CN1316000A - Stabilized variants of i(streptomyces) subtilisin inhibitor - 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 Streptomyces subtilisin inhibitors Cross-reference to related applications

This application claims the benefit of US Provisional Patent Application No. 60/091,911, filed July 7,1998.

field of invention

The present invention relates to variants of Streptomyces subtilisin inhibitors (SSI) and inhibitors having homology to SSI (SSI-like inhibitors). These variants are useful in cleaning and personal hygiene compositions in combination with enzymes, especially proteases. The invention also relates to cleaning and personal hygiene compositions comprising the variants and genes encoding these variants.

Background of the invention

Enzymes constitute the largest class of naturally occurring proteins. One class of enzymes includes proteases that catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins is useful for the incorporation of naturally occurring and engineered proteins into cleaning compositions, especially in relation to laundry applications. Furthermore, others have incorporated these proteases into personal hygiene compositions, albeit in lesser attempt. However, during storage of these compositions and even during expression of the proteases, these proteases are often degraded by themselves or possibly other enzymes present in the composition. As a consequence of degradation, these cleansing and personal hygiene compositions are limited in their ability to achieve the desired enhancement.

Therefore, it would be beneficial to add to the composition an inhibitor of protease activity to limit autohydrolysis by proteases and degradation by other enzymes. It would be of great value to provide a reversible protease inhibitor so that when the composition is used for cleaning or is diluted in a cleaning environment, proteases are no longer inhibited and can hydrolyze protein stains. Furthermore, these inhibitors must be stable enough to fully exert their inhibitory function.

Synthetic protease inhibitors or stabilizers have been published for this use, especially in the laundry environment. For example, Panandiker et al., U.S. Patent No. 5,422,030, assigned to The Procter & Gamble Co., issued June 6, 1995, disclose aromatic borate esters for use in stabilizing laundry cleaning compositions. In addition, Bruno et al. in US Patent No. 4,566,985, assigned to Applied Biochemists, Inc., issued January 28, 1986, proposed the use of benzamidine hydrochloride as an enzyme inhibitor. These synthetic inhibitory pathways may extend storage time, but may be more expensive, and it may not improve isolation yield due to proteolysis in the fermenter.

Recognizing these deficiencies, those in the art have experimented with proteinaceous protease inhibitors that stabilize enzymes in cleaning compositions. Nature provides proteinaceous protease inhibitors to regulate proteases in vivo. However, due to the tendency of these naturally occurring proteinaceous protease inhibitors to be unstable, their commercial use in protease-containing cleaning and personal hygiene vehicles may be somewhat limited.

Protein-based protease inhibitors are typically long peptide chains that bind to the active site of a protease and inhibit its activity. These inhibitors are generally classified into several families (Families I to IX) according to the homology of the primary amino acid sequence (see Laskowski et al., "Protein Inhibitors of Proteinases", Annual Review of Biochemistry, Vol. 49, pp. 593-626 (1980)). These inhibitors include inhibitors commonly referred to as Family VI inhibitors, which include eglin and barley chymotrypsin inhibitors, and family III inhibitors, which include Streptomyces subtilis inhibitors (SSI) and plasminostretin.

These inhibitors tend to bind specific proteases and bind others weakly. This facilitates the consideration of inhibitors for a particular protease. For this reason, "protease/polypeptide inhibitor pairs" are often discussed in the art. An example of a known protease/polypeptide inhibitor pair is subtilisin BPN'/SSI. See for example Mitsui et al., "Crystal Structure of a Bacterial Protein Proteinase Inhibitor (Streptomyces Subtilisin Inhibitor) at 2.6_Resolution", Journal of Molecular Biology, Vol.131, pp.697-724 (1979) and Hirono et al., "Crystal Structure at 1.6_ Resolution of the Complex of Subtilisin BPN'with Streptomyces Subtilisin Inhibitor", Journal of Molecular Biology, Vol.178, pp.389-413(1984).

SSI is stable in the presence of Subtilisin BPN' as long as the amount of the inhibitor is sufficient to inhibit all protease activity. However, it is believed that inhibitors with high protease affinity will not dissociate from them when diluted in the wash environment. See WO 92/03529, Mikkelson et al., designated Novo Nordisk A/S, published March 5, 1992.

However, if the binding constant (Ki) of an inhibitor allows certain enzymatic activity in a cleaning composition containing an enzyme/inhibitor pair, the inhibitor and the enzyme in the composition may be hydrolyzed. Therefore, it would be of great value to find SSI variants or other inhibitors that are suitably stable in the presence of proteases and cleaning and personal hygiene compositions. Furthermore, these inhibitors preferably have a preferred Ki for the particular protease to be inhibited. This Ki must allow inhibition of proteases in the final composition during storage. However, when these cleaning and personal hygiene compositions are diluted in the wash environment, the protease and inhibitor must dissociate to produce uninhibited protease activity.

Kojima et al., "Inhibition of Subtilisin BPN'by ReactionSite P1 Mutants of Streptomyces Subtilisin Inhibitor", Journal of Molecular Biology, Vol.109, pp.377-382 (1991) made use of Subtilisin BPN' to produce P1 positions of several SSIs ( 73) variants and their Ki were measured. As another example, Mikklsen et al. disclose mutations in family VI inhibitors that are said to have lower binding activity. WO 93/17086, Nielsen et al., designated Novo Nordisk A/S, published September 2, 1993, discloses mutations in plasminostreptin which are said to have lower binding activity.

However, the stability of these protease inhibitors has been problematic. WO 98/13387, Correa et al. , designated The Procter & Gamble Co., published April 2, 1992 (corresponding to U.S. Patent Application Serial No. 60/026,944), discloses variants that provide enhanced stability.

Despite the variety of approaches described in the art, there remains a continuing need for more stable and effective protease inhibitors for use in cleaning and personal hygiene compositions. The inventors have surprisingly found that SSI inhibitors, SSI-like inhibitors and their variants are readily hydrolyzed between positions 63 and 64 corresponding to SSI when expressed and/or in cleaning and personal hygiene compositions. Accordingly, the invention herein provides modified SSI inhibitor variants and SSI-like inhibitor variants which are modified in particular at position 63 by an alternative amino acid residue. These substitutions confer greater stability on protease inhibitors. These inhibitors are also beneficial in that they bind proteases at preferred levels as defined herein. Accordingly, the present invention provides protease inhibitor variants of proteinaceous ingredients which have higher proteolytic stability, particularly in cleaning and personal hygiene compositions, which are also more protease-resistant than the related parental inhibitors. low affinity.

Summary of Invention

The invention provides variants of the parent amino acid sequence having a modified amino acid sequence comprising an amino acid substitution at position 63 corresponding to SSI, the parent amino acid sequence being selected from the group consisting of SSI, SSI-like inhibition A population consisting of agents, SSI variants, and SSI-like inhibitor variants. These variants are useful for such purposes as inhibition of proteases, especially during their storage and expression. The invention also relates to genes encoding these variants and to cleaning and personal hygiene compositions containing these variants.

Detailed description of the invention

The essential components of the invention are described herein. Also included herein are non-limiting descriptions of various optional and preferred components, which are useful in embodiments of the present invention.

The present invention may comprise, consist of, or consist essentially of any required or optional components, ingredients and/or limitations as described herein.

All percentages and ratios are by weight unless otherwise indicated. All percentages are calculated on the basis of the total composition unless otherwise indicated.

Reference is made herein to trade names of materials, including proteases and optional components, but not limited thereto. The present inventors do not wish to limit the materials to those labeled as specific commercial products. Materials equivalent to those of the cited trade names (eg, ® ) may be substituted for use in the compositions herein.

All levels of components, ingredients or compositions refer to the active level of such components, ingredients or compositions and do not include impurities, such as residual solvents or by-products, which may be present in commercial sources.

All documents cited herein, including all patents, patent applications, and printed publications, are hereby incorporated by reference in their entirety.

Abbreviations used herein will be used to describe amino acids. Table 1 provides a list of abbreviations used herein.

Table 1 amino acid three letter abbreviation single letter abbreviation Alanine alas A arginine Arg R Asparagine Asn N aspartic acid Asp D. cysteine Cys C Glutamine Gln Q glutamic acid Glu E. Glycine Gly G Histidine His h Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine met m Phenylalanine Phe f proline Pro P serine Ser S threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Definition

The term "mutation" as used herein refers to changes in gene sequences and amino acid sequences resulting from those gene sequences. Mutations can be deletions, substitutions or additions of amino acid residues in the wild-type or parental sequence.

As used herein, the term "parent" refers to a wild-type or variant protease, protease inhibitor, protein or polypeptide without an amino acid substitution at position 63 corresponding to SSI (i.e., the amino acid substitution at position 63 occurs naturally) . An example of one of these parents is the known Streptomyces subtilisin inhibitor (SSI) (SEQ ID NO: 1). SSI is further described in 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). Amino acid numbering used herein is from Ikenaka et al. The present inventors also used a synthetic SSI gene designed to be rich in adenine and thymine, just like the DNA of B. subtilis. By constructing an expression plasmid, this synthetic gene encodes four additional amino acid residues at the amino-terminus of the polypeptide. 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 polypeptide produced by an organism that has not been mutated, and herein specifically refers to a protease or a protease inhibitor.

The term "variant" as used herein refers to a protein or polypeptide, here specifically a protease inhibitor or protease, which has an amino acid sequence different from a parent protease inhibitor or a parent protease, respectively.

Variants of the Invention

The present inventors have discovered variants of protease inhibitors that are more stable in conditions such as in vitro and in the presence of cleaning and personal hygiene composition materials. Furthermore, these variants are also more stable in vivo, which increases the production of proteases from the organism.

The inventors also found variants that exhibit preferred binding constants (K _i ), which allow for proper inhibition of proteases during growth, harvesting, purification, storage and washing. This preferred combination provides better stability and longer shelf life.

The variants of the present invention have improved stability towards proteases, inhibit proteases in cleaning and personal hygiene compositions, but dissociate from them upon dilution in the wash environment.

The variants of the present invention have a modified amino acid sequence of the parent amino acid sequence, where the modified amino acid sequence includes an amino acid substitution corresponding to position 63 of Streptomyces subtilisin (i.e. referred to herein as SSI), where the parent amino acid sequence selected from SSI, SSI-like inhibitors, SSI variants and SSI-like inhibitor variants. The amino acid substitution at position 63 corresponding to SSI can be any amino acid residue that results in greater stability compared to the parental amino acid sequence. The amino acid substitution at position 63 corresponding to SSI is most preferably by isoleucine. One such variant is designated "L63I". The variant is indicated first by the original amino acid as it appears in the parental amino acid sequence, followed by the position number and third by the substituted amino acid. Thus, L63I refers to the replacement of leucine (L) occurring at the 63rd amino acid position (position 63) of the natural inhibitor SSI by isoleucine (I). The position numbers are the same as Ikenaka et al. above. (SEQ ID NO: 1) corresponding, and ignoring the 4 additional amino acid residues present at the amino terminus of the synthetic SSI (SEQ ID NO: 1). All other substitutions listed herein are represented in this manner.

The variants herein are not limited to SSI with 63 bits replaced. The substitution at position 63 can also be carried out on the parent amino acid sequence (including of course the nucleotide sequence encoding the amino acid sequence), and the parent itself is an SSI variant, an SSI-like inhibitor or a variant of an SSI-like inhibitor. More preferred parental amino acid sequences include SSI and SSI variants. Most preferred parental amino acid sequences are SSI variants. Examples of disclosure of SSI variants are Kojima et al., "Inhibition of Subtilisin BPN' by Reaction Site P1 Mutants of Streptomyces Subtilisin Inhibitor", Journal of Molecular Biology, Vol.109, pp.377-382 (1991); Tamura et al., " Mechanisms of Temporary Inhibition in Streptomyces Subtilisin Inhibitor Induced by anAmino Acid Substitution, Typtophan 86 Replaced by Histidine", Biochemistry, Vol.30, pp.5275-5286(1991); JO 3099-099-A, assigned to Tsum Issued September 12, 1989; Mikkelson et al., U.S. Patent No. 5,674,833, assigned to Novo Nordisk A/S, granted October 7, 1997; WO 93/17086, Nielsen et al., assigned to Novo Nordisk A/S, Published September 2, 1993. Variants of other SSIs are disclosed in WO 98/13387 of U.S. Patent Application Serial No. 60/026,944, Correa et al., assigned to The Procter & Gamble Co., published April 2, 1992, and these variants are collectively referred to herein as " Inhibitor Class A". Preferred variant SSIs (used here as parent amino acid sequences) are variants within group A of inhibitors. More preferred SSI variants for use as parental amino acid sequences are listed here in Tables 2-6 below. Likewise, all position numbers correspond to eg Ikenaka et al. SSI as described.

Table 2

Non-limiting examples of parental amino acid sequences with single site substitutions parent 1 D83C parent 4 M73D parent 34 M73P

Table 3 Non-limiting examples of parental amino acid sequences with two-site substitutions parent 2 M73D+D83C parent 3 M73D+D83C parent 5 M70Q+D83C parent 29 M73P+S98D parent 30 M73P+S98E parent 31 M73P+S98A

Table 4 Non-limiting examples of parental amino acid sequences with three-position substitutions parent 6 M73P+D83C+S98A parent 7 M73P+Y75A+D83C parent 8 M73P+D83C+S98V parent 9 M70Q+M73P+D83C parent 10 M73P+V74A+D83C parent 11 M73P+M74F+D83C parent 12 M70Q+D83C+S98A parent 13 G47D+M70Q+D83C parent 14 G47D+D83C+S98A parent 15 G47D+M73P+D83C parent 16 G47D+M73D+D83C parent 27 M73Q+D83C+S98D parent 28 M73P+D83C+S98E

Table 5 Non-limiting examples of parental amino acid sequences with four-position substitutions parent 17 M70Q+M73P+V74F+D83C parent 18 M70Q+M73P+V74W+D83C parent 19 M70Q+M73P+D83C+S98A parent 20 G47D+M73P+V74F+D83C parent 21 G47D+M73P+V74W+D83C parent 22 G47D+M73P+D83C+S98A parent 32 G47D+M73P+D83C+S98D parent 33 G47D+M73P+D83C+S98E

Table 6 Non-limiting examples of parental amino acid sequences with five-position substitutions parent 23 G47D+M70Q+M73P+V74F+D83C parent 24 G47D+M70Q+M73P+V74W+D83C parent 25 G47D+M73P+V74F+D83C+S98A parent 26 G47D+M73P+V74W+D83C+S98A

Thus, non-limiting examples of variants of the invention may be described as variant 1, variant 2, etc., for example, wherein variant 1 may be represented as L63 ^* +D83C, where " ^* " represents any non-SSI position 63 Amino acids of the original amino acids, variant 1-1 can be expressed as L63I+D83C. The more preferred variants among them are listed in Table 7, and the 63rd positions of these variants are all replaced by isoleucine.

Table 7

Non-limiting examples of preferred variants of the invention Variant 1 L63 ^* +D83C Variant 4 L63 ^* +M73D Variant 1-I L63I+D83C Variant 4-I L63I+M73D variant 2 L63 ^* +M73D+D83C Variant 3 L63 ^* +M73P+D83C Variant 5 L63 ^* +M70Q+D83C Variant 2-I L63I+M73D+D83C Variant 3-I L63I+M73P+D83C Variant 5-I L63I+M70Q+D83C Variant 6 L63 ^* +M73P+D83C+S98A Variant 7 L63 ^* +M73P+Y75A+D83C Variant 8 L63 ^* +M73P+D83C+S98V Variant 9 L63 ^* +M70Q+M73P+D83C Variant 10 L63 ^* +M73P+V74A+D83C Variant 11 L63 ^* +M73P+V74F+D83C Variant 12 L63 ^* +M70Q+D83C+S98A Variant 13 L63 ^* +G47D+M70Q+D83C Variant 14 L63 ^* +G47D+D83C+S98A Variant 15 L63 ^* +G47D+M73P+D83C Variant 16 L63 ^* +G47D+M73D+D83C Variant 6-I L63I+M73P+D83C+S98A Variant 7-I L63I+M73P+Y75A+D83C Variant 8-I L63I+M73P+D83C+S98V Variant 9-I L63I+M70Q+M73P+D83C Variant 10-I L63I+M73P+V74A+D83C Variant 11-I L63I+M73P+V74F+D83C Variant 12-I L63I+M70Q+D83C+S98A Variant 13-I L63I+G47D+M70Q+D83C Variant 14-I L63I+G47D+D83C+S98A Variant 15-I L63I+G47D+M73P+D83C Variant 16-I L63I+G47D+M73D+D83C Variant 17 L63 ^* +M70Q+M73P+V74F+D83C (table continued) Variant 18 L63 ^* +M70Q+M73P+V74W+D83C Variant 19 L63 ^* +M70Q+M73P+D83C+S98A Variant 20 L63 ^* +G47D+M73P+V74F+D83C Variant 21 L63 ^* +G47D+M73P+V74W+D83C Variant 22 L63 ^* +G47D+M73P+D83C+S98A Variant 17-I L63I+M70Q+M73P+V74F+D83C Variant 18-I L63I+M70Q+M73P+V74W+D83C Variant 19-I L63I+M70Q+M73P+D83C+S98A Variant 20-I L63I+G47D+M73P+V74F+D83C Variant 21-I L63I+G47D+M73P+V74W+D83C Variant 22-I L63I+G47D+M73P+D83C+S98A Variant 23 L63 ^* +G47D+M70Q+M73P+V74F+D83C Variant 24 L63 ^* +G47D+M70Q+M73P+V74W+D83C Variant 25 L63 ^* +G47D+M73P+V74F+D83C+S98A Variant 26 L63 ^* +G47D+M73P+V74W+D83C+S98A Variant 23-I L63I+G47D+M70Q+M73P+V74F+D83C Variant 24-I L63I+G47D+M70Q+M73P+V74W+D83C Variant 25-I L63I+G47D+M73P+V74F+D83C+S98A Variant 26-I L63I+G47D+M73P+V74W+D83C+S98A Variant 27-I L63I+M73P+D83C+S98D Variant 28-I L63I+M73P+D83C+S98E Variant 29-I L63I+M73P+S98D Variant 30-I L63I+M73P+S98E Variant 31-I L63I+M73P+S98A Variant 32-I L63I+G47D+M73P+D83C+S98D Variant 33-I L63I+G47D+M73P+D83C+S98E Variant 34-I L63I+M73P

Other preferred parent amino acid sequences herein include variants with substitutions at position 62 corresponding to SSI. The substitution at position 62 can be any amino acid except the naturally occurring amino acid in the parent (for SSI, the naturally occurring amino acid is alanine). Preferred substitutions at position 62 are selected from Lys, Arg, Glu, Asp, Thr, Ser, Gln, Asn and Trp, more preferred substitutions are selected from Lys, Arg, Glu, Asp, Thr, Ser, Gln and Asn, more preferably Preferred substitutions are selected from Lys, Arg, Glu and Asp, more preferred substitutions are selected from Lys and Arg, most preferred is Lys. The preferred amino acid sequence has a substitution at position 62 in addition to those listed in Tables 2-6. An example of such a parent is designated parent X-A62 ^* , where X corresponds to the parent exemplified in Tables 2-6. Thus, the parental 6-A62 ^* corresponds to A62 ^* +M73P+D83C+S98A. Similarly, parental 6-A62K corresponds to A62K+M73P+D83C+S98A. Similarly, one variant exemplified herein is variant 6-I-A62 ^* , which corresponds to A62 ^* +L63I+M73P+D83C+S98A. Thus, variant 6-I-A62K corresponds to A62K+L63I+M73P+D83C+S98A. Table 8 lists other preferred variants of the invention in this way.

Table 8 Non-limiting examples of other preferred variants of the invention Variant 1-A62 ^* A62 ^* +L63 ^* +D83C Variant 4 - A62 ^* A62 ^* +L63 ^* +M73D Variant-I-A62 ^* A62 ^* +L63I+D83C Variant 4-I-A62 ^* A62 ^* +L63I+M73D Variant 4-I-A62K A62K+L63I+M73D Variant 4-I-A62R A62R+L63I+M73D Variant 2 - A62 ^* A62 ^* +L63 ^* +M73D+D83C Variant 3 - A62 ^* A62 ^* +L63 ^* +M73P+D83C Variant 5-A62 ^* A62 ^* +L63 ^* +M70Q+D83C Variant 2-I-A62 ^* A62 ^* +L63I+M73D+D83C Variant 3-I-A62 ^* A62 ^* +L63I+M73P+D83C Variant 5-I-A62 ^* A62 ^* +L63I+M70Q+D83C Variant 2-I-A62K A62K+L63I+M73D+D83C Variant 2-I-A62R A62R+L63I+M73D+D83C Variant 3-I-A62K A62K+L63I+M73P+D83C Variant 3-I-A62R A62R+L63I+M73P+D83C Variant 5-I-A62K A62K+L63I+M70Q+D83C Variant 5-I-A62R A62R+L63I+M70Q+D83C Variant 6-A62 ^* A62 ^* +L63 ^* +M73P+D83C+S98A Variant 7-A62 ^* A62 ^* +L63 ^* +M73P+Y75A+D83C Variant 8-A62 ^* A62 ^* +L63 ^* +M73P+D83C+S98V Variant 9-A62 ^* A62 ^* +L63 ^* +M70Q+M73P+D83C Variant 10-A62 ^* A62 ^* +L63 ^* +M73P+V74A+D83C Variant 11-A62 ^* A62 ^* +L63 ^* +M73P+V74F+D83C Variant 12-A62 ^* A62 ^* +L63 ^* +M70Q+D83C+S98A Variant 13-A62 ^* A62 ^* +L63 ^* +G47D+M70Q+D83C Variant 14-A62 ^* A62 ^* +L63 ^* +G47D+D83C+S98A Variant 15-A62 ^* A62 ^* +L63 ^* +G47D+M73P+D83C Variant 16-A62 ^* A62 ^* +L63 ^* +G47D+M73D+D83C Variant 6-I-A62 ^* A62 ^* +L63I+M73P+D83C+S98A Variant 6-I-A62K A62K+L63I+M73P+D83C+S98A Variant 6-I-A62R A62R+L63I+M73P+D83C+S98A Variant 7-I-A62 ^* A62 ^* +L63I+M73P+Y75A+D83C

Variant 7-I-A62K A62K+L63I+M73P+Y75A+D83C Variant 7-I-A62R A62R+L63I+M73P+Y75A+D83C Variant 8-I-A62 ^* A62 ^* +L63I+M73P+D83C+S98V Variant 8-I-A62K A62K+L63I+M73P+D83C+S98V Variant 8-I-A62R A62R+L63I+M73P+D83C+S98V Variant 9-I-A62 ^* A62 ^* +L63I+M70Q+M73P+D83C Variant 9-I-A62K A62K+L63I+M70Q+M73P+D83C Variant 9-I-A62R A62R+L63I+M70Q+M73P+D83C Variant 10-I-A62 ^* A62 ^* +L63I+M73P+V74A+D83C Variant 10-I-A62K A62K+L63I+M73P+V74A+D83C Variant 10-I-A62R A62R+L63I+M73P+V74A+D83C Variant 11-I-A62 ^* A62 ^* +L63I+M73P+V74F+D83C Variant 11-I-A62K A62K+L63I+M73P+V74F+D83C Variant 11-I-A62R A62R+L63I+M73P+V74F+D83C Variant 12-I-A62 ^* A62 ^* +L63I+M70Q+D83C+S98A Variant 12-I-A62K A62K+L63I+M70Q+D83C+S98A Variant 12-I-A62R A62R+L63I+M70Q+D83C+S98A Variant 13-I-A62 ^* A62 ^* +L63I+G47D+M70Q+D83C Variant 13-I-A62K A62K+L63I+G47D+M70Q+D83C Variant 13-I-A62R A62R+L63I+G47D+M70Q+D83C Variant 14-I-A62 ^* A62 ^* +L63I+G47D+D83C+S98A Variant 14-I-A62K A62K+L63I+G47D+D83C+S98A Variant 14-I-A62R A62R+L63I+G47D+D83C+S98A Variant 15-I-A62 ^* A62 ^* +L63I+G47D+M73P+D83C Variant 15-I-A62K A62K+L63I+G47D+M73P+D83C Variant 15-I-A62R A62R+L63I+G47D+M73P+D83C Variant 16-I-A62 ^* A62 ^* +L63I+G47D+M73D+D83C Variant 16-I-A62K A62K+L63I+G47D+M73D+D83C Variant 16-I-A62R A62R+L63I+G47D+M73D+D83C Variant 17-A62 ^* A62 ^* +L63 ^* +M70Q+M73P+V74F+D83C Variant 18-A62 ^* A62 ^* +L63 ^* +M70Q+M73P+V74W+D83C Variant 19-A62 ^* A62 ^* +L63*+M70Q+M73P+D83C+S98A Variant 20-A62 ^* A62 ^* +L63 ^* +G47D+M73P+V74F+D83C Variant 21-A62 ^* A62 ^* +L63 ^* +G47D+M73P+V74W+D83C Variant 22-A62 ^* A62 ^* +L63 ^* +G47D+M73P+D83C+S98A Variant 17-I-A62 ^* A62 ^* +L63I+M70Q+M73P+V74F+D83C Variant 17-I-A62K A62K+L63I+M70Q+M73P+V74F+D83C Variant 17-I-A62R A62R+L63I+M70Q+M73P+V74F+D83C Variant 18-I-A62 ^* A62 ^* +L63I+M70Q+M73P+V74W+D83C Variant 18-I-A62K A62K+L63I+M70Q+M73P+V74W+D83C Variant 18-I-A62R A62R+L63I+M70Q+M73P+V74W+D83C Variant 19-I-A62 ^* A62 ^* +L63I+M70Q+M73P+D83C+S98A Variant 19-I-A62K A62K+L63I+M70Q+M73P+D83C+S98A

Variant 19-I-A62R A62R+L63I+M70Q+M73P+D83C+S98A Variant 20-I-A62 ^* A62 ^* +L63I+G47D+M73P+V74F+D83C Variant 20-I-A62K A62K+L63I+G47D+M73P+V74F+D83C Variant 20-I-A62R A62R+L63I+G47D+M73P+V74F+D83C Variant 21-I-A62 ^* A62 ^* +L63I+G47D+M73P+V74W+D83C Variant 21-I-A62K A62K+L63I+G47D+M73P+V74W+D83C Variant 21-I-A62R A62R+L63I+G47D+M73P+V74W+D83C Variant 22-I-A62 ^* A62 ^* +L63I+G47D+M73P+D83C+S98A Variant 22-I-A62K A62K+L63I+G47D+M73P+D83C+S98A Variant 22-I-A62R A62R+L63I+G47D+M73P+D83C+S98A Variant 23-A62 ^* A62 ^* +L63 ^* +G47D+M70Q+M73P+V74F+D83C Variant 24-A62 ^* A62 ^* +L63 ^* +G47D+M70Q+M73P+V74W+D83C Variant 25-A62 ^* A62 ^* +L63 ^* +G47D+M73P+V74F+D83C+S98A Variant 26-A62 ^* A62 ^* +L63 ^* +G47D+M73P+V74W+D83C+S98A Variant 23-I-A62 ^* A62 ^* +L63I+G47D+M70Q+M73P+V74F+D83C Variant 23-I-A62K A62K+L63I+G47D+M70Q+M73P+V74F+D83C Variant 23-I-A62R A62R+L63I+G47D+M70Q+M73P+V74F+D83C Variant 24-I-A62 ^* A62 ^* +L63I+G47D+M70Q+M73P+V74W+D83C Variant 24-I-A62K A62K+L63I+G47D+M70Q+M73P+V74W+D83C Variant 24-I-A62R A62R+L63I+G47D+M70Q+M73P+V74W+D83C Variant 25-I-A62 ^* A62 ^* +L63I+G47D+M73P+V74F+D83C+S98A Variant 25-I-A62K A62K+L63I+G47D+M73P+V74F+D83C+S98A Variant 25-I-A62R A62R+L63I+G47D+M73P+V74F+D83C+S98A Variant 26-I-A62 ^* A62 ^* +L63I+G47D+M73P+V74W+D83C+S98A Variant 26-I-A62K A62K+L63I+G47D+M73P+V74W+D83C+S98A Variant 26-I-A62R A62R+L63I+G47D+M73P+V74W+D83C+S98A Variant 27-I-A62K A62K+L63I+M73P+D83C+S98D Variant 27-I-A62R A62R+L63I+M73P+D83C+S98D Variant 28-I-A62K A62K+L63I+M73P+D83C+S98E Variant 28-I-A62R A62R+L63I+M73P+D83C+S98E Variant 29-I-A62K A62K+L63I+M73P+S98A Variant 29-I-A62R A62R+L63I+M73P+S98A Variant 30-I-A62K A62K+L63I+M73P+S98D Variant 30-I-A62R A62R+L63I+M73P+S98D Variant 31-I-A62K A62K+L63I+M73P+S98E Variant 31-I-A62R A62R+L63I+M73P+S98E

Other parental amino acid sequences useful in the present invention that are SSI variants include variants with a single substitution at position 98 corresponding to SSI and variants with double substitutions at positions 98 and 62. Table 9 lists preferred parental amino acid sequences in this class.

Table 9

Non-limiting examples of parental amino acid sequences parent 32 A62K+S98Q parent 33 A62K+S98D parent 34 A62K+S98E parent 35 A62R+S98Q parent 36 A62R+S98D parent 37 A62R+S98E parent 38 S98A parent 39 A62K+S98A parent 40 A62R+S98A parent 41 S98Q parent 42 S98D parent 43 S98E Relevant examples of variants of the invention are listed in Table 10 below.

Table 10

Non-limiting examples of variants of the invention Variant 32 L63I+A62K+S98Q Variant 33 L63I+A62K+S98D Variant 34 L63I+A62K+S98E Variant 35 L63I+A62R+S98Q Variant 36 L63I+A62R+S98D Variant 37 L63I+A62R+S98E Variant 38 L63I+S98A Variant 39 A62K+L63I+S98A Variant 40 A62R+L63I+S98A Variant 41 L63I+S98Q Variant 42 L63I+S98D Variant 43 L63I+S98E

SSI can exist in binary form. Thus, without being bound by theory, it is also possible to provide stabilized dimeric SSIs with enhanced resistance to excess proteases. Preferred such stabilized dimeric SSIs include two monomers covalently bonded together. This bond can be an ester, amide, disulfide or other bond commonly found in amino acids or their side chains. "Covalent dimerization" and "covalent stabilization" herein refer to monomers so covalently bound as dimers. More preferred dimerization occurs via disulfide bonds. The variants of the present invention are intended to include variants that exist as dibodies, whether they associate by intramolecular or intermolecular forces.

Other useful parent amino acid sequences herein include SSI-like inhibitors (commonly referred to as SSI-like (SIL) proteins) and SSI-like inhibitor variants. Background information on SSI-like inhibitors can be found in Laskowski et al., "Protein Inhibition of Proteases", Annual Review of Biochemistry, Vol. 49, pp. 593-626 (1980). Preferred SSI-like inhibitors and SSI have more than about 50% of the same amino acid sequence, more preferably more than 65% of the same sequence, more preferably more than 70% of the same sequence, wherein the more preferred inhibitors can be classified as Family III inhibitors. See Laskowski et al. above. literature. Examples of such SSI-like inhibitors include SIL10 (sequence provided in SEQ ID NO:4), SIL13 (SEQ ID NO:5) and SIL14 (SEQ ID NO:6), which are further described in Terabe et al., "Three Novel Subtilisin-TrypsinInhibitors from Streptomyces:Primary Structures and Inhibitor Properties", Journal of Biochemistry, Vol.116 pp.1156-1163 (1994), such examples also include SIL2 (sequence provided in SEQ ID NO: 9), SIL3 (SEQ ID NO :10) and SIL4 (SEQ ID NO: 11), their further descriptions are found in Taguchi et al., "Comparative Studies on the Primary Structure and Inhibitory Properties of Subtilisin-trypsin Inhibitors from Streptomyces", European Journal of Biochemistry, Vol.220, pp.911 -918 (1994). Two other examples of such SI-like inhibitors include STI1 (sequence provided in 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 SSI-like inhibitor is known as plasminostreptin (sequence provided in SEQ ID NO: 12), which is further described in Sugino et al., "plasminostreptin, a ProteinProtease Inhibitors Produced by Streptomyces antifibrinostreptin", The Journal of Biological Chemistry, Vol.253, No. 5, pp. 1546-1555 (1978). Another SSI-like inhibitor is SLPI (sequence provided in SEQ ID NO: 13), which is further described in Ueda et al., "aProtein Protease Inhibitors Produced by Streptomyces lividans66 Exhibits Inhibitory Activities Towards Both SubtilisinBPN' and Trypsin", Journal of Biochemistry, Vol.112, pp.204-211 (1993). Another SSI-like inhibitor is SACI (sequence provided in SEQ ID NO: 14), which is further described in Tanabe et al., "Primary Structure and Reactive Site of Streptoverticillium Anticoagulant (SAC), a Novel Protein Protease Inhibitors Produced by Streptoverticillium cinnamoneum Subsp.cinnamoneum" , Journal of Biochemistry, Vol.115, pp.752-761 (1994). Another SSI-like inhibitor is SILI (sequence provided in SEQ ID NO: 15), which is further described in Kojima et al., "Primary Structure and Inhibitory Properties of a Protease Inhibitor Produced by Streptomycescacaoi", Biochemica et Biophysica Acta, Vol.1207, pp. 120-125 (1994). Other SSI-like 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 Inhibitors-Like Proteins Are Distributed Widely in Streptomycetes", Applied and Environmental Microbiology, pp.4338-4341 (Dec.1993), and Suzuki et al., "Partial Amino Acid Sequence of an alkaline Vaginal Protease, Aggressive Inhibitor, 5 Biolog, 4 pp. 629-634 (1981). As known to those skilled in the art, there are other SSI-like inhibitors described in the art.

Variants of SSI-like inhibitors may also be used herein as parent amino acid sequences. These variants include those selected from the SSI-like inhibitors described above, the amino acid sequence of which contains one or more mutations. All of the substitutions exemplified in the variants mentioned herein can be made at the corresponding positions of the SSI-like inhibitor to provide a parent amino acid sequence. Other non-limiting examples of SSI-like inhibitor variants that can be used as parental amino acid sequences are disclosed in Nielson et al., WO 93/17086, assigned to Novo Nrdisk A/S, granted September 2, 1993.

As is known to those skilled in the art, naturally encoded position 63 (example) of an SSI-like inhibitor, a variant thereof, or a variant of SSI may not correspond to position 63 of SSI. Therefore, sequence numbering may need to be adjusted to locate the 63 bits corresponding to SSI (example), as is well known in the art. Alignments of sequences can be found in references cited herein as well as other references in the art.

Preferred variants of the present invention exhibit a _K such that the variant inhibits almost all proteases (preferably inhibits about 60% of proteases, more preferably inhibits about 99%) in cleaning and personal hygiene compositions, Instead, it can dissociate from the inhibitor after dilution and/or during washing.

For example, when the stoichiometric ratio of the variant to the protease is 2:1, preferred inhibitors have a K _i for the protease of about 10 ⁻¹² M to 10 ⁻⁴ M, more preferably 10 ⁻¹⁰ M to 10 ^-6 M, most preferably 10 ^-8 M to 10 ^-7 M. Of course, if the size of the washing equipment or the concentration of the product changes, K _i must be adjusted accordingly. Prediction of a useful _Ki can be made by the skilled artisan without taking into account factors such as dilution of the composition at the time of use, dependence of the binding constant on the temperature of the relevant cleaning method used, the stoichiometric ratio of the variant to the protease Excessive experimentation with similar parameters.

Since the variant is ultimately encoded by DNA in vivo, this DNA can be used to define the variant sequence. The DNA encoding the variant may be used in any number of plasmids and/or expression systems, including in vivo and in vitro expression systems, such as plants (preferred for use in biotechnology, including tobacco, oilseed crops such as rapeseed, soybean and and the like, cereals such as corn, barley, oats, other vegetables such as tomato, potato and similar plants) and microbial expression systems, the latter including fungi such as yeast, and bacteria such as Bacillus subtilis, Escherichia coli and other similar bacteria. The preferred expression system is a microorganism, more preferably a natural bacterium, most preferably Bacillus subtilis or Escherichia coli, and Bacillus subtilis is even better.

The DNA encoding the variant may be incorporated into a plasmid or phage active in cells, and may also be incorporated directly into the genome of the organism used to clone or express the variant of the invention.

It should be understood that the skilled artisan, given the teachings of the present invention, will recognize that the DNA encoding one variant of the present invention can be introduced into the same plasmid, phage or chromosome as other variants of the present invention. In addition, the plasmid, phage or chromosome may also encode a protease, including a fusion protein comprising an inhibitor and/or protease as part of it, whether or not it is inhibited by the variants of the invention.

The skilled artisan will also understand that the DNA described above is also contemplated, and that RNA transcripts of the DNA are disclosed. The skilled artisan can know the RNA sequence by examining the DNA sequence without experimentation.

The invention also relates to the genes encoding these variants.

It is contemplated that the skilled artisan can prepare antibodies to the variants of the invention. These antibodies can be prepared by known methods.

For example, the variants of the invention can be injected into suitable mammals such as mice, rabbits and other similar animals. A suitable regimen involves repeated injections of the immunogen in the presence of an adjuvant, on a schedule that increases antibody production in the serum. Measurement of titers of immune sera can be performed with the variants of the invention as antigens by standard immunoassay procedures in the art.

Antisera can be used directly, or monoclonal antibodies can be obtained by collecting peripheral blood lymphocytes or the spleen of immunized animals and immortalizing the antibody-producing cells, followed by identification of suitable antibody-producing cells using standard immunoassay methods.

Polyclonal or monoclonal antibodies can be used to detect expression of the invention by standard detection methods. As such, it is contemplated that these antibodies can be used to prepare kits for measuring expression levels, among other things.

These antibodies may also be conjugated to scintigraphic labels, such as technetium 99 or 1-131, or to fluorescent labels using standard conjugation methods. Labeled antibodies can also be used in competition assays, such as kinetic assays, to measure K _i .

As is known in the art, there will be occasional errors in DNA and amino acid sequences. Therefore, those of ordinary skill in the art can find any mistakes and make appropriate corrections through conventional techniques when repeating the inventor's work from the content disclosed herein.

                                     

The following examples are not intended to limit the claimed invention, but to provide guidance to the skilled artisan on how to make and use the invention. Given the guidance of these examples, the other disclosure herein, and other information readily available to those skilled in the art, a skilled artisan can make and use the present invention. For the sake of brevity, repeated references to methods in the art and known art have been omitted, since they would be within the knowledge of the skilled artisan.

Variants can be prepared by mutating the nucleotide sequence encoding a parental amino acid sequence, thus producing a variant with a modified amino acid sequence. These methods are known in the art, one of which is shown below.

Escherichia coli dut ^- ung ^- strain CJ236 was transformed with a phagemid containing a gene corresponding to the amino acid sequence of the parent and VCSM13 helper phage was used to generate a single-stranded uracil-containing DNA template (Kunkel et al., "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection", Methods in Enzymology, Vol.154, pp.367-382 (1987), see Yuckenberg et al. for modification, "Site Directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectors", Mutagenesis-APractical Approach, McPherson, MJed., pp. 27-48(1991)). Primer site-specific mutagenesis can be performed by a modification of the method of Zoller and Smith (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 Acids Research, Vol. 10, pp. 6487-6500 (1982)) were used to generate all variants (essentially as described in Yuckenberg et al., supra).

Oligonucleotides were produced using a 380B DNA synthesizer. The mutation reaction product was transformed into E. coli strain MM294 (American Type Culture Collection E. coli 33625). All mutations were confirmed by DNA sequencing and the isolated DNA was transformed into the Bacillus subtilis expression strain PG632 (Sauders et al., "Optimization of the Siganal-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., "Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an in vitro-DerivedDeletion Mutation", Journal of Bacteriology, Vol .160, pp.15-21(1984)).

Variants were prepared as follows. Cultivate the Bacillus subtilis cells containing the objective plasmid in a medium containing 29g/l of tryptone, 29g/l of yeast extract and 5g/l of sodium chloride, supplemented with 1.25% maltrin M100 (Grain Processing Corporation, Muscatine, IA), 100 mM HEPES pH 7.5, 80 μM MnCl ₂ , 50 μM kanamycin. Cultures were incubated at 37°C for 24 hours.

Purification of variants is first performed by centrifugation to remove cells. Then 1N hydrochloric acid was added to lower the pH to around 4. Insoluble material was pelleted by centrifugation. Usually the variant can be found in the supernatant. The supernatant was dialyzed against 20 mM sodium acetate, pH 4. Variants typically precipitate out at this step. The pellet was resuspended in Tris base and analyzed for inhibition of the Y217L derivative of subtilisin BPN'. In some cases, the variants remained soluble after these precipitation steps. In this case, the soluble fraction can be separated by S Sepharose column with 20 mM sodium acetate, pH 4. Samples were eluted with increasing concentrations of NaCl and analyzed for protease inhibition. In all cases, variant-containing fractions were dialyzed against 1 mM Tris, pH 8.0 prior to use.

Features of variants of the invention

SSI inhibits proteases, especially subtilisin BPN' and the Y217L variant of subtilisin BPN'. Taking SSI as an example, the inhibitory activity of the variants of the present invention was measured as follows. The SSI was mixed with the protease and incubated for 15 minutes at room temperature in the presence of 0.1 M Tris pH 8.6, 10 mM _CaCl2 . Protease activity was then measured according to the method of DelMar et al., Analytical Biochemistry, Vol. 99, pp. 316-320 (1979). 10 ul of N-succinyl-ala-ala-Pro-Phe-p-azaniline (20 mg/ml) was added to initiate the reaction. Reaction rates were measured by an increase in absorption at 410 nm, which reflects inhibition of the protease.

Where a variant of the invention is desired to be combined with a protease (suitable proteases are described below) in a cleaning or personal hygiene composition, its stability in the product environment is also tested. The stability of variants can be tested by measuring protease activity over time. If the variant is stable, the level of protease activity will be more constant. But if the variant is disrupted, protease activity will be elevated. In this example, the variant was mixed with 1.1 nmol of a subtilisin BPN' variant with the Y217L substitution. Water was added to make the volumes of each sample consistent. Form the complex for 10 minutes in a liquid detergent complex configured as follows: components weight percentage C _14-15 Alkyl (Ethoxy 2,25) Sulfonic Acid 18.0 C _12-13 Alkyl Ethyl Oxide 2.0 C ₁₂ -N-Methylglutamine 5.0 citric acid 4.0 ethanol 3.5 Monoethanolamine 2.0 1,2 propanediol 7.0 sodium formate 0.6 Tetraethylenepentamine ethyl oxide 1.18 Soil release Polymer 0.25 Silicone Foam Inhibitors 0.10 brightener 0.10 water, NaOH Add balance to 100%

This composition occupies one-third of the total sample volume. Mix 15 μl of sample with 975 μl of 0.1 M, pH 8.6 Tris HCl and 0.01 M CaCl ₂ . The dilution was incubated for 30 minutes at room temperature. Substrate was added after incubation and the amount of protease was measured. Degradation of the variants was detected by an increase in protease activity after several weeks. This degradation can be directly compared with the corresponding data of SSI.

K _i for variants are calculated as follows. The variants were mixed with 600 μg/ml N-succinyl-ala-ala-Pro-Phe-p-azaniline in 990 μl of 50 mM pH 8 Tris solution. A selected protease is added to initiate the reaction (suitable proteases are described below). The rate of hydrolysis was followed for 20 minutes. A steady rate was observed over the final 10 to 15 minutes. According to the equation in Goldstein, "The Mechanism of Enzyme-Inhibitor-Substrate Reactions", Journal of General Physiology, Vol.27, pp.529-580 (1944), the rate was compared with the rate in the case of no variant and calculated K _i .

The cleaning composition of the present invention

In another embodiment of the invention, effective amounts of one or more variants of the invention are included in cleaning compositions useful for cleaning surfaces to be freed from peptide contamination. These cleaning compositions include, but are not limited to, fabric cleaning compositions, hard surface cleaning compositions, light duty cleaning compositions including dish cleaning compositions, and automatic dish washer detergent compositions.

The cleaning compositions herein comprise an effective amount of one or more variants of the present invention and a cleaning composition carrier comprising a protease. As used herein, an "effective amount of a variant" or other similar terms means the amount of the variant necessary to obtain the requisite proteolytic activity in a particular cleaning composition. The effective amount can be readily determined by one of ordinary skill in the art on the basis of a number of factors such as, for example, the particular variant used, the cleaning application, the particular combination of cleaning compositions, whether a liquid or a dry form is desired (e.g. Granular, columnar) composition and other similar factors. Preferred cleaning compositions contain from 0.0001% to 10% of one or more variants of the invention, more preferred cleaning compositions contain from 0.001% to 1%, most preferred cleaning compositions contain from 0.01% to 0.1%. Several examples of various cleaning compositions that may be applied to this variant are discussed in detail below.

The variants of the invention can be used in cleaning compositions to inhibit proteases during storage, thus protecting the proteases from degradation by themselves or from other sources, such as other enzymes present in the composition. Thus, an essential component of the compositions of the invention is a protease inhibited by the variants of the invention. The protease can be from animals, plants, more preferably from microorganisms. Preferred proteases include proteases whose inhibitor is SSI. Such proteases include those produced by Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharius, Bacillus licheniformis, Bacillus lentus and Bacillus subtilis. Preferred among these proteases include sublisin BPN, sublisin BPN', sublisin Carlsberg, sublisin DY, sublisin 309, proteinase K, and thermitase, which includes A/ ^Salcalase® (Novo Industries, Copenhagen, Denmark), ^Esperase® (Novo Industries), Sayinase ^_ (Novo Industries), Maxatase ^_ (Gist-Brocades, Delft, Netherlands), Maxacal ^_ (Gist-Brocades), Maxapem15 ^_ (Gist-Brocades) and variants of the above. More preferred proteases for use herein include proteases from Bacillus amyloliquefaciens and variants thereof. The most preferred wild type protease is sublisin BPN'.

Variants of sublisin BPN', collectively referred to herein as "protease group A", are useful as proteases and are disclosed in U.S. Patent No. 5,030,378, Venegas, July 9, 1991, and are characterized by sublisin BPN' having the following mutations Amino acid sequence (sequence represented by SEQ ID NO:3).

(a) Gly at position 166 is replaced by Asn, Ser, Lys, Arg, His, Gln, ala or Glu; Gly at position 169 is replaced by Ser; Met at position 222 is Gln, Phe, His, Asn, Glu , ala or Thr; or

(b) Gly at position 160 is replaced by ala, and Met at position 222 is replaced by ala.

Other variants of sublisin BPN', hereinafter collectively referred to herein as "protease group B", are useful herein as proteases and are disclosed in European Patent No. EP-B-251,446, assigned to Genencor International, Inc., January 1988 Published on the 7th, authorized on December 28, 1994, characterized by wild sublisin BPN' amino acid sequence with mutations at 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,Met124,Gly127,Gly128,Pro129,Leu135,Lys170,Tyr171,Pro172,Asp197,Met199,Ser204,Lys213,Gyr21; or The loci listed above were found in single or multiple combinations with the following mutations, Asp32, Ser33, Tyr104, ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222.

Other sublisin BPN' variants useful herein as proteases, hereinafter collectively referred to as "Protease Group C", are described in WO 95/10615, assigned to Genencor International Inc., published April 20, 1995, and characterized by Wild sublisin BPN' amino acid sequence with mutation, the mutation is a combination of Asn76 and one or more of the following mutations 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 sublisin BPN' variants useful herein as proteases, hereinafter collectively referred to as "Protease Group D", are described in U.S. Pat. No. 4,760,025, Estell, et al., Jul. 26, 1988, characterized by having one or A plurality of mutated wild sublisin BPN' amino acid sequences, whose mutation sites are selected from the group consisting of Asp32, Ser33, His64, Tyr104, Asn155 Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222.

More preferred proteases for use herein are selected from the group consisting of ^alcalase_ , sublisin BPN', protease group A, protease group B, protease group C, protease group D. Most preferred proteases are selected from protease group D.

The composition of the present invention comprises 0.0001% to 1% by weight of active protease, the preferred composition comprises 0.0005% to 0.2%, and the most preferred composition comprises 0.002% to 0.1%. Mixtures of proteases may also be included. Of course, the weight percent of protease in the cleaning combination will vary with the level of water, builder level, and other similar conditions in the final composition. For example, in preferred granular detergents, the composition preferably contains from 0.064 mg/g to 0.64 mg/g protease.

In a preferred embodiment, the preferred molar ratio of variant to protease in the cleaning composition (variant to protease) is from 3:1 to 1:1, more preferably from 3:1 to 1.5:1, most preferably The ratio is 2:1.

In addition to the variants of the present invention, the cleaning compositions of the present invention also include a cleaning composition carrier comprising one or more cleaning composition materials compatible with the variant and/or protease. As used herein, the term "cleaning composition material" refers to any material selected for a particular type of desired cleaning composition and product form (e.g., liquid, granule, stick, spray, stick, paste, gel), The material should be compatible with the variant used in the composition. The particular choice of cleaning composition material can be readily made depending on the material to be cleaned and the desired form of the composition in use for cleaning the environment. As used herein, the term "compatible" means that the cleaning composition material does not reduce the inhibitory activity of the variant and/or the proteolytic activity of the protease to such an extent that the protease would not be as effective as is required for typical use. Specific cleaning composition materials are exemplified in detail below.

The variants of the present invention can be used in a variety of detergent compositions where rich lather and good cleaning activity are desired. As such, the present variants can be used with a variety of traditional ingredients in fully formulated hard surface cleaners, dish cleaning compositions, fabric laundry compositions, and others. These compositions can be in liquid, granular, cylindrical and other forms. These compositions can be formulated as "concentrated" detergents containing from 30% to 60% by weight surfactant.

The cleaning compositions herein may preferably contain various surfactants (eg, anionic, nonionic or zwitterionic surfactants), as appropriate. These surfactants are typically present at levels from 5% to 35% of the composition.

Non-limiting examples of surfactants useful herein include conventional C ₁₁ -C ₁₈ alkylbenzene sulfonic acids and primary and random alkyl sulfuric acids, C ₁₀ -C ₁₈ secondary (2,3) alkyl sulfuric acids, The molecular formula is CH ₃ (CH ₂ ) _x (CHOSO ₃ ) ^- M ⁺ CH ₃ and CH ₃ (CH ₂ ) _y (CHOSO ₃ M ⁺ )CH ₂ CH ₃ , where x and (y+1) are at least 7 An integer, more preferably an integer of at least 9, M is a water-soluble cation, especially a sodium ion, non-limiting examples also include C ₁₀ -C ₁₈ alkyl alkoxysulfuric acid (especially E0 1-5 Oxysulfuric acid), C ₁₀ -C ₁₈ alkyl alkoxy carboxylic acids (especially EO 1-5 ethoxy carboxylic acids), C ₁₀ -C ₁₈ alkyl polyglycosides and their corresponding sulfated polyglycosides , C ₁₂ -C ₁₈ α-sulfonated fatty acid esters, C ₁₂ -C ₁₈ alkyl and alkylphenol alkoxylates (especially ethoxylates or ethoxylated/propoxylated mixtures), C ₁₂ -C ₁₈ Betaines and thiobetaines ("sultaines"), C ₁₀ -C ₁₈ amine oxides and other analogues. Alkylalkoxysulfuric acids (AES) and alkylalkoxycarboxylic acids (AEC) are preferred here. Combinations of these surfactants with amine oxides and/or betaines or saltaines are also preferred, depending on the requirements of the formulator. Other useful conventional surfactants are listed in the standard text. Particularly useful surfactants include _C10 - _C18 N-methylglucamides, which are disclosed in US Patent No. 5,194,639, Connor et al., registered March 16,1993.

Other quite broad components of the cleaning compositions of the present invention include other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, and for liquid formulations, solvents. If additional suds enhancement is desired, suds generators such as _C10 - _C16 alkanolamides (alkolamides) can be added to the composition, typically at a level of from about 1% to about 10%. C ₁₀ -C ₁₄ monoethanol or diethanolamides are a typical class of such suds generators. It is also beneficial to use such suds generators with high sudsing co-surfactants such as the amine oxides, betaines and thiobetaines mentioned above. If desired, soluble magnesium salts can be added to generate more foam, such as _MgCl2 , _MgSO4 , etc., generally at a concentration of about 0.1% to about 2%.

The liquid detergent compositions herein may contain water or other solvents as carriers. Low molecular weight primary or secondary alcohols are suitable for use, such as methanol, ethanol, propanol and isopropanol. For soluble surfactants, monohydric alcohols are preferred, but polyols containing about 2 to 6 carbon atoms and about 2 to 6 hydroxyl groups (e.g., 1,3-propanediol, 1,2-ethylene glycol, glycerin and 1,2-propanediol). The compositions may contain from about 5% to about 90% of these carriers, typically from about 10% to about 50%.

The detergent compositions herein are preferably formulated for cleaning operations in water having a pH of between 6.8 and 11. Final products are typically formulated within this range. Techniques used to control pH at recommended usage levels include, for example, the use of buffers, bases and acids. These processes are well known to those skilled in the art.

In formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may prefer to use builders at levels from about 5% to about 50% by weight. Typical builders include 1-10 micron zeolites, polycarboxylic acids such as citric and oxosuccinic acids, layered silicates, phosphates and others. Other conventional builders are listed in standard formulary lists.

Likewise, the formulator may wish to employ various additional enzymes in these compositions, such as cellulases, lipases, amylases and proteases, typically at levels of from about 0.001% to about 1% by weight. A variety of cleaning and fabric enzymes are known in the laundry detergent art.

A variety of bleaching compounds, such as percarbonates, perborates and the like, can be used in these compositions, typically at levels of from about 1% to about 15% by weight. These compositions, if desired, can also contain bleach activators, such as tetraacetylethylenediamine, nonanoyloxybenzenesulfonic acid and other similar compounds, which are also known in the art. It is typically used in an amount of about 1% to about 10% by weight.

These compositions can also use soil release agents: especially oligoester anionics, chelating agents, especially aminophosphonates and ethylenediamine succinates, clay removers, especially ethylenediamine succinate, Tetraethylenepentylamine oxide, dispersants, especially polyacrylates and polyasparatates, brighteners, especially anionic brighteners, foam inhibitors, especially silicones and secondary alcohols, textiles Softening agents, especially smectite clays, and the like, may be present in an amount of about 1% to about 35% by weight. Standard recipe lists and published patents contain numerous detailed descriptions of these traditional materials.

Enzyme stabilizers can also be used in cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably at levels of about 1% to about 10%), sodium formate (preferably at levels of about 0.1% to about 1%), and calcium formate (preferably at levels of about 0.1% to about 1%).

Other useful cleaning compositions include clay removers, dispersants, brighteners, suds suppressors and fabric softeners.

The variants of the invention are useful in hard surface cleaning compositions. As used herein, "hard surface cleaning composition" is a liquid or granular detergent composition for cleaning hard surfaces such as floors, walls, bathroom tiles, and the like. Hard surface cleaning compositions typically contain surfactants and water-soluble sequestering builders. However, surfactants are sometimes not used in certain specialty products such as window spray cleaners because they may leave filmy and/or spotty marks on glass surfaces.

Surfactants may be present at levels as low as 0.1% of the compositions herein, but typically the compositions comprise from about 0.25% to about 10%, more preferably from about 1% to about 5%.

The present compositions typically contain from about 0.5% to about 50%, more preferably from 1% to about 10%, of detergency builder.

A more preferred pH is about 7 to 12. If pH adjustment is required, conventional pH adjusting agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used.

A solvent may be included in the composition. Useful solvents include glycol ethers such as diethylene glycol monohexyl ether, diethylene glycol monobutyl ether, ethylene glycol monobutyl ether, ethylene glycol monohexyl ethers, propylene glycol monobutyl ether, dipropylene glycol monobutyl ether, useful solvents also include diols such as 2,2,4-trimethyl-1,3-pentanediol and 2 -Ethyl-1,3 hexanediol, examples of the solvent are not limited thereto. . These solvents are typically used at levels of from about 0.5% to about 15%, more preferably at levels of from about 3% to about 11%.

In addition, when the composition is applied "full effect" to the surface without rinsing the surface, a strong volatile solvent such as isopropanol or ethanol may be used with the present composition to aid in faster evaporation of the composition from the surface. When used, volatile solvents are typically present in the composition at a level of from about 2% to about 12%.

Variations of the present invention are also useful in cleaning compositions contained in: U.S. Patent Provisional Application No. 60/079,477, Rubingh et al., filed March 26, 1998; U.S. Patent Provisional Application No. 60 /079,397, Rubingh et al., filed March 26, 1998; U.S. Patent Application No. 09/048,174, Weisgerber et al., filed March 26, 1998; U.S. Patent Application No. 09/088912, requiring Priority over U.S. Patent Provisional Application No. 60/079,477, Weisgerber et al., filed June 2, 1998.

Hard surface cleaning compositions are illustrated by the following examples.

Example 1-6

Liquid Hard Surface Cleaning Composition example 1 Example 2 Example 3 Example 4 Example 5 Example 6 variant 6-I protease 0.05% 0.50% 0.02% 0.03% 0.30% 0.05% EDTA - - 2.90% 2.90% - - Sodium citrate - - - - 2.90% 2.90% Sodium C ₁₂ alkylbenzene sulfonate 1.95% - 1.95% - 1.95% - Sodium _C12 Alkyl Sulfate - 2.20% - 2.20% - 2.20% Sodium C ₁₂ (ethoxy)sulfate - 2.20% - 2.20% - 2.20% C ₁₂ dimethylamine oxide - 0.50% - 0.50% - 0.50% Sodium cumene sulfonate 1.30% - 1.30% - 1.30% - Hexylethoxyethoxyethanol 6.30% 6.30% 6.30% 6.30% 6.30% 6.30% water 90.4% 88.3% 87.53% 85.87% 87.25% 85.85%

All recipes adjusted to pH7

In Examples 1-6, the variants recited in Tables 7, 8, 10 and the preferred variants cited herein among other variants were used in place of variant 6-1 with essentially similar results.

In another embodiment of the invention, dish cleaning compositions comprise one or more variants according to the invention. As used herein, "dish cleaning composition" refers to all forms of compositions for cleaning dishes, including, but not limited to, granular and liquid forms. The dish cleaning compositions of the present invention are illustrated by the following examples.

Example 7-10

Liquid Dish Stain Remover Example 7 Example 8 Example 9 Example 10 variant 7-I-A62K/protease 0.05% 0.50% 0.02% 0.40% C ₁₂ -C ₁₄ N-methylglucamide 0.90% 0.90% 0.90% 0.90% _C12 Ethoxy(1)sulfuric acid 12.0% 12.0% 12.0% 12.0% 2-Methylundecanoic acid 4.50% 4.50% 4.50% 4.50% C ₁₂ ethoxy(2) carboxylic acid 4.50% 4.50% 4.50% 4.50% _C12 Alcohol Ethoxylates (4) 3.00% 3.00% 3.00% 3.00% C ₁₂ amine oxide 3.00% 3.00% 3.00% 3.00% Sodium propylbenzene sulfonate 2.00% 2.00% 2.00% 2.00% ethanol 4.00% 4.00% 4.00% 4.00% Mg ²⁺ (such as MgCl ₂ ) 0.20% 0.20% 0.20% 0.20% Ca ²⁺ (such as CaCl ₂ ) 0.40% 0.40% 0.40% 0.40% water 65.45% 65% 65.48% 65.1%

All formulations were adjusted to pH7.

In Examples 7-10, the variants recited in Tables 7, 8, 10 and the preferred variants cited herein among other variants were used in place of variant 7-I-A62K with essentially similar results.

The liquid fabric cleaning compositions of the present invention are illustrated by the following examples.

Example 11-13

Liquid Fabric Cleaning Composition Example 11 Example 12 Example 13 variant 2-I/protease 0.05% 0.03% 0.30% Sodium C ₁₂ -C ₁₄ Alkyl Sulfate 20.0% 20.0% 20.0% 2-Butyl octanoic acid 5.0% 5.0% 5.0% Sodium citrate 1.0% 1.0% 1.0% C ₁₀ Alcohol Ethoxylates (3) 13.0% 13.0% 13.0% Monoethanolamine 2.50% 2.50% 2.50% Water/propylene glycol/ethanol (100:1:1) 58.45% 58.47% 58.20% In Examples 11-13, the variants recited in Tables 7, 8, 10 and among other variants the preferred variants cited herein were used in place of variant 2-1 with essentially similar results.

Personal Hygiene Compositions

A variant of the invention is also suitable for use in personal hygiene compositions selected from leave-on and rinse-off conditioners, shampoos, leave-on and rinse-off acne compositions, face washes and treatments, body washes , soaps, foaming and non-foaming facial cleansers, cosmetics, hand, face, and body washes and moisturizers, leave-on facial moisturizers, cosmetic and cleansing wipes, oral hygiene compositions, and contact lens hygiene compositions. The personal hygiene compositions of the present invention comprise an effective amount of one or more variants of the present invention and a personal hygiene carrier comprising a protease. Effective amount variations, including preferred limitations thereof, are described herein with respect to cleaning compositions.

For ease of illustration, variants of the present invention in the presence of a protease are suitable for compositions described in the following references: U.S. Patent No. 5,641,479, Linares et al., issued June 24, 1997 (skin cleansers); 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 on July 30, 1996 (skin cleanser); U.S. Patent No. 5,510,050, Dunbar et al., issued on April 23, 1996 (skin cleanser); U.S. Patent No. 5,612,324, Guang lin et al. , issued March 18, 1997 (anti-acne preparations); U.S. Patent No. 5,587,176, Warren et al., issued December 24, 1996 (anti-acne preparations); U.S. Patent No. 5,549,888, Venkateswaran et al., issued 1996 August 27, 1995 (anti-acne preparations); U.S. Patent No. 5,470,884, Corless et al., issued November 28, 1995 (anti-acne preparations); U.S. Patent No. 5,650,384, Gordon et al., issued July 22, 1997 U.S. Patent No. 5,607,678, Moore et al., issued March 4, 1997 (body wash); U.S. Patent No. 5,624,666, Coffindaffer et al., issued April 29, 1997 (hair conditioner and and/or shampoo); U.S. Patent No. 5,618,524, Bolich et al., issued April 8, 1997 (conditioner and/or shampoo); U.S. Patent No. 5,624,666, Coffindaffer et al., issued April 29, 1997 ( Conditioner and/or shampoo); U.S. Patent No. 5,612,301, Inman, issued March 18, 1997 (conditioner and/or shampoo); U.S. Patent No. 5,573,709, Wells, issued November 12, 1996 ( Hair Conditioner and/or Shampoo); U.S. Patent No. 5,482,703, Pings, issued January 9, 1996 (Hair Conditioner and/or Shampoo); U.S. Patent No. Re. 34,584, Grote et al., Reissued 1994 April 12 (conditioners and/or shampoos); U.S. Patent No. 5,641,493, Date et al., issued June 24, 1997 (cosmetics); U.S. Patent No. 5,605,894, Blank et al., issued February 1997 25 (cosmetics); U.S. Patent No. 5,585,090, Yoshioka et al., issued December 17, 1996 (cosmetics); U.S. Patent No. 4,939,179, Chenev et al., issued July 3, 1990 (hands, face and Body Wash); U.S. Patent No. 5,607,980, McAtee et al., issued March 4, 1997 (Hand, Face, and Body Wash); U.S. Patent No. 4,045,364, Richet et al., issued August 30, 1977 (Cosmetics and cleaning wipes); European Patent Application, EP 0619 074, Touchet et al., published October 12, 1994 (Cosmetic and cleaning wipes); U.S. Patent No. 4,975,217, Brown-Skrobot et al., issued 1990 December 4, 1992 (cosmetics and cleaning wipes); U.S. Patent No. 5,096,700, Seibel, issued March 17, 1992 (oral cleaning compositions); U.S. Patent No. 5,028,414, Sampathkumar, issued July 2, 1991 (oral cleaning compositions); U.S. Patent No. 5,028,415, Benedict et al., issued July 2, 1991 (oral cleaning compositions); U.S. Patent No. 5,028,415, Benedict et al., issued July 2, 1991 (oral Cleaning compositions); U.S. Patent No. 4,863,627, Davies et al., issued September 5, 1989 (contact lens cleaning solutions); U.S. Patent No. Re 32,672, Huth et al., reissued May 24, 1988 (contact Lens Cleaning Solution); US Patent No. 4,609,493, Schafer, issued September 2, 1986 (Contact Lens Cleaning Solution).

Variations of the present invention are also useful in personal hygiene compositions contained in: U.S. Patent Provisional Application No. 60/079,477, Rubingh et al., filed March 26, 1998; U.S. Patent Provisional Application No. 60/079,397, Rubingh et al., filed March 26, 1998; U.S. Patent Application No. 09/048,174, Weisgerber et al., filed March 26, 1998; U.S. Patent Application No. 09/088912, Claims priority over US Patent Provisional Application No. 09/048,174, Weisgerber et al., filed June 2, 1998.

In a preferred embodiment, the preferred molar ratio of variant to protease in the personal hygiene composition (variant to protease) is from 3:1 to 1:1, more preferably from 3:1 to 1.5:1, most preferably A preferred ratio is 2:1.

To further illustrate the oral cleaning compositions of the present invention, one or more variants of the present invention and one or more proteases are included in a composition for removing protein stains from teeth or dentures. As used herein, "oral cleaning composition" refers to dentifrice, toothpaste, tooth gel, tooth powder, mouthwash, mouth spray, oral gel, chewing gum, lozenge, sachet, tablet, biogel, prophylactic cream , dental treatment solutions and the like. Preferred oral cleansing compositions contain from about 0.0001% to about 20% by weight of one or more variants of the present invention and a protease and a personal hygiene carrier, more preferred variants are present in an amount from about 0.001% to about 10%, more preferred variant levels are from about 0.01% to about 5%.

Typically, the personal hygiene carrier component of the oral cleansing component of the oral cleansing composition is generally present in an amount of about 50% to about 99.99% by weight, preferably in an amount of about 65% to about 99.99%, more preferably in an amount of about 65% to about 99%.

Personal hygiene carrier 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 composition types, carrier components and optional components that can be used in oral cleaning compositions are disclosed in the references cited above herein.

In another embodiment of the present invention, a denture cleaning composition for cleaning dentures outside the oral cavity comprises one or more variants of the present invention. These denture cleaning compositions contain from about 0.0001% to about 50% by weight of one or more variants of the present invention and-and a protease and a personal hygiene carrier, with the preferred variants present in an amount of from about 0.001% to About 35%, more preferably the variant content is from about 0.01% to about 20%. Various forms of denture cleaning compositions are known in the art, such as effervescent tablets and the like (see eg US Pat. No. 5,055,305, Young), which are generally suitable for one or more variants for removing protein stains from dentures.

In another embodiment of the present invention, contact lens cleaning compositions contain one or more of the variants of the present invention. The contact lens cleaning compositions preferably contain from about 0.01% to about 50% by weight of one or more variants of the present invention and a protease and a personal hygiene carrier, more preferably the variants comprise about 0.01% to about 20%, more preferred variant levels are from about 1% to about 5%. Various forms of contact lens cleaning compositions are known in the art, such as tablets or liquids, etc., which are generally suitable for one or more variants for removing proteinaceous stains from contact lenses.

Embodiments of the contact lens cleaning compositions of the present invention are illustrated by Examples 14-17.

Example 14-17

contact lens cleaning composition Example 14 Example 15 Example 16 Example 17 variant 9-I/protease 0.01% 0.5% 0.1% 2.0% glucose 50.0% 50.0% 50.0% 50.0% Nonionic Surfactant (Polyoxyethylene Polyoxypropylene Copolymer) 2.0% 2.0% 2.0% 2.0% Anionic surfactant (polyoxyethylene-alkylphenylether sodium sulfricester) 1.0% 1.0% 1.0% 1.0% Sodium chloride 1.0% 1.0% 1.0% 1.0% Borax 0.30% 0.30% 0.30% 0.30% water 45.69% 45.20% 45.60% 43.70%

In Examples 14-17, the variants recited in Tables 7, 8, 10 and among other variants the preferred variants cited herein were used in place of variant 9-1 with essentially similar results.

Examples 18-21 illustrate the use of variants of the invention in body cleansing products:

Example 18-21

body cleansing products Example 18 Example 19 Example 20 Example 21 water 62.62% 65.72% 57.72% 60.72% Disodium EDTA 0.2% 0.2% 0.2% 0.2% glycerin 3.0% 3.0% 3.0% 3.0% Polyquaternium 10 0.4% 0.4% 0.4% 0.4% Sodium Laureth Sulphate 12.0% 12.0% 12.0% 12.0% Cocamide MEA 2.8% 2.8% 2.8% 2.8% lauraphoacetate sodium salt 6.0% 6.0% 6.0% 6.0% myristic acid 1.6% 1.6% 1.6% 1.6% Magnesium Sulfate Heptahydrate 0.3% 0.3% 0.3% 0.3% (table) continued Trihydroxystearin Glyceric Acid 0.5% 0.5% 0.5% 0.5% PEG-6 caprylic acid/capric triglycerides 3.0% - - - Sucrose polyesters of cotton fatty acid 3.0% - - - Sucrose polyesters of behenate fatty acid 3.0% - 4.0% - Petrolatum - 4.0% 8.0% - mineral oil - - - 6.0% DMDM hydantoin 0.08% 0.08% 0.08% 0.08% variant 14-I/protease 0.1% 2.0% 2.0% 5.0% citric acid 1.40% 1.40% 1.40% 1.40%

In Examples 18-21, the variants recited in Tables 7, 8, 10 and among other variants the preferred variants cited herein were used in place of variant 14-I with substantially similar results.

Examples 22-25 illustrate the use of variants of the invention in facial cleansing products:

Example 22-25

cleansing products Example 22 Example 23 Example 24 Example 25 water 66.52% 65.17% 68.47% 68.72% Disodium EDTA 0.1% 0.1% 0.2% 0.2% citric acid - - 1.4% 1.4% Sodium Laureth-3 Sulfate 3.0% 3.5% - - Sodium Laureth-4Carboxylate 3.0% 3.5% - - Laureth-12 1.0% 1.2% - - Polyquaternium 10 - - 0.4% 0.4% Polyquaternium 25 0.3% 0.3% - - glycerin 3.0% 3.0% 3.0% 3.0% (table) continued Sodium Lauroamphoacetate - - 6.0% 6.0% Lauric acid 6.0% 6.0% 3.0% 3.0% myristic acid - - 3.0% 3.0% Magnesium Sulfate Heptahydrate 2.3% 2.0% 2.0% 2.0% Triethanolamine 4.0% 4.0% 4.0% 4.0% Glyceryl trihydroxystearate 0.5% 0.5% 0.5% 0.5% Sucrose polyesters of behenate fatty acid 2.0% 2.0% - - Sucrose polyesters of cotton fatty acid 3.0% 2.0% - - PEG-6 caprylic acid/capric triglycerides - - - 2.0% Petrolatum - - 4.0% - mineral oil - - - 2.0% Cocamidopropyl betaine 2.0% 3.0% 1.8% 1.8% lauryl dimethylamine oxide 1.0% 1.2% 1.2% 1.2% Dex panthenyl 1.0% 0.25% 0.25% - DMDM hydantoin 0.08% 0.08% 0.08% 0.08% variant 24-I/protease 1.0% 2.0% 0.5% 0.5% spices 0.2% 0.2% 0.2% 0.2%

In Examples 22-25, the variants recited in Tables 7, 8, 10, and among other variants, preferred variants cited herein were used in place of variant 24-1 with substantially similar results.

Examples 26-27 illustrate the use of variants of the invention in leave-on skin moisturizing compositions:

Example 26-27

leave-on skin moisturizing composition Example 26 Example 27 glycerin 5.0% - stearic acid 3.0% - C _11-13 isoparaffins 2.0% - Ethylene glycol stearate 1.5% - Propylene Glycol - 3.0% mineral oil 1.0% 10.0% sesame oil - 7.0% Petrolatum - 1.8% Triethanolamine 0.7% - cetyl acetate 0.65% - Glyceryl Stearate 0.48% 2.0% TEA stearate - 2.5% cetyl alcohol 0.47% - lanolin - 1.8% DEA-Cetyl Phosphate 0.25% - Methylparaben 0.2% 0.2% Propylparaben 0.12% 0.1% Carbomer934 0.11% - Disodium EDTA 0.1% - variant 13-I/protease 0.1% 0.5% water 84.32% 71.1%

In Examples 26-27, the variants recited in Tables 7, 8, 10, and among other variants, preferred variants cited herein were used in place of variant 13-I with substantially similar results.

Example 28 illustrates the use of variants of the invention in cleaning wipe compositions:

Example 28

cleaning wipe composition Propylene glycol 1.0% ammonium lauryl sulfate 0.6% Succinic acid 4.0% sodium succinate 3.2% ^Triclosan_ 0.15% variant 20-I/protease 0.05% water 91.0%

A woven absorbent sheet comprising cellulose and/or polyester is impregnated with the above composition in an amount of 250% by weight of the absorbent sheet.

In Example 28, the variants recited in Tables 7, 8, 10 and the preferred variants cited herein among other variants were used in place of variant 20-1 with essentially similar results.

Sequence Listing <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>15<170>PatentIn Ver.2.0<210>1<211>113<212>PRT<213>Streptomyces albogriseolue<400>1Asp Ala Pro Ser Ala Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Val1 5 10 15G1y Lys Gly Val Ser Ala Thr Thr Ala Ala Pro Glu Arg Ala Val Thr

20 25 25 30Leu Thr Cys Ala Pro Gly Pro Ser Gly Thr His Pro Ala Ala Gly Ser

35 40 45Ala Cys Ala Asp Leu Ala Ala Val Gly Gly Asp Leu Asn Ala Leu Thr

50 55 60ARG GLY GLU ASP Val Met Cys Pro Met Val Tyr ASP Pro Val Leu65 70 75 80thr Val Val Trn GLN GLY LYS ARG Valr Glu ARG Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val

85 90 95Phe Ser Asn Glu Cys Glu Met Asn Ala His Gly Ser Ser Val Ala Phe

100                 105                 110Phe<210>2<211>117<212>PRT<213>白浅灰链霉菌(Streptomyces albogriseolus)<400>2Ala Gly Glu Phe Asp Ala Pro Ser Ala Leu Tyr Ala Pro Ser Ala Leu1               5                  10                  15Val Leu Thr Val Gly Lys Gly Val Ser Ala Thr Thr Ala Ala Pro Glu

20 25 30Arg Ala Val Thr Leu Thr Cys Ala Pro Gly Pro Ser Gly Thr His Pro

35 40 45Ala Ala Gly Ser Ala Cys Ala Asp Leu Ala Ala Val Gly Gly Asp Leu

50 55 60asn Ala Leu ThR ARG GLY GLU ASP Val Met Cys Pro Met Val Tyr ASP65 70 80pro Val Leu ThR Val Val TRN GLN GLY LYS ARG Val Val Ser

85 90 95 Tyr Glu Arg Val Phe Ser Asn Glu Cys Glu Met Asn Ala His Gly Ser

100 105 110Ser Val Phe Ala Phe

115 <210> 3 <211> 275 <212> PRT <213> Bacillus amyloliquetaciens (400> 3ALA Gln Ser Val Val Val Ser Gln Ile LYS ALA Leu1 5 10 15his Ser Gln Gln Gln Gln Gln Gln Gln Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp

20 25 30Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala

35 40 45Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His

50 55 60Gly Thr His Vel Ala Gly Thr Val Ala Leu Asn Serle Gly65 70 75 80VAL Leu Gly Val Ala Sero Seru Val Lys Val Leu

85 90 95Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu

100 105 110Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala

130 135 140SER GLE VAL VAL VAL VAL ALA ALA Ala Gly Asn GLU GLY Thr Ser Gly145 150 155 160SER Servr Val Gly Tyr Pro Serle Ala

165 170 175Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val

180 185 190Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr

155 200 205Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser

210 215 220Pro his value ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His His Pro Asn225 230 235 240TRP THR Gln Val Serg Serg Ser Leu asn Thr THR LYSS

245 250 255Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala

260 265 270Ala Ala Gln

275 <210> 4 <211> 107 <212> PRT <213> Streptomycess Thermotolerans <400> 4tyr Ala Pro Seru Val Leu ThR Val GLY GLY GLY GLU Series Glu Arg Ala Val Thr Leu Thr Cys Ala Pro Lys

                                                                                                                         

35 40 45Gly 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 THR Val GLY Val TRP65 70 75 80GLN GLY LYS ARG Valr Glu ARG THR PHE Ser ASP CYS MET

85 90 95Lys Asn Ala Tyr Gly Thr Gly Val Phe Ser Phe

100 105 <210> 5 <211> 109 <212> PRT <213> Streptomycess Galbus <400> 5SER Leu Tyr Ala Leu Val Leu Thr Met GLY GLU1 5 10 15SER Ala Ala Ala Val Ser Pro Ala Arg Ala Val Thr Leu Asn Cys Ala

20 25 25 30Pro Ser Ala Ser Gly Thr His pro Ala Pro Ala Leu Ala Cys Ala Glu

35 40 45Leu Arg Ala Ala Gly Gly Asp Leu Asp Ala Leu Ala Gly Pro Ala Asp

50 55 60thr Val Cys Thr Lya Gln Tyr Ala Pro Val Val ILS THR Val ASP GLY55 70 80VAL TRN GLN GLY LYS ARG Val Sar Tyr Phe Ala asn Gly

85 90 95Cys Val Lys Asn Ala Ser Gly Ser Ser Val Phe Ala Phe

100 105 <210> 6 <211> 107 <212> PRT <213> Streptomycess Azureus <400> 6tyr Ala Pro Seru Val Leu ThR Val GLU GLU GLY GLU Serite Pro Glu Arg Ala Val Thr Leu Thr Cys Ala Pro Arg

20 25 30Pro Ser Gly Thr His Pro Val Ala Gly Ser Ala Cys Ala Glu Leu Arg

35 40 45Gly Val Gly Gly Asp Val His Ala Leu Thr Ala Thr Asp Gly Val Met

 50                  55                  60Cys Thr Lys Gln Tyr Asp Pro Val Val Val Thr Val Asp Gly Val Trp65                  70                  75                  80Gln Gly Arg Arg Val Ser Tyr Glu Arg Thr Phe Ser Asn Glu Cys Val

85 90 95Lys Asn Ala Tyr Gly Ser Gly Val Phe Ala Phe

100 105 <210> 7 <211> 110 <212> PRT <213> Streptomycess Lividans <400> 7SER Leu Tyr Ala Pro Seru Val Leu ThR Val GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLY GLE Ala Ala Pro Leu Arg Ala Val Thr Leu Thr Cys Ala

20 25 30Pro Thr Ala Ser Gly Thr His Pro Ala Ala Ala Ala Ala Cys Ala Glu

35 40 45Leu Arg Ala Ala His Gly Asp Pro Ser Ala Leu Ala Ala Glu Asp Ser

50 55 60VAL MET CYS THR ARG GLU TYR ALA PRO Val Val Val THR Val ASP GLY65 75 80VAL TRN GLN GLN GLY ARG Leu Serg THR PHLA Aan Glu

85 90 95Cys Val Lys Asn Ala Gly Ser Ala Ser Val Phe Thr Phe Glu

100 105 110 <210> 8 <111> 110 <212> PRT <213> Streptomycess Longisporus <400> 8Ala Sero Tyr Ala Sera La Leu Val Leu Thr Val Gly1 5 10 15Thr Sera Ala Ala Ala Ala Thr Pro Leu Arg Ala Val Thr Leu Asn Cys

                                                                                                                           

35 40 45Asp Leu Arg Gly Val Gly Gly Asp Ile Asp Ala Leu Lys Ala Arg Asp

50 55 60Gly Val Ile Cysn LYS Leu Tyr ASP PRO VAL VAL VAL VAL VAL VAL VAL VAL VAL VAL ASP65 70 80GLY VAL TRN GLN GLS ARG VAL GLU ARG THR PHLY GLY ASN

85 90 95Glu Cys Val Lys Asn Ser Tyr Gly Thr Ser Leu Phe Ala PRe

100 105 110 <210> 9 <111> 113 <212> PRT <213> Streptomycess Parvulus <400> 9thr Ala Ala Ala Ala Pro Seru Val Leu Val Ile1 5 10 15gly Gln Gln Glu. Ser Ala Ala Ala Thr Ser Pro Leu Arg Ala Val Thr

20 25 25 30Leu Thr Cys Ala Pro Lys Ala Thr Gly Thr His Pro Ala Ala Asp Ala

35 40 45Ala Cys Ala Glu Leu Arg Arg Ala Gly Gly Asp Phe Asp Ala Leu Ser

50 55 60ALA ALA ASP GLY VAL MET CYS THR ARG GLU TYR ALA PRO Val Val Val65 70 75 80thr Val Val Val Trn GLN GLY ARG Leu Serg THR

65 90 95Phe Ala Asn Glu Cys Val Lys Asn Ala Gly Ser Ala Ser Val phe Thr

100 105 110phe <210> 10 <211> 107 <212> PRT <213> STREPTOMYCES CoelOlor <400> 10Tyr Ala Pro Seru Val Leu ThR Val GLY GLY GLY GLY GLY GLY GLY GLY GLA1 5 10 15ALA THR ALA Ala Pro Leu Arg Ala Val Thr Leu Thr Cys Ala Pro Thr

20 25 30Ala Ser Gly Thr His Pro Ala Ala Asp Ala Ala Cys Ala Glu Leu Arg

35 40 45Ala Ala His Gly Asp Pro Ser Ala Leu Ala Ala Asp Asp Ala Val Met

50 55 60CYS Thr ARG Glu Tyr Ala Pro Val Val Val THR Val ASP GLY VAL TRP65 70 75 80GLN GLY ARG Leu Serg ThR PHE ALA Asn Glu Cys Val Val

85 90 95Lys Asn Ala Gly Ser Ala Ser Val Phe Thr Phe

100 105 <210> 11 <211> 116 <212> PRT <213> Streptomycess Lavendulae <400> 11ALA Pro Ala Ala Ala Ala Ala Pro Seru Val1 5 10 15leu Thr Ile Gly His Gly Gly Ala Ala Ala Thr Ala Thr Pro Glu Arg

20 25 25 30Ala Val Thr Leu Thr Cys Ala Pro Thr Ser Ser Ser Gly Thr His Pro Ala

35 40 45Ala Ser Ala Ala Cys Ala Glu Leu Arg Gly Val Gly Gly Asp Phe Ala

50 55 60ALA Leu Lys Ala ARG ASP Val TRP CYS ASN LEU TYR ASP PRO65 70 75 80VAL VR Ala Gln GLN GLN GLN GLN ARG Val Serg Val Ser Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr

85 90 95 Glu Arg Thr Phe Gly Asn Ser Cys Glu Arg Asp Ala Val Gly Gly Ser

100 105 110Leu Phe Ala Phe

115 <210> 12 <211> 109 <212> PRT <213> Streptomycess Antifibrinolyticus <400> 12Gly Leu Tyr Ala Leu Val Leu THR MET GLY HIS GLY Asn1 5 10 15SER ALA THRA THRA THRA THRA THRA THRA THRA THRA THRA THRA THRA THRA THE Val Asn Pro Glu Arg Ala Val Thr Leu Asn Cys Ala

20 25 30Pro Thr Ala Ser Gly Thr His Pro Ala Ala Leu Gln Ala Cys Ala Glu

35 40 45Leu Arg Gly Ala Gly Gly Asp Phe Asp Ala Leu Thr Val Arg Gly Asp

50 55 60VAL Ala Cys THR LYS GLN PHE ASP Pro Val Val Val THR Val ASP GLY65 75 80VAL TRN GLN GLY LYS ARG Valr Glu ARG THA ALA Asn Glu asn Glu

85 90 95Cys Val Lys Asn Ser Tyr Gly Met Thr Val Phe Thr Phe

100 105 <210> 13 <211> 107 <212> PRT <213> Streptomycess Lividans (400> 13tyr Ala Pro Seru Val Leu Thr Val GLY GLU SERA1 5 L0 15ALA THR ALA ALA Pro Leu Arg Ala Val Thr Leu Thr Cys Ala Pro Thr

20 25 30Ala Ser Gly Thr His Pro Ala Ala Ala Ala Ala Cys Ala Glu Leu Arg

35 40 45Ala Ala His Gly Asp Pro Ser Ala Leu Ala Ala Glu Asp Ser Val Met

50 55 60CYS Thr ARG Glu Tyr Ala Pro Val Val Val THR Val ASP GLY VAL TRP65 70 75 80GLN GLY ARG Leu Serg ThR PHE ALA Asn Glu Cys Val Val

85 90 95Lys Asn Ala Gly Ser Ala Ser Val Phe Thr Phe

100 105 <210> 14 <211> 110 <212> PRT <213> Streptoverticillium Cinnamoneum <400> 14ser Leu Tyr Ala Leu Val Leu THR Ile Gln Gln GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN Ala Ala Ala Ala Gly Ile Gln Arg Ala Val Thr Leu Thr Cys Met

20 25 25 30Pro Lys Ala Asp Gly Thr His Pro Asn Thr Arg Gly Ala Cys Ala Gln

35 40 45Leu Arg Leu Ala Gly Gly Asp Phe Glu Lys Val Thr Lys Ile Lys Glu

50 55 60Gly Thr Ala Cys THR ARG GLU TRP Asn Pro Ser Val Val THR ALA GLU65 70 75 80GLY Val TRU GLY ARG Val Serg THR PHRA ALA ALA Asnn

85 90 95Pro Cya Glu Leu Lys Ala gly Lys Gly Thr Val Phe Glu Phe

100 105 110 <210> 15 <111> 110 <212> PRT <213> Streptomycess Cacaoi <400> 15ser Lsu Tyr Ala Val Val Val Ile Ser Lysr Gln GLN GLA1 5 10 15SER ALA Asp Ala Pro Ala Gln Arg Ala Val Thr Leu Arg Cys Leu pro

                                                                                  

35 40 45Glu Ala Gly Gly Asp Pro Ala Ala Leu Pro Arg Tyr Val Glu Asp Thr

 50                  55                  60Gly Arg Val Cys Thr Arg Glu Tyr Arg Pro Val Thr Val Ser Val Gln65                  70                  75                  80Gly Val Trp Asp Gly Arg Arg Ile Asp His Ala Gln Thr Phe Ser Asn

85 90 95 Ser Cys Glu Leu Glu Lys Gln Thr Ala Ser Val Tyr Ala Phe

100 105 110

Claims (10)

1. A variant characterized by a modified amino acid sequence of a parent amino acid sequence, wherein the modified amino acid sequence is characterized by an amino acid substitution at position 63 corresponding to SSI, wherein the parent amino acid sequence is selected from the group consisting of SSI, SSI-like inhibitors , SSI variants, and SSI-like inhibitor variants. 2. The variant of claim 1, wherein the amino acid substitution at position 63 corresponding to SSI is made with isoleucine. 3. A variant according to any preceding claim, wherein the parent amino acid sequence is selected from the group consisting of SSI and SSI variants. 4. A variant according to any preceding claim which exhibits a _K such that the variant: (a) inhibiting a protease in a composition comprising the variant and the protease; and (b) Dissociation from proteases after dilution. 5. A variant according to any preceding claim which exhibits a _K1 of from about 10 ^"12M to about 10" ^4M . 6. A variant of any preceding claim selected from the group consisting of: (a) L63I+D83C; (b) L63I+M73D; (c) L63I+M73D+D83C; (d) L63I+M73P+D83C; ( e) L63I+M70Q+D83C; (f) L63I+M70Q+M73P+V74F+Dg3C; (g) L63I+M70Q+M73P+V74W+D83C; (h) L63I+M70Q+M73P+D83C+S98A; (i) L63I+G47D+M73P+V74F+D83C; (j)L63I+G47D+M73P+V74W+D83C; (k)L63I+G47D+M73P+D83C+S98A; (l)L63I+G47D+M70Q+M73P+V74F+D83C ; (m)L63I+G47D+M70Q+M73P+V74W+D83C; (n)L63I+G47D+M73P+V74F+D83C+S98A; (o)L63I+G47D+M73P+V74W+D83C+S98A; (p)A62 ^* +L63I+D83C; (q)A62 ^* +L63I+M73D; (r)A62 ^* +L63I+M73D+D83C; (s)A62 ^* +L63I+M73P+D83C; (t)A62 ^* +L63I+M70Q+ D83C; (u)A62 ^* +L63I+M73P+D83C+S98A ^; (v)A62 ^* +L63I+M73P+Y75A+D83C; (w)A62* +L63I+M73P+D83C+S98V; (x)A62 ^* + L63I+M70Q+M73P+D83C; (y)A62 ^* +L63I+M73P+V74A+D83C; (z)A62 ^* +L63I+M73P+V74F+D83C; (aa)A62 ^* +L63I+M70Q+D83C+S98A; (bb)A62 ^* +L63I+G47D+M70Q+D83C; (cc)A62 ^* +L63I+G47D+D83C+S98A; (dd)A62 ^* +L63I+G47D+M73P+D83C; (ee)A62 ^* +L63I+ G47D+M73D+D83C; (ff)A62 ^* +L63I+M70Q+M73P+V74F+D83C; (gg)A62 ^* +L63I+M70Q+M73P+V74W+D83C; (hh)A62 ^* +L63I+M70Q+M73P+ D83C+S98A; (ii)A62 ^* +L63I+G47D+M73P+V74F+D83C; (jj)A62 ^* +L63I+G47D+M73P+V74W+D83C; (kk)A62 ^* +L63I+G47D+M73P+D83C+ S98A; (ll)A62 ^* +L63I+G47D+M70Q+M73P+V74F+D83C; (mm)A62 ^* +L63I+G47D+M70Q+M73P+V74W+D83C; (nn)A62 ^* +L63I+G47D+M73P+ V74F+D83C+S98A; (oo)A62 ^* +L63I+G47D+M73P+V74W+D83C+S98A; (pp)L63I+A62K+S98Q; (qq)L63I+A62K+S98D; (rr)L63I+A62K+S98E ;(ss)L63I+A62R+S98Q;(tt)L63I+A62R+S98D;(uu)L63I+A62R+S98E;(vv)L63I+S98A;(ww)L63I+M73P+D83C+S98D;(xx)L63I +M73P+D83C+S98E; (yy)L63I+M73P+S98D; (zz)L63I+M73P+S98E; (aaa)L63I+M73P+S98A; (bbb)A62K+L63I+M73P+D83C+S98D; A62R+L63I+M73P+D83C+S98D; (ddd)A62K+L63I+M73P+D83C+S98E; (eee)A62R+L63I+M73P+D83C+S98E; (fff)A62K+L63I+M73P+S98A;(ggg) A62R+L63I+M73P+S98A; (hhh)L63I+G47D+M73P+D83C+S98D; (iii)L63I+G47D+M73P+D83C+S98E; and (jjj)L63I+M73P. 7. A variant of any preceding claim selected from the group consisting of: (a) A62K+L63I+M73P+D83C+S98Q; (b) A62K+L63I+M73P+D83C+S98D; (c) A62K+L63I+ M73P+D83C+S98E; (d) A62K+L63I+S98Q; (e) A62K+L63I+S98D; (f) A62K+L63I+S98E; (g) A62R+L63I+M73P+D83C+S98Q; (h) A62R +L63I+M73P+D83C+S98D; (i)A62R+L63I+M73P+D83C+S98E; (j)L63I+M73P+D83C+S98A. (k)A62R+L63I+S98Q; (l)A62R+L63I+S98D ; (m)A62R+L63I+S98E; (n)L63I+S98A; (o)A62K+L63I+M73P+D83C+S98D; (p)A62R+L63I+M73P+D83C+S98D; (q)A62K+L63I+ M73P+D83C+S98E; (r)A62R+L63I+M73P+D83C+S98E; (s)A62K+L63I+M73P+S98A; and (t)A62R+L63I+M73P+S98A. 8. A mutated gene encoding a variant according to any preceding claim. 9. A composition comprising a variation of any preceding claim and a carrier selected from the group consisting of a cleaning composition carrier and a personal hygiene composition carrier. 10. 9. A composition according to claim 9, wherein the ratio of variant to protease is between about 3:1 and about 1:1.