MXPA01000302A - Stabilized variants of streptomyces - Google Patents

Abstract

The present invention relates to variants of Streptomyces subtilisin inhibitor (SSI) and those inhibitors having homology to SSI. Such variants are useful in conjunction with enzymes, particularly proteases, in cleaning compositions and personal care compositions. The variants comprise an amino acid substitution at position 63 corresponding to SSI. Such variants provide greater proteolytic stability in cleaning compositions and personal care compositions. The present invention also relates to cleaning compositions and personal care compositions comprising the present variants, as well as genes encoding the variants.

Description

STABILIZED VARIANTS OF THE STREPTOMYCES SUBTIL1SIN INHIBITOR RECIPROCAL REFERENCE TO RELATED REQUEST This application claims the benefit of the provisional application E.U.A. No.60 / 091, 911, filed July 7, 1998.

FIELD OF THE INVENTION The present invention relates to variants of the subtilisin inhibitor Streptomyces (SSI) and to those inhibitors which have homology to SSI (SSI-type inhibitors). Said variants are useful in conjunction with enzymes, particularly proteases, in cleaning compositions and personal care compositions. The present invention also relates to cleaning compositions and personal care compositions comprising the present variants, as well as genes coding for said variants.

BACKGROUND OF THE INVENTION Enzymes make up the largest class of proteins that occur naturally. One class of enzyme includes proteases that catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has been exploited by incorporating proteinases manipulated with protein and occurring naturally in cleaning compositions, particularly those relevant for laundry applications. In addition, although they have been explored to a lesser degree, others have been incorporated into said proteases in personal care compositions. During storage of the composition or even expression of the protease, however, the protease frequently degrades on its own or may degrade other enzymes present in the composition. As a result of this degradation, cleaning and personal care compositions have a limited ability to achieve intentional increased performance. Therefore, it would be beneficial to incorporate an inhibitor of protease activity in compositions to limit protease autolysis and other degradations. It would be convenient to provide reversible inhibitors of the protease, so that with the dilution of the composition during cleaning, or in the cleaning medium, the protease is no longer inhibited, but is available to hydrolyze the protein spots. In addition, said inhibitors must be stable enough to adequately perform their inhibitory function. Synthetic protease inhibitors or stabilizers have been described for such uses, particularly in the laundry medium. For example, Patent E.U.A. No. 5,422,030, Panandiker, et al., Assigned to The Procter & Gamble Co., issued June 6, 1995, discloses aromatic borate esters for stabilizing enzymes in laundry compositions. In addition the Patent E: U: A. No. 4,566,985, Bruno et al., Assigned to Applied Biochemists, Inc., and issued January 28, 1986, proposes the use of benzamidine hydrochloride as an enzyme inhibitor. Such synthetic approaches to inhibition can provide longer shelf life, but can be expensive and can not improve the performance of the insulation due to proteolysis in the fermenter. Recognizing these drawbacks, those skilled in the art have experimented with proteinaceous protease inhibitors to stabilize enzymes in cleaning compositions. Nature provides protease inhibitors to regulate protease in vivo. However, because these protein protease inhibitors that occur naturally tend to be unstable, their commercial use in the presence of proteases and cleaning and personal care vehicles may be somewhat limited. Protein protease inhibitors are typically long chain peptides that bind to the active site of a protease and inhibit its activity. These inhibitors have been typically classified into several families (family I to IX) based on the homologies of primary amino acid sequences (See, Laskowski et al., "Protein Inhibitors of Proteinases", Annual Review of Biochemistry, Vol. 49, pp. 593-626 (1980)). Included in these inhibitors are those commonly referred to as the VI inhibitor family, including the chymotrypsin inhibitor -.- ^ - alíH-IJ-i-tÉ and inhibitors of the III family, such as the inhibitor (SSI) of subtilisin Streptomyces and plasminostreptin. Such inhibitors tend to bind to certain proteases better than others. Therefore it is convenient to consider the inhibitor with a specific protease in mind. For this reason, the technique often describes "inhibitor protease / peptide pairs". An example of a known protease inhibitor / peptide pair is subtilisin BPN7SSI. See for example, Mitsui et al., "Crystal Structure of a Bacterial Protein Proteinase Inhibitor (Streptomyces Subtilisin Inhibitor) at 2.6 A Resolution", Journal of Molecular Bioloqy, Vol. 131, pp. 697-724 (1979) and Hirono et al., "Crystal Structure at 1.6 A Resolution of the Complex of Subtilisin BPN" with Streptomyces Subtilisin Inhibitor, "Journal of Molecular Bioloqy, Vol. 178, pp. 389-413 (1984). SSI is stable in the presence of subtilisin BPN ', as long as the inhibitor is present in sufficient amounts to inhibit all protease activity. However, it has been suggested that inhibitors that have high affinity for the protease do not dissociate with dilution in the washing medium. See WO 92/03529i Mikkelson et al., Assigned to Novo_Nordisk A / S, and published on March 5, 1992. However, if the binding constant (K,) of an inhibitor provides some protease activity in the composition of cleaning that contains the enzyme / inhibitor pair, the inhibitor, as well as the enzymes in the composition, can be hydrolyzed. Therefore, it would be convenient to find SSI variants or other inhibitors that are adequately stable in the presence of the protease as well as in cleansing and personal care compositions. In addition, these inhibitors preferably have a preferred K, for the particular protease that is to be inhibited. Said K i should allow the inhibition of the protease in the final composition and during its storage. However, with the dilution of the cleaning or personal care composition or during the cleaning procedure, the protease and the inhibitor must be dissociated, allowing the activity of the non-inhibited protease. Kojima et al., "Inhibition of Subtilisin BPN 'by Reaction Site Pl Mutants of Streptomyces Subtilisin Inhibitor", Journal of Biochemistry, Vol. 109, pp. 377-382 (1991), performed said inhibition and measured the K i of various variants of the SSI P1 position (position 73) using subtilisin BPN '. As another example, Mikkelsen et al., Describes mutations in inhibitors of the VI family, which are known to reduce binding affinity. WO 93/17086, Nielsen et al. Assigned to Novo Nordisk A / S, and published on September 2, 1993, describe mutations in plasminostreptin, which is known to reduce binding affinity. However, the stability of said protease inhibitors has been problematic. WO 98/13387, Correa et al., Assigned to The Procter & Gamble Co., published on April 2, 1998, (corresponding to the patent application E.U.A Series No. 60 / 026,944) refers to the variants that are described as those that provide increased stability. Despite the variety of approaches described in the art, there is a continuing need for more stable and effective protease inhibitors useful in cleaning and personal care compositions. The present inventors have surprisingly discovered that SSI inhibitors, SSI-type inhibitors, and variants thereof are easily hydrolyzed between positions 63 and 64 corresponding to SSI, for example during expression and / or in cleaning compositions and of personal care. Accordingly, the present inventors provide in the present invention variants of SSI inhibitors and SSI-type inhibitors that are modified, inter alia, at position 63 by an amino acid residue substituent. Said substitution imparts an increased stability to the protease inhibitor. Such inhibitors are also convenient because they bind the protease to preferred levels as defined herein. The present invention therefore provides protein protease inhibitor variants having greater proteolytic stability, particularly in cleaning and personal care compositions, and a lower affinity for the protease than the corresponding source inhibitor.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides variants having a modified amino acid sequence of an origin amino acid sequence, wherein the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to SSI, and wherein the origin amino acid sequence is selected from the group consisting of SS1, SS1 type inhibitors, SS1 variants, and variants of SS1 type inhibitors. Such variants are useful, for example, for inhibiting proteases, particularly during storage or during expression. The present invention also relates to genes coding for said variants and cleaning and personal care compositions comprising said variants.

DETAILED DESCRIPTION OF THE INVENTION The essential components of the present invention are described herein. Also included are non-limiting descriptions of various optional and preferred components useful in the embodiments of the present invention. The present invention may comprise, consist of, or consist essentially of, any of the required or optional components, ingredients and / or limitations that are described herein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated. Hereby commercial names are mentioned for materials including, but not limited to, proteases and optional components. The inventors herein are not intended to be limited by materials under a certain trade name. The materials -É ÉllHIittilMMMIi equivalents (for example, those obtained from a different source under a different name or catalog (reference number) to those indicated by the trade name can be substituted and used in the compositions herein. , or levels of composition are taken as reference the active level of that component, ingredient or composition, and exclude impurities, for example, residual solvents or residues, that may be present in commercially available sources All the documents referred to herein, including All patents, patent applications and printed publications are hereby incorporated by reference in their entirety As used herein, abbreviations will be used to describe amino acids Table 1 provides a list of abbreviations used herein: TABLE I Definitions As used herein, the term "mutation" refers to alterations in the gene sequences and in the amino acid sequences produced by those gene sequences. Mutations may be deletions, substitutions, or additions of amino acid residues to the wild type or origin sequence. As used herein, the term "origin" refers to a protease, protease, protein or peptide inhibitor, wild type or variant, with no amino acid substitution at position 63 corresponding to SS1 (ie, substitution of amino acid in position 63 that occurs naturally). An example of one of these origins is an inhibitor known as the Subtilisin Streptomyces (SSI) inhibitor (represented by SEQ ID NO: I). SS1 is further described by Ikenaka et al., "Amino Acid Sequence of an Alkaline Proteinase Inhibitor (Streptomyces Subtilisin Inhibitor) from Streptomyces albogriseoulus S-3253", Journal of Biochemistry, Vol. 76, pp. 1191-1209 (1974). As used herein, the amino acid numbering of SS1 is that of Ikenaka et al. The present inventors also use a synthetic SS1 gene, designed to have a rich content of adenine and thymine, as is the DNA of B. subtilis. This synthetic gene codes for four amino acid residues at the amino terminus of the peptide due to the methods of constructing the expression plasmid. This modified amino acid sequence, including these four additional amino acids, is represented by SEQ ID NO: 2. As used herein, the term "wild-type" refers to a protein or peptide, which is specifically a protease inhibitor or protease, produced by non-mutated organisms. As used herein, the term "variant" means a protein or peptide that is herein specifically a protease or protease inhibitor, having an amino acid sequence that differs from the protease inhibitor source or protease, respectively.

Variants of the present invention The present inventors have discovered variants of protease inhibitors that are more stable, for example in vitro and in the presence of materials of cleaning composition and for personal care. In addition, said variants may be more stable in vivo as well, thereby increasing the performance of the organism's protease. The present inventors have also discovered variants that exhibit preferred binding constants (K i), which provide for adequate inhibition of the protease during growth, culture, purification, and storage during the cleaning process. Said preferred union provides better stability and a longer shelf life. The variants of the present invention have improved stability to the proteases, and inhibit the protease in a cleaning or personal care composition, but dissociate with the dilution in the cleaning medium. The variants herein have a modified amino acid sequence of an origin amino acid sequence, wherein the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to the subtilisin inhibitor Streptomyces (which is referred to herein as SS1), and wherein the origin amino acid sequence is selected from SS1 inhibitors, SS1 type, SS1 variants, and SS1 type inhibitor variants. The substitution at position 63 corresponding to SS1 can be with any amino acid residue that imparts an increased stability in relation to the origin amino acid sequence. Most preferably, the substitution at position 63 corresponding to SS1 which is with isoleucine. Said variant may be represented as "L63I". In describing this variant, the original amino acid is presented in the origin amino acid sequence and is provided first, the second position number, and the third substituted amino acid. Therefore, L63I means that the leucine (L) that appears as the position of amino acid sixty-three (position 63) in the SSl native inhibitor is replaced with isoleucine (I). The numbering of the position corresponds to that of Ikenaka et al .. supra (SEQ ID NO: I), and ignores the four additional amino acid residues present in the amino terminal of the synthetic SS1 (SEQ ID NO: 2). Such representations for other substitutions listed here occur consistently. The variants herein are not limited to the SSl substituted at position 63. Instead, the substitution at position 63 can also be performed on the origin amino acid sequences (including, of course, the nucleotide sequences coding for that amino acid sequence) wherein the origin is itself a variant of SS1, an inhibitor similar to SS1, or a variant of SS1 type inhibitors. The most preferred source amino acid sequences include SS1 and SS1 variants. SS1 variants have been described in, for example, Kojima et al., "Inhibition of Subtilisin BPN 'by Reaction Site P1 Mutants of Streptomyces Subtilisin Inhibitor," Journal of Biochemistry, Vol. 109. pp. 377-382 (1991); Tamura et al., * tl ^ to ^^^ __ ^^^^^^^ | MHItfaM || tt ^ M "Mechanisms of Temporary Inhibition in Streptomyces Subtilisin Inhibitor Induced by an Amino Acid Substitution, Tryptophan 86 Replaced by Histidine", Biochemistry, Vol. 30 pp. 5275-5286 (1991); JO 3099-099-A, assigned to Tsumura & Co., published on September 12, 1989; Mikkelsen et al., Patent E.U.A. No. 5,674,833, assigned to Novo Nordisk A / S, issued October 7, 1997; and WO 93/17086, Nielsen et al., assigned to Novo Nordisk A / S, published September 2, 1993. Other variants of SS1 have been described in the patent application E.U.A. Series No. 60 / 026,944, Correa et al., Corresponding to WO 98/13387, Correa et al., Assigned to The Procter & Gamble Co., published April 2, 1998, said variants herein have been collectively described as "The Inhibitor Group A". The preferred variants of SS1 (for use as originating amino acid sequences herein) are those of Inhibitor Group A. The most preferred variants that are useful as the originating amino acid sequences herein are listed in the following tables 2-6 . Again, all position numberings correspond to SSl as described by Ikenaka et al.

TABLE 2 Non-limiting examples of origin amino acid sequences having a single substitution TABLE 3 Non-limiting examples of source amino acid sequences having double substitutions TABLE 4 Non-limiting examples of origin amino acid sequences having triple substitutions TABLE 5 Non-limiting examples of origin amino acid sequences having four-fold substitutions TABLE 6 Non-limiting examples of origin amino acid sequences that have guintuple substitutions Therefore, non-limiting examples of the variants of the present invention can be described as Variant 1, Variant 2, etc., in which, for example, Variant 1 can be represented as L63 * + D83C, in which "*" represents any amino acid different from that which occurs naturally in the position corresponding to 63 in SSl, and in which Variant 1-1 can be represented as L63I + D83C. Accordingly, the preferred variants of the present invention are listed in the following Table 7. Even more preferred among those variants listed in Table 7 are those which have isoleucine as a substituent at position 63.

TABLE 7 Non-limiting examples of preferred variants of the present invention fifteen Other preferred origin amino acid sequences of the The present invention includes those comprising a substitution at position 62 corresponding to SS1. The substitution at position 62 can be any amino acid residue different from that naturally occurring at the Origin (in the case of SS1, the amino acid residue that n¡ - ^^ «u occurs naturally is alanine). Preferably, the amino acid substituent at position 62 is selected from Lys, Arg, Glu, Asp, Thr, Ser, Gln, Asn, and Trp, most preferred Lys, Arg, Glu, Asp, Thr, Ser, Gln, and Asn, even more preferred Lys, Arg, Glu, and Asp, even still more preferred Lys and Arg and most preferred Lys. The preferred source amino acid sequences in the present invention have a substitution at position 62 in addition to the substitutions listed in Tables 2-6. Examples of such origins are designated as Origin X-A62 *, in which the "X" corresponds to the Origin exemplified in Tables 2-6. In this way, Origin 6 - A62 * corresponds to A62 * + M73P + D83C + S98A. Similarly, Origin 6 -A62K corresponds to A62K + M73P + D83C + S98A. Likewise, an exemplified variant of the present invention is Variant 6-I-A62 *, which corresponds to A62 * + L63I + M73P + D83C + S98A. Therefore, Variant 6 - I - A62K corresponds to A62K + L63I + M73P + D83C + S98A. In this regard, Table 8 lists other preferred variants of the present invention.

TABLE 8 Non-limiting examples of preferred variants of the present invention ^^^ Other preferred origin amino acid sequences (which are variants of SS1) useful in the present invention include those having a single substitution in position 98 corresponding to SS1 and those having a double substitution, one in position 62 and another in position 98. Table 9 lists the preferred source amino acid sequences in this class.

TABLE 9 Non-limiting examples of origin amino acid sequences The corresponding examples of variants of the present invention are listed in the following table 10.

TABLE 10 Non-limiting examples of variants of the present invention -MHMSÜßlllÉi ^ ¡^ Éaám? The SSl can exist in a dimeric form. Therefore, without being limited by theory, it is possible that by stabilizing the dimeric SSl an increased resistance to the protease for excess protease is provided. Preferably, this stabilized dimeric SSl variant consists of two SS1 variant monomers linked together covalently. This can be through ester, amido, disulfide, or other links, which commonly occur in amino acids and their side chains. Therefore "covalent dimerization" and "covalent stabilization" refer to such covalently bonded monomers, which form the dimer. Preferably, this dimerization occurs through disulfide bonds. The variants of the present invention are intended to include those that exist in dimeric form, either by intramolecular or intermolecular forces. Other source amino acid sequences that are useful in the present invention include the SS1-type inhibitors (often referred to as SS1-type proteins (SIL)) and variants of the SS1-type inhibitors. Background information that relates to SSl-type inhibitors can be found in Laskowski et al., "Protein Inhibitors of Proteases", Annual Review of Biochemistry. Vol. 49, pp. 593-626 (1980). Preferred SSl-type inhibitors have more than about 50%, preferably more than about 65%, and more preferred more than about 70% amino acid sequence identity with SSI, preferably wherein the inhibitor can be classified as an inhibitor of the III family.

See Laskowski et al., Supra. Examples of such SS1 type inhibitors include SIL10 (whose sequence is provided as SEQ ID NO: 4), SIL13 (SEQ ID NO: 5), and SIL14 (SEQ ID NO: 6), each of which is further described in Terabe et al., "Three Novel Subtilisin-Trypsin Inhibitors from Streptomyces: Primary Structures and Inhibitory Properties", Journal of Bíochemistrv, Vol. 116, p. 1156-1163 (1994), and SIL2 (whose sequence is provided as SEQ ID NO: 9), SIL3 (SEQ ID NO: 10), and SIL4 (SEQ ID NO: 11), each of which is further described by Taguchi et al., "Comparative Studies on the Primary Structures and Inhibitory Properties of Subtilisin-Trypsin Inhibitors from Streptomyces", European Journal of Biochemistry, Vol. 220, p. 911-918 (1994). Two other examples of such SS1-type inhibitors include STI1 (whose sequence is provided as SEQ ID NO: 7) and STI2 (SEQ ID NO: 8), which are further described in Strickler et al., "Two Novel Streptomyces Protein Protease. Inhibitors ", The Journal of Biological Chemistry, Vol. 267, No. 5, pp. 3236-3241 (1992). Another SS1-type inhibitor is known as plasminostreptin (whose sequence is provided as SEQ ID NO: 12), which is further described in Sugino et al., "Plasminostreptin, a Protein Proteinase Inhibitor Produced by Streptomyces antifibrinolyticus", The Journal of Biological Chemistry , Vol. 253, No. 5, pp. 1546-1555 (1978). Even another SS1 type inhibitor is SLPI (whose sequence is provided as SEQ ID NO: 13), which is also described in Ueda et al., "A Protease Inhibitor Produced by Streptomyces lividans 66 Exhibits Inhibitory Activities Toward Both Subtilisin BPN 'and Trypsin ", Journal of Biochemistry. Vol. 112, pp. 204-211 (1993). Even another SS1 type inhibitor is SAC I (whose sequence is provided as SEQ ID NO: 14), which is also described in Tanabe et al., "Primary Structure and Reactive Site of Streptoverticillium Anticoagulant (SAC), to Novel Protein Inhibitor of Blood Coagulation Produced by Streptoverticillium cinnamoneum subsp. cinnamoneum ", Journal of Biochemistry, Vol. 115, p. 752-761 (1994). Even another SS1 type inhibitor is SIL1 (whose sequence is provided as SEQ ID NO: 15), which is also described in Kojima et al., "Primary Structure and Inhibitory Properties of a Proteinase Inhibitor Produced by Streptomyces cacao?", Biochimica et al. Biophvsica Acta, Vol. 1207, pp. 120-125 (1994) Other SSl type inhibitors are discussed in Taguchi et al., "High Frequency of SSI-Like Protease Inhibitors Among Streptomyces", Bioscience, Biotechnology and Biochemistry, Vol. 57, pp. 522-524 (1993), Taguchi et al., "Streptomyces Subtilisin Inhibitor-Like Proteins Are Distributed Widely in Streptomycetes", Applied and Environmental Microbiology, pp. 4338-4341 (December 1993), and Suzuki et al. al., "Partial Amino Acid Sequence of an Alkaline Protease Inhibitor," Aqricultural Biological Chemistry, Vol 45, pp 629-634 (1981) As will be understood by those skilled in the art, even other SS1 type inhibitors are described in the art. Inhibitor variants can also be used SS1 type as the origin amino acid sequences in the present invention. Such variants include those that have one or more mutations in the amino acid sequence of a selected SS1-type inhibitor such as those described "? Jj" previously in the present invention. Among others, all of the substitutions exemplified in the variants shown in the present invention can also be made at the corresponding positions in SS1 type inhibitors to provide an origin amino acid sequence. Other non-limiting examples of variants of the SS1-type inhibitors that can be used as the amino acid sequences are described in Nielsen et al .. WO 93/17086, assigned to Novo Nordisk A / S, published on September 2, 1993. As one skilled in the art will understand, position 63 (for example) of an inhibitor of type SSl, variant of the same or variant of SSl, using the original numbering, could not correspond to position 63 of SSl. Accordingly, as is readily understood in the art, it may be necessary to adjust the numbering of the sequence to locate the position corresponding to that of position 63 (for example) of SSl. Alignments of the sequences are easily found in the references cited in the present invention, as well as in other references in the art. Preferably, the present variants have a Ki value that allows the variant to inhibit almost all of the protease (preferably more than about 60%, more preferred about 99%) in the compositions for cleaning or for personal care, but that allows it to dissociate from the protease after dilution and / or during the cleaning procedure. For example, where a 2: 1 stoichiometry of variant to protease is used, inhibitors preferably have a KI against * é "*? x. protease from about 10"12 M to about 10" 4 M, more preferred from about 10'10 M to about 10"6 M, and even more preferred from about 10 ~ 8 M to about 10" 7 M. Of course, in the case in which the dimensions of the washing machine change or the concentrations of the products, the K, is adjusted accordingly. The prediction of a useful range of K is easily determined by the person skilled in the art without undue experimentation considering parameters such as the dilution of the composition during use, the temperature dependence of the binding constant in relation to the temperature of the method of cleaning used, the stoichiometry of inhibitor to protease, and the like. As the variant is finally encoded in vivo by the DNA, this DNA can be used to define the sequence of the variant. The DNA, which codes for the variant, can be used in any number of plasmids and / or expression systems, including in vitro expression systems and in vivo systems, such as plants, (preferably those used in biotechnology, including tobacco, oilseed plants such as rapeseed, soybeans and the like, grains such as corn, barley, oats, other vegetables such as tomatoes, potatoes and the like) and microorganisms, including fungi such as yeasts and bacteria such as Bacillus, E coli and the like. Preferably the expression system is a microorganism, more preferred is bacterial in nature, preferably E. eoli or Bacillus, and even more preferred is Bacillus.

The DNA encoding the protein can be incorporated into a plasmid or phage, active in the cell, or can be incorporated directly into the genome of the organism that is used in the cloning or expression of the variants of the present invention. It should be understood that those skilled in the art, given the instructions of this invention, will appreciate that the DNA used to code for the present variant could be placed on the same plasmid, phage or chromosome as with other variants of the invention. In addition, such plasmids, phages or chromosomes could also code for proteases, including fusion proteins that include as part of the fusion protein an inhibitor and / or protease, which could be inhibited or not by the variant of the present invention. It is also well understood by those skilled in the art that the DNA described above also contemplates, and describes the RNA transcript of the DNA. The person skilled in the art can, of course, without need of experimentation, know the RNA sequence, inspecting the DNA sequence. The present invention also relates to the genes and / or DNA encoding the variants of the present invention. It is also contemplated that one skilled in the art could wish to prepare antibodies for the variants of the present invention. These antibodies can be prepared using known methods. rt-taM-i-M-Til ^ For example, the variants of the present invention can be injected into appropriate mammalian subjects such as mice, rabbits and the like. Appropriate protocols involve the repeated injection of the immunogen in the presence of adjuvants in accordance with a program that promotes the production of antibodies in serum. The titres of the immune serum can be easily measured using methods for known immunological tests, which are currently normal in the art, using the proteins of the invention as antigens. The antiserum obtained can be used directly or monoclonal antibodies can be obtained by cultivating lymphocytes from the spleen peripheral blood of immunized animals and immortalizing the antibody producing cells, followed by the identification of the appropriate antibody producers using standard testing techniques. immunological Polyclonal or monoclonal preparations are then useful in monitoring the expression of the invention using standard test methods. It is therefore anticipated that a kit can be prepared using these antibodies for one of them to determine the level of expression and the like. Such antibodies can also be coupled to markers such as scintigraphic markers, for example technetium 99 or 1-131, or to fluorescent labels, using standard coupling techniques. The labeled antibodies can also be used in competitive tests, such as kinetic tests to determine Ki. As recognized in the art, there are occasional errors in DNA and amino acid sequence determination methods. As a result, one skilled in the art reproducing the work of the inventors of the present invention from the description thereof could discover any of the sequence determination errors using routine skills, and make the changes as appropriate.

Method of preparation and use The following examples are not intended to limit the invention claimed in any way, but rather provide the person skilled in the art with guidance on how to prepare and use the invention. Given the guidance of the examples, the other description in the present invention, and the information readily available to those skilled in the art, one skilled in the art can prepare and use the invention. To summarize, the exhaustive description of the technique and the methods known therein are eliminated, since they fall within the general knowledge of the person skilled in the art. Variants can be prepared by mutating the nucleotide sequences encoding an origin amino acid sequence, which results in variants having modified amino acid sequences. Such methods are known in the art; One such method is the following: A phagemid containing the gene corresponding to the origin amino acid sequence to transform Escherichia coli dut-ung-strain CJ236 is used and a single-strand DNA template containing uracil is produced using the auxiliary phage VCSM13 (Kunkel et al., " Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection ", Methods in Enzymoloqy, Vol. 154, pp. 367-382 (1982) as modified by Yuckenberg et al.," Site-Directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectors ", Directed Mutagenesis - A Practical Approach, McPherson, MJ ed., pp. 27-48 (1991). Site-directed mutagenesis with modified primer was used from the Zoller and Smith method (Zoller, MJ and M). Smith, "Oligonucleotide - Directed Mutagenesis Using M13 - Derived Vectors: An Efficient and General Procedure for the Production of Point Mutations in Any Fragment of DNA", Nucleic Acid Research, Vol. 10, pp. 6487-6500 (1982) to produce all the s variants (essentially as presented by Yuckenberg et al., supra). The oligonucleotides are prepared using a 380B DNA synthesizer (Applied Biosystems Inc.). The reaction products of mutagenesis are transformed into Escherichia coli strain MM294 [American Type Culture Deposit (ATCC) E. coli 33625]. All mutations are confirmed by DNA sequence determination and the isolated DNA is transformed into the expression strain PG632 of Bacillus subtilis (Saunders et al., "Optimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34- amino acid Fragment of Human Parathyroid Hormone ", Gene. Vol. 102, pp. 277-282 (1991) and Yang et al.," Clone? ng of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Créate an in vitro - Derived Deletion Mutation ", Journal of Bacterioloqy, Vol 160, pp. 15-21 (1984) The preparations of the variant are made as follows: Bacillus subtilis cells containing the plasmid of interest are grown in medium with 20 g / l of tryptone, 20 g / l of yeast extract and 5 g / l of sodium chloride supplemented with 1.25% malnutrin M100 (Grain Processing Corporation, Muscatine, IA), 100 mM HEPES, pH 7.5, 80 μM of MnCl2 and 50 μM of kanamycin cultures are incubated for 24 hours s at 37 ° C. The variant is purified by first removing the cells by centrifugation. The pH is allowed to reduce to approximately 4, adding 1 N HCl. The insoluble material is formed into pellets with centrifugation. Typically, the variant is found in the supernatant. Subsequently, the supernatant is subjected to dialysis against 20mM of sodium acetate with pH4. Typically, the variant is precipitated in this step. Both precipitates are resuspended on Tris base and tested for the inhibition of a Y217L derivative of subtilisin BPN '. In some cases, the variant remains soluble through these precipitation steps. In that case, the soluble fraction is separated on a Sepharose S column, run in 20mM sodium acetate with pH4. The sample is eluted with increased concentrations of sodium chloride and tested for inhibition.

In all cases, the fractions containing the variant are dialyzed against 1 mM Tris pH 8.0 before use.

Characterization of Valancetes of the Present Invention SS1 inhibits, inter alia, subtilisin BPN 'and a variant Y217L of subtilisin BPN'. The inhibition activity of the present variants is measured as follows, using SSl as an example. SS1 is mixed with protease and incubated for 15 minutes at room temperature in the presence of 0.1M Tris, pH 8.6, 10mM CaCl2. The protease activity is then measured using the method of DelMar et al., Analytical Biochemistry. Vol. 99, pp. 316-320, (1979). Addition of 10 μl of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide 20 mg / ml) initiates the reaction. The reaction rate is measured by the increase in absorbance at 410 nm which indicates the inhibition of the protease. The inhibitory properties of the variants of the present invention are determined in the same way. Because it is desired to incorporate a variant of the present invention with a protease in the compositions for cleaning or for personal care, (suitable proteases are also described herein), the stability in the environment of the product is also evaluated. The stability of a variant can be monitored by measuring the activity of the protease over time. If the variant is stable, the level of protease activity will be constant. However, if the variant is destroyed, the protease activity will increase. In this example, the variants are mixed with 1.1. nmoles of a variant of subtilisin BPN 'with a substitution of Y217L. Water is added so that the volumes of all the samples are the same. A complex is allowed to form for 10 minutes in a liquid detergent composition made according to the following formula: This composition constitutes one third of the total volume of the sample. Mix 15 μl of sample with 975 μl of 0.1 M Tris HCl, pH 8.6, 0.01 M CaCl2. This dilution is incubated for 30 minutes at room temperature. After incubation, the substrate is added, and the amount of protease is measured. The degradation of the variant part is detected by the increase in protease activity after several weeks. Such degradation can be compared directly to that of, for example, SSl. The K i of a fusion protein is determined as follows. The varainte and 600 μg / ml of succinyl-Ala-Ala-Pro-Fe-p-nitroanilide are mixed in 990 μl of a 50 mM Tris solution, pH8. The reaction is initiated by the addition of a selected protease (suitable proteases are described herein). The hydrolysis rate is observed for twenty minutes. A constant speed is observed during the last ten to fifteen minutes. This speed, compared to the speed in the absence of the variant, is used to calculate the Ki according to Goldstein's equations "The Mechanism of Enzyme-Inhibitor-Substrate Reactions", Journal of General Phvsioloqy. Vol. 27, pp. 529-580, 1944).

Cleaning compositions of the present invention In another embodiment of the present invention, an effective amount of one or more of the variants herein, is included in cleaning compositions useful for cleaning a variety of surfaces that require the removal of peptide stain. . Such cleaning compositions include, but are not limited to, fabric cleaning compositions, hard surface cleaning compositions, light duty cleaning compositions including dishwashing cleaning compositions and automatic dishwashing detergent compositions. The cleaning compositions of the present invention comprise an effective amount of one or more of the fusion proteins of the present invention and a vehicle of the cleaning composition, which vehicle includes a protease. As used herein, an "effective amount of variant", or similar, refers to the amount of variant necessary to achieve the required proteolytic activity in the specific cleaning composition. Said effective amounts are easily assured by those skilled in the art and are based on various factors, such as the particular variant used, the cleaning application, the specific composition of the cleaning composition and whether a liquid or dry composition is desired. (for example, granulated or in bar), and the like. Preferably, the cleaning compositions comprise from about 0.0001% to about 10%, most preferably from about 0.001% to about 1%, and still most preferably from about 0.01% to about 0.1% of one or more variants of the present invention. Various examples of various cleaning compositions, wherein the variants may be employed, are described in greater detail below. The present variants are useful in cleaning compositions for inhibiting a protease in and during storage, thereby protecting the protease from autolysis or hydrolysis from other sources, such as other sources such as additional enzymes present in the composition. Accordingly, an essential ingredient in the cleaning compositions herein is a protease from which a present variant is inhibited. The protease can be of animal, vegetable or, preferably, microorganism origin. Preferred proteases include those proteases for which SS1 is an inhibitor. Such proteases include, for example, those produced by the microorganisms Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus and Bacillus subtilis. Among such proteases, preferred ones include, for example, subtilisin BPN, subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, and thermitase, including A / S Alcalase® (Novo Industries, Copenhagen, Denmark), Esperase® (Novo Industries), Savinase® (Novo Industries), Maxatase® (Gist Brocades, Delft, The Netherlands), Maxacal® (Gist Brocades), Maxapem 15® (Gist Brocades), and the variants of the above. Especially preferred proteases for use in the present invention include those obtained from Bacillus amyloliquefaciens and variants thereof. The most preferred wild-type protease is subtilisin BPN '. The subtilisin variants BPN ', collectively referred to hereinafter as "Group A Proteases", are useful as the proteases of the present invention and are described in the patent E.U.A. No. 5,030,378, Venegas. July 9, 1991, as characterized by the amino acid sequence of subtilisin BPN '(whose sequence is represented as SEQ ID NO: 3) with the following mutations: (a) Gly at position 166 is replaced with Asn, Ser , Lys, Arg, His, Gln, Ala or Glu; Gly in position 169 is replaced with Ser; and Met at position 222 is substituted with Gln, Phe, His, Asn, Glu, Ala or Thr; or (b) Gly at position 160 is replaced with Ala, and Met at position 222 is replaced with Ala.

Additional variants of subtilisin BPN ', collectively referred to hereafter as "Group B Proteases", are useful as the proteases of the present invention and are described in European Patent EP-B-251, 446 , assigned to Genencor International, Inc., published on January 7, 1998 and issued on December 28, 1994, as characterized by the wild-type BPN 'amino acid sequence with mutations in one or more of the following positions : Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67, Ser87, Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Methyl 24, Gly127, Gly128, Pro129, Leu135, Lys170, Tyr171, Pro172, Asp197, Meti 99, Ser204, Lys213, Tyr214, Gly215, and Ser221; or two or more of the positions listed above combined with Asp32, Ser33, Tyr104, Alai 52, Asn155, Glu156, Giy166, Gly169, Phe189, Tyr217 and Met222. Other preferred variants of subtilisin BPN 'useful as the proteases of the present invention are collectively referred to hereafter as "Group C Proteases", and are described in WO 95/10615, assigned to Genencor International, Inc., published on April 20, 1995, as characterized by the wild-type BPN 'amino acid sequence with a mutation in the Asn76 position, in combination with mutations in one or more other positions that are selected from the group consisting of: Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265 and Ala274. Other preferred variants of subtilisin BPN 'useful as the proteases of the present invention are collectively referred to hereafter as "Group D Proteases", and are described in the patent E.U.A. No.4, 760, 025, Estell et al., July 26, 1988, as characterized by the wild-type BPN 'amino acid sequence with mutations at one or more of the amino acid positions that are selected from the group consists of: Asp32, Ser33, His64, Tyr104, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222. The most preferred proteases as used in the present invention are selected from the group consisting of Alcalase®, subtilisin BPN ', Group A Protease, Group B Protease, Group C Protease and Group D Protease. The most preferred protease is selected from Group D Proteases. Preferably, the cleaning compositions comprise from about 0.0001% to about 1%, more preferably from about 0.0005% to about 0.2% and more preferably from about 0.002% to about 0.1% by weight of the composition, of active protease. Mixtures of proteases can also be included. Of course, the percentage by weight of protease in the cleaning composition will vary depending on the water content, builder content and the like, of the finished composition. For example, it is preferred that in granular detergent, about 0.064 mg / g to about 0.64 mg / g of protease in the composition, it is desirable. In a preferred embodiment, the preferred molar ratio of variant to protease (variant to protease ratio) in cleaning compositions is from about 3: 1 to about 1: 1, most preferably from about 3: 1 to about 1.5 : 1, and more preferably around 2: 1. In addition to the present variants, these cleaning compositions also comprise a vehicle for cleaning composition comprising one or more of the materials for cleaning composition compatible with the variant and / or protease. The term "material for cleaning composition", as used in the present invention, means any material selected for the particular type of cleaning composition desired and the shape of the product (e.g., liquid, granulate, in tablet, for sprinkling, in bar, in paste, gel, etc.) whose materials are also compatible with the variant used in the composition. The specific selection of the materials for the cleaning composition is easily made by considering the material to be cleaned, the desired form of the composition for the cleaning conditions during use. The term "compatible", as used in the present invention, means that the materials for the cleaning composition do not reduce the inhibitory activity of the variant and / or the proteolytic activity of the protease to such an extent that the protease is not effective as desired during normal use situations. The specific materials for cleaning composition are exemplified in greater detail later in the present invention. The variants of the present invention can be used in a variety of detergent compositions in which high foam formation and good cleaning activity are desired. Therefore, the variants can be used with various conventional ingredients to provide fully formulated hard surface cleaners, dishwashing compositions, fabric washing compositions and the like. Such compositions may be in the form of liquids, granules, sticks and the like. Such compositions can be formulated as "concentrated" detergents containing as much as about 30% to about 60% by weight of surfactants. The cleaning compositions of the present invention may contain, optionally, and preferably, various surfactants (for example, anionic, nonionic or zwitterionic surfactants). Such surfactants are typically present at levels from about 5% to about 35% of the compositions. Non-limiting examples of surfactants useful in the present invention include the conventional Cn-Ciß alkylbenzene sulphonates and primary sulfates, the secondary (2,3) C 0 -C 8 alkyl sulfates of the formulas CH 3 (CH 2) x (CHOS03"M +) CH3 and CH3 (CH2) and (CHOS03" M +) CH2CH3 in which xy (y +1) are integers of at least about 7, preferably of at least about 9, and M is a cation conferring solubility in water, especially sodium, the alkylalcoxy sulfates of Cío-Cía (especially the ethoxysulfates with EO 1-5), the alkylalkoxycarboxylates of C 10 -C 18 (especially the ethoxycarboxylates with EO 1-5), the alkyl polyglycosides of C 10 -C 18 and their corresponding sulphated polyglycosides, C12-C18 α-sulfonated fatty acid esters, C12-Cis alkyl-alkoxylates and alkylphenol-alkoxylates (especially ethoxylates and ethoxy / mixed propoxy), betaines and sulfobetaines (sultaines) of C12-C18, s C10-C18 amine oxides and the like. Preferred in the present invention are alkylalkoxy sulfates (AES) and alkylalkoxycarboxylates (AEC). The use of such surfactants in combination with amine oxide and / or betaine or sultaine surfactants is also preferred, depending on the desires of the formulator. Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-C18 N-methylglucamides described in US Pat. No. 5,194,639, Connor et al., Issued March 16, 1993. A wide variety of other ingredients useful in the present cleaning compositions include, for example, other active ingredients, vehicles, hydrotropes, processing aids, dyes or pigments. , and solvents for liquid formulations. If a further increase in foam is desired, foam enhancers such as C10-C16 alcolamides can be incorporated into the compositions, typically from about 1% to about 10%. The C-io-C monoethanol and the diethyl amides illustrate a typical class of such foam enhancers. The use of said foam enhancers with adjunct surfactants of high foaming effect such as amine oxides, betaines and sultaines mentioned above are also convenient. If desired, the soluble magnesium salts such as MgCl 2, MgSO 4, and the like, can be added at levels of, typically from about 1% to about 2% to provide an additional foaming effect. The liquid detergent compositions herein may contain water and other solvents as carriers. The low molecular weight primary and secondary alcohols exemplified by methanol, ethanol, propanol, e / so-propanol are suitable. Monohydric alcohols are preferred to solubilize surfactants, however polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (eg, 1,3-propanediol, ethylene glycol) , glycerin, and 1-2 propanediol may also be used.The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of said vehicles.Detergent compositions herein will preferably be formulated in such a way that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11. The finished products are typically formulated on this scale.The techniques for controlling the pH at recommended use levels they include the use of, for example, pH, alkali and acid regulators, which are well known to those in the technical field. When the hard surface cleaning compositions and cleaning compositions for fabrics of the present invention are formulated, the formulator may wish to employ various builders at levels of from about 5% to about 50% by weight. Typical builders include zeolites, 1-10 microns, polycarboxylates such as citrate and oxydisuccinates, phosphates, layered silicates, and the like. Other conventional detergency builders are listed on standard forms. Likewise, the formulator may desire to employ several additional enzymes, such as cellulases, lipases, amylases and proteases in said compositions, typically at levels of from about 0.001% to about 1% by weight. Various enzymes for the care of fabrics and detersives are well known in the laundry detergent art. Various bleaching compounds, such as percarbonates, perborates and the like, can be used in such compositions typically from about 1% to about 15% by weight. If desired, said compositions may also contain bleach activators such as tetraacetylethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art.

Technically use levels vary from about 1% to about 10% by weight. Dirty release agents, especially those of the anionic oligoester type, chelating agents, especially the aminophosphonates and ethylenediamindisuccinates, clay soil removal agents, especially tetraethylenepentaminethoxylate, dispersing agents, especially polyacrylates and polyaspartates, brighteners, especially anionic brighteners, suppressants of foam, especially silicones and secondary alcohols, fabric softeners, especially smectite clays, and the like, all of them can be used in said compositions at levels ranging from about 1% to about 35% by weight. The standard forms and published patents contain multiple detailed descriptions of such conventional materials. The enzyme stabilizers can also be used in cleaning compositions. Said enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1% to about 1%). Other useful materials for the cleaning compositions include clay soiling removal agents, dispersing agents, brightening agents, suds suppressors and fabric softeners.

The present variants are useful in compositions for cleaning hard surfaces. As used herein, "hard surface cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tiles, and the like. Hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering detergent builder. However, in certain specialized products such as aerosol window cleaners, surfactants are sometimes not used because they can produce a film residue and / or scratch effect on the surface of the glass. The surfactant component, when present, may comprise as much as 0.1% of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%, most preferably from about 1% to about 5% of the surfactant. Typically, the compositions will contain from about 0.5% to about 50% of a builder, preferably from about 1% to about 10%. Preferably, the pH should be in the range of about 7 to about 12. Conventional pH adjusting agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if such adjustment is necessary.

Solvents can be included in the compositions. Useful solvents include, but are not limited to, glycol ethers, such as diethylene glycol monohexyl ether, diethylene glycol monobutyl ether, ethylene glycol monobutyl ether, ethylene glycol monoexrylic ether, propylene glycol monobutyl ether, dipropylene glycol monobutyl ether, and diols. such as 2,2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-exanediol. When such solvents are used, they are typically present at levels of from about 0.5% to about 15%, and most preferably from about 3% to about 11%. Additionally, highly volatile solvents such as / so-propanol or ethanol can be used in the present compositions to facilitate the more rapid evaporation of the composition of the surfaces, when the surface is not rinsed after the application of "total resistance" of the composition to the surface. When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions. The present variants are also useful for being included in the cleaning compositions described in the following U.S. Patent Applications. Provisional Series No. 60 / 079,477, Rubingh et al., filed March 26, 1998; Patent application E.U.A. provisional series No. 60/079397, Rubinqh et al., filed March 26, 1998; Patent application E.U.A. series No. 09 / 048,174, Weisqerber et al .. filed on March 26, 1998; and patent application E.U.A. Series No. 09/088912, which claims priority to the patent application E.U.A. series No.09 / 048, 174, Weisqerber et al., filed June 2, 1998. The hard surface cleaning compositions of the present invention are illustrated by the following examples.

EXAMPLES 1-6 Liquid hard surface cleaning compositions All the formulas are adjusted to a pH7. In Examples 1-6, the variants mentioned in Tables 7, 8 and 10, and the preferred variants mentioned herein, among others, are substituted by the 6-I variant, with substantially similar results. In another embodiment of the present invention, the dishwashing compositions comprise one or more variants of the present invention. As used herein, "dishwashing composition" refers to all forms of dishwashing compositions including, but not limited to, granular and liquid forms. The dishwashing compositions of the present invention are illustrated by the following examples.

EXAMPLES 7-10 Liquid dishwashing detergent All the formulas are adjusted to pH7. In Examples 7-10, the variants that are mentioned in Tables 7,8 and 10, and the preferred variants mentioned herein, among others, are replaced by the 7-1-A62K variant, with substantially similar results . The liquid fabric cleaning compositions of the present invention are illustrated by the following examples.

EXAMPLES 11-13 Liquid compositions for fabric cleaning In Examples 11-13, the variants indicated in Tables 7, 8 and 10, and the preferred variants indicated herein, inter alia, are substituted by the 2-I variant, with substantially similar results.

Compositions for personal care The present variants are also suitable for use in personal care compositions selected from, for example, hair conditioners that are left on and rinsed, shampoos, acne compositions left on the skin and rinse, facial milk and conditioners, bath gels, soaps, facial cleansers that make and foam and without foam, cosmetics, lotions and moisturizers for hands, facials and body, facial moisturizers left on the skin, cosmetic towels and for cleaning, oral care compositions, and contact lens care compositions. The present personal care compositions comprise an effective amount of one or more variants of the present invention and a personal care vehicle, which personal care vehicle includes a protease. Effective amounts of variants, including preferred limitations, are described herein with respect to cleaning compositions. Suitable proteases, including those that are preferable, are described herein with respect to cleaning compositions. To illustrate them, the present variants are suitable for inclusion, together with a protease, in the compositions described in the following references: Pat. E.U. No. 5,641, 479, Linares et al., Issued June 24, 1997 (skin cleansers); (skin cleansers; U.S. Patent No. 5,599,549, Wivell et al., issued February 4, 1997; (skin cleansers); U.S. Patent No. 5,585,104; Ha et al; issued December 17, 1996; (skin cleansers); U.S. Patent No. 5,540,852; Kefauver et al; issued July 30, 1996; (skin cleansers); U.S. Patent No. 5,510,050; Dunbar et al; issued April 23, 1996; cleansers), U.S. Pat. No. 5,612,324, Guanq Lin et al, issued March 18, 1997 (anti-acne preparations) (anti-acne preparations), U.S. Pat. No. 5,587,176, Warren et al: issued 24 December 1996 (Anti-acne preparations Pat. EU No. 5,549,888, Venkateswaran, issued August 27, 1996 (anti-acne preparations), US Pat. No. 5,470,884; Corless et al; Issued on November 28, 1995; (anti-acne preparations); Pat. E.U. No. 5,650,384; Gordon et al; issued on July 22, 1997; (shower gels) (gels for bath); Pat. E.U. No. 5,607,678; Moore et al; Issued on March 4, 1997; (shower gels); Pat. E.U. No. 5,624,666; Coffindaffer et al; issued on April 29, 1997; (hair conditioners and / or shampoos) (conditioners and / or shampoos for hair); Pat. E.U. No. 5,618,524; Bolich et al; Issued on April 8, 1997; (hair conditioners and / or shampoos); Pat. E.U. No. 5,573,709; Wells, issued November 12, 1996; (hair conditioners and / or shampoos); Pat. E.U. No. 5,482,703; Pinqs; Issued on January 9, 1996; (hair conditioners and / or shampoos); Pat. E.U. No. Re. 34,584; Grote et al; reissued on April 12, 1994; (hair conditioners and / or shampoos); Pat. E.U. No.5,641, 493; Date et al; Issued on June 24, 1997; (Cosmetics) (cosmetics); Pat. E.U. No. 5,605,894; Blank et al; Issued on February 25, 1997; (cosmetics); Pat. E.U. No. 5,585,090, Yoshioka et al; Issued on December 17, 1996; (cosmetics); Pat. E.U. No. 4,939,179; Chenev et al; Issued on July 3, 1990; (hand, face, and / or body lotions) (lotions for hands, face and / or body); Pat. E.U. No. 5,607,908; Me Atee et al; Issued on March 4, 1997; (hand, face, and / or body lotions); Pat. E.U. No. 4, 045,364; Richter et al; Issued on August 30, 1977; (cosmetic and cleansing wipes) (cosmetic and cleaning towels); European Patent Application EP 0 619 074, Touched et al: published October 12, 1994; (cosmetic and cleansing wipes); Pat. E.U. No. 4,975,217; Brown-Skrobot et al; Issued on December 4, 1990; (cosmetic and cleansing wipes); Pat. E.U. No. 5,096,700; Seibel issued on March 17, 1992; (oral cleaning compositions) (compositions for oral cleansing); Pat. E.U. No. 5,028,414, Sampathkumar. Issued on July 2, 1991; (oral cleaning compositions); Pat. E.U. No. 5,028,415, Benedict et al; Issued on July 2, 1991; (oral cleaning compositions); Pat. E.U. No. 4,863,627; Davies et al; September 5, 1989; (contact lens cleaning solutions) (cleaning solutions for contact lenses); Pat. E.U. No. Re 32,672; Huth et al; reissued on May 24, 1988; (contact lens cleaning solutions); and Pat. E.U. No. 4,609,493. Schaffer: issued on September 2, 1986 (contact lens cleaning solutions). The present variants are also useful for inclusion in the personal care compositions described in the following: U.S. Patent Application. Provisional, Series No., 60 / 079,475; Rubinqh et al; filed on March 26, 1998; patent application E.U. Provisional Series No. 60 / 079,397; Rubinqh et al: filed on March 26, 1998; patent application E.U. Provisional Series No. 09 / 048,174; Weischerber et al; filed on March 26, 1998; and Patent Application E.U. provisional series no. 09/088912, claiming priority to the US patent application. provisional series No. 09 / 048,174, Weisqerber et al; filed on June 2, 1998. In a preferred embodiment, the preferred molar ratio of variant to portease (variant to protease ratio) in the personal care compositions is from about 3: 1 to about 1: 1, more preferably about from 3: 1 to about 1.5: 1, and more preferably 2: 1. To further illustrate the oral cleansing compositions of the present invention, one or more variants of the present invention and one or more proteases are included in useful compositions to remove protein spots of teeth or dentures. As used herein, "oral cleansing compositions" refer to dentifrices, toothpastes, dental gels, dental powders, mouth rinses, mouth sprays, mouth gels, chewing gums, lozenges, pads, tablets, biogels, pastes. for prophylaxis, solutions for dental treatments and the like. Preferably, compositions for oral cleansing comprise from about 0.0001% to about 20% of one or more variants of the present invention, together with a protease, more preferably from about 0.001% to about 10%, still more preferably from about 0.01% to about 5%, by weight of the composition, and a vehicle of personal care composition. Typically, the personal care vehicle components of oral cleansing compositions will generally comprise from about 50% to about 99.99%, preferably from about 65% to about 99.99%, more preferably from about 65% to about 99% in weight for the composition. The personal care vehicle components and optional components that may be included in the oral cleansing compositions of the present invention are well known to those skilled in the art. A wide variety of types of compositions, vehicle components as well as optional components, useful in compositions for oral cleansing, are described in references cited herein above. In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside the oral cavity comprise one or more variants of the present invention. Said denture cleaning compositions comprise one or more variants of the present invention together with a protease, preferably from about 0.0001% to about 50%, more preferably from about 0.001% to about 35%, with even greater preference of about 0.01% to about 20% by weight of the composition, and a vehicle for personal care. Various denture cleaning composition formats, such as effervescent tablets and the like, are well known in the art (See, for example, U.S. Patent No. 5,555,305, Younq). and are generally suitable for the incorporation of one or more variants to remove protein spots from the dentures. In another embodiment of the present invention, the contact lens cleaning compositions comprise one or more variants of the present invention. Said contact lens cleaning compositions comprise one or more variants, preferably from about 0.01% to about 50% of one or more variants, more preferably from about 0.01% to about 20%, and still more preferably from around from 1% to about 5% by weight of the composition, and a vehicle for personal care. The various formats for the contact lens cleaning composition such as tablets, liquids and the like are well known to those skilled in the art and are generally suitable for the incorporation of one or more variants of the present invention to remove protein spots from the contact lenses. The embodiment of the contact lens cleaning composition of the present invention is illustrated in examples 14-17.

EXAMPLES 14-17 Cleaning Solution for Contact Lenses In Examples 14-17, the variants mentioned in Tables 7, 8 and 10, and the preferred variants mentioned herein, among others, are replaced by the 9-I variant with substantially similar results.

EXAMPLES 18-21 Body Cleaning Products In Examples 18-21, the variants mentioned in Tables 7, 8 and 10, and the preferred variants mentioned herein, inter alia, are substituted by variant 14-1 with substantially similar results. Examples 18-21 illustrate the use of the present variants in facial cleansing products: EXAMPLES 22-25 Facial Cleaning In Examples 22-25, the variants mentioned in Tables 7, 8 and 10, and the preferred variants mentioned herein, among others, are replaced by the 24-I variant with substantially similar results. Examples 26-27 illustrate the use of the present variants in moisturizing compositions left on the skin: EXAMPLES 26-27 Moisturizing composition left on the skin In Examples 26-27, the variants mentioned in Tables 7, 8 and 10, and the preferred variants mentioned herein, among others, are substituted by variant 13-1 with substantially similar results. Example 28 illustrates the use of the present variants in cloth cleaning compositions: EXAMPLE 28 Composition for cleaning with cloth The above composition is impregnated onto a woven absorbent sheet consisting of cellulose and / or polyester at about 250% by weight of the absorbent sheet. In example 28, the variants mentioned in tables 7, 8 and 10, and the preferred variants mentioned herein, among others, are replaced by variant 20-I with substantially similar results.

LIST OF SEQUENCES < 110 > Saunders, Charles W. Correa, Paul E. Sun, Yiping Rubingh, Donn N. < 120 > Stabilized variants of Streptomyces Subtilisin Inhibitor < 130 > Stabilized variants < 140 > < 141 > < 150 > 60/091, 911 < 151 > 1998-07-07 < 160 > fifteen < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 113 < 212 > PRT < 213 > Streptomyces albogriseolus < 400 > 1 Asp Ala Pro Ser Ala Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Val i S 10 1S Gly Lys Gly Val Ser Wing Thr Thr Wing Wing Pro Glu Arg Wing Val Thr 20 25 30 Leu Thr Cys Wing Pro Gly Pro Ser Gly Thr His Pro Wing Wing Gly Ser 0 45 Wing Cys Wing Asp Leu Wing Wing Val Gly Gly Asp Leu Asn Wing Leu Thr • 50 55 60 Arg Gly Glu Asp Val Met Cys Pro Met Val Tyr Asp Pro Val Leu Leu 65 70 75 80 Thr Val Asp Gly Val Trp Gln Gly Lys Arg Val Ser Tyr Glu Arg Val 85 90 95 Phe Ser Asn Glu Cys Glu Met Asn Wing His Gly Ser Ser Val Wing Phe 100 105 110 Phe < 210 > 2 < 211 > 117 < 212 > PRT < 213 > Streptomyces albogriseolus < 400 > 2 Wing Gly Glu Phe Asp Wing Pro Being Wing Leu Tyr Wing Pro Being Wing Leu 10 15 Val Leu Thr Val Gly Lys Gly Val Ser Wing Thr Thr Ala Wing Pro Glu 20 25 30 Arg Wing Val Thr Leu Thr Cys Ala Pro Gly Pro Ser Gly Thr His Pro 35 40 45 «T &aagAas-, ~ Ala Ala Gly Be Ala Cys Ala Asp Leu Ala Ala Val Gly Gly Asp Leu 50 55 60 Asn Ala Leu Thr Arg Gly Glu Asp Val Met Cys Pro Met Val Tyr Asp dS 70 75 80 Pro Val Leu Leu Thr Val Asp Gly Val Trp Gln Gly Lys Arg Val Ser 85 90 95 Tyr Glu Arg Val Phe Ser Asn Glu Cys Glu Met Asn Wing His Gly Ser 100 105 110 Ser Val Phe Ala Phe 115 < 210 > 3 < 211 > 275 < 212 > PRT < 213 > Bacillus amyloliquefaciens < 400 > 3 Ala Gln Ser Val Pro Tyr Gly Val Ser Gln He Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Wing Val He Asp 25 30 Ser Gly He Asp Ser Ser His Pro Asp Leu Lys Val Wing Gly Gly Wing 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 ßO Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser He Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Wing Asp Gly Ser Gly Gln Tyr Being Trp He He Asn Gly He Glu 100 105 110 Trp Wing Wing Asn Asn Met Asp Val As As Met Le Gly Gly US 120 125 Pro Ser Gly Ser Ala Ala Leu Ala Ala Ala Asp Val Lys Wing Val Wing 130 135 140 Ser Gly Val Val Val Val Wing Wing Wing Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Being Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val He Ua 165 170 175 Val Gly Wing Val Asp Being Ser Asn Gln Arg Wing Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Wing Pro Gly val Ser He Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Wing Tyr Asn Gly Thr Ser Met Wing Ser 210 215 220 Pro His Val Wing Ala Wing Ala Wing Leu He Leu Ser Lys His Pro Asn 225 230 23 S 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Is- 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu He Asn Val Gln Wing 260 265 270 Wing Wing Gln 275 < 210 > 4 < 211 > 107 < 212 > PRT < 213 > Streptomyces thermotolerans . , -.- ytáÉíS ??: -. ,. ,. i, -? < 400 > 4 Tyr Ala Pro Be Ala Leu Val Leu Thr Val Gly His Gly Glu Be Ala 1 5 10 as He Ala Ala Thr Pro Glu Arg Ala to Thr Leu Thr Cys Ala Pro Lys 20 25 30 Ala Ala Gly Thr His Pro Ala Ala Ala Ala Ala Cys Ala Glu Leu Arg 35 40 45 Gly Val Gly Gly Asp Phe Asp Ala Leu Thr Ala Arg Asp Gly Val Met 50 55 60 Cys Thr Lys Gln Tyr Asp Pro Val Val Val Val Val Val Glu Gly Val Trp 65 70 75 80 Gln Gly Lys Arg Val Ser Tyr Glu Arg Thr Phe Ser Asn Asp Cys Met 85 90 95 Lys Asn Wing Tyr Gly Thr Gly Val Phe Ser Phe 100 105 < 210 > 5 < 211 > 109 < 212 > PRT < 213 > Streptomyces galbus < 400 > 5 Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Met Gly His Gly Glu 1 5 10 15 Be Wing Wing? Val Ser Pro Wing Arg Wing Val Thr Leu Asn Cys Wing 20 25 30 Pro Sex Wing Being Gly Thr His Pro Wing Pro Wing Leu Wing Cys Wing Glu 40 45 Leu Arg? La? The Gly Gly Asp Leu Asp Ala Leu Ala Gly Pro Ala? Sp 50 55 60 Thr Val cys Thr Lys Gln Tyr? The Pro Val Val He Thr Val? Sp Gly 65 70 75 80 Val Trp Gln Gly Lys Arg Val Ser Tyr Glu Arg Thr Phe? The Asn Gly 85 90 95 Cys Val Lys Asn Wing Ser Gly Ser Ser Val Phe Wing Phe 100 ios < 210 > 6 < 211 > 107 < 212 > PRT < 213 > Streptomyces azureus < 400 > 6 Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly Glu Gly Glu Ser Ala 1 5 10 15 Wing Wing Wing Thr Pro Glu Arg Wing Val Thr Leu Thr Cys Wing Pro Arg 2S 30 Pro Ser Gly Thr His Pro Val Wing Gly Ser Wing Cys Wing Glu Leu Arg 35 40 45 Gly Val Gly Gly Asp Val His Ala Leu Thr Ala Thr Asp Gly al Mee 50 55 60 Cys Thr Lys Gln Tyr Asp Pro Val Val to Thr Val Asp Gly Val Trp 65 70 75 80 Gln Gly Arg Arg Val Ser Tyr Glu Arg Thr Phe Ser Asn Glu Cys Val 85 90 95 Lys Asn Wing Tyr Gly Ser Gly Val Phe Wing Phe 100 105 < 210 > 7 < 211 > 110 < 212 > PRT < 213 > Streptomyces lividans < 400 > 7 Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly Glu 1 5 10 ÍS Ser Ala Ala Thr Ala Ala Pro Leu Arg Ala Val Thr Leu Thr Cys Ala 20 25 30 Pro Thr Ala Ser Gly Thr His Pro Ala Ala Ala Ala? The Cys? The Glu 35 40 45 Leu Arg? La? La His Gly? Sp Pro Ser? La Leu Ala? Glu? Sp Ser 50 55 60 Val Met Cys Thr? Rg Glu Tyr Ala Pro Val Val Val Thr Val Asp Gly 65 70 75 80 Val Trp Gln Gly Arg Arg Leu Ser Tyr Glu Arg Thr Phe Wing Asn Glu 85 90 95 Cys Val Lys Asn Wing Gly Ser Wing Val Phe Thr Phe Glu 100 105 110 < 210 > 8 < 211 > 110 < 212 > PRT < 213 > Streptomyces longisporus < 400 > 8 Ala Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly 1 5 10 15 Thr Be Ala Ala Ala Ala Thr Pro Leu? Rg? The Val Thr Leu Asa Cys 20 25 30? The Pro Thr? The Ser Gly Thr His Pro? The Pro Ala Leu? The Cys? The 35 40 4S Asp Leu Arg Gly val Gly Gly Asp He Asp Wing Leu Lys Wing Arg Asp SO 55 60 Gly Val He Cys Asn Lys Leu Tyr A = p Pro Val Val Val Thr Val Asp 65 70 75 80 Gly Val Trp Gln Gly Lys Arg Val Ser Tyr Glu Arg Thr Phe Gly Asn 85 90 95 Glu Cys Val Lys Asn Ser Tyr Gly Thr Ser Leu Phe Ala Phe 100 105 HO < 210 > 9 < 211 > 113 < 212 > PRT < 213 > Streptomyces parvulus < 400 > 9 Thr Ala Pro Wing Being Leu Tyr Wing Pro Being Wing Leu Val Leu Thr He 1 5 10 15 Gly Gln Gly Glu Wing Wing Wing Thr Ser Pro Leu Arg Wing Val Thr 20 25 30 Leu Thr Cys Wing Pro Lys Wing Thr Gly Thr His Pro Ala Ala Asp Ala 35 40 45 Ala Cys Ala Glu Leu Arg Arg Ala Gly Gly Asp Phe Asp Ala Leu Ser 50 55 60 Ala Ala Asp Gly Val Mee Cys Thr Arg Glu Tyr Ala Pro Val Val Val 65 70 75 80 Thr Val Asp Gly Val Trp Gln Gly Arg Arg Leu Ser Tyr Glu Arg Thr 85 90 95 Phe Wing Asn Glu Cys Val Lys Asn Wing Gly Ser Wing Val Phe Thr 100 105 110 Phe < 21O > 10 < 211 > 107 < 212 > PRT < 213 > Streptomyces coelicolor < 400 > 10 Tyr Ala Pro Ser? Leu Val Leu Thr Val Gly His Gly Glu Ser? La 1 5 10 15 Ala Thr Ala Ala Pro Leu Arg Ala Val Thr Leu Thr Cys Ala Pro Thr 25 30 Wing Ser Gly Thr His Pro Wing Wing? Sp? The? The Cys Wing Glu Leu Arg 3S 40 45 Wing Wing His Gly Asp Pro Wing Wing Leu Wing Wing Asp Asp Wing Val Met 50 55 60 Cys Thr Arg Glu Tyr Ala Pro Val Val Val Thr Val Asp Gly Val Trp 65 70 75 80 Gln Gly Arg Arg Leu Ser Tyr Glu Arg Thr Phe Wing Asn Glu Cys Val 85 90 95 Lys Asn Wing Gly Ser Wing Being Val Phe Thr Phe 100 IOS < 210 > 1 1 < 211 > 116 < 212 > PRT < 213 > Streptomyces lavendulae < 400 > eleven Wing Pro Asp Wing Wing Pro Wing Being Leu Tyr Wing Pro Being Wing Leu Val 1 5 10 15 Leu Thr He Gly His Gly Gly Wing Wing Wing Thr Wing Thr Pro Glu Arg 20 25 30 Wing Val Thr Leu Thr Cys Wing Pro Thr Ser Ser Gly Thr His Pro Wing 35 40 45 Wing Wing Wing Cys Wing Glu Leu Arg Gly Val Gly Gly Asp Phe Wing 50 55 60 Wing Leu Lys Wing Arg Asp Asp Val Trp Cys Asn Lys Leu Tyr Asp Pro 65 70 75 80 Val Val Val Thr Ala Gln Gly Val Trp Gln Gly Gln Arg Val Ser Tyr 85 90 95 Glu Arg Thr Phe Gly Asn Ser Cys Glu Arg Asp Wing Val Gly Gly Ser 100 105 110 Leu Phe Wing Phe 115 < 210 > 12 < 211 > 109 < 212 > PRT < 213 > Streptomyces antifibrinolyticus < 400 > 12 Gly Leu Tyr Ala Pro Be Ala Leu Val Leu Thr Met Gly His Gly Asn 1 S 10 is Be Ala Ala Thr Val? Sn Pro Glu Arg Ala Val Thr Leu Asn Cys Ala 20 25 30 Pro Thr Ala Ser Gly Thr His Pro? The Ala Leu Gln Ala Cys Ala Glu 35 40 45 Leu Arg Gly? The Gly Gly Asp Phe? sp? the Leu Thr Val? rg Gly Asp 50 55 60 Val? the Cys Thr Lys Gln Phe? sp Pro Val Val Val Thr Val? sp Gly SS 70 75 80 Val Trp Gln Gly Lys Arg Val Ser Tyr Glu Arg Thr Phe? the? sn Glu 85 90 95 Cys Val Lys? sn Ser Tyr Gly MeC Thr Val Phe Thr Phe 100 IOS < 210 > 13 < 211 > 107 < 212 > PRT < 213 > Streptomyces lividans < 400 > 13 Tyr Ala Pro Be Ala Leu Val Leu Thr Val Gly His Gly Glu Be Ala 1 5 10 15 Ala Thr Ala Ala Pro Leu Arg Ala Val Thr Leu Thr Cys Ala Pro Thr 20 25 30 Ala Ser Gly Thr His Pro Ala Ala Ala Ala Ala Ala Cys Ala Glu Leu Arg 35 40 45 Ala Ala His Gly Asp Pro Be Ala Leu Ala Ala Glu Asp Ser Val Met 50 55 60 Cys Thr Arg Glu Tyr Ala Pro Val Val Val Thr al Asp Gly Val Trp 65 70 75 80 Gln Gly Arg Arg Leu Ser Tyr Glu Arg Thr Phe Wing Asn Glu Cys Val 85 90 95 Lys Asn Wing Gly Ser Wing Val Phe Thr Phe 100 105 < 210 > 14 < 211 > 110 < 212 > PRT < 213 > Streptoverticillium cinnamoneum < 400 > 14 Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr He Gly Gln Gly Asp 1 S 10 15 Be Ala? La? The Wing Gly He Gln Arg Wing Val Thr Leu Thr Cys Met 20 25 30 Pro Lys Wing Asp Gly Thr His Pro Asn Thr? Rg Gly Wing Cys Wing Gln 35 40 45 Leu Arg Leu Wing Gly Gly Asp Phe Glu Lys Val Thr Lys He Lys Glu 50 55 60 Gly Thr Wing Cys Thr Arg Glu Trp Asn Pro Ser Val Val Thr Ala Glu 65 70 7S 80 Gly Val Trp Glu Gly? Rg Arg Val Ser Phe Glu Arg Thr Phe Wing Asn 85 90 95 Pro Cya Glu Leu Lys Wing Gly Lys Gly Thr Val Phe Glu Phe 100 105 110 < 210 > 15 < 211 > 1 10 < 212 > PRT < 213 > Streptomyces cacaoi < 400 > fifteen Ser Leu Tyr Ala Pro Ser Ala Val Val lie Lie Lys Thr Gln Gly Ala 1 5 10 15 Be Ala Asp? Pro Gln Arg Ala Val Thr Leu Arg Cys Leu Pro 20 25 30 Val Gly Gly? Sp His Pro? The Pro Glu Lys? The Cys? The Ala Leu Arg 35 40 45 Glu? The Gly Gly Asp Pro? Ala Leu Pro? Rg Tyr Val Glu Asp Thr so 55 60 Gly Arg Val Cys Thr Arg Glu Tyr Arg Pro Val Thr Val Ser Val Gln 65 70 75 80 Gly Val Trp Asp Gly Arg Arg He Asp His Wing Gln Thr Phe Ser Asn 85 90 95 Ser Cys Glu Leu Glu Lys Gln Thr Wing Ser Val Tyr Wing Phe 100 105 110 * s * * »« ^ »i» Ms é ^

Claims (10)

NOVELTY OF THE INVENTION CLAIMS

1. - A variant characterized by a modified amino acid sequence of an origin amino acid sequence, wherein the modified amino acid sequence is characterized by an amino acid substitution at position 63 corresponding to SS1, and wherein the origin amino acid sequence is selected from the a group consisting of SSl, SSI-like inhibitors, SSl variants, and variants of SSI-like inhibitors.

2. The variant according to claim 1, further characterized in that the substitution of the amino acid at position 63 corresponding to SS1 is with isoleucine.

3. The variant according to any of the preceding claims, further characterized in that the origin amino acid sequence is selected from the group consisting of SS1 and SS1 variants.

4. The variant according to any of the preceding claims which shows a K, such that the variant: a) inhibits a protease in a composition comprising the variant and the protease; and b) it is separated from the protease with the dilution.

5. - The variant according to any of the preceding claims which shows a Kl of IO.12 M to approximately 10.4 M.

6. The variant according to any of the preceding claims selected from the group consisting of: (a) L631 + D83C; (b) L631 + M73D; (c) L631 + M73D + D83C; (d) L631 + M73P + D83C; (e) L631 + M70Q + D83C; (f) L631 + M70Q + M73P + V74F + D83C; (g) L631 + M70Q + M73P + V74W + D83C; (h) L631 + M70Q + M73P + D83C + S98A; (i) L631 + G47D + M73P + V74F + D83C; (j) L631 + G47D + M73P + V74W + D83C; (k) L631 + G47D + M73P + D83C + S98A; (I) L631 + G47D + M70Q + M73P + V74F + D83C; (m) L631 + G47D + M70Q + M73P + V74F + D83C; (n) L631 + G47D + M73P + V74F + D83C + S98A; (o) L631 + G47D + M73P + V74W + D83C + S98A; (p) A62 * + L631 + D83C; (q) A62 * + L631 + M73D; r) A62 * + L631 + M73D + D83C; (s) A62 * + L631 + M73P + D83C; (t) A62 * + L631 + M70Q + D83C; (u) A62 * + L631 + M73P + D83C + S98A; (v) A62 * + L631 + M73P + Y75A + D83C; (w) A62 * + L631 + M73P + D83C + S98V; (x) A62 * + L631 + M70Q + M73P + D83C; (y) A62 * + L631 + M73P + V74A + D83C; (z) A62 * + L631 + M73P + V74F + D83C; (aa) A62 * + L631 + M70Q + D83C + S98A; (bb) A62 * + L631 + G47D + M70Q + D83C; (ce) A62 * + L631 + G47D + D83C + S98A; (dd) A62 * + L631 + G47D + M73P + D83C; (ee) A62 * + L631 + G47D + M73D + D83C; (ff) A62 * + L631 + M70Q + M73P + V74F + D83C; (gg) A62 * + L631 + M70Q + M73P + V74W + D83C; (hh) A62 * + L631 + M70Q + M73P + D83C + S98A; (ii) A62 * + L631 + G47D + M73P + V74F + D83C; (J) A62 * + L631 + G47D + M73P + V74W + D83C; (kk) A62 * + L631 + G47D + M73P + D83C + S98A; (II) A62 * + L631 + G47D + M70Q + M73P + V74F + D83C; (mm) A62 * + L631 + G47D + M70Q + M73P + V74W + D83C; (nn) A62 * + L631 + G47D + M73P + V74F + D83C + S98A; (oo) A62 * + L631 + G47D + M73P + V74W + D83C + S98A; (pp) L631 + A62K + S98Q; (qq) L631 + A62K + S98D; (rr) L631 + A62K + S98E; (ss) L631 + A62R + S98Q; (tt) L631 + A62R + S98D; (uu) L631 + A62R + S98E; (w) L631 + S98A; (ww) L631 + M73P + D83C + S98D; (xx) L631 + M73P + D83C + S98E; (yy) L631 + M73P + S98D; (zz) L631 + M73P + S98E; (aaa) L631 + M73P + S98A; (bbb) A62K + L631 + M73P + D83C + S98D; (ccc) A62R + L631 + M73P + D83C + S98D; (ddd) A62K + L631 + M73P + D83C + S98E; (eee) A62R + L631 + M73P + D83C + S98E; (fff) A62K + L631 + M73P + S98A; (ggg) A62R + L631 + M73P + S98A; (hhh) L631 + G47D + M73P + D83C + S98D; (iii) L631 + G47D + M73P + D83C + S98E; (¡J) L631 + M73P.

7. The variant according to any of the preceding claims selected from the group consisting of: (a) A62K + L631 + M73P + D83C + S98Q; (b) A62K + L631 + M73P + D83C + S98D; (c) A62K + L631 + M73P + D83C + S98E; (d) A62K + L631 + S98Q; (e) A62K + L631 + S98D; (f) A62K + L631 + S98E; (g) A62K + L631 + M73P + D83C + S98Q; (h) A62K + L631 + M73P + D83C + S98D; (i) A62K + L631 + M73P + D83C + S98E; (j) L631 + M73P + D83C + S98A; (k) A62R + L631 + S98Q; (I) A62R + L631 + S98D; (m) A62R + L631 + S98E; (n) L631 + S98A; (o) A62K + L631 + M73P + D83C + S98D; (p) A62R + L631 + M73P + D83C + S98D; (q) - ^ a toaé ^ A62K + L631 + M73P + D83C + S98E; (r) A62R + L631 + M73P + D83C + S98E; (s) A62K + L631 + M73P + S98A; and (t) A62R + L631 + M73P + S98A.

8. A mutant gene that codes for a variant according to any of the preceding claims.

9. A composition comprising a variant according to any of the preceding claims and a vehicle selected from the group consisting of a cleaning composition vehicle and a personal care composition vehicle.

10. The composition according to claim 9, further characterized in that the variant to protease ratio is from about 3: 1 to about 1: 1.