably, R 1 is methyl, R 2 is straight C 1-3 alkyl or Ciß-iß alkyl or an alkenyl sequence such as coconut alkyl or mixture thereof, and Z is a derivative of a reduced sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction. Highly preferred are the anionic surfactants including the alkoxylated alkyl sulfate surfactants. Examples of these are the water soluble salts or acids of the formula RO (A) mS? 3M wherein R is unsubstituted alkyl C? OC-24 or a hydroxyalkyl group having a C? 0-C4 alkyl component, preferably a C 2 -C 20 alkyl or a hydroxyalkyl, more preferably a C 2 -C 8 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which may be, for example, a metal cation (eg, sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or a cation of substituted ammonium. Alkyl ethoxylated sulfates as well as sulfates alkyl propoxylates are contemplated here. Specific examples of substituted ammonium cations include methyl-, dimethyl-, trimethylammonium cations and quaternary ammonium cations such as tetramethylammonium cations and dimethyl piperidine and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures of them, and the like. Exemplary surfactants are C ?2-C ?8 alkylpolyethoxylated sulfate (1.0) (C ?2-C ?8E (1.0) M), C ?2-C ?8 alkylpolyethoxylated sulfate (2.25) (C ?2-C ?8E) (2.25) M), and C? 2-C? 8 alkyl polyethoxylated sulfate (3.0) (C12-C? 8 (3.0) M), and C? 2-C? 8 alkyl polyethoxylated sulfate (4.0) (C? 2- C? 8E (4.0) M), where M is conveniently selected from sodium and potassium. Suitable anionic surfactants to be used are the alkyl sulfonated ester surfactants which include linear esters of C8-C2o carboxylic acids (eg, fatty acids) which are sulfonated with gaseous SO3 in accordance with "The Journal of the American Society of Petroleum Chemicals "52 (1975), pp. 323-329. Suitable initial materials will include natural fat substances _cotno_ derived from tallow, palm oil, etc. The preferred alkyl sulfonated ester surfactant, especially for laundry applications, includes alkyl sulfonated ester surfactants of the structural formula: II Tr - si - wherein R3 is a C8-C20 hydroxycarbyl, preferably an alkyl, or combination thereof, and M is a cation which forms a water-soluble salt with the alkyl sulfonate ester, the salts of suitable formed cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethone-amine, and triethanolamine. Preferably, R 3 is C 1 -C 6 alkyl, and R 4 is methyl, ethyl or isopropyl. Especially preferred are methylsulphonated esters wherein R3 is C_o-C_6 alkyl. Other suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of the ROS03M formula wherein R is preferably a C 0 -C 2 hydroxyvalent, preferably an alkyl or hydroxyalkyl having a C 10 -C 20 alkyl component, more preferably an alkyl or hydroxyalkyl C? 2-Ci8, and M is H or a cation, for example, a metal alkali cation (for example sodium, potassium, lithium), or ammonium or substituted ammonium (for example methyl-, dimethyl-, cation- , and trimethylammonium and quaternary ammonium cations such as tetramethyl ammonium and dimethyl piperidine cations and quaternary cations derived from alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like). Typically, C12-C16 alkyl sequences are preferred for low wash temperatures (for example below 50 ° C) and Cie-Ciß alkyl sequences are preferred for high temperature washing (for example above 50 ° C). Other anionic surfactants used for detersive or abstergent purposes may also be included in laundry detergent compositions of the present invention. These may include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono-di- and triethanolamine salts) of soap, C8-C22 primary and secondary alkenesulfonates, C8-C2 olefinsulfonates, polycarboxylic acids sulphonates prepared by sulfonation of the pyrolyzed product * from the iron alkalies of metal citrates, for example, as described in the British patent specification No. 0,082,179, the alkylpiglicoletersulfate (containing above 10 moles of ethylene oxide); alkylglycerol sulfonates, acyl glycerol fatty sulphonates, fatty oleic acid glycerol sulfates, alkyl phenol sulfates of ethylene ether ether, paraffin sulphonates, alkyl phosphates, such as the acyl group, N-acyl taurates, alkyl succinates and sulfosucinates, sulfosuccinate monoesters (especially saturated and unsaturated C2 ~ Cia) and diesters of sulfosucinates (especially saturated and unsaturated C? 2-C? 8 monoesters) and sulfosuccinate diesters (especially saturated and unsaturated C6-C? ), acyl sarcosinates, alkylpolysaccharide sulfates such as alkylpolyglucoside sulfates (the non-sulfated nonionic compositions are described below), primary branch alkyl sulphates, and alkyl polyethoxy carboxylates such as those of the RO (CH? CH70) formula ) -CH2COO-M + wherein R is a C8-C22alkyl is an integer from 1 to 10, and M is a cationic salt formed. Resin acids and hydrogenated resinous acids are also suitable, such as turpentine resin, hydrogenated turpentine resin, and resin acids and hydrogenated resin acids present in or derived from wood pulp oil. The alkylbenzene sulfonates are highly preferred. Especially preferred are (straight sequences) the linear alkyl benzene sulfonates (LAS) wherein the alkyl group preferably comprises from 10 to 18 carbon atoms. Additional examples are described in "Active Surface Agents and Detergents" (vol.I and II by Schwartz, Perry and Berch). A variety of such surfactants are also generally shown in US 3,929,678, (Column 23, line 58 through Column 29, line 23, incorporated herein by reference). When included therein, the laundry detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants. The laundry detergent compositions of the present invention may also contain cationic, ampholytic, zionic, and semipolar surfactants, as well as nonionic and anionic surfactants and others than those already described herein. The cationic abstergent surfactants suitable for use in the detergent, laundry compositions of the present invention are those having a long chain hydroxycarbyl group. Examples of such cationic surfactants include ammonium surfactants such as halogenated alkyltrimethylammonium, these surfactants having the formula: [R2 (OR3) and] [R4 (OR3) and] 2R5N +? - wherein R 2 is an alkyl or alkylbenzyl group having about 8 to about 18 carbon atoms in the alkyl chain, each R 3 is selected from the group which. consists of -CH2CH2-, -CH2CH (CH3) -, -CH2CH (CH2OH) -, CH2CH2CH2-, and mixtures thereof; each R4 is selected from the group consisting of C1-C4 alkyl, hydroxyalkyl d.-C4, benzene ring structures formed by joining the two groups R4, -CH2CHOHCHOHCOR6CHOHCH2OH, wherein R6 is any hexose or hexose polymer having a molecular weight less than 1000, and hydrogen when y is not 0; R5 is the same as R4 or is an alkyl sequence, wherein the total number of carbon atoms or R2 plus R5 is not more than about 18; each Y is from 0 to approximately 10, and the sum of the values of Y is from 0 to approximately 15; and X is any compatible anion. The highly preferred cationic surfactants are water-soluble quaternary ammonium compositions used in the present compound having the formula: wherein Ri is Cs-Cie alkyl, each of R2, R3 and R4 is independently C4-C4 alkyl, hydroxy at q? i 1 or C1-C4, benzyl, and - (C? H.4o)? H where X has a value from 2 to 5, and X is an anion. No more than one of R2, R3 or R4 will be benzyl. The preferred alkyl long chain for Ri is C 2-Ci 5, particularly wherein the alkyl group is a mixture of long sequences derived from coconut or kernel palm fat or is synthetically derived from a constructed olefin or the synthesis of OXO alcohols.

Preferred groups for R2R3 and R4 are methyl and hydroxy ethyl groups and the anion X can be selected from halide, methosulfate, acetate and phosphate ions. Examples of the suitable ammonium quaternary compositions of the formula (i) to be used herein are: Coconut trimethyl ammonium chloride or bromide; coconut or methyldihydroxyethyl ammonium bromide or chloride; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; chloride or bromide dimethyl hydroxyethyl ammonium C12-15; coconut or dimethyl hydroxyethyl ammonium bromide; methyl sulphate myristyl trimethyl ammonium; chloride or bromide lauryl dimethyl (ethenoxy) 4 ammonium; ester colino (compositions of the formula (i) where Rx is CH2-CH2-O-C-C12-14 alkyl and R2R3R4 are methyl) di-alkylimidazolines (compositions of the formula (i)].

Other used cationic surfactants are also disclosed in US 4,228,044 and in EP 000 224. When included in these, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 25%, preferably from about 1% to about 8% weight of each cationic surfactant. Amfolitic surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of the secondary and tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight- or branched-sequence. One of the aliphatic substituents comprises at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one comprises a water-soluble anionic group, for example, carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants. When included herein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10%, by weight of said ampholytic surfactant. Zwiterionic surfactants are also suitable for use in laundry detergent compositions - these surfactants can be broadly described as secondary and tertiary amine derivatives, heterocyclic secondary and tertiary amine derivatives, or quaternary ammonium derivatives, quaternary phosphonium or tertiary sulfonium compositions. See US 3,929,678 (column 19, line 38 through column 22, line 48) for examples of zwitterionic surfactants. When included within, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such z-ionic surfactants. Non-ionic semipolar surfactants are a special category of non-ionic surfactants which include water-soluble amine oxides containing an alkyl part of about 10 to about 18 carbon atoms and 2 parts selected from the group consisting of alkyl groups and groups hydroxyalkyl containing about 1 to about 3 carbon atoms; water-soluble phosphine oxides containing an alkyl part of about 10 to about 18 carbon atoms and 2 parts selected from the group consisting of alkyl groups and hydroxyalkyl groups containing about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing an alkyl portion of about 10 to about 18 carbon atoms and a portion selected from the group consisting of alkyl and hydroalkyl portions of about 1 to about 3 carbon atoms. The non-ionic ipolar detergent surfactants include the amine oxide surfactant which has the formula: wherein R3 is an alkyl, hydroxyalkyl, or an alkylphenyl group or mixtures thereof containing about 8 to about 22 carbon atoms; R4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof; X is from 0 to about 3: and each R 5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing about 1 to about 3 ethyl oxide groups. The R5 groups may be linked to one another, for example, through an oxygen atom, or nitrogen to form a ring structure. These amine oxide surfactants in particular include alkyl dimethyl amine Cι-Ciß and C8-C ?2 alkoxy ethyldihydroxy ethylamine oxides. When included herein, the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10%, by weight of such semi-polar nonionic surfactants.

Builder system The compositions according to the present invention may also contain an architecture system. Any conventional architectural system is suitable for use herein, including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphosphonates, particularly ethylene diamine tetramethylene phosphonic acid and diethylene triamine pentamethylene phosphonic acid. . However less preferred for obvious environmental reasons, phosphate builders can also be used here. Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as the hydrated zeolite A, X, B, HS or MAP. Other suitable inorganic builder materials are layered silicate, for example SKS-6 (HOECHST). SKS-6 is a crystalline stratified silicate consisting of sodium silicate (Na2Si205). Suitable polycarboxylates containing a carboxy group including lactic acid, glycolic acid and ether and derivatives thereof as shown in Belgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of sucinic acid, malonic acid, diacetic acid (ethylenedioxy), maleic acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German. Offenle-enschrift 2,446,686 and 2,4476,487, US 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623. Polycarboxylates containing three carboxy groups including, in particular, water-soluble citrates, aconitrates and citraconates as well as succinate derivatives such as carboxymethyloxysucinate described in British Patent No. 1, 379, 241, the described lactoxysucinates. in Dutch application 7205873, and oxypolycarbolylate materials such as 2-oxa-l, 1,3-propane tricarboxylates described in British Patent No. 1,387,447. The polyoxycarboxylates containing four carboxyl groups including the oxydisucinates shown in British Patent No. 1, 261, 829, tetracarboxylates 1,1,2,2, -ethane, tetracarboxylates 1, 1, 3, 3-propane containing sulfo substituents included the sulfosucinate derivatives shown in British Patents Nos. 1,398,421 and 1,398,422 and in US 3,936,448, and the sulfonated pyrolysed citrates described in British Patent no. No. 1,082,179, While polycarboxylates containing phosphonium substituents are shown - in British Patent No. 1, 439, 000. The alicyclic and heterocyclic polycarboxylates include cyclopentane-cis tetracarboxylates., cis-cis, pentacarboxylates, cyclopentadiene, tetracarboxylates, 2, 3, 4, 5-tetrahydro-furan-cis, cis, cis-discarboxylates, 2,5-tetrahydro-furan-cis, tetracarboxylates 2, 2, 5, 5, -tetrahydrofuan, hexacarboxylates 1,2,3,4,5,6, -hexane and the carboxymethyl derivatives of the polyhydric alcohols such as sorbitol, mannitol and xylitol. Polycarboxylates include the melific acid, pyromellitic acid and phthalic acid derivatives shown in British Patent No. 1,425,343.

Of the above, the preferred polycarboxylates are the hydroxycarboxylates which contain above three carboxy groups per molecule, more particularly the citrates. Preferred builder systems for use in the present compound include a mixture of a water-insoluble aluminosilicate builder such as zeolite A or a layered silicate (SK- &), and a water-soluble carboxylate chelating agent such as acid citric. A chelator suitable for inclusion in the detergent compositions according to the invention is the disucinic acid of ethylenediamine-N, N '- (EDDS) or the alkali metal, alkaline earth metal, ammonium, or ammonium salts substituted therefrom, or mixtures from them. The preferred compositions EDDs are in the form of free acids and the sodium and magnesium salts thereof. Examples of said preferred sodium salts of EDDS include Na2EDDS and Na4EDDS. Examples of said preferred magnesium salts of EDDS include MgEDDS and Mg2 EDDS. Magnesium salts are most preferred for inclusion in the compositions according to the invention. Preferred builder systems include a mixture of a water-insoluble aluminosilicate builder such as zeolite A, and a water-soluble carboxylate chelating agent such as citric acid. Other builder materials that can be part of the builder system for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as organic phosphonates, Amino polyalkylene phosphonates and amino polycarboxylates. Other suitable water-soluble organic salts are homo- or copolymer acids or their salts, in which the polycarboxylic acid includes at least two carboxyl radicals separately from one another by not more than two carbon atoms. Polymers of this type are shown in GB-A1, 596, 756. Examples of such salts are the polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, said copolymers having a molecular weight of from 20,000 to 70,000, especially about 40,000. The builder salts are usually included in amounts of 5% to 80% by weight of the composition. Preferred builder levels for liquid detergents are from 5% to 30%.

Enzymes Preferred detergent compositions, in addition to the preparation of enzymes of the invention, include other enzymes which provide cleaning performance and / or beneficial care of the fabrics. Such enzymes include other proteases, lipases, cutinases, amylases, celluloses, peroxidases, oxidases (for example laccases).

Proteases: Any other protease suitable for use in alkaline solutions can be used. Suitable proteases include those of animals, plants or of microbial origin. Those of microbial origin are preferred. Chemically or genetically modified mutations are included. The protease can be a serine protease, preferably a microbial alkaline protease or a trisine-like protease. Examples of the alkaline proteases are the subtisilins, especially those derived from Bacillus, for example, the subtisiline Novo, the sublsilin Carlsberg, the subtisilin 309, the subtisilin 147 and the subtisilin 168 (described in WO 89/06279). Examples of the trypsin-like protease are trypsin (for example, porcine or bovine original) and Fusarium protease described in WO 89/06270. __ The commercially available protease enzymes include those sold under the brand names Alcalase, Savinase, Primase, Durazim, and Esperase by Novo Nordisk A / S (Denmark), those sold under the brand names Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the brand names Opticleas and Optimase by Solvay Enzimes. The protease enzymes can be incorporated into the compositions according to the invention at a level of from 0.00001% to 2% of the enzyme protein by weight of the compound, preferably at a level of from 0.0001% to 1% of the protein of the enzyme by weight of the compound, more preferably at a level of from 0.001% to 0.5% of the protein of the enzyme by weight of the compound.

Lipases: Any suitable lipase for use in alkaline solutions can be used. Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutations are included. Examples of the lipases used include a Humicola lanuginosa lipase, for example, as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, for example, as described in EP 238 023, a Candida lipase, such as a C. Antarctic lipase, for example, the A or B Antarctic lipase described in EP 214, 761, a Pseudomonas lipase such as a lipase, P. alcaligenes and P. pseudoalcaligenes, for example, as described in EP 218 272, a lipase P.cepacia, for example, as described in EP 331 376, a lipase P. Stutzari, for example, as shown in GB 1,372,034, a lipase P. Fluorescens, a Bacillus lipase, for example, a lipase B. subtilis (Dartois et al., (1993), Biochemistry and Biophysics Act 1131, 253-260), a lipase B. stearothermophilus (JP 64/744992) and a lipase B. pumilus (W0_91 / 16422). In addition, a number of cloned lipases may be used, which include the lipase Penicillium camemberti described by Yamaguchi et al., (1991), Gen 103, 61-67), the lipase Geomicum candium (Shimada, Y. Et al., (1989), J. Biochem., 106, 383-388), and several Rhizopus lipases such as a lipase R. Delemar (Hass, MJ et al. (1991), Gen 109, 117-113), a lipase R. Niveus (Kugimiya et al., (1992), Biosci, Biotech, Biochem 56, 716-719) and a R. oryzae lipase. Another type of lipolytic enzymes such as cutinases can also be used, for example, a cutinase derived from Psudomonas mendocin _ as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (for example that described in WO 90 / 09446). Particularly suitable lipases are lipases such as Ml Lipase ™, Luma fast ™ and Lipomax ™ (Genencor), Lipolase ™ and Lipolase Ultra ™ (Novo Nordisk A / S), and Lipase P "Amano" (Amano Pharmaceutical Co. LTD. ). Lipases are usually incorporated into the detergent composition at a level from 0.00001% to 2% enzyme protein by weight of the compound, preferably at a level from 0.0001% to 1% of the enzyme protein by weight of the compound, more preferably at a level from 0.001% up to 0.5% enzyme protein by weight of the compound, even more preferably at a level from 0.01% to 0.2% enzyme protein by weight of the compound.

Amylases: Any amylase (a and / or ß) suitable for use in alkaline solutions can be used. Suitable amylases include those of bacterial or fungal origin. Chemically and genetically modified mutations are included. The included amylases, for example, the α-amylases obtained from a special strain of B. licheniformis, described in more detail in GB 1,296,839. The commercially available amylases are Duramyl ™, Termanyl ™, Funga yl ™ and Ban ™ (available from Novo Nordisk A / S) and Rapidase ™ and Maxamyl P ™ (available from Genecor). The amylases are normally incorporated in the detergent composition at a level from 0.00001% to 2% of the enzyme protein by weight of the compound, preferably at a level from 0.0001% to 1% protein of the enzyme by weight of the compound, more preferably at a level from 0.001% to 0.5% of the enzyme protein by weight of the compound, even more preferably at a level from 0.01% to 0.2% of the enzyme protein by weight of the compound.

Celluloses: Any suitable cellulose to be used in alkaline solutions can be used. Suitable celluloses include those of bacterial or fungal origin. Chemically or genetically modified mutations are included. Suitable celluloses are shown in US 4,435,307 which shows celluloses of fungi produced from Humicola Insolens. Especially suitable celluloses are celluloses which have benefits in the care of color. Examples of such celluloses are the celluloses described in European patent application No. 0 495 257. Commercially available celluloses include Celluzyme ™ produced from a strain of Humicola insolens, (Novo NordiskA / S), and KAC-500 (B) ™ (Kao Corporation). The celluloses are normally incorporated in the detergent composition at a level from 0.00001% to 2% of the enzyme protein by weight of the compound, preferably at a level from 0.0001% to 1% of the enzyme protein by weight of the compound, more preferably at a level from 0.001% to 0.5% of the enzyme protein by weight of the compound, even more preferably at a level from 0.01% to 0.2% of the enzyme protein by weight of the compound.

Peroxidases / Oxidases: the peroxidase enzymes are used in combination with hydrogen peroxide or a source thereof (for example a percarbonate, a perborate or a persulfate). Oxidases enzymes are used in combination with oxygen. Both types of enzymes are used for "bleaching solution", for example to prevent the transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a washing solution, preferably together with an enhancing agent such as described in for example W0 94/12621 and W0 95/01426. Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. Chemically or genetically modified mutations are included. Enzymes of peroxidases and / or oxidases are normally incorporated in the detergent compound at a level from 0.00001% to 2% of the enzyme protein by weight of the compound, preferably at a level from 0.0001% to 1% of the protein of the enzyme by weight of the compound, more preferably at a level from 0.001% to 0.5% of the protein of the enzyme by weight of the compound, even more preferably at a level from 0.01% to 0.2% of the protein of the enzyme by weight of the compound . The mixtures of the enzymes mentioned above are even included, in particular a mixture of a protease, an amylase, a lipase and / or a cellulose. The enzyme of the invention, or any other enzyme incorporated in the detergent compound, is normally incorporated in the detergent compound at a level from 0.00001% to 2% of the enzyme protein by weight of the compound, preferably at a level of 0.0001% up to 1% of the enzyme protein by weight of the compound, more preferably at a level from 0.001% to 0.5% of the protein of the enzyme by weight of the compound, even more preferably at a level from 0.01% to 0.2% of the protein of the enzyme in weight of the compound.

Bleaching agents: Optional additional ingredients for detergents that may be included in the detergent compositions of the present invention include bleaching agents such as PB1, PB4 and percarbonate with a particle size of 400-800 microns. These bleaching agent components can include one or more oxygenated bleaching agents and, depending on the bleaching agent chosen, one or more bleach activators. When oxygenated bleaching compositions will typically be present at a level from about 1% to about 25%. In general, bleaching compositions are optionally added components in non-liquid formulations, for example granular detergents. The bleach agent component to be used herein may be any of the bleaching agents used in detergent compositions including oxygenated bleach as well as others known in the art. The bleaching agent suitable for the present invention may be an activated or non-activated bleaching agent. A category of bleaching agent that can be used encompasses percarboxylic acid, bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium hexahydrate onoperoxyphthalate, the magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydecanedioic acid. Such bleaching agents are shown in US 4,483,781, US 740,446, EP 0 133 354 and US 4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in US 4,634,551. Another category of bleaching agents that can be used include halogenated bleaching agents. Examples of hypohalite bleaching agents, for example, include trichloro isocyanuric acid and the sulfonamides of sodium and potassium dichloroisocyanurate and N-chloro and N-Bromoalkano. Such materials are usually aggregates of 0.5-10% by weight of the finished product, preferably 1-5% by weight. Agents liberated from hydrogen peroxide can be used in combination with bleach activators such as tetraacetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS, described in US 4,412,934), 3,5-trimethylhexanoloxybenzenesulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG), which are perhydrolides to form a peracid as the active bleaching species, leading to improve the bleaching effect. In addition, bleach activators (C8 (6-octanamido-caproyl) oxybenzenesulfonate, C9 (6-nonanamido capryl) oxybenzenesulfonate and CIO (6-decanamido caproyl) oxybenzenesulfonate or mixtures thereof are very suitable. acylated citrate esters such as those shown in European Patent Application No. 91870207.7 Bleaching agents used include peroxyacids and bleaching systems containing bleach activators and peroxygen bleach compositions for use in cleaning compositions in accordance with The invention is described in the application USSN 08 / 136,626, The hydrogen peroxide can also be present by adding an enzymatic system (for example an enzyme and a substrate thereof) which is capable of generating hydrogen peroxide at the start or during the washing and / or filling process Such enzyme systems are shown in the European Patent Application EP 0 537 3 81. Other bleaching agents than oxygenated bleaching agents are also known in the art and can even be used. One type of non-oxygenated bleaching agent of particular interest includes photoactivated bleaching agents such as zinc sulphonate and / or aluminum phthalocyanines. These materials can be deposited on top of the substrate during the washing process. By irradiation with light, in the presence of oxygen, such as by hanging clothes out of washing in daylight, the sulphonated zinc phthalocyanine is activated and, consequently, the substrate is bleached. The preferred zinc phthalocyanine and a photoactivated bleaching process are described in US 4,033,718. Typically, the detergent composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc phthalocyanine. The bleaching agents may also contain a manganese catalyst. The manganese catalyst may be, for example, one of the compositions described in "Catalyst efficient manganese for low bleaching temperatures", Nature 369, 1994. _ pp 637-639.

Foam suppressors: Another optional ingredient is a foam suppressor, exemplified by silicones, and a mixture of silica-silicones. The silicones can generally be expressed by alkylated polysiloxane materials, while the silica is normally used in finely divided forms exemplified by aerogels and silica Xerogels and hydrophobic silica of various types. These materials can be incorporated as particles, in which the foam suppressant is advantageously freely incorporated in a water-soluble or water-dispersible, substantially non-active surface detergent vector. Alternatively, the foam suppressant can be dissolved or dispersed in a liquid vector and spray applied to one or more of the other components. A preferred foam controller silicone agent is shown in US 3,933,672. Other foam suppressors particularly used are self-emulsifying foam suppressor silicones, described in the German patent application DTOS 2,646,126. An example of how a compound is DC-544, commercially available from Dow Corning, which is a copolymer of siloxane-glycol. The preferred foam controlling agents are those of the foam suppressor system containing a mixture of silicone oils and 2-alkyl alkanols. Suitable 1-alkyl alkanols are 2-butyl octanol which are commercially available under the brand name Isofol 12 R. Such foam suppressor systems are described in European Patent Application EP 0 593 841. The foam controlling agents of Particularly preferred silicones are described in European Patent Application No. 92201649.8. Said compositions may contain a mixture of silicone / silica in combination with vaporized non-porous silica such as Aerosil®. The suppressors described above are normally employed at levels from 0.001% to 2% by weight of the compound, preferably from 0.01% to 1% by weight.

Other components: Other components used in detergent compositions may be employed such as stain-dispersing agents, stain-releasing agents, optical brighteners, abrasives, bactericides, discoloration inhibitors, coloring agents, and / or encapsulated or non-encapsulated perfumes. Particularly suitable encapsulated materials are water soluble capsules which consist of a polysaccharide matrix and polyhydroxy compositions such as those described in GB 1,464,616. Other suitable water-soluble encapsulated materials comprise dextrins derived from the non-gelatinized starch acids, the esters of substituted diacarboxylic acids such as those described in US 3,455,838. These acid-ester dextrins are preferably prepared from each starch such as waxed corn, waxed sorghum, sago, tapioca and potato. Suitable examples of such encapsulation of the materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material consists of a modified starch of corn and glucose. The starch is modified by the addition of substituted monofunctional groups such as octeyl sucinic acid anhydride. Suitable antiredeposition and slurry agents here include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or copolymeric polycarboxylic acids or their salts. Polymers of this type include polyacrylates and acrylic maleic anhydride copolymers previously mentioned as builders, either copolymers of maleic anhydride with ethylene, methylvinyl ether or methacrylic acid, maleic anhydride as builders of at least 20 mole percent of the copolymer. These materials are normally used at levels from 0.54% to 10% by weight, more preferably from 0.75% to 8%, more preferably from 1% to 6% by weight of the. compound. The preferred optical brighteners are anions in character, examples of these are 4,4'-bis- (2-ditanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2: 2'-disodium disulfonate, 4, 4'-bis- disulfonate ( 2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2: 2-disodium, 4,4'-bis- (2,4-dianilino-s-triazin-6-ylamino) stilbene-2-disulfonate : 2 'disodium, sulfonate 4', '' -bis (2,4-dianilino-s-triazin-6-ylamino) stilbene-2-monosodium, Disulfonate 4,4'-bis- (2-anilino-4- ( N-methyl-N-2-hydroxyethylamino) -s-triazin-6-ylamino) stilbene-2, 2'-disodium, 4,4'-bis- (4-phenyl-2, 1,3-triazole-2-disulfonate) -yl) -stilbene-2, 2 'disodium, disulfonate 4,' bis (2-anilino-4- (l-methyl-2-hydroxyethylamino) -s-triazin-6-ylamino) stilbene-2, 2"disodium, sulfonate 2 (stilbene-4"- (naphtho-1 ', 2': 4, 5) -1, 2, 3, -triazole-2" -sodium and biphenyl 4,4'-bis (2-sulfostyril). Polymeric materials used are polyethylene glycols, particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and more preferably about 4000. These are used at levels of about 0.20% to 5% more preferably from 0.25% to 2.5% by weight. These polymers and the previously mentioned homo- or polycarboxylate copolymer salts are valuable for improving the maintenance of whiteness, the deposition of ashes in the fabric, and the cleaning behavior in clay, proteinization and oxidability of the stains in the presence of impurities of transition metals.

The stain releasing agents used in the compositions of the present invention are conventionally copolymers and terpolymers of terephthalic acid with ethylene glycol and / or propylene glycol units in various arrangements. Examples of such polymers are shown in US 4,116,885 and 4,711,730 and EP 0 272 033.A the preferred polymer in particular in accordance with EP 0 272 033 has the formula: (CH3 (PEG) 43) 0.75 (POH) o._5_ (T-P0) 2.8 (T-PEG) 0.4.T (POH) 0.2s ((PEG) 43CH3) 0.75 where PEG is - (OC2H4) 0-, PO is (OC3H60) and T is (pOOC6H4CO).

Also widely used are polyesters modified as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol, the end groups consist primarily of sulfobenzoate and secondarily of mono esters of ethylene glycol and / or 1,2-propanediol . The objective is to obtain a polymer covered at both ends by sulfobenzoate groups, "primarily", in the present context more of said copolymers here, will be covered at the ends by sulfobenzoate groups. However, some copolymers will be less than fully covered, and therefore their end groups may consist of ethylene glycol monoester and / or 1,2-propanediol, hence the "secondarily" of such species. The selected polyesters even comprise about 46% by weight of the dimethyl terephthalic acid, about 16% by weight of 1,2-propanediol, about 10% by weight of ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight. weight of sulfoisophthalic acid, and has a molecular weight of about 3,000. The polyesters and their method of preparation are described in detail in EP 311 342.

Softening agents: fabric softening agents may also be incorporated into the laundry detergent compositions according to the present invention. These agents can be of the inorganic or organic type. The inorganic softening agents are exemplified by the esclase muds shown in GB-A1 400898 and in US 5,019,292. Organic fabric softening agents include the water insoluble tertiary amines as shown in GB-A1 514 276 and EP 0 011 340 and combinations thereof with C 12 -C 14 mono quaternary ammonium salts are shown in EP-B-0 026 528 and long sequence amides are shown in EP 0 242 919. Other organic ingredients used in fabric softener systems include high molecular weight polyethylene oxide materials as shown in EP 0 299 575 and 0 313 146. The levels of esclaimed clay they are usually in the range of 5% to 15%, more preferably from 8% to 12% by weight, with the material being added as a dry mixed component for the remainder of the formulation. Organic fabric softening agents such as insoluble tertiary amines or long sequence amide materials are incorporated at levels from 0.5% to 5% by weight, usually from 1% to 3% by weight while polyethylene oxide materials from High molecular weight and water soluble cationic materials are aggregated at levels from 0.1% to 2%, usually from 0.15% to 1.5% by weight. These materials are usually added to the powdered dry part of the compound, although in some instances they may be more convenient to add as a dry mixed particle, or sprayed as liquid melted into the other solid components of the compound.

Polymeric agents that inhibit color transfer: Compositions for detergents in accordance therewith may also contain from 0.001% to 10%, preferably from 0.01% to 2%, more preferably from 0.05% to 1% by weight of the polymeric agents color transfer inhibitors. Said color transfer inhibitory polymeric agents are normally incorporated within the detergent compositions in order to inhibit the transfer of colors from the dyed fabrics onto the fabrics washed with them. These polymers have the ability to form complexes or to absorb fugitive colors from the washing of dyed fabrics before the dyes have the opportunity to become attached to other articles in the wash. The polymeric agents inhibitors of the color transfer are polymers N-oxide polyamine copolymers of N-vinyl-pyrollidone and N-vinylimidazolepolymers of polyvinylpyrrolidone, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. The addition of such polymers also enhances the development of the enzymes according to the invention. The detergent composition according to the invention may be in liquid, paste, gels, sticks, or granular form.

Non-dust granulates, for example, such as those shown in US 4,106,991 and 4,661,452 (both for Novo Industries A / S) and may occasionally be covered by methods known in the art. Examples of waxed covered materials are poly (ethylene oxide) (polyethylene glycol, PEG) products with average molecular weights of 1000 to 2000; the ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol comprises from 12 to 20 carbon atoms and in which there are from 15 to 80 units of ethylene oxide; fatty alcohols; the fatty acids; and the mono- and di- and triglyceride of the fatty acids. Examples of formed film covering materials suitable for fluid bed techniques are given in GB 1483591. The granular compositions according to the present invention can also be in "compact form", for example they can have a relatively higher density. than conventional granular detergents, for example the form 550 to 950 g / 1; in such a case, the granular detergent compositions in accordance with the present invention will contain a small amount of "inorganic filler salt", compared to conventional granular detergents; Typical filler salts are salts of sulfates and ferrous alkali metal chlorides, typically sodium sulfate; The "Compact" detergent typically comprises no more than 10% filler salt. The liquid compositions according to the present invention can also be in "concentrated form", in such case, the liquid detergent compositions according to the present invention will contain a small amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergents is less than 30%, more preferably less than 20%, more preferably less than 10% by weight of the detergent compositions. The compositions of the invention can, for example, be formulated as compositions for hand washing and machine washing detergents that include additive washing compositions and compositions suitable for use in the pretreatment of soiled fabrics, in additional rinse softening compositions of the fabric, and in compositions for use in domestic cleaning operations on difficult surfaces and dishwashing operations. The following examples are mentioned to exemplify the compositions of the present invention, but not necessarily mentioned to limit or otherwise define the scope of the invention. In the compositions for detergents, the identifications of the abbreviated components have the following meanings: LAS: C12 linear sodium alkyl sulfonate alkyl benzene TAS: Alkyl Fatty Sodium Sulfate XYAS: Sodium sulfate C? -C? And alkyl SS: Secondary Surfactant Soap of 2-butyl octanoic acid formula 25EY: A linear primary alcohol C12-C15 predominantly condensed with an average of Y moles of ethylene oxide 45EY: A linear primary alcohol C? 4-C15 predominantly condensed with an average of Y moles of ethylene oxide XYEZS: The alkyl sodium sulfate C _? - C? Y condensed with an average of Z moles of ethylene oxide per mole Non-ionic: An ethoxylated / propoxylated mixed fatty alcohol C13-C15 with an average degree of exoxylation of 3.8 and an average degree of propoxylation of 4.5 sold under the trademark Plurafax LF404 by BASF Gmbh CFAA: alkyl N-methylglucamide C12-C14 TFAA: alkyl N-methylglucamide Ciß-Ciß Silicate: Amorphous sodium silicate (Si02: Na20 range = 2.0) NaSKS-6: Stratified crystalline silicate of formula d- Na2Si205 Carbonate: Anhydrous sodium carbonate Phosphate: Sodium tripolyphosphate MA / AA: Maleic acid / acrylic copolymer 1: 4, average molecular weight of approximately 80,000 Polyacrylate: A polyacrylate homopolymer with an average molecular weight of 8,000 sold under the trademark PA- = by BASF Gmbh Zeolite A: Hydrated sodium aluminosilicate of formula Nal2 (A102Si02) i2.27H2? which has a primary particle of size in the range of 1 to 10 micrometers Citrate: tri-sodium citrate dihydrate Citrus: Citric acid Perborate: Perborate sodium anhydride monohydrate bleach, empirical formula NaB02.H2? 2 PB4: Perborate sodium anhydride tetrahydrate Percarbonate: Anhydrous sodium percarbonate bleach of empirical formula 2Na2C03.3H202 TAED: Tetraactyl ethylene diamine CMC: Sodium carboxymethyl cellulose DETPMP: Penta diethylene triamine (methylenphosphonic acid), marketed by Monsanto under the trademark Dequest 2060 PVP: polyvinylpyrrolidone polymer EDDS: Ethylenediamine-N, N '-disusinic acid, [S, S] isomer in the sodium salt form Foams: 25% Paraffin wax Mpt 50 ° C, 17% hydrophobic silica, 58% Suppressor: Paraffinic oil Granular foam: 12% silicone / silica, 18% stearyl alcohol, 70% suppressant: granular starch Sulfate: Sodium anhydrous sulfate HMWPEO: High molecular weight polyethylene oxide TAE 25: Ethoxylated fatty alcohol Detergent Example 1 A granular fabric cleaning composition according to the invention can be prepared as follows: Sodium Sulfonate Linear Ci2 alkyl benzene 6.5 Sodium Sulfate 15.0 Zeolite A 26.0 Sodium Nitroacetate 5.0 Enzyme of the invention 0.1 PVP 3.0 TAED 3.0 Boric Acid 4.0 Perborate 18.0 Phenolic sulfonate 0.1 Minor Up to 100 * Detergent Example II A compact granular fabric cleaning composition (density 800 g / 1) according to the invention can be prepared as follows: 45AS 8.0 25E3S 2.0 25E3 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0 Citrus acid 3.0 Carbonate 7.0 MA / AA 5.0 CMC 0.4 Enzyme of the invention 0.1 TAED 6.0 Percarbonate 22.0 EDDS 0.3 Granular foam suppressor 3.5 Water / minors up to 100% Detergent Example III The granular fabric cleaning compositions according to the invention which are used especially in the washing of dyed fabrics are prepared as follows: LAS 10.7 TAS 2.4 TFAA-4.0 45AS 3.1 10.0 45E7 4.0 25E3S-3.0 68E11 1.8 25E5 - 8.0 Citrate 15.0 7.0 Carbonate - 10.0 Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 - 9.0 MA7AA 5.0 5.0 DETPMP 0.2 0.8 Enzyme of the invention 0.10 0.05 Silicate 2.5 - Sulfate 5.2 3.0 PVP 0.5 _ Polyoxide (4-vinylpyridine) 0.2 -N- / co-olimero of vinyl imidazole and vinyl-pyrrolidone Perborate 1.0 Phenolic sulfonate .02 Water / minors up to 100% Detergent Example IV _ The granular fabric cleaning compositions according to the invention which provides the ability to "Soften through washing" can be prepared as follows: 45AS-10.0 LAS 7.6 - 68AS 1.3 - 45E7 4.0 - 25E3 - 5.0 Coconut-alkyl-1.4- 1.0 dimethyl hydroxyethyl ammonium citrate 5.0 3.0 Na-SKS-6 - 11.0 Zeolite A 15.0 15.0 MA / AA 4.0 4.0 DETPMP 0.4 0.4 Perborate 15.0 - Percarbonate - 15.0 TAED 5.0 5.0 Greda smectite 10.0 10.0 HMWPEO - 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0 Carbonate 10.0 10.0 Granular foam suppressor 1.0 4.0 CMC 0.2 4.0 Water / Children Up to 100% Detergent Example V The heavy-duty liquid fabric cleaning compositions according to the invention can be prepared as follows: I II THE form of acid - 25.0 Citric acid 5.0 2.0 25AS acid form 8.0 - 25AE2S acid form 3.0 - 25 E7 8.0 - CFAA 5.0 - DETPMP 1.0 1.0 Fatty acid 8.0 - Oleic acid - 1.0 Ethanol 4.0 6.0 Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coconut-alkyl- - 3.0 dimethyl hydroxy-ethyl ammonia Greda smectite - 5.0 PVP 2.0 - Water / Children Up to 100% APPLICATIONS IN THE LEATHER INDUSTRY A subtyla of the invention can be used in the leather industry, in particular for use in hair removal. In said application a variant of the subtilase of the invention is preferably used in an enzymatic compound which also comprises another protease. For a more detailed description of other suitable proteases see the section on enzymes suitable for use in a detergent compound (see above).

APPLICATIONS IN THE WOOL INDUSTRY A sublay of the invention can be used in the wool industry, in particular for use in cleaning clothes comprising wool. In said application a variant of the subtyla of the invention is preferably used in an enzymatic compound which also comprises another protease. For a more detailed description of other suitable proteases see the section on enzymes suitable for use in a detergent compound (see above).

The invention is described in more detail in the following examples which are not in any way intended to limit the scope of the invention as claimed.

MATERIALS AND METHODS Strains; B. subtilis DB1885 (Diderichseb et al., 1990). B. lentus 309 and 147 are specific strains of Bacillus luntus, deposited with the NCIB and in accordance with the accession numbers NCIB 10309 and Í0147, and described in the US patent NO. 3,723,250 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 American Patent Application Series No. 039,298 .

Plasmids: pJS3: E. coli - B. subtilis launch vector containing a synthetic gene coding for subtyla 309. (Described by Jacobo Schi0dt et al in Proteins and Peptides letters 3: 39-44 (1996)). pSX222: B, expressed vector subtilis (Described in W = 96/34946).

General methods of biology ffiolecula ?. : Unless otherwise indicated, DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular Cloning: A manual laboratory, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (Eds.) "Current Protocols in Molecular Biology." Jonh Wiley and sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Biological Molecular Methods for Bacilli." John iley e Hijos , 1990). Enzymes for DNA manipulations were used according to the specifications of the suppliers.

Enzymes for DNA manipulations Unless otherwise indicated, all.

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

Prsteolitic activity In the context of this invention, the proteolytic activity is expressed in Kilo of Novo Protease Units (KNPU). The activity is determined to a standard enzyme (SAVINASE®), and the determination is based on the digestion of a solution of dimethyl casein (DMC) by the proteolytic enzyme under standard conditions, for example 50 ° C, pH 8.3, time of reaction 9 min., measurement time 3 min. A folder AF 220/1 is available on questions to Novo Nórdísk A / S, Dínaf? Árcá, the folder is included here for reference. A GU is a Glycine Unit, defined as the activity of the proteolytic enzyme which, under standard conditions, for a time of 15 minutes of incubation at 40 ° C, with casein N-acetyl as a substrate, produces an amount of the NH2 group equivalent to 1 mole of glycine. Enzymatic activity can also be measured using the PNA assay, in accordance with the reaction with the soluble substrate sucinylan-alanine-alanine-proline-phenylalanine-para-nitrophenol, which is described in the Journal of the American Chemical Society in Oil, Rothgeb, TM, Goodlander, BD, Garrison, PH, and Smith, LA, (1998).

FéiSfiéntaeióñ: Fermentation of subtyiase enzyme was performed at 30 ° C on a rotary shaking table (300 r.p.m.) in a 500 ml Erlenmeyer flask containing a medium of 100 ml BPX for 5 days. Consequently, to prepare, for example, 2 liters of broth, 20 Erlenmeyer flasks were fermented simultaneously.

Medium: BPX: Composition (per liter) Potato starch 100g Barley 50g Soybean meal 20a Na2HP04 X 12 H20 9g Pluronic O.lg Sodium Casinate lOg The starch in the medium is liquefied with a-amiiase 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 NaHCQ3 to Q.1M.

E-TEMPLES EXAMPLE Construction and expression of the Enzyme Variants: Directed mutagenesis & n.

The on-site directed subtype 309 variants were made by "Single Site Removal (USE)" or the "Uracil-USE" technique described respectively by Deng et al. (Anal Biochem 200: 81-88 (1992)) and Markvardsen et al. (Biotechnics 18 (3): 371-372 (1995)). The standard plasmid was pJS3, or an analogue thereof, which contains a variant of subtylase 309, for example USE mutagenesis was performed on analogous pJS3, which contains a gene encoding the T134A variant with an oligonucleotide targeting the Q137L construct of the variant that results in a variant of Subtilasa 309 final T134A + Q137L. The subtylase 309 variants constructed in pJS3 were then subcloned into the expression plasmid B. subtilis pSX222, using the restriction enzymes Kpnl and Mlul.

Mütagénesls located randomly The total strategy, used to perform the randomized local mutagenesis was: A mutagenic primer (oligonucleotide) was synthesized, which corresponds to the part of the DNA sequence to be mutagenized except for the nucleotides corresponding to the corresponding amino acid codon to be mutagenized Subsequently, the resulting mutagenic primer was used in a reaction in PCR with a suitable opposite primer. The resulting PCR fragment was purified and digested and cloned into an E. coli -B firing vector. subtilis. Alternatively and if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a suitable second counter primer to allow digestion and cloning of the mutagenized region within the trigger vector. The PCR reactions were performed under normal conditions. Following this strategy, a randomly located library was constructed in SAVINASE, where both positions T134 and Q137 were completely randomized. An oligonucleotide was synthesized with 25% of each of the four bases (N) in the first and second bases in the amino acid codons that seek to be mutagenized. The third nucleotide (the fluctuating base) was synthesized in the codoens with 50% G / 50% C (S) to avoid two (TAA, TGA) of the three stop codons. The mutagenic primer (5-G AAC GCC TCT AGA AGT CGC GCT ATT AAC AGC SNN CTC GAG SNN GGC ACT TGG CGA AGG GCT TCC-3 ^ (antisensitive)) was used in a PCR reaction with a suitable counter-primer (e.g. 'GAA CTC GAT CCA GCG ATT TC 3' (sensitive)) and plasma pJS3 as standard. This resulting PCR product was cloned into the firing vector pJS3, by the use of the restriction enzymes HindIII and Xbal. The randomly located library in the pJS3 construct was then subcloned into the expression plasmid B. subtilis pSX222, using the restriction enzymes Kpnl and Mlul. The prepared library contains approximately 100,000 individual clones / library.

Ten colonies chosen at random were sequenced to confirm the designated mutations.

To purify the variant of the subtyla of the invention, the expression plasmid B. subtilis pSX222 comprising a variant of the invention was transformed into a competent B. subtilis strain, and was fermented as described above in a medium containing 10 μg / ml of chloramphenicol (CAM). • EXAMPLE 2 Purification of the variants of the enzyme: This procedure refers to the purification of a fermentation at 2 liters scale of the enzyme of Subtilisin 147, the enzyme of Subtilisin 309 or mutations of them. Approximately 1.6 liters of fermentation broth were centrifuged at 5000 r.p.m. for 35 minutes in a 1 liter laboratory glass. 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 Amico CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate was centrifuged and filtered prior to absorption at room temperature in a Bacitracin affinity column at pH7. The protease was dissolved from the Bacitracin column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer with 0.01 of dimethylglutaric acid., 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7. Protease activity fractions from the Bacitracin purification step were combined and applied to a 750 ml Sephadex G25 column (5 cm day) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.2M boric acid and 0.002m calcium chloride adjusted to pH 6.5. The fractions with proteolytic activity of the Sephadex G25 column were combined and applied to a 750 ml Sephadex G25 column (5 cm dia.) Equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.2 M boric acid and 0.002 m calcium chloride adjusted at pH 6.5. The protease was diluted using a linear gradient of 0-0.1 M sodium chloride in 2 liters of the same buffer (sodium chloride 0-0.1 M in case of subtilisin 147).

In a final purification step, the protease containing fractions from the Sepharose CM column were combined and concentrated in an ultrafiltration cell.

Amicon equipped with a GR81PP membrane (from Danish Sugar Factories Inc.). By using techniques of Example 1 for the above construction and isolation procedure, the following subtilisin 309 variants were produced and isolated: T134A + Q137L T134S + Q137L T134A + Q137E Q137F Q137L T134V + Q137T T134V + Q137L T134C + Q137S T134A + Q137C Q137C; And Q137D.

EXAMPLE 3 Washing performance of detergent compositions comprising the enzyme variants The following examples provide results of a number of wash tests that were performed under the indicated conditions.

EXPERIMENTAL CONDITIONS Table VI; Experimental conditions for the evaluation of subtilisin 309 variants.

The detergent used is a simple model formulation. The pH is adjusted to 10.5 which is within the normal range for a powder detergent. The composition of the model 95 detergent is as follows: 25% STP (Na5P3O?) 25% Na2S04 10% Na2C03 20% LAS (Nansa 80S) 5.0% Tenside Nonionic (Dobanol 25-7) 5.0% Na2Si205 0.5% Carboxymethylcellulose (CMC) 9.5% Water The hardness of the water was adjusted by the addition of CaCl2 and MgCl2 (Ca2 +: Mg2 + = 2: 1) to deionized water (see also Surfactant Theory in Consumer Products, Technology and Application, Springer Verlag 1986). The pH of the detergent solution was adjusted to pH 10.5 by the addition of HCl.

The reflectance (R) measurements on the test materials were made at 460 nm using a Macbeth ColorEye 7000 photometer (Macbeth, Division of Kollmorgen Instruments Corporation, Germany). The measurements were made in accordance with the manufacturer's protocol.

The washing development of the subtilisin 2309 variants was evaluated by calculating a performance factor: Rvariant Rbla P = R savmasa - Rbla P: Performance factor Rivarante Reflectance of the washed test material with the variant Rsavinase • reflectance of the washed test material with Savinase Rbla Reflectance of the washed test material without enzyme The claimed Subtilisin 309 variants, all, have improved wash performance, compared to the Sabinse® - for example P > 1.

The variants are divided into improved classes designated with capital letters: Class A 1 < P < 1.5 Class B 1.5 < P < 2 Class C P > 2 Table V: Variants of subtilisin 309 and improved classes.

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

Claims (24)

1. l.A variant of the subtylase enzyme which has improved washing performance in detergent, characterized in that it comprises modification or modifications in the position or positions 134 and / or 137 (in the BASBPN numbering).
2. 2. A variant of the subtylase enzyme having improved washing performance in detergents, characterized in that it comprises at least one modification chosen from the group comprising (in the BASBPN numbering): 134A + 137L 134S + 137L 134A + 137E 137F 137L 134V + 137T 134V + 137L 134C + 137S 134A + 137C 137C 137D; or a variant comprising one or more conservative modifications in any of the above-mentioned variants (for example a conservative modification of a variant 134A (small aa) + 137L including variants such as 134G (small aa) + 137L, 134S (small aa) + 137L, 134T (small aa) +1371, and 134M (small aa) + 137L).
3. 3. The subtilase enzyme variant according to claim 2, characterized in that the modification is chosen from the group comprising (in BASBPN numbering): T134A + Q137L T134S + Q137L T134A + Q137E Q137F Q137L T134V + Q137T T134V + Q137L T134C + Q137S T134A + Q137C Q137C Q137D; or a variant containing one or more conservative modifications in any of the above-mentioned variants (for example a conservative modification of a variant T134A (small aa) + Q137L including variants such as T134G (small aa) + Q137L, T134S (small aa) + Q137L), T134T (small aa) + Q137L, and T134M (small aa) + Q137L).
4. 4. The variant of any of claims 1 to 3, characterized in that the original subtyla is selected from the sub-group I-SI.
5. 5. The variant of claim 4, characterized in that the original subtylase is selected from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR or the functional variants thereof that retain the characteristic of sub-group I-SI.
6. 6. The variant of any of claims 1 to 3, characterized in that the original subtylase is selected from the sub-group I-S2.
7. 7. The variant of claim 6, characterized in that the original subtylase is selected from the group comprising BLS147, BLS309, BAPB92, TVTHER and BYSYAB or the functional variants thereof which retain the characteristic of subgroup I-S2.
8. 8. The variant of any of the claims "above, characterized in that said modification (s) is (are) combined with one or more modification (s) in any other position (s).
9. 9. The variant of claim 8, characterized in that said modification (s) are (are) combined with the modification (s) in one or more of the positions 27, 36, 57, 76, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224, 235 and 274.
10. 10. The variant of claim 9, characterized in that said subtilase belongs to sub-group I-S2 and the additional change mentioned is chosen from the group containing K27R, * 36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D , N123S, Y167A, Y167I, R170S, R170L, R170N, Q206E, N218S, M222S, M222A, T224S, K235L, Y
11. ll. The variant of claim 10 characterized in that it comprises any of the variants V104N + S101G, - K27R + V104Y + N123S + T274A, - or N76D + V104A, - or other combinations. of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, V104A), in combination with any one or more substitutions, deletions and / or insertions mentioned in any of claims 1 to 10.
12. 12. The subtilase variant of any of the preceding claims, characterized in that said modification (s) is (are) combined with the modification (s) in one or more of the positions 129, 131, 133, and 194.
13. 13. The variant of claim 12, characterized in that said subtilase belongs to sub-group I-S2 and said additional change is chosen from the group containing P129K, P131H, A133P, A133D and A194P.
14. 14. an isolated DNA sequence, characterized in that it encodes a variant of the subtilase of any of claims 1 to 13.
15. 15. An expression vector characterized in that it comprises an isolated DNA sequence of claim 14.
16. 16. A microbial host cell, characterized in that it is transformed with an expression vector of claim 15.
17. 17. The microbial host of claim 16 / characterized in that it is a bacterium / preferably a Bacillus, especially B. lentus.
18. 18. The microbial host of claim 16 / characterized in that it is a fungus or yeast / preferably a filamentous fungus, especially an Aspergillus.
19. 19. a method for producing a variant of any of claims 1 to 13, characterized in that a host of any of claims 16 to 18 is cultured under conditions conducive to the expression and secretion of said variant, and the variant is recovered.
20. 20. a composition characterized in that it comprises a variant of the subtilase according to any of claims 1 to 13.
21. 21. The composition according to claim 20, characterized in that it additionally comprises a cellulose, -lipase, • cutinase, -oxidoreductase, -other protease, or an amylase.
22. 22. The composition according to claim 20 or 21, characterized in that the composition is a detergent composition.
23. 23. The use of a subtyla variant according to any of claims 1 to 13 or an enzyme composition according to any of claims 20 to 22 in a laundry detergent and / or a dish washer.
24. 24. a process for the identification of a variant of the protease that presents improved washing performance in detergents, characterized in that it effects a mutation in the DNA encoding a subtylase enzyme or its pre or preproenzyme in one or more of the positions corresponding to the amino acids (in the BASBPN numbering): T134A + Q137L T134S + Q137L T134A + Q137E Q137F Q137L T134V + Q137T T134V + Q137L T134C + Q137S T134A + Q137C Q137C Q137D; Or a variant containing one or more of the conservative modifications in any of the variants mentioned above; transforming a Bacillus strain with said mutated DNA; selecting strains that produce said protease variants; fermentation / growth of such strain; recovery of said protease variant, and tests for improved wash performance in detergents.