MXPA00001890A - 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 The present invention relates to novel mutant protease enzymes or enzyme variants useful in the formulation of detergent compositions and exhibiting improved performance in detergent washing; compositions of cleaners and detergents containing said enzymes; mutated genes that code for the expression of said enzymes when they are inserted into an appropriate host cell or organism; and said host cells transformed in this way and capable of manifesting said variants of the enzymes.

BACKGROUND OF THE INVENTION In the detergent industry, enzymes have been implemented for more than 30 years in formulations for washing. The enzymes used in said formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. The most important commercial enzymes are proteases.

An increasing number of commercially used proteases are designed variants of protease types of proteins REF .: 32789 that are produced naturally, for example DURAZYM® (Novo Nordisk A / S), RELASE® (Novo Nordisk A / S), MAXAPEM ® (Gist-Brocades NV), PURAFECT® (Genencor International, Inc).

Additionally numerous protease variants are mentioned in the art, such as in EP 130756 (GENENTECH) (corresponding to the reissited North American Patent No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht (1985) -Vature 318 375-376; Thomas, Russell, and Fersht (1987) J. Mol. Biol. 193 803-813; Russel and Fersht Na ture 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011 (PROCTER &GAMBLE COMPANY); WO 95/30010 (PROCTER &GAMBLE COMPANY); WO 95/29979 (PROCTER &GAMBLE); US 5,543,302 (SOLVAY S.A.); EP 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK A / S); WO 91/00345 (NOVO NORDISK A / S); EP 525 610 Al (SOLVAY); WO 94/02618 (GIST-BROCADES N.V.); and WO 96/34946 (NOVO NORDISK A / S).

However, although numerous useful protease variants have been described, there is still a need for new and improved protease variants for numerous industrial uses.

Therefore, an object of the present invention is to provide designed variants of improved protein protease, especially for use in the detergent industry.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have intensively studied numerous of the possible combinations of V95 residues, G97 and A98 from SAVINASE®, and identified a number of variants with an improved increase in wash performance.

For further details reference is made here to working examples (see below).

Accordingly, the present invention relates in its first aspect to a subtylase protease variant which has improved performance in washing in detergents, comprising the modification (s) in the (s) position (s). , 97 and / or 98.

In a second aspect the invention relates to a variant of subtylase enzyme which has an improved performance in detergent washing, comprising at least one modification selected from the group comprising: 95T + 97T + 98E 97E + 98V; or a variant comprising one or more conservative modifications in any of the above-mentioned variants (for example a moderate modification of a variant of 97E (a.a.a. acid) + 98V includes a variant such as 97D (a.a.a. acid) + 98V.).

In a third aspect, the invention relates to an isolated sequence of the DNA encoding a variant of the invention.

In a fourth aspect the invention relates to an expression vector comprising an isolated sequence of the DNA encoding a subtyla variant of the invention.

In a fifth aspect the invention relates to the production of subtilisin enzymes of the invention by inserting an expression vector according to the fourth aspect within a microbial host, cultivating the host to manifest the desired subtylase enzyme, and recovering the product enzyme.

Although the invention relates to a composition comprising a subtyla variant of the invention.

Finally the invention relates to the use of a mutant enzyme for a number of industrial uses, in particular for use in cleaning compositions and cleaning compositions comprising mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes.

DEFINITIONS Before discussing this invention with additional details, the following terms must first be defined.

Amino acid nomenclature A = Ala Alaniña V = Val Valine L = Leu = Leucine I lie Isoleucine P Pro Proline F Phe Phenylalanine W Trp Triptophan M Met Methionine G Gly Glycine S Serine Thr Threonine c Cys Cystine And Tyr Tyrosine N Asn Asparagine Q Gln Glutamine D Asp Acid Aspartic E Glu Glutamic Acid K Lys Lysine R Arg Arginine H His Histidine X Xaa Any Amino Acid Nomenclature of nucleic acids A = Adenine G = Guanine C Cytosine T Thymine (only in DNA) U Uracil (only in RNA) Nomenclature of variants To describe the various variants of enzymes produced or contemplated according to the invention, the following nomenclature has been adapted for ease of reference: Original position (s) of amino acid (s) amino acid (s) substituted (s) Accordingly the substitution of glutamic acid by glycine at position 195 is designated as: GLy 195 Glu or G195E an elimination of glycine in the same position is: Gly 195 * or G195 * and the insertion of an amino acid residue such as lysine is: GLy 195 GLyLys or G195GK Where a deletion compared to the sequence used for numbering is indicated, an insertion in such a position is indicated as: * 36 Asp or * 36D for the insertion of an aspartic acid at position 36 Multiple mutations are separated by more, ie: Arg 170 Tyr + Gly 195 Glu or R170Y + G195E representing mutations at positions 170 and 195 replacing tyrosine and glutamic acid with arginine and glycine, respectively.

Proteases The enzymes that divide the amide bond into protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms, W.H. Freeman and Company, San Francisco, Chapter 3).

Numeration of amino acid / residue positions If not mentioned otherwise, the numbering used here corresponds to the subtyle sequence BPN '(BASBPN). For an additional description of the BPN sequence 'see Siezen et al., Protein Engng. 4 (1991) 719-737 and Figure 1.

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

The bacterial serine proteases have molecular weights in the range of 20.00 to 45,000 Daltons. They are inhibited by diidopropylfluorophosphate. They hydrolyse simple terminal esters and are similar in activity to eukaryotic chymotrypsin, which is also a serine protease. A nearer term, alkaline protease, which covers a subgroup, reflects the high pH optimum of some of the serine proteases, from a pH of 9.0 to 11.0 (for review, see Priest (1977) Bacterioligical Rev. 41 711-753).

Subtilases A subgroup of serine proteases tentatively designated as subtilases have been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as sub-ilysin type proteases. A subtilisin was previously defined as a serine protease produced by Gram positive bacteria or fungi, and according to Siezen et al., It is now a subtylase group. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. Reference is made here to Siezen et al. And to FIG. 1 for a more detailed description of such subtilases and their amino acid sequences.

A subgroup of these 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 IS2 is recognized by Siezen et al. (Supra). The subset of proteases I-S2 are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), Subtilisin 309 (SAVINASE®, NOVO DORDISK A / S), subtilisin 147 (ESPERASE®, NOVO NORDISK A / S), and alkaline elastase YaB.

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

Original subtyla The term "original subtylase" is a subtyla defined according to Siezen et al. (Protein Engineering 4: 719-737 (1991)). For more details see the description of "SUBTILASAS" immediately above. Also, an original subtylase may be an isolated subtyla from a natural source, where subsequent modification has been made while the characteristic of a subtyla remains. Alternatively, the term "original subtylase" may be referred to as "subtylae of the naturally occurring type".

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

Subtylase variant In the context of this invention, the term variant subtylase or mutated subtylase means a subtyla that has been produced by an organism that manifests a mutant gene derived from an original microorganism that possessed an original or parent gene and which produced a corresponding original enzyme, the original gene being mutated in order to produce the mutant gene from which said mutated subtylase protease is produced when it manifests in an appropriate host.

Homologous Subtylase Sequences SAVINASA® subtyla specific amino acid residues are identified herein by modification for obtaining a subtyla variant of the invention.

However, the invention is not limited to modifications of this particular subtilase, but extends to other original subtylas (natural state type), which have a primary structure homologous to that of SAVINASE®.

In order to identify other homologous subtilases, within the scope of this invention, an alignment of said subtylase (s) is performed to a group of previously aligned subtilases maintaining the previous alignment constant. A comparison is made to 18 highly conserved residues in subtilases. The 18 highly conserved residues are shown in Table I (see Siezen et al. For more details regarding such conserved residues).

Table I 18 highly conserved residues in subtilases Position: Residue Conserved 23 G 32 D 34 G 39 H 64 H 65 G 66 T 70 G 83 G 125 S 127 G 146 G 154 G 155 N 219 G 220 T 221 S 225 P After the alignment, the allowances for the necessary insertions and eliminations are identified in order to maintain the proper alignment of homologous residues. Said homologous residues can be modified according to the invention.

Using the computer alignment program CLUSTALW (version 1.5, April 1995) (Thompson, JD, Higgins, DG and Gibson, TJ (1994) Nucleic Acids Research, 22: 4673-4680.), With an open penalty GAP of 10.0 and a GAP with an extension penalty of 0.1, using the BLOSUM30 protein weight matrix, the alignment of a given subtilas to a group of previously aligned subtilases is done using the option of the Alignment Profile of the program. For a given subtyla that is within the scope of the invention, preferably 100% of the 18 highly conserved residues should be retained. However, alignment of more than or equal to 17 of the 18 residues, or as little as 16 of such conserved residues is also adequate to identify homologous residues. Conservation should be maintained in the subtilases of the catalytic trio Asp32 / His64 / Ser221.

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

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

It is obvious that a similar or larger table covering other homologous subtilases can easily be produced by a person skilled in the art.

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

Performance in washing The ability of the enzyme to catalyze the degradation of several substrates of natural origin present in the objects that are going to be cleaned by lasting, for example, washing, often refers to its washing ability, washability, detergency, or performance in washing. Throughout this application the term wash performance will be used to include this property.

Isolated Sequence of DNA The term "isolated", when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic environment and is therefore free of other foreign or undesirable coding sequences, and is in a form suitable for use in protein production systems by genetic engineering. Such isolated molecules are those that are separated from the natural environment and include cDNA and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include non-translated regions of 5 'and 3' natural origin such as promoters and terminators. The identification of associated regions may be apparent to someone with common experience in the art (see for example, Dynany Tijan, Nature 316: 774-78, 1985). The term "an isolated DNA sequence" can alternatively be termed "a cloned DNA sequence".

Isolated protein When applied to a protein, the term "isolated" indicates that the protein is in a condition different from that of its native 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 preferred higher at 99% purity, as determined by SDS-PAGE. The term "isolated protein" can be referred to alternatively by the term "purified protein".

Homologous impurities The term "homologous impurities" means any impurity (eg, a polypeptide other than the polypeptide of the invention) which originates from the homologous cell from which the polypeptide of the invention is originally obtained.

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

Substrate The term "substrate" used in connection with a substrate for a protease should be construed in its broadest form as comprising a compound that contains at least one peptide ligation capable of being hydrolyzed by a protease subtilisin.

Product The term "product" used in connection with a product derived from the enzymatic reaction of a protease should be interpreted in the context of this invention, to be interpreted to include the products of a hydrolysis reaction involving a subtylase protease. A product can be the substrate in a subsequent reaction-hydrolysis.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows an alignment of a number of homologous subtilases, which are aligned with 18 highly conserved residues in subtilases. The 18 highly conserved residues are highlighted in bold. All show subtilases, except JP170, having 100% identity in said specific residues. JP170 has an "N" instead of a "G" in conserved residues G146.

DETAILED DESCRIPTION OF THE INVENTION Subtilase variants with improved wash performance The present inventors have identified variants with improved wash performance in BLS309 (SAVINASE®).

Accordingly, one aspect of the invention relates to a subtyla enzyme variant, wherein the modification is selected from the group comprising: V95T + G97T + A98E G97E + A98V; or a variant comprising one or more moderate modifications in any of the above-mentioned variants (eg, a moderate modification of a variant G97E (a.a.a. acid) + A98V includes a variant such as G97D (a.a.a. acid) + A98V.).

Numerous subtilase variants of the invention are tested here, showing improved wash 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 moderate amino acid only gives a small change in the characteristic of the enzyme.

Table III below lists groups of moderate amino acids.

TABLE III Moderate amino acid substitutes Basic: arginine lysine histidine Acidic: asymptotic acid, glutamine, glutamine, leucine, isoleucine valine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine Accordingly, subtylase variants such as 97E + 98V and 97D + 98V will have a similar improved wash performance.

In addition, subtylase variants such as G97E + A98V and G97D + A98V will also have similar improved wash performance.

Based on the present description of subtilase variants, for a person skilled in the art, it is routine work to identify additional appropriate moderate substitutions in order to obtain variants of subtilases with improved washing performance.

In aspects of the invention, the subtilases of interest are those that belong to subgroups I-SI and I-S2.

In relation to subgroup I - A preferred original subtylase is selected from the group comprising ABSS168, BASBPN, BSSDY, and BLDCAR or their functional variants which retain the characteristic of subgroup I-SI.

In relation to subgroup I-S2 a preferred original subtylase is selected from the group comprising BLS147, BLS309, BAPB92, TVTHER and BYSYAB or their functional variants which retain the characteristic of subgroup I-S2.

The present invention also comprises one or more modifications in the above mentioned positions in combination with any other modification, to the amino acid sequence of the original enzyme. Especially combinations with other modifications known in the art are considered to provide improved properties to the enzyme. The art describes a number of subtilase variants with improved properties and several of those are mentioned in the "Background of the Invention" section (see above). These references are described herein as references for identifying a subtyla variant, which may advantageously be combined with a subtyla variant of the invention.

Such combinations comprise the positions: 222 (improved stability to oxidation), 218 (improved thermal stability), substitutions at Ca binding sites that stabilize the enzyme, for example, position 76, and many others that appear in the known art.

In additional aspects a subtyla variant of the invention can be advantageously combined with one or more modifications at any of the positions: 27, 36, 57, 76, 97, 101, 104, 120, 123, 167, 170, 206, 218, 222, 224, 235 and 274.

Specifically the variants BLS309 and BAPB92 are considered suitable for 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, the 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), combination with any one of one or more of the modifications mentioned above exhibit improved properties.

Still further, the subtilase variants of the main aspects of the invention are preferably combined with one or more modifications in any of positions 129, 131, 133 and 194, modifications preferably as 129K, 131H, 133D and 194P, and with greater preferences modifications such as P129K, P131H, A133H, A133P, A133D and A194P. Any of these modifications may give a higher level of expression of a subtyla variant of the invention.

PRODUCTION OF MUTILATIONS IN SUBTILASE GENES Many methods for the cloning of a subtyla of the invention and for the introduction of mutations in genes (for example, subtyla genes) are well known in the art.

In general, standard procedures for gene cloning and the introduction of mutations (randomized and / or site-directed) into said genes can be used in order to obtain a subtyla variant of the invention. For further descriptions of convenient techniques reference is made here to working examples (see below) and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab-, Cold Spring Harbor, NY; Ausubel F. M. et al. (Eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C.R., and Cutting, S.M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990); and WO 96/34946.

EXPRESSION VECTORS A recombinant expression vector comprising a DNA construct encoding the enzyme of the invention can be any vector that can conveniently be subjected to recombinant DNA procedures, and the selection of the vector will often depend on the host cell in which will be introduced. Therefore, it can be a vector of autonomous replication, that is, a vector that exists as an extrachromosomal entity, the replica that is independent of the chromosomal replica, for example, a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, integrates into the genome of the host cell in part or in its entirety and replicates together with the chromosome (s) in which it has been integrated.

The vector is preferably an expression vector in which the coding of the DNA sequence for the enzyme of the invention is in operable form linked to additional segments required for DNA transcription. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "in operable linked form" indicates that the segments are arranged in such a way that they function together for the intended purposes, for example the transcription is initiated in a promoter and proceeds through the coding of the DNA sequence for the enzyme .

The promoter can be any DNA sequence that exhibits transcription activity in the selected host cell and can be derived from coding gene proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in host cells include the maltogenic amylase gene promoter Bacillus s tearothermophilus, the Bacillus licheniformi s alpha-amylase gene, the Bacill us amyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkaline protease gene, or the xylosidase gene Bacillus pumi li s, or promoters of fag Lambda PR or PL or promoters of E. coli lac, trp or tac.

The DNA sequence encoding the enzyme of the invention can also, if necessary, be operably connected to an appropriate terminator.

The recombination vector may further comprise a DNA sequence that allows the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, for example, a gene whose product complements a defect in the host cell, or a gene encoding a resistance to, for example, antibiotics such as kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycin, or similar, or resistance to heavy metals or herbicides.

To direct an enzyme of the present invention into the secretory pathway of host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre-sequence) must be provided in the recombinant vector. The secretory signal sequence binds with the DNA coding sequence of the enzyme in the correct reading frame. The secretory signal sequences are commonly positioned 5 'to the DNA coding sequence of the enzyme. The secretory signal sequence may be that normally associated with the enzymes or may be from a gene encoding another secreted protein.

The methods used to ligate the coding of DNA sequences for the present enzyme, the promoter and optionally the terminator and / or the secretory sequence, respectively, or to assemble these sequences by PCR amplification schemes, and to insert them into vectors containing the information necessary for the replication or integration are well known to persons skilled in the art (see, for example, Sambrook et al., in the cited work).

GUEST CELL The DNA sequence encoding the present enzyme introduced into the host cell can be either homologous or heterologous to the host in question. If it is homologous to the host cell, that is, produced by the host cell in nature, it will typically be operably linked to another promoter sequence or, if applicable, to another secretory signal sequence and / or terminator sequence, unlike when it is in its natural environment. The term "homologs" is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Therefore, the DNA sequence may come from another organism, or it may be a synthetic sequence.

The host cell wherein the DNA constructs or the recombinant vector of the invention is introduced can be any cell that is capable of producing the present enzyme and includes, bacteria, yeast, fungi, larger eukaryotic cells.

Examples of bacterial hosts that by culture are capable of producing the enzyme of the invention are gram-positive bacteria such as bacillus species, such as species B. Subtilis, B. Licheniformis, B. Lentus, B. Brevis, B. Stearothermophilus, B. Alkalophilus, B. Amyloliquefaciens, B. Coagilans, B. Circulans, B. Lautus, B. Megatherium or B. Thuriniensis, or Streptomyces species, such as S. Lividans or S. Murinus, or gram-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 (see Sambrook et al., Supra).

When the enzyme is expressed in bacteria such as E. Coli, the enzyme can be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or perhaps directed to the periplasmic space by a sequence of bacterial secretion. In the previous case, the cells are lysed and the granules are recovered and denatured, after which the enzyme is homogenized by dilution with the denaturing agent. In the latter case, the enzyme can be recovered from the periplasmic space by breaking the cells, for example, by sonic or osmotic shock, to release the content of the periplasmic space and recover the enzyme.

When the enzyme is expressed in gram-positive bacteria such as bacilli or Streptomyces species, 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 FOR PRODUCING SUBTILASE The present invention provides a method for the production of an isolated enzyme according to the invention, wherein an appropriate 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 result in the heterologous recombinant production of the enzymes of the invention.

For this reason it is possible to produce a highly purified subtyla composition characterized by being free of homologous impurities.

In this context, homologous impurities mean any impurity (for example, polypeptides different from the enzymes of the invention) that originate from the homologous cell from which the enzyme of the invention is obtained.

The medium used to cultivate the transformed host cells can be any conventional means suitable for the culture of the host cell in question. The expressed subtylase can conveniently be secreted into the culture medium and can be recovered there by well known methods including the separation of cells by centrifugation or filtration, precipitation of proteinaceous components from the medium through a salt such as ammonium sulfate, followed by chromatographic such as ion exchange chromatography, affinity chromatography, or the like.

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

In addition, the invention relates to a composition of an enzyme comprising a subtyla variant of the invention.

A summary of preferred industrial applications and corresponding compositions of preferred enzymes are described below.

This summary is not intended in any way to be a complete list of appropriate applications of a subtyla variant of the invention. A subtyla variant of the invention can be used in other industrial applications known in the art which includes the use of protease, in particular of subtilasa.

COMPOSITIONS OF DETERGENTS COMPRISING MUTATING ENZYMES The present invention comprises the use of mutant enzymes of the invention in cleaning compositions and in detergents, and such compositions comprise the mutant subtilisin enzymes. Such cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO95 / 30011 for further descriptions of variants of compositions suitable for cleaning and detergents.

Additional references are made herein to working examples that show improvements in the washing function for numerous subtyla variants of the invention.

DESCRIPTION OF THE DETERGENT AND EXAMPLES Surfactant System The detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant may be selected from nonionic and / or anionic and / or cationic and / or ampholytic and / or zwitterionic surfactants and / or semipolar.

The surfactant is typically present at a level of 0.1% to 60% by weight.

The surfactant is preferably formulated to be compatible with the components of the enzymes present in the composition. In liquid or gel compositions the surfactant is most preferably formulated in a manner that promotes, or at least does not degrade, the stability of the enzyme in these compositions.

Preferred systems to be used in accordance with the present invention comprise as surfactant one or more of the nonionic and / or anionic surfactants described herein.

The polyethylene, polypropylene, and polybutylene oxides condensed from alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present invention, with condensates being preferred as polyethylene oxides. These compounds include the condensation products of the alkyl phenols having an alkyl group containing from 6 to 14 carbon atoms, preferably from 8 to 14 carbon atoms, either in a straight chain or branched chain configuration with the alkylene oxide . In a preferred aspect, the ethylene oxide is present in an amount equal to about 2 to about 25 moles, more preferably about 3 to about 15 moles, of ethylene oxide per alkyl phenol template. Commercially available nonionic surfactants of this type include Igepal ™ CO-630, marketed by the GAF Corporation; and Triton ™ X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (eg, alkyl phenol ethoxylates).

The condensation products of primary and secondary alcohols of about 1 to 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 may be linear or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. The condensation products of alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide are preferred. mol of alcohol. About 2 to about 7 moles of ethylene oxide and more preferably 2 to 5 moles of ethylene oxide per mole of alcohol are present from said product condensation. Examples of commercially available nonionic surfactants of this type include Tergitol ™ 15-S-9 (the linear alcohol condensation product Qu-Cis with 9 moles of ethylene oxide), Tergitol ™ 24 -L- 6 NMW (the product of condensation of primary alcohol C? -C? with 6 moles of ethylene oxide with a very narrow molecular weight distribution), both marketed by Union Carbide Corporation; Neodol ™ 45-9 (the linear alcohol condensation product C14-C15 with 9 moles of ethylene oxide), Neodol ™ 23-3 (the C12-C13 linear alcohol condensation product with 3.0 moles of ethylene oxide), Neodol ™ 45- (the linear alcohol condensation product C? 4-C15 with 7 moles of ethylene oxide), Neodolt 45-5 (the linear alcohol condensation product C? 4-C? 5 with 5 moles of oxide of ethylene) marketed by Shell Chemical Company, Kyro ™ EOB (the linear alcohol condensation product C13-C? 5 with 9 moles of ethylene oxide), marketed by Procter & Gamble Company, and Genapol LA 050 (the linear alcohol condensation product C? 2-C? With 5 moles of ethylene oxide) marketed by Hoechst. The preferred range of HLB is these products is 8-11 and more preferably 8-10.

Also useful as nonionic surfactants of the surfactant systems of the present invention are the alkylpolysaccharides described in US 4,565,647, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide. , for example, a polyglucoside, hydrophilic group containing from about 13 to about 10, preferably from about 1.3 to about 3, more preferably from about 1.3 to about 2.7 units of saccharides. Any reducing saccharide containing 5 or 6 carbon atoms can be used, for example, portions of glucose, galactose and galactosyl can be substituted by glucosyl portions (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions). Thus giving a glucose or galactose as opposed to a glycoside or galactoside). The intersaccharide linkages can be, for example, between the position of the additional saccharide unit and positions 2-, 3-, 4-, and / or 6- in the preceding saccharide units.

Preferred alkyl polyglycosides have the formula R20 (CnH2nO) t (glycosyl) x Wherein R2 is selected from the group consisting of alkyls, alkylphenyls, hydroxyalkyls, hydroxyalkyl phenyls, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, 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, more preferably from about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol alkylpolyethoxy alcohol is first formed and then reacted with glucose, or a source of glucose, to form the glucoside (linkage at position 1-). The additional glycosyl units can then be linked between their 1- position and the positions of the preceding glycosyl units 2-, 3-, 4-, and / or 6-, preferably and predominantly at the 2- position.

The condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as additional non-ionic surfactant systems of the present invention. The hydrophobic portion of these compounds should preferably have a molecular weight of about 1500 to about 1800 and should exhibit insolubility to water. The addition of polyoxyethylene portions to this hydrophobic portion tends to increase the water solubility of the entire molecule, and the liquid character of the product is retained to the point where the polyoxyethylene content is about 50% of the total weight of the product of condensation, which corresponds to the condensation of up to 40 moles of ethylene oxide. Examples of compounds of this type are certain of the commercially available surfactants such as Pluronic ™, marketed by BASF.

Also suitable for use as nonionic surfactant systems of the present invention are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylene diamine. The hydrophobic portion of these products consists of the product of the reaction of ethylene diamine and excess propylene oxide, and generally has a molecular weight of about 2500 to about 3000. This hydrophobic portion is condensed with ethylene oxide to the extent that the condensation product contains from 40% to 80% by weight of polyoxyethylene and has a molecular weight of about 11,000. Examples of these types of nonionic surfactants include certain commercially available compounds such as Tetronic ™, marketed by BASF.

Preferred for use as the non-ionic surfactant of the surfactant systems of the present invention are the polyethylene oxide condensates of alkylphenols, condensation products of primary and secondary aliphatic alcohols with about 1 to 25 moles of ethylene oxide, alkyl polysaccharides, and its mixtures Most preferred is C8-C alkyl? ethoxylated phenols with 3 to 15 ethoxy groups and ethoxylated C8-C18 alcohols (preferably average C02) with 2 to 10 ethoxy groups, and mixtures thereof.

The highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the formula: R * N // ¡I R1 where R1 is H, or R1 is a C ^, 2 -hydroxyethyl, 2-hydroxyethyl, 2-hydroxypropyl hydrocarbyl or a mixture thereof, R2 is a C5-C31 hydrocarbyl and Z is a polyhydroxyhydrocarbyl with a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or their alkoxylated derivatives. Preferably R1 is methyl, R2 is a linear C ??-15 alkyl or Ciß-iß alkyl or an alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction.

Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants. Examples of these are water-soluble salts or acids of the formula RO (A) mS03M where R is an unsubstituted C? 0-C 24 alkyl or hydroxyalkyl group containing a C? 0-C2 alkyl component, preferably a C12-C2a alkyl or hydroxyalkyl, more preferably an alkyl or hydroalkyl CL2-C? 8, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 to about 6, with more preference between 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.), an ammonium or substituted ammonium cation. Also contemplated here are alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates. Specific examples of substituted ammonium cations include methyl- cations. dimethyl-, trimethyl-ammonium and quaternary ammonium cations such as tetramethyl-ammonium cations and dimethyl-piperidinium and those derivatives of alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like. Examples of surfactants are polyethoxylate sulfate (1.0) of C 1 -C 18 alkyl (Ci 2 -C 8 E (1.0) M), polyethoxylate sulfate (2 25) of C 12 -C 18 alkyl (C 12 -C 8 E (2.25) M), and polyethoxylate sulfate (3.0) of C ?2-C18 alkyl (C ?2-C ?8E (3.0) M), and polyethoxylate sulfate (4.0) of C12-C ?8 alkyl (d2-C ?8E (4.0) M) , where M is conveniently selected from sodium and potassium.

Anionic surfactants suitable for use are surfactants of alkyl ester sulfonates including esters of C8-C2o carboxylic acids (eg, fatty acids) which are sulfonated are gaseous S03 according to "The Journal of the American Oil Chemists Society", 52 ( 1975), pp. 323-329. Suitable starting materials could include natural fatty substances such as those derived from animal fat, palm oil, etc.

The alkyl ester sulfonate surfactants, especially for laundry applications, comprise alkyl ester sulfonate surfactants of the structural formula: O L RJ CH - C - OR4 S03M where R is a C8-C20 hydrocarbyl, preferably an alkyl, or combinations thereof, R4 is a C? -C6 hydrocarbyl, preferably an alkyl, or combinations thereof, and M is a cation forming a water soluble salt with the sulfonate of alkyl ester. Suitable salt formation cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethanolamine, and triethanolamine. Preferably R3 is a C 0 -C 0 alkyl, and R 4 is methyl, ethyl or isopropyl. Especially preferred are the methyl ester sulfonates wherein R3 is a C? 0-C? S alkyl? Other suitable anionic surfactants include alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO 3 M wherein R is preferably a C 0 -C 24 hydrocarbyl, preferably an alkyl or hydroalkyl having a C 10 -C 20 component, more preferably a C 2 -C 8 alkyl or hydroalkyl, and M is H or a cation, for example, an alkali metal cation (eg, sodium, potassium, lithium), or ammonium or substituted ammonium (eg, methyl cations) -, dimethyl-, and trimethylammonium and quaternary amionium cations such as tetramethyl ammonium and dimethyl piperidinium cations and ammonium cations derived from alkylamines such as ethyleneamine, diethylamine, triethylamine, and mixtures thereof, and the like). Typically, 2-6C-6 alkyl chains are preferred for low wash temperatures (eg, below 50 ° C) and C ?6-C18 alkyl chains for high wash temperatures (eg, above 50 ° C). ).

Other useful surfactants for detersive purposes can also be included in the laundry detergent compositions of the present invention. These may include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono-, di-, and triethanolamine) of soap, C8-C22 primary or secondary alkanesulfonates, C8-C24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline rare earth metal citrates, for example, as described in the British patent specification No. 1,082,179, C8-C24 alkyl polyglycol ether sulphates (containing up to 10 moles of ethyl acid); alkyl glycerol sulfonates, acyl glycerol fatty sulphonates, oleyl glycerol fatty sulfates, ethylene alkyl alkyl phenol ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as acyl isethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated C 12 -C 8 monoesters) and sulfosuccinate diesters (especially saturated and unsaturated C 6 -C 2 diesters), acyl sarcosinates, alkylpolysaccharide sulfates such as alkyl polyglycoside sulfates (the non-sulfated nonionic compounds which are described below), primary branched alkyl sulfates, and alkyl polyethoxy carbocylates such as those of the formula RO (CH2CH20) k-CH2C00-M + where R is a C8-C22 alkyl, k is an integer from 1 to 10, and M is a cation of soluble salt formation. Also suitable are resinous acids, hydrogenated resinous acids and resinous acids, hydrogenated resinous acids present in or derived from animal fat oil.

Alkylbenzene sulfonates are highly preferred. Alkyl (linear chain) benzene (LAS) sulfonates are especially preferred where the alkyl group preferably contains from 10 to 18 carbon atoms.

Additional examples are described in "Surface Active Agents and Detergents" (Vol. I and II by Schwartz, Perry and Berch). A variety of such surfactants are also generally described in US 3,929,678 (Column 23, line 58 to column 29, line 23, incorporated herein by reference).

When included, the laundry detergent compositions of the present invention typically comprise from 1% to 40%, preferably from 3% to 20% by weight, of said anionic surfactants.

The laundry detergent compositions of the present invention may contain cationic, ampholytic, zwitterionic and semi-polar surfactants, as well as non-ionic and / or anionic surfactants other than those described herein.

The cationic detersive surfactants suitable for use as laundry detergent compositions of the present invention are those having a long chain hydrocarbyl groups. Examples of such cationic surfactants include ammonium surfactants such as alkyltrimethylammonium halides, and those surfactants having the formula: [R2 (OR3)] and] [R4 (OR3) and] 2R5N + X- where R2 is an alkyl group or benzyl alkyl having about 8 to 18 carbon atoms in the alkyl chain, each R3 is selected from the group consisting of -CH2CH2-, -CH2CH (CH3) - -CH2CH (CH2OH) -, -CH2CH2CH2-, and mixtures thereof; each R 4 is selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 4 hydroalkyl benzyl ring structures formed by joining two R 4 groups, -CH 2 CHOHCHOHCOR 6CHOHCH 2 OH, where Re is any hexose or hexose polymer having a lower molecular weight of about 1000, and hydrogen when it is not 0; R5 is the same as R4 or is an alkyl chain, where the total number of carbon atoms or R2 plus R5 is not more than 18; each y is from 0 to about 10, and the sum of the values of y from 0 to about 15; and x is any compatible cation.

The highly preferred cationic surfactants in the present composition are the water-soluble quaternary ammonium compounds having the formula: R1R2R3R4N + X "(i) where R is a C8-C6alkyl, each R2, R3 and R is independently a C?-C4 alkyl / C?-C4 hydroalkyl, benzyl, and (C2H40) xH where x has a value of 2 to 5, and x is an anion. No more than one R2, R3 or R4 must be benzyl.

The preferred length of the alkyl chain for Ri is C 12 -C 15, particularly where the alkyl group is a mixture of chain lengths derived from coconut or grain fat or is derived synthetically by olefin construction or synthesis of 0X0 alcohols.

Preferred groups for R2R3 and R4 are methyl and hydroxyethyl groups and the anion X can be selected from the halide, metasulfate, acetate and phosphate ions.

Examples of quaternary ammonium compounds of the formula (i) for use herein are: coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C12-15 hydroxyethyl ammonium dimethyl chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; Methyl Trimethyl Ammonium Methyl Sulfate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide; colino esters (compounds of the formula (i) where R ± is alkyl CH2-CH2-0-C-C12_? 4 and R2R3R4 are methyl) L O dialkyl imidazolines [compounds of the formula (i)] Other cationic surfactants useful herein are also described in US 4,228,044 and in EP 000 224.

When included, the laundry detergent compositions of the present invention typically comprise from 0.2% to 25%, preferably from 1% to 8% by weight, of such cationic surfactants.

Ampholytic surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives or secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary or tertiary amines in which the aliphatic radical can be linear or branched, One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains 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, the laundry detergent compositions of the present invention typically comprise of 0. 2% to 15%, preferably 1% to 19% by weight of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in laundry detergent compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or quaternary ammonium derivatives, quaternary phosphonium compounds or tertiary sulfonium compounds. See US 3,929,678 (column 19, line 38 to column 22, line 48) for example of zwitterionic surfactants.

When included, the laundry detergent compositions of the present invention typically comprise of 0. 2% to 15%, preferably from 1% to 10% by weight of such zwitterionic surfactants.

Semi-polar non-ionic surfactants with a special category of non-ionic surfactants including water-soluble amine oxides containing portions of about 10 to about 18 carbon atoms and 2 selected portions of the group consisting of alkyl groups and hydroxyalkyl groups containing 1 at about 3 carbon atoms; soluble phosphine oxides containing water an alkyl moiety of about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety of about 10 to about 18 carbon atoms and a selected portion of the group consisting of alkyl and hydroxyalkyl portions 1 one 3 carbon atoms.

The non-ionic semipolar detergent surfactants include the amine oxide surfactants having the formula: O t R3 (OR) xN (R5) 2 where R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms; R4 is an alkylene or hydroalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof; x 0 to about 3: and each R5 is an alkyl group or hydroxyalkyl containing 1 to about 3 carbon atoms or a group of polyethylene oxide containing 1 to about 3 ethylene oxide groups. The R5 groups can be linked to one another, for example, through an on or nitrogen atom, to form a ring structure.

These surfactants include amine oxides including oxides of C? 0-C? 8 dimethyl amine oxides and C8-Ci2 alkoxy ethyl dihydroxy ethyl amine.

When included, the compositions of laundry detergent of the present invention typically comprise from 0.2% to 15%, preferably 1% to 10% by weight of such semipolar nonionic surfactants.

Construction system The compositions according to the present invention may additionally comprise a construction system. Any construction system is suitable for use here including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylene diamine tetraacetate, metal ion sequestrants such as tetramethylene phosphonic acid and diethylene triamine pentamethylene phosphonic acid. Although they are less preferred for environmental reasons, phosphate builders can also be used here.

Suitable builders may be inorganic ion exchange materials, commonly an inorganic hydrated aluminosilicate material, more particularly a synthetic hydrated zeolite such as a hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic builder material is layered silicate, for example, SKS-6 (Hoechst). SKS-6 is a layered silicate consisting of sodium silicate (Na2Si205).

Suitable polycarboxylates containing a carboxy group include lactic acid, glycolic acid and its ether derivatives as described in Belgian Patents Nos. 831 368, 821 369 and 821 370. Polycarboxylates containing two carboxy groups include water-soluble salts of succinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycolic acid, tartaric acid, tartronic acid and fumaric acid, as well as ether carboxylates described in German application 2,446,686, and 2,446,487, US 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623. Polycarboxylates containing three carboxy groups include, in particular, water-soluble citrates, aconitrates and citraconates as well as succinate derivatives such as the carboxymethyloxysuccinates described in British Patent No. 1,379,241, Lactoxysuccinates described in the Dutch Solicitus 7205873, and oxypolycarboxylates such as the tricarboxylates 2-oxa-l, 1,3-propane described in British Patent No. 1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinates described in British Patent No. 1,261,829, 1, 1,2, 2-ethane tetracarboxylates, 1, 1, 3, 3, -propane carboxylates containing sulfo substituents include the derivatives of sulfosuccinates described in British Patent Nos. 1,389,421 and 1,398,422 and in US 3,936,448, and the sulfonated pyrolysed citrates described in British Patent No. 1,082,179, while polycarboxylates containing phosphonic substituents are described in British Patent 1,439,000.

The alicyclic and heterocyclic polycarboxylates include cyclopentane-cis, cis-cis-tetracarboxylates, cyclopentadienido pentacarboxylates, 2,3,4,5, -tetrahydrofuran-cis, cis, cis-tetracarboxylates, 2,5, -tetrahydro-furan-cis, discarboxylates , 2,2,5,5-tetrahydrofuran-tetracarboxylates, 1,2,3,4,5,6-hexane-hexacarboxylates and carboxylmethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylotol. Aromatic polycarboxylates include melific acid, pyromellitic acid and the italic acid derivatives described in British Patent No. 1,425,343.

Of the above, preferred polycarboxylates are hydrocarboxylates containing up to three carboxy groups per molecule, more particularly citrates.

Preferred builder systems for use in the present compositions include a mixture of water insoluble aliminosilicate builder, such as zeolite A or layered silicate (SKS-6), and a water soluble carboxylate chelating agent, such as citric acid .

A suitable chelator for inclusion in the detergent compositions according to the present invention is ethylenediamine-N, N'-disuccinic acid (EDDS) the alkali metal, alkaline earth metal, ammonium, or substituted ammonium salts, or mixtures thereof. The preferred EDDS compounds are those of acid free form and their sodium or magnesium salts. Examples of such preferred EDDS sodium salts include Na2EDDS and NaEDDS. Examples of such preferred EDDS magnesium salts include MgEDDS and Mg2 EDDS. Magnesium salts are most preferred to be included in the compositions according to the invention.

Preferred cosurizing 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 the co-polymeric acids and their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from one another by not more than two carbon atoms.

Polymers of this type are described in GB-A-1,596,756. Examples of such salts are polyacrylates of molecular weight of 2000-500 and their copolymers with maleic anhydride, such copolymers having a molecular weight of about 20,000 to 70,000, especially about 40,000.

Builder salts are usually included in amounts of 5% to 80% by weight of the composition. The preferred levels of builders for liquid detergents are from 5% to 30%.

Enzymes Preferred detergent compositions, in addition to the preparation of enzymes of the invention, comprise other enzyme (s) that provide the cleaning function and / or benefits in the care of fabrics.

Such enzymes include other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g., laccases).

Proteases: Any other protease suitable for use in alkaline solutions can be used. Suitable proteases include those of animal origin, vegetable or microbial. The microbial origin is preferred. Chemically or genetically modified mutants are included. The protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacilli, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (for example of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.

Preferred commercially available protease enzymes include those sold under the tradenames Alcalase, Savinasa, Primasa, Durazym, and Esperase from Novo Nordisk A / S (Denmark), those sold under the tradename Maxatasam Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the trade name Opticlean and Optimase by Solvay Enzimes. Protease enzymes can be incorporated in the compositions according to the invention at a level of 0.00001% to 2% of the enzyme protein by weight of the composition, preferably at a level of 0.0001% to 1% of enzyme protein per weight of composition , more preferably at a level of 0.001% to 0.5% of the enzyme protein of the composition, even more preferably at a level of 0.01% to 0.2% enzyme protein by weight of the composition.

Lipases: Any suitable lipase can be used for its use in alkaline solutions. Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.

Examples of useful lipases include a Humicola lanuginosa lipase, for example, as described in EP 258 068 and EP 238 023, a Rhizomucor miehei lipase, for example, as described in EP 238 023, a Candida lipase, such as a C lipase. Antarctic, for example, the A or B Acarbatic lipase described in EP 214 761, a Pseudomonas lipase, for example, as described in EP 218 272, a lipase P, cepacia, for example as described in EP 331 376 , a lipase P. stutzeri, for example as described in GB 1,372,034, a lipase P. fluorescens, a lipase Bacillus, for example, a lipase B. Subtilisa (Dartois et al., (1993), Biochemica et Biophysica acta 1131, 253 -260), a lipase B. stearothermophilus (JP 64/744992) and a lipase B. pumilus (WO 91/16422).

In addition, several cloned lipases may be useful, including Penicillium camenbertii lipase described by Yamaguchi et al. (1991), Gene 103, 61-67, the lipase Geotricum candidum (Schimada, Y. et al., (1989), J.

Biochem. , 106, 383-388), and several Rhizopus lipases such as a R. delemar lipase (Hass, MJ et al., (1991), Gene 109, 117-113), a R. niveus lipase (Kugimiya et al., (1992 ), Biosci, Biotech, Biochem 56, 716-719) and a R. oryzae lipase.

Other types of lipolytic enzymes, such as cutinases, for example, a cutinase derived from Pseudomonas mendocin as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (eg, described in WO 90/09446) may also be useful. ).

Especially suitable lipases are lipases such as Ml Li «paseTM, Luma FastTM and Li • pomaxTM (/ Genencor \), Li > poilaseTM and Lipolase Ultra ™ (Novo Nordisk A / S), and Lipase P "Amano" (Amano Pharmaceutical Co. Ltd.).

Lipases are normally incorporated into the detergent composition at a level of 0.00001% to 2% protein enzyme by weight of the composition, preferably at a level of 0.0001% to 1% of the protein enzyme by weight of the composition, more preferably at a level of 0.001% to 0.5% protein enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein enzyme by weight of the composition.

Amylases: Any amylase (a and / or ß) suitable for use in alkaline solutions can be used. Appropriate amylases include those of bacterial or fungoid origin. Chemically or genetically modified mutants are included. Amylases include, for example, α-amylases obtained from a special strain of B. licheniformis, described in more detail in GB 1,296,839. The commercially available amylases are Duramyl ™, Termamyl ™, Fungamyl ™, and BAN ™ (available from Novo Nordisk A / S) and Rapidase ™ and Maxamyl P ™ (available from Genencor).

The amylases incorporated are generally incorporated into the detergent composition at a level of 0.00001% to 2% protein enzyme by weight of the composition, preferably at a level of 0.0001% to 1% of the enzyme protein by weight of the composition, more preferably at a level of 0.001% to 0.5% protein enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein enzyme by weight of the composition.

Cellulases: Any appropriate cellulase can be used for use in alkaline solutions. Suitable celluloses include those of bacterial and fungal origin. Chemically or genetically modified mutants are included. Appropriate cellulases are described in US 4,435,307, which describes fungoid cellulases produced from Humicola insolens. Cellulases that have benefits in the care of color are especially appropriate. Examples of such cellulases are cellulases described in European Patent Application No. 0 495 257.

Commercially available cellulases include Celluzime ™ produced by a strain of Humicla insolens, (Novo Nordisk A / S), and KAC-500 (B) ™ (Kao Corporation).

The cellulases are normally incorporated in the detergent composition at a level of 0.00001% to 2% protein enzyme by weight of the composition, preferably at a level of 0.0001% to 1% of the protein enzyme by weight of the composition, more preferably at a level of 0.001% to 0.5% protein enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein enzyme by weight of the composition.

Peroxidases / Oxidases: Peroxidase enzymes are used in combination with hydrogen peroxide or one of its sources (for example, a percarbonate, perborate or persulfate). Oxidases enzymes are used in combination with oxygen. Both types of enzymes are used for "bleaching solutions", ie to prevent the transfer of a textile dye from a colored fabric to another fabric when said fabrics are washed together in a wash liquor, preferably together with a highlighting agent as described above. described in, for example WO 94/12621 and WO 95/01426. Suitable peroxidases / oxidases include of plant, bacterial or fungoid origin. Chemically or genetically modified mutants are included.

The peroxidase and / or oxidase enzymes are usually incorporated in the detergent composition at a level of 0.00001% to 2% protein enzyme by weight of the composition, preferably at a level of 0.0001% to 1% of the enzyme protein by weight of the composition, more preferably at a level of 0.001% to 0.5% protein enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein enzyme by weight of the composition.

The mixtures of the enzymes mentioned above are included here, in particular a mixture of protease, an amylase, a lipase and / or a cellulase.

The enzyme of the invention, or any other enzyme incorporated in the detergent composition, is normally incorporated into the detergent composition at a level of 0.00001% to 2% protein enzyme by weight of the composition, preferably at a level of 0.0001% at 1% protein enzyme by weight of the composition, more preferably at a level of 0.001% to 0.5% protein enzyme by weight of the composition, even more preferably at a level of 0.01% to 0.2% protein enzyme by weight of the composition.

Bleaching agents: Additional optional detergent ingredients may be included in the detergent compositions of the present invention which include bleaching agents such as PB1, PB4 and percarbonate with a particle size of 40-800 microns. These bleaching agent components may include one or more oxygenated bleaching agents and, depending on the bleaching agent selected, one or more bleach activators.

When oxygenated bleaching compounds are present, they will typically be present at levels of 1% to 25%. In general, bleaching compounds are optional addition components in non-liquid formulations, for example, granular detergents.

The bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygenated bleach as well as others known in the art.

The bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.

A category of oxygenated bleaching agent that can be used includes percarboxylic acid bleaching agents and their salts. Acceptable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents are described 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-pxoperoxycaproic acid as described in US 4,634,551.

Another category of bleaching agents that can be used include halogenated bleaching agents. Examples of hypohalide bleaching agents, for example, include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulfonamides.

Said materials are normally added of 0.5-10% by weight of the finished product, preferably 1-5% by weight.

The hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzene sulfonate (NOBS, described in US 4,412,934), 3,5-trimethylhexsanoloxybenzene sulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG), which are perhydrolyzed to form a peracid as the active bleaching species, resulting in an improved bleaching effect. In addition, the bleach activators C8 (6-octanamido-caproyl) oxybenzene sulfonate, C9 (6-nonanamido caproyl) oxybenzene sulfonate and CIO (6-decanamido caproyl) oxybenzene sulfonate or mixtures thereof are very suitable. Acylated citrate esters are also suitable as activators as described in European Patent No. 91870207.7.

Bleaching agents, including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleach compounds for use in cleaning compositions according to the invention are described in USSN Application 08 / 136,626.

Hydrogen peroxide may be present by the addition of an enzymatic system (i.e., an enzyme and a substrate) that is capable of generating hydrogen peroxide at the start or during the washing and / or the rinsing process. Said enzymatic systems are described in European Patent Application EP 0 537 381.

Other bleaching agents other than oxygenated bleaching agents are also known in the art. One type of non-oxygenated bleaching agent of particular interest includes photoactivated bleaching agents such as zinc sulfonate and / or aluminum phthalocyanines. These materials can be deposited on the substrate during the washing process. With the irradiation of the light, in the presence of oxygen, such as in the hanging of clothes for drying with sunlight, the sulphonated zinc phthalocyanine is activated and, consequently, the substrate is bleached. Preferred zinc phthalocyanines and a photoactivity bleaching process are described in US 4,033,718. Typically, the detergent composition may contain 0.025% to 1.25% by weight of sulfonated zinc phthalocyanine.

The bleaching agents may also comprise a manganese catalyst.The manganese catalyst may, for example, be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pages 637-639.

Foam suppressors: Another optional ingredient is a foam suppressant, exemplified by silicone blends, and silica-silicone. Silicones can generally be represented by alkylated polysiloxane materials, while silica is usually used in finely divided forms exemplified by silica and silica xerogels and hydrophobic silica of various types. These materials can be incorporated as particles, in which the foam suppressant is advantageously incorporated in a water-soluble detergent support water-soluble, dispersible in water and substantially without surface activity. Alternatively, the foam suppressant may be dissolved or dispersed in a liquid carrier and applied by spraying on one or more of the other components.

A preferred silicone foam controlling agent is described in US 3,933,672. Other particularly useful foam suppressors are self-emulsifying silicone foam suppressors, described in the German Patent Application DTOS 2,646,126. An example of such a compound is DC-544, commercially available from Dow Corning, which is a siloxane-glycol copolymer. Especially preferred foam controlling agents are the suds suppressor systems comprising a mixture of silicone oils and 2-alkyl alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol which is commercially available under the trade name Isofol 12R.

Such foam suppressor systems are described in European Patent Application EO 0 593 841.

Especially preferred silicone foam controlling agents are described in European Patent Application No. 92201649.8. Said compositions may comprise a mixture of silicone / silica in combination with nonporous fuming silica such as Aerosil.RTM.

The aforementioned foam suppressors are normally employed at levels of 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.

Other components: Other components used in detergent compositions can be used as slime suspension agents, soil release agents, optical brighteners, abrasives, bactericides, discoloration inhibitors, coloring agents, and / or encapsulated and non-encapsulated perfumes.

Particularly suitable encapsulating materials are water-soluble capsules consisting of a matrix of polysaccharide and polyhydroxy compounds as described in GB 1,464,616.

Other suitable water-soluble encapsulating materials comprise dextrins derived from non-gelatinized starch acid esters of substituted dicarboxylic acids as described in US 3,455,838. These acid dextrin esters are preferably prepared from such starches as waxy maize, waxy sorghum, sago, tapioca and potato. Appropriate examples of such materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material consists of a modified corn starch and glucose. The starch is modified by the addition of substituted monofunctional groups such as octenyl succinic acid anhydride.

Suitable anti-redeposition and sludge suspension agents include cellulose derivatives such as methyl cellulose, carboxymethyl cellulose and hydroxymethyl cellulose, and homo- or co-polymeric polycarboxylic acids and their salts.

Polymers of this type include the polyacrylates and copolymers of maleic anhydride-acrylic acid previously mentioned as builders, as well as copolymers of maleic anhydride with ethylene, methylvinyl ether or methacrylic acid, the maleic anhydride constituting at least 20 mol% of the copolymer. These materials are normally used at levels of 0.5% to 10% by weight, more preferably from 0.75% to 8%, much more preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic in character, examples of which are 4,4'-bis- (2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2: 2'-disulfonate disodium, 4, 4 '-bis- (2-morpholino-4-anilino-s-triazino-6-ylamino-stilbene-2: 2'-disodium disodium, 4,4'-bis- (2,4-dianilino-s-triazino-6) -ylamino) stilbene-2: 2'-disodium disulfonate, 4'4"-bis- (2,4-dianilino-s-triazino-6-ylamino) stilbene-2-monosodium sulphonate, 4,4'-bis- (2-anilino-4- (N-methyl-N-2-hydroxyethylamino) -s-triazino-6-ylamino) stilbene-, 2'-disulfonate disodium, 4,4'-bis- (4-phenyl-2, 1,3-triazol-2-yl) -stilbene-2,2'-disulfonate disodium, 4,4'-bis (2-anilino-4- (1-methyl-2-hydroxyethylamino) -s-triazine-6-ylamino) stilbene-2, 2 'disodium disulfonate, 2 (stilbil-4"- (naphtho-1', 2 ': 4, 5) -1, 2, 3, -triazol-2" -sulfonate of sodium and 4, 4'-bis (2-sulphostiryl) biphenyl sodium.

Other useful polymeric materials are the polyethylene glycols, particularly those of molecular weight of 1000-10000, more particularly from 2000 to 8000 and more particularly of about 4000. They are used in levels of 0.20% to 5%, more preferably 0.25% to 2.5% by weight. These polymers and the previously mentioned homo- or co-polymeric polycarboxylate salts are valuable for the improvement of the maintenance of whiteness, the deposition of ash in the fabrics and the cleaning function on the earthy, protein and oxidizable earths in the presence of Transition metal impurities.

Land releasing agents useful in compositions of the present invention are conventionally copolymers or thermopolymers of terephthalic acid with ethylene glycol and / or propylene glycol units in various arrangements. Examples of such polymers are described in US 4,116,885 and 4,711,730 and EP 0 272 033. A particularly preferred polymer according to EP 0 272 033 has the formula: (CH3 (PEG) 43) 0.75 (POH) o.2s [T-PO) 2.8 (T-PEG) o.4] T (POH) 0.25 ((PEG) 43CH3) 0.75 where PEG is - (02H4) 0- , PO is (OC3H60) and T is (pOOC6H4CO) Modifying polyesters such as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1,2-propanediol are also very useful, the terminal groups consist mainly of sulfobenzoates and secondarily of monoesters of ethylene glycol and / or 1,2-propanediol. The objective is to obtain a polymer covered at both ends by sulfobenzoate groups, "mainly", here in the current context most of the copolymers will be covered at their ends by sulfobenzoate groups. However, some copolymers will be less than fully covered, and therefore their terminal groups may consist of ethylene glycol monoesters and / or 1,2-propanediol, therefore they consist "secondarily" of said species.

The polyesters selected here contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of 1,2-propanediol, about 10% by weight of ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about of 15% by weight of sulfoisophthalic acid, and have a molecular weight of about 3000. The polyesters and their method of preparation are described in EP 311 342.

Softening agents: Fabric softening agents may also be incorporated into laundry detergent compositions according to the present invention. These agents can be of the organic or inorganic type. Inorganic softening agents are exemplified with the smectite clays described in GB-A-1 400898 and in US 5,019,292. Organic softening agents include water-insoluble tertiary amines as described in GB-A-1 514 276 and in EP 0 011 340 and their combination with quaternary ammonium C C 2-C 14 mono salts described in EP-B-0 026 528 and amides with two long chains as described in EP 0 242 919. Other useful organic ingredients of fabric softening systems include high molecular weight polyethylene oxide materials as described in EP 0 299 575 and 0 313 146.

The smectite clay levels are usually in the range of 5% to 15%, more preferably from 8% to 12% by weight, the material being added as a dry component mixed to the remainder of the formulation. Fabric softening agents such as water-insoluble tertiary amines or long two-chain amide materials are incorporated at levels of 0.5 to 5% by weight, normally from 1% to 3% by weight while polyethylene oxide materials of High molecular weight and water-soluble cationic materials are added at levels from 0.1% to 2%, usually from 0.15% to 1.5% by weight. These materials are usually added to the portion of the spray dried composition, although in some cases it may be more convenient to add them as a dry mixed particle, or spray it as a molten liquid over the other solid components of the composition.

Polymeric color transfer inhibiting agents: In accordance with the present invention, detergent compositions can also comprise from 0.001% to 10%, preferably from 0.01% to 2%, more preferably from 0.05% to 1% by weight of agents polymeric inhibitors of color transfer. Said color transfer inhibiting polymeric agents are normally incorporated within the detergent compositions in order to inhibit the transfer of color on colored fabrics to the fabrics with which they are washed. These polymers have the ability to complex or adsorb fugitive dyes from washing of colored fabrics before the dyes have the opportunity to bind or other articles in the wash.

Especially suitable color transfer inhibiting polymeric agents are N-oxide polyamine polymers, copolymers of N-vinyl-pyrrolidone and N-vinylimidazole, polymers of polyvinylpyrrolidone, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.

The addition of such polymers also enhances the performance of the enzymes according to the invention.

According to the invention, the detergent composition can be in the forms of liquid, paste, gels, sticks or granules.

Granules that do not form powders can be produced, for example, as described in US 4,106,991 and 4,661,452 (both by Novo Industri A / S) and can optionally be covered by methods known in the art. Examples of materials for waxy coatings are poly (ethylene oxide) products (polyethylene glycol, PEG) with average molecular weights of 1000 to 20,000; nonylphenols ethoxylated with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film forming materials for coatings suitable for application by fluid bed techniques are given in GB 1483591.

The granular compositions according to the present invention may also be in "compact form", that is, they may have a relatively higher density than conventional detergent granules, ie, from 550 to 950 g / 1; in such a case, the granular detergent compositions according to the present invention may contain a smaller amount of "inorganic filler salt", compared to conventional granular detergents; typical filler salts are alkaline earth metal salts or sulphates and chlorides, typically sodium sulfate; a "compact" detergent typically comprises no more than 10% filler salt. The liquid compositions according to the present invention may also be in "concentrated form", in such case, the liquid detergent compositions according to the present invention may contain a lower amount of water, compared to conventional liquid detergents. Typically, the water content of the concentrated liquid detergent is less than 30%, more preferably less than 20%, much more preferably less than 10% by weight of the detergent compositions.

The compositions of the invention can for example be formulated as detergent compositions for hand or machine washing including additive laundry compositions and compositions suitable for use in the pretreatment of soiled fabrics, additive compositions of fabric softening rinses, and compositions for use. in general in the home in hard surface cleaning operations and dishwashing operations.

The following examples are intended to exemplify the compositions of the present invention, but do not necessarily mean limitations or definitions of the scope of the invention.

In detergent compositions, the abbreviations identifying the components have the following meaning: LAS: Alkyl Ci2 benzen sulfonate Sodium TAS: Animal fat Sodium alkyl sulfate XYAS; Alkyl C ?? -C? Sodium sulfate SS: Secondary soap surfactant of the formula 2-butyl octanoic acid 25EY; A C 1 -C 2 predominantly linear primary alcohol condensed with an average of Y moles of ethylene oxide 45EY: A predominantly linear C 4 -C 15 primary alcohol condensed with an average of Y moles of ethylene oxide XYEZS Alkyl C? X-Ci? Sodium sulphate condensed with an average of Z moles of ethylene oxide per mole Nonionic: C Alcohol 3-C ?5 fatty alcohol mixed (ethoxylated / propoxylated) with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5 sold under the trade name of Plurafax LF404 by BASF Gmbh CFAA: C12-C14 alkyl N-methyl glucamide CMC: sodium carboxymethyl cellulose DETPMP: Penta (methylene phosphonic acid) diethylene triamine, marketed by Monsanto under the trade name Dequest 2060 PVP: Polyvinylpyrrolidone polymer EDDS Ethylene-diamine-N, N 'acid disuccinic, [S, S] isomer in the form of sodium salt Suds 25% paraffinic wax Fusion point 50 ° C, 17% hydrophobic silica, 58% suppressant: paraffinic acid Sulphate: Anhydrous sodium sulfate HMPEO: polyethylene oxide high molecular weight TAE 25: Ethoxylated animal fat alcohol (25) Detergent Example I A granular fabric cleaning composition according to the invention can be prepared as follows: Alkyl Ci2 Sodium benzensulfonate 6.5 sodium sulfate 15.0 Zeolite A 26.0 Sodium Nitrilotriacetate 5.0 Enzyme of the invention 0.1 PVP - 0.5 TAED 3.0 Boric acid 4.0 Perborate 18.0 Phenol sulfonate 0.1 Minors up to 100 Detergent Example II A compact granular composition (density 800 g / 1) for fabric cleaning 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 Citric 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 Granular compositions for cleaning fabrics according to the invention, which especially useful in washing colored fabrics can be 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 MA / AA 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 _ Oxide of -N- poly (4-vinylpyridino) / 0.2 copolymer of vinylimidazole and vinylpyrrolidone Perborate 1.0 Phenol sulfonate 0.2 Water / minors up to 100% Detergent Example IV Granular compositions for cleaning fabrics according to the present invention that provide the "softener by washing" capability can be prepared as follows: 45AS-10.0 LAS 7.6 68AS 1.3 45E7 4.0 25E3 - 5.0 Ammonium Chloride Coco-alkyl-dimethyl 1.4 1.0 hydroxyethyl 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 TAED-Smellite Clay 10.0 10.0 HMWPEO 0 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 6.0 Carbonate 10.0 10.0 Granular foam suppressor 1.0 4.0 CMC 0.2 0.1 Water / minors Up to 100% Detergent Example V Compositions of a heavy duty liquid in the cleaning of fabrics according to the invention can be prepared as follows: I II THE acid form - 25.0 Citrus acid 5.0 2. .0 25AS acid form 8.0 - 25AE2S acid form 3.0 - 25AE7 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 Ammonium chloride coco- • alkyl dimethyl-3. .0 hydroxyethyl Clay smectite - 5. .0 PVP 2.0 - Water / minors Up to 100% APPLICATION IN THE INDUSTRY OF THE LEATHER A subtyla of the invention can be used in the leather industry, in particular for use in skin depilations. In said application a subtylase variant of the invention is preferably used in an enzymatic composition which additionally comprises another protease. for a more detailed description for other appropriate proteases see the section relating to suitable enzymes for use in a detergent composition (see above).

APPLICATIONS IN THE WOOL INDUSTRY A subtyla of the invention can be used in the wool industry in particular for use in the cleaning of clothing consisting of wool. In said application a subtylase variant of the invention is preferably used in an enzymatic composition which additionally comprises another protease. for a more detailed description for other appropriate proteases see the section relating to suitable enzymes for use in a detergent composition (see above).

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

MATERIALS AND METHODS Species: B. subtilis DN1885 (Diderichsen et al., 1990).

B. lentus 309 and 147 are specific varieties of Bacillus lentus, deposited with the NCIB and agreed accession numbers NCIB 10309 and 10147, and described in U.S. 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), r ", m + were prepared by conventional methods and is also described in the US Patent Application Serial No. 039,298.

Plasmids: pJS3: E. coli - B. subtilis transport vector containing an encoding of a synthetic gene for subtyla 309. (Described by Jacob Schi0dt et al in Protein and Peptide Letters 3: 39-44 (1996)). pSX222: B. subtilis expression vector (Described in WO 96/34946).

General methods of molecular biology: Unless otherwise indicated, DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausabel, F.M. and collaborators (editors) "Current protocole in Molecular Biology. "John Wiley and Sons, 1995; Harwood, C.R., and Cutting, S. M. (ed.)" Molecular Biological Methods for Bacillus. "John Wiley and Sons, 1990) Enzymes for DNA manipulations were used according to the specifications of the suppliers.

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

Proteolytic Activity In the context of this invention, proteolytic activity is expressed in units of Kilo NOVO Protease (KNPU). The activity is determined in relation to a standard enzyme (SAVINASEÓ), and the determination is based on the digestion of a solution of dimethyl casein (DMC) by the proteolytic enzyme at standard conditions, ie 50 ° C, pH 8.3, time reaction time of 9 min., measurement time of 3 min. An AF 220/1 folder is available upon request from Novo Nordisk A / S, Denmark, whose folder is included here as a reference. A GU is a unit of glycine, defined as the proteolytic activity which, under standard conditions, during 15 minutes of incubation at 40 ° C, with N-acetyl casein as substrate, produces an amount of the NH2-group equivalent to 1 mmol of glycine. Enzymatic activity can also be measured using the PNA assay, according to the reaction with the soluble substrate succinyl-alanine-alanine-proline-fignile-alanine-for-nitrophenol, which is described in the Journal of the American Oil Chemists Society, Rothgeb , TM, Goodlander, BD, Garrison PH, and Smith, LA, (1988).

Fermentation: The fermentation of subtylase enzymes was carried out at 30 ° C on a rotary shaking table (300 r.p.m.) in 500 ml Erlenmeyer flasks. with screen containing 100 ml. of BPX as a medium for 5 days. Consequently, for the preparation, for example of a 2 liter preparation broth, the fermentation was carried out in 20 Erlenmeyar flasks simultaneously.

Medium: BPX: Composition (per liter) Potato starch 100g Ground barley 50g Soybean meal 20g Na2HP0 X 12 H20 9g pluronic 0. lg Sodium caseinate lOg The starch in the medium is liquefied with -amylase and the medium is sterilized by heating at 120 ° C for 45 minutes. After sterilization the pH of the medium is adjusted to 9 with the addition of 0.1 M NaHCO3.

EXAMPLES EXAMPLE 1 Construction and expression of the Enzyme Variant: Mutagenesis if directed: The directed subtylase 301 site variants were elaborated using the techniques of "single site elimination (USE)" or "Uracil-USE", respectively described by Deng and collaborators (Anal. Biochem. 200: 81-88 (1992)) and Markvardsen et al. (BioTechniques 18 (3): 371-372 (1995)). The base plasmid was pJS3, or an analog thereof containing a variant of Subtilasa 309, for example, USE mutagenesis was performed on the pJS3 analog containing a gene encoding the G97E variant with an oligonucleotide directed to the construction of an A98V variant resulting in in a final variant G97E + A98V Subtylase 309. Subtylase variants 309 constructed in pJS3 were then cloned into the expression plasmid B. subtilis pSX222, using Kpnl and Mlul resynthesis enzymes.

Randomized localized mutagenesis: The overall strategy used to perform the randomly localized mutagenesis was: A mutagenic initiator (oligonucleotide) was synthesized which corresponds to the part of the DNA sequence to be mutagenized except for the corresponding nucleotide (s) ( s) to the amino acid codon (s) to be mutagenized. Subsequently, the resulting mutagenic initiator was used in a PCR reaction with a suitable antagonist initiator. The resulting PCR fragment was purified and treated by digestion and cloned into an E transport vector. coli -B. subtilus. Alternatively and if necessary, the resulting PCR fragment is used in a second PCR reaction as an initiator with a suitable second antagonist to allow digestion and cloning of the mutagenized region to the transport vector. PCR reactions are performed under normal conditions. Following this strategy, a randomly located library was constructed in SAVINASE, where positions V95, G97 and A98 were completely randomized. An oligonucleotide was synthesized with 25% of each of the four bases (N) in the first and second bases in amino acid codons to be mutagenized. The third nucleotide (the staggering base) in codons was synthesized with -50% G / 50% C (S) to avoid two (TAA, TGA) of the three codon stations. The mutagenic primer (5"-GC GCT GAG CTA TAC GCT GTT AAA NNS CTA NNS NNS AGC GGT TCA GGT TCG GTC-3" (sennsible)) was used in a PCR reaction with a suitable anti-sensitive antagonist primer, located downstream of the Mlul site in pJS3, and the plasmid pJS3 as base. This resulting PCR product was cloned into the transport vector pJS3 by the use of restriction enzymes Blpl and Mlul.

The randomly located library constructed in pJS3 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.

In order to purify a subtyla variant of the invention, the expression plasmid B. subtilis psX222 comprising a variant of the invention was transformed into a B species. competent subtilis and was fermented as described above in a medium containing 10 μg / ml Chloramphenicol (CAM) EXAMPLE 2 Purification of the Enzyme Variants: This procedure is related to the purification of a 2 liter fermentation scale of the enzyme Subtilisin 147, the enzyme Subtilisin 309 or its mutants. Approximately 1.6 liters of the preparation broth were centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The creams were adjusted to a pH of 6.5 using 10% acetic acid and in a Seitz Supra S100 dish filter.

The filtrates were concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate was centrifuged and filtered before absorption at room temperature in a Bacitracin affinity column at a pH of 7. The protease was eluted from the Bacillin column at room temperature using 25% 2-propanol and 1 M chloride. sodium in a buffer solution with 0.01 M of dimethyl glutaric acid, 0.1 M of boric acid and 0.002 M of calcium chloride adjusted to a pH of 7. The fractions with protease activity of the purification stage in Bacitracin were combined and applied to a 750 ml Sephadex G25 column (5 cm diameter), equilibrated with a buffer containing 0.01 M of dimethylglutaric acid, 0.2 M of boric acid, and 0.002 M of calcium chloride adjusted to a pH of 6.5. The protease was eluted using the same buffer (0-0.2 M sodium chloride in the case of Subtilisin 147). In a final purification step, the protease containing fractions from the CM Sepharose column were combined and concentrated in an Amicon ultrafiltration cell equipped with a GR81PP membrane (from Danish Sugar Factories Inc.). Using the techniques of Example 1 for the construction and the above isolation procedure, the following variants of subtilisin 309 were produced and isolated: V95T + G97T + A98E; And G97E + A98V.

EXAMPLE 3 Performance in Washing of Detergent Compositions Comprising Enzyme Variants The following examples provide results of various washing tests that were conducted under the indicated conditions.

EXPERIMENTAL CONDITIONS Table VI Experimental conditions for the evaluation of subtilisin variants 309 The detergent used is a simple model formulation. The pH is adjusted to 10.5 which is within the normal range for a powder detergent. The composition of the model 95 detergent is as follows: % STP (Na5P3? 10) 25% Na2S04 10% Na2C03 20% LAS (Nansa 80S) 5.0% Nonionic tenside (Dobanol 25-7) 5.0% Na2Si205 0.5% Carboxymethylcellulose (CMC) 9.5% Water The hardness of the water was adjusted by adding CaCl2 and MgCl2 (Ca2 +: Mg + = 2: 1) in deionized water (see also Surfactants in Consumer Products - Theory, Technology and Application, Springer Verlag 1986), the pH of the detergent solution was adjusted to 10.5 by the addition of HCl.

The reflectance measurements (R) in the test material were made at 460 nm using a Macbeth ColorEye 7000 photometer (Macbeth, Division of Kollmorgen Instruments Corporation, Germany). The measurements were made according to the manufacturers protocol.

The washing performance of the subtilisin 309 variants were evaluated by calculating a performance factor: arxante - R "-BBllaanco P = R S: avmasa" RBlanco P: Rvarian performance factor: Reflectance of the washed test material with the R. savinasa variant • Reflectance of the washed test material with Savinasa® RB? Reflectance of the washed test material without enzymes.

All Subtilisins 309 claimed to have improved wash performances compared to Savinasa® - ie 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: Subtilisin 309 variants 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 the conventional one for the manufacture of the objects to which it relates.

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

Claims (21)

1. A variant of subtylase enzyme that has an improved washing performance in detergents, characterized in that it comprises the modification (s) in the position (s) 95, 97 and / or 98 (in the BASBPN numbering).
2. A variant of subtylase enzyme which has an improved washing performance in detergents, characterized in that it comprises at least one modification selected from the group consisting of (in the BASBPN numbering): 95T + 97T + 98E 97E + 98V; or a variant comprising one or more moderate modifications in any of the aforementioned variants (eg, a moderate modification of a 97E variant (a.a.a. acid) + 98V includes a variant such as 97D (a.a.a. acid) + 98V.).
3. A subtylase enzyme variant according to claim 2, characterized in that the modification is selected from the group consisting of (in the BASBPN numbering): V95T + G97T + A98E G97E + A98V; or a variant comprising one or more moderate modifications in any of the aforementioned variants (for example, a moderate modification of a variant G97E (a.a.a. acid) + A98V includes a variant such as G97D (a.a.a. acid) + A98V.).
4. The variant of any of claims 1 to 3, characterized in that the original subtylase is selected from the subgroup I-SI.
5. The variant of claim 4, characterized in that the original subtylase is selected from the group comprising ABSS168, BASBPN, BSSDY, and BLSCAR or its functional variants that retain the characteristics of sub-group I-SI.
6. The variant of any of claims 1 to 3, characterized in that the original subtyla is selected from the subgroup I-S2.
7. The variant of claim 6, characterized in that the original subtylase is selected from the group consisting of BLS147, BLS309, BAPB92, TVHER and BYSYAB or its functional variants that retain the characteristic of subgroup I-S2.
8. The variant of any of the preceding claims, characterized in that said modification (s) is combined with one or more modifications in any other position.
9. The variant of claim 8, characterized in that said modification (s) is combined with 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. . The variant of claim 9, characterized in that said subtilase belongs to subgroup I-S2 and said additional change is selected from the group comprising K27R, * 36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, M123S, Y167A, Y167I, R170S, R170L, R170N, Q206E, N218S, M222S, M222A, T224S, K235L, and T274A.
11. . 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. . The subtyla variation of any of the preceding claims, characterized in that said modification (s) is combined with modifications in one or more of positions 129, 131, 133 and 194.
13. . The variant of claim 12, characterized in that said subtilase belongs to subgroup I-S2 and said additional change is selected from the group consisting of P129K, P131H, A133P, A133D and A194P.
14. . An isolated DNA sequence characterized in that it encodes a subtyla variant of any of claims 1 to 13.
15. . An expression vector characterized in that it comprises an isolated DNA sequence of claim 14.
16. . A host microbial cell characterized in that it is transformed by 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 that contribute to the expression and secretion of said variant, and the variant is recovered.
20. 20. A composition characterized in that it comprises a subtilase variant according to claims 1 to 13.
21. 21. The composition according to claim 20, characterized in that it additionally comprises a cellulose, lipase, cutinase, oxireductase, another protease, or an amylase. . The composition according to claim 20 or 21, characterized in that the composition is a detergent composition. . The use of a subtyla vanant according to claims 1 to 13 or an enzymatic composition according to any of claims 20 to 22 characterized in that the use is in a laundry and / or dishwashing detergent. . A process for the identification of a protease vanant exhibiting an improved washing performance in detergents, characterized in that it comprises carrying out a mutation in the DNA with coding of a subtylase enzyme or its pre or preproenzyme in one or more of the positions that correspond to an amino acid (in the BASBPN numbering): V95T + G97T + A98E G97E + A98V; Or a variant comprising one or more moderate modifications in any of the aforementioned variants, - transformation of a Bacillus strain with said mutated DNA; selection of strains qt? e produce such protease variants; fermentation / growth of such strains; recovery of said protease variant; and testing in detergents for improved wash performance.