MXPA01000304A - Proteases fused with variants of streptomyces subtilisin inhibitor - Google Patents

Abstract

The present invention relates to fusion proteins wherein the fusion protein comprises a protease part;and a variant part, wherein the variant part has a modified amino acid sequence of a parent amino acid sequence, wherein the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to SSI, and wherein the parent amino acid sequence is selected from the group consisting of SSI, SSI-like inhibitors, variants of SSI, and variants of SSI-like inhibitors. Such fusion proteins are useful in cleaning compositions and personal care compositions. The present invention also relates to cleaning compositions and personal care compositions comprising the present fusion proteins, as well as DNA encoding the fusion proteins.

Description

FUSED PROTEASES WITH VARIANTS OF STREPTOMYCES SUBTILISIN INHIBITOR CROSS REFERENCE TO RELATED REQUESTS This application claims the benefit of the Provisional Application E.U.A. Serial No. 60/091, 904, filed July 7, 1998.

FIELD OF THE INVENTION The present invention relates to fusion proteins of: (1) proteases and (2) subtilisin inhibitor variants of Streptomyces (SSI) and to those inhibitors that have homology to SSI (SSI type inhibitors). Such fusion proteins are useful in cleaning compositions and in personal care compositions. The present invention also relates to cleaning compositions and personal care compositions comprising the present fusion proteins, as well as the genes encoding the fusion proteins.

BACKGROUND OF THE INVENTION Enzymes are the largest class of proteins found in Nature. One class of enzymes includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has been exploited by incorporating in the cleaning compositions proteases that occur naturally and proteases obtained by genetic manipulation of proteins, particularly those that are important for laundry applications. In addition, although it has been explored to a lesser degree, others have incorporated such proteases into personal care compositions. However, during the storage of the composition or even during the expression of the protease, the protease often degrades on its own or may degrade to other enzymes present in the composition. As a result of this degradation, the cleaning and personal care compositions have limited capacity to achieve the intended improved performance. It is therefore beneficial to incorporate in the compositions a protease activity inhibitor to limit the autolysis of the protease and other degradation. It would be advantageous to provide reversible inhibitors of the protease, so that after dilution of the composition during cleaning, or in the cleaning environment, the protease is no longer inhibited, but rather is available to hydrolyze the protein spots. In addition, such inhibitors must be stable enough to adequately perform their inhibitory function. Those skilled in the art have experimented with proteinaceous protease inhibitors to stabilize the enzyme in cleaning compositions. Nature provides protein protease inhibitors to regulate the 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 vehicles for personal care and cleaning may be somewhat limited. Protein protease inhibitors are typically long peptides that bind to the active site of a protease and inhibit its activity. These inhibitors have typically been classified into several families (families I through IX) based on their primary amino acid sequence homologies (See Laskowsky et al., "Protein Inhibitors of Proteinases", Annual Review of Biochemistry, Vol. 49, pp. 593-626 (1980)). Included within these inhibitors are those commonly referred to as inhibitors of Family VI, including inhibitors of barium chymotrypsin and eglin, and inhibitors of the III family, such as the inhibitor of Streptomyces subtilisin (SSI), 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 frequently discusses "peptide inhibitor / protease pairs". An example of a known protease / peptide 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 A Resolution", Journal of Molecular Biologv, Vol. 131, p. 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 Bioloav. 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 after being diluted in the wash environment. See WO 92/03529, Mikkelson et al., Assigned to Novo Nordisk A / S, published on March 5, 1992. However, if the binding constant (K) of an inhibitor provides some protease activity in the cleaning composition containing the enzyme / inhibitor pair, the inhibitor, as well as the enzymes in the composition, could be hydrolyzed. Therefore, it would be advantageous to find SSI variants or other inhibitors that are appropriately stable in the presence of protease as well as compositions for cleaning and personal care. In addition, preferably these inhibitors have a preferred K, for the particular protease to be inhibited. Such K i must allow the inhibition of the protease in the final composition and during its storage. However, after diluting the composition for cleaning or for personal care or during the cleaning procedure, the protease and the inhibitor must be dissociated, allowing the activity of the non-inhibited protease. However, the stability of such protease inhibitors has been problematic. WO 98/13387, Correa et al., Assigned to The Procter & amp;; Gamble Co., published on April 2, 1998 (corresponding to patent application U.S. Serial No. 60 / 026,944) describes variants which are described as providing increased stability. In addition, the manufacture of proteases, including those that are useful in cleansing and personal care compositions, present their own unique problems. For example, production may be limited by autolysis during the fermentation or purification process. Unfortunately, the addition of protease inhibitors to the fermentation broth or to the mixture for purification requires the purchase and addition of an excess of inhibitor. The addition of inhibitor at this stage may also be untimely because hydrolysis of the protease may occur before the feasible addition of the inhibitor. In addition, the addition of the inhibitor could actually reduce the yield of the protease. As an example of addition in the fermentation step, German Patent Specification 2,131, 451, assigned to Nagase & Co., published December 30, 1971, describes a process for producing alkaline protease. It is said that this procedure requires the addition of water-soluble borate as an inhibitor. It is said that this borate improves the filtration activity and, therefore, the protease yield. However, it is recognized that, at certain levels, borate can actually slow the production of the enzyme. Joeraensen et al .. WO 93/13125, assigned to Novo Nordisk A / S, published on July 8, 1993, describe a process for producing a "protein susceptible to inactivation" in a fluid production medium "continuously protecting and reversible "the protein against inactivation during the production stage, deprotecting the protein and recovering the protein product. It is disclosed that such a procedure is useful to obtain increased yields of the protein by reversibly inactivating the protein. However, such procedures may require the addition of exogenous materials that could be expensive, inefficient, may require additional processing and make the process difficult to control. Saunders et al .. WO 98/13483, assigned to The Procter & Gamble Co., published on April 2, 1998, faces the need to inhibit protease in vivo by providing fusion proteins. Hartman et al .. WO 97/15670, assigned to Arris Pharmaceutical Corp., published on May 1, 1997, mention the use of fusion proteins. Such fusion proteins could be useful for providing inhibitor / protease pairs with significant cost savings and increased yields. By producing stoichiometric amounts of inhibitor concurrently with the protease, early autolysis could be reduced or eliminated early in the production phase of the protease. However, the protease inhibitor by itself could be adequately stable to achieve this purpose. It has been surprisingly discovered that SSI inhibitors, SSI inhibitors and variants thereof are hydrolyzed between positions 63 and 64 corresponding to SSI. Accordingly, the inventor of the present invention provides inhibitor / protease fusion proteins in which the inhibitors are variants of SSI, SSI inhibitors and SSI inhibitors which are modified, inter alia, at position 63 by a residue of amino acid substituent. Such substitution imparts increased stability to the protease inhibitor. The present inventor has incorporated such inhibitors into fusion proteins in the present invention, whereby the aforementioned in vivo protease degradation problem is solved. The present invention therefore provides fusion proteins comprising inhibitors that have greater proteolytic stability, lower affinity to the protease than that of the original inhibitor, and which facilitate reduced autolysis of the protease.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides fusion proteins comprising: (a) a part of protease; (b) a variant part, in which the variant part has a modified amino acid sequence of a progenitor amino acid sequence, in which the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to SSI, and in the which the progenitor amino acid sequence is selected from the group consisting of SSI, SSI-type inhibitors, SSI variants, and SSI-type inhibitor variants and, optionally (c) a linking part in which when the linking part is present, the protease part and the variant part are covalently bound through the linking part. The protease part preferably includes those proteases for which SSI is an inhibitor. Such proteases include, for example, those produced by the microorganisms Bacillus alkalophilus, Bacillus amyloliquefaclens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus and Bacillus subtilis. The present invention also relates to the genes coding for such fusion proteins and to the cleansing and personal care compositions comprising such fusion proteins.

DETAILED DESCRIPTION OF THE INVENTION The essential components of the present invention are described in the present invention. Also included are non-limiting descriptions of the 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 described in the present invention. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages were calculated based on the total composition unless otherwise stated. In the present invention reference is made to trade names for the materials including, but not limited to, proteases and optional components. The inventors of the present invention are not intended to be limited to a certain trade name. Equivalent materials (eg, those obtained from a different source under a different name or catalog number (reference)) can be substituted for those referenced by the trade name and used in the compositions herein. invention. All the levels of the components, ingredients or composition are in reference to the active level of that component, ingredient or composition, and do not include impurities, for example residual solvents or by-products that may be present in commercially available sources. All documents referred to in the present invention, including all patents, patent applications and printed publications are incorporated in their entirety in the present invention for reference. As used in the present invention, abbreviations will be used to describe the amino acids. Table I provides a list of the abbreviations used in the present invention: TABLE I Definitions As used in the present invention, the term "fusion protein" has a recognized significance in the art, ie, two proteins are expressed as a chain of amino acids, typically under the control of a regulatory element. For example, fusion proteins have been used in recent years for various applications (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press (1989). Expression vectors can be commercially available to utilize fusion technology in the production of a protein of interest.A discussion on fusion proteins comprising SSI variants can also be found in Saunders et al., US patent application no. series 60 / 026,947, which corresponds to Saunders et al .. WO 98/13483, assigned to The Procter & amp;; Gamble Co., published April 2, 1998. As used in the present invention, the term "mutation" refers to alterations in the sequences of the genes and in the amino acid sequences produced by those sequences of the genes. The mutations can be deletions, substitutions or additions of amino acid residues to the progenitor or wild-type sequence. As used in the present invention, the term "progenitor" refers to a protease, protease inhibitor, protein, or wild-type or variant peptide, which do not have 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 progenitors is an inhibitor known as Streptomyces Subtilisin Inhibitor (SSI) (represented by SEQ ID NO: 1). SSI is also described by Ikenaka et al., "Amino Acid Sequence of an Alkaline Proteinase Inhibitor from Streptomyces albogriseolus S-3253, Journal of Biochemistry, Vol. 76, pp. 1191-1209 (1974). As used in the present invention, the amino acid numbering of SSI is that of Ikenaka et al .. The inventors of the present invention also used a synthetic gene for SSI, designed to be enriched for adenine and thymine, as is the DNA of B. subtilis The sequence of this synthetic SSI gene is represented by SEQ ID NO: 1. This synthetic gene codes for four extra amino acid residues at the amino terminus of the peptide due to the methods of plasmid construction This sequence of modified amino acids, including these four additional amino acids, is represented by SEQ ID NO: 2. As used in the present invention, the term "silves type tre "refers to a protein, specifically in the present invention, a protease inhibitor or a protease, having an amino acid sequence that is different from the protease sequence or the progenitor protease inhibitor sequence, respectively.Fusion Proteins of the Present Invention The inventors of the present invention have discovered fusion proteins comprising: (a) a protease part (for simplicity's sake, also referred to in the present invention as a protease); (b) a variant part for reasons of simplicity, also referred to in the present invention as a variant); and, optionally, (c) a linking part in which, when the linking part is present, the protease part and the variant part are covalently linked through the linking part. The variant has a modified amino acid sequence of a progenitor amino acid sequence, in which the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to SSI, and in which the progenitor amino acid sequence is selected from the group consists of SSI, SSI-type inhibitors, SSI variants, and variants of SSL-type inhibitors. The fusion proteins of the present invention are beneficial, inter alia, due to the stability of the variant. Without intending to be limited to theory, the fusion proteins of the present invention minimize or avoid the potential risks of one protein being expressed at a higher rate than the other. The fusion proteins of the present invention allow the synthesis of molar amounts selected specifically from protease and inhibitor (variant) simultaneously. For example, the fusion protein can be constructed to provide an equimolar amount of inhibitor (variant), or twice the molar amount of inhibitor (variant) to protease, and the like. Therefore, it is possible to avoid autolysis of the protease as soon as possible, that is, right after translation in vivo. The fusion protein may contain one or more protease portions, the same or different, as long as the fusion protein contains at least one part of protease and one part of variant. Most preferably, the fusion protein has a part of protease, a part of variant, with a linker being optional but preferred. It is also contemplated that the same cell will produce other proteases and / or inhibitors, in addition to the fusion protein, either on the same plasmid as the gene for the fusion protein, on a different co-existing plasmid in the cell, a plasmid while the gene for the fusion protein is chromosomal, or as part of the chromosome of the cell. In addition, either a specific inhibitor for a different protease, or a protease with the specific inhibitor for the protease in the fusion protein, can be produced. In addition, the fusion protein can be expressed in combination with one or more additional inhibitors, which inhibitor could be the same as the part of the variant constituting the fusion protein.

Protease Parts As already indicated, the present invention relates to fusion proteins comprising a part of protease (protease). Accordingly, an essential component of the fusion protein is a protease from which a part of the variant present (variant) is inhibited. The protease can be of animal, vegetable or, preferably, microorganism origin. Preferred proteases include those proteases for which SSI is an inhibitor. Such proteases include, for example, those produced by the microorganisms Bacillus alkalophillus, Bacillus amyloliquefaciens, Bacillus amylosaccharícus, Bacillus licheniformis, Bacillus lentus and Bacillus subtilis. Among such proteases the 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 previous ones. 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 BPN 'variants, collectively referred to hereafter as "Group A Proteases", are useful as the proteases of the present invention and are described in US Pat. No. 5,030,378, Veneqas, 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 the position substitutes 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 granted 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, Gly166, 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 "C Group 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 that consists 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 "D Group 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 consisting of : Asp32, Ser33, His64, Tyr104, Asn155, Glu 156, 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.

Part of variant In addition to the protease part, the fusion proteins of the present invention also comprise a variant part (variant). The variants of the present invention are protease inhibitors having an amino acid sequence of a progenitor amino acid sequence, wherein the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to the subtilisin inhibitor of Streptomyces (referred to in the present invention as SSI), and in which the progenitor amino acid sequence is selected from the group consisting of SSI, SSI-type inhibitors, SSI variants and variants of SSI-type inhibitors. Such variants can be fused to the protease in vivo. Preferably the variant is resistant to hydrolysis by the corresponding protease part. The substitution at position 63 corresponding to SSI can be with any amino acid residue that imparts increased stability relative to the progenitor amino acid sequence. More preferably, the substitution at position 63 corresponding to SSI is with isoleucine. Such variant can be represented as "L63I". In describing this variant, the original amino acid that is present in the progenitor amino acid sequence is first given, the position number is given second and the substituted amino acid is given third. Therefore, L63I means that the leucine (L) which appears at position 63 of amino acid (position 63) in the original SSI inhibitor is replaced with Isoleucine (I). The numbering of the positions corresponds to that of Ikenaka et al., Supra (SEQ ID NO: 1) and ignores the four additional amino acid residues present at the amino-terminal end of the synthetic SSI (SEQ ID NO: 2). Such representations for other substitutions listed in the present invention are presented in a consistent manner. The variants of the present invention are not limited to SSI substituted at position 63. Conversely, substitution at position 63 can also be made in the progenitor sequences (including, of course, the nucleotide sequences encoding that sequence of amino acids) in which the progenitor itself is a variant of SSI, an SSI-type inhibitor or a variant of SSI-type inhibitors. The most preferred progenitor amino acid sequences include SSI and SSI variants. The most preferred progenitor amino acid sequences are variants of SS The SSI 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., "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 on October 7, 1997; and WO 93/17086, Nielsen et al., assigned to Novo Nordisk A / S, published on September 2, 1993. Other variants have been described in the U.S. Patent Application. No. 60 / 026,944, Correa et al., Corresponding to WO 98/13387, Correa et al., Assigned to The Procter & Gamble Co., published on April 2, 1998, such variants being described collectively in the present invention as "Group A of Inhibitors". The preferred SSI variants (to be used in the present invention as the progenitor amino acid sequences) are those from Group A of Inhibitors. The most preferred variants which are useful as the progenitor amino acid sequences in the present invention are listed in the following tables 2-6. Again, all position numberings correspond to SSI as described by Ikenaka et al.

TABLE 2 Non-limiting examples of proqenitor amino acid sequences having a single substitution TABLE 3 Non-limiting examples of progenitor amino acid sequences that have double substitutions TABLE 4 Non-limiting examples of progenitor amino acid sequences that have triple substitutions TABLE 5 Non-limiting examples of progenitor amino acid sequences which have four-fold substitutions TABLE 6 Non-limiting examples of progenitor 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 SSI, and where 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 the 63-position.

TABLE 7 Non-limiting examples of preferred variants of the present invention Other preferred progenitor amino acid sequences of the present invention include those that comprise a substitution at position 62 corresponding to SSI. The substitution at position 62 can be any amino acid residue different from that naturally occurring in the parent (in the case of SSI, the naturally occurring amino acid residue 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 progenitor 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 progenitors are designated Progenitor X-A62 *, in which the "X" corresponds to the parent exemplified in Tables 2-6. In this way, Progenitor 6 - A62 * corresponds to A62 * + M73P + D83C + S98A. Similarly, Progenitor 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 sequences of preferred variants of the present invention 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 Version 29-I-A62K A62K + L63I + M73P + S98A Version 29-I-A62R A62R + L63I + M73P + S98A Version 30-I-A62K A62K + L63I + M73P + S98D Other preferred progenitor amino acid sequences (which are variants of SSI) useful in the present invention include those having a single substitution at position 98 corresponding to SSI and those having a double substitution, one at position 62 and another at position 98. Table 9 lists the preferred progenitor amino acid sequences in this class.

TABLE 9 Non-limiting examples of progenitor 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 SSI can exist in a dimeric form. Therefore, without being limited by theory, it is possible that stabilizing dimeric SSIs will provide an increased protease resistance to excess protease. Preferably, this stabilized dimeric SSI variant consists of two SSI variant monomers linked together covalently. This can be through ester bonds, amido, disulfide, or other bonds, 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 progenitor amino acid sequences that are useful in the present invention include SSI type inhibitors (often referred to as SSI (SIL) type proteins) and SSI type inhibitors. Background information regarding SSI type inhibitors can be found in Laskowski et al., "Protein Inhibitors of Proteases", Annual Review of Biochemistry. Vol. 49, pp. 593-626 (1980). Preferred SSI-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 SSI 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 Biochemistry. Vol. 116, pp. 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, pp. 911-918 (1994). Two other examples of such SSI 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 SSI-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 Bioloqical Chemistrv , Vol. 253, No. 5, pp. 1546-1555 (1978). Even another SSI 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 livldans 66 Exhibits Inhibitory Activities Toward Both Subtilisin BPN 'and Trypsin ", Journal of Biochemistry. Vol. 112, pp. 204-211 (1993). Even another SSI 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, pp. 752-761 (1994). Even another SSI 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 cacaof, Biochimica et Biophvsica Acta Vol 1207, pp. 120-125 (1994) Other SSI inhibitors are discussed in Taguchi et al., "High Frequency of SSI-Like Protease Inhibitors Among Streptomyces," Bioscience, Biotechnology, and Biochemistry, Vol. pp. 522-524 (1993), Taguchi et al., "Streptomyces Subtilisin Inhibitor-Like Proteins Are Distributed Widely in Streptomycetes," Applied and Environmental Microbioloqv, pp. 4338-4341 (December 1993), and in Suzuki et 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 SSI inhibitors are described in the art. You can use variants of the inhibitors SSI type as the progenitor 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 SSI-type inhibitor such as those described above in the present invention. Among others, all substitutions exemplified in the variants shown in the present invention can also be made at the corresponding positions in the SSI-type inhibitors to provide a sequence of progenitor amino acids. Other non-limiting examples of variants of the SSI-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 SSI-type inhibitor, variant thereof or SSI variant, using the original numbering, may not correspond to position 63 of SSI. 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 SSI. 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 variants of the present invention have a Ki value that allows the variant to inhibit almost the entire protease (preferably more than about 60%, more preferred about 99%) in the compositions for cleaning or for care personal, but allowing it to dissociate from the protease after dilution and / or during the cleaning procedure. Preferred variants have a K, from about 10"12 M to about 10" 4 M, more preferred from about 10"10 M to about 10" 6 M, and more preferably from about 10"8 M to about 10". 7 M. Of course, in the case that the dimensions of the washing machine or the concentrations of the products change it, the K2 is adjusted accordingly. The prediction of a useful range of K i 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.

Linker In addition to the protease part and the variant part, the fusion protein may optionally comprise a linker. Preferably the fusion protein comprises a linker part. The linker part is a hydrolysable linker amino acid chain which separates the protease part from the variant part, in which the protease part and the variant part are covalently linked via the linker part. The person skilled in the art can construct the linking part to achieve several different objectives. For example, the amino acid residues of the linker part could be designed to be a good substrate for hydrolysis. In addition, the amino acid sequence could be designed to facilitate the post-translational separation of the protease part and the variant part, or to optimize the position of the variant part relative to the binding site or active site of the variant. part of protease. It is preferred that the optional linker part have a length of about twenty amino acid residues. Preferably, the linking part can be easily cut by the protease part.

In the case in which the fusion protein does not comprise a linking part, the protease part and the variant part are directly linked by covalent bonds.

Other characteristics of the fusion protein Because the fusion protein is ultimately encoded by the DNA in vivo, the DNA can be used to define the sequence of the fusion protein. The DNA, which codes for the fusion protein, can be used in any number of plasmids and / or expression systems, including in vitro expression systems and in vivo expression systems such as plants, (preferably those used in biotechnology. , including tobacco, oilseed producing plants such as rapeseed, soybeans and the like, grain-producing plants 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. coli or Bacillus, and even more preferred is Bacillus. The DNA encoding the fusion protein can be incorporated into a plasmid or phage, active in the cell, or it can be incorporated directly into the genome of the organism which is used in the cloning or expression of the fusion protein 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 fusion protein could be placed on the same plasmid, phage or chromosome as well as 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 fusion protein protease. 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 fusion proteins of the present invention. In a preferred embodiment of the present invention, the fusion proteins are expressed jointly from the same expression system with one or more protease inhibitors, preferably some other protease inhibitor. Preferably, the additional protease inhibitor is a variant of the protease inhibitors selected from SSI, SSI type inhibitors, SSI variants and SSI type inhibitors. More preferred, the additional protease inhibitor is a variant that carries, independently, the same definition as that of the "variant part" discussed in the present invention, including preferred limitations. Most preferably, the additional protease inhibitor is the same variant as the variant part of the fusion protein. Accordingly, the inventors of the present invention provide expression systems comprising the DNA encoding the fusion protein and, optionally, one or more additional protease inhibitors. Preferably the expression system is a living, more preferred organism of bacterial nature. It is also contemplated that one skilled in the art could wish to prepare antibodies for the fusion proteins of the present invention. These antibodies can be prepared using known methods. For example, the fusion proteins 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 harvesting lymphocytes from the spleen peripheral blood of immunized animals and immortalizing the antibody producing cells using standard immunological test techniques. 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 labels, for example technetium 99 or 1-131, or to fluorescent labels, using standard coupling techniques. The labeled antibodies can also be used in kinetic tests to determine the K. Competitive tests, such The fusion proteins of the present invention can also comprise additional "parts" that provide a desired function such as, for example, cellulose binding domains, lipases, amylases and cellulases. 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. For brevity, 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. The variant parts (variants) can be prepared by mutating the nucleotide sequences encoding a progenitor 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 progenitor 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 phage helper VCSM13 (Kunkel et al., " Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection ", Methods in Enzymology, 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 to the 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.," Cloning 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 genes that code for the variant can be fused with a gene for protease.A standard method is to genetically manipulate the restriction sites in the appropriate place in each gene Restriction digestion can be performed, and the restriction fragments can be ligated, for example with T4 DNA ligase The binding mixture can be used to transform either E. coli or B. subtilis , depending on the nature of the plasmids. For example, a gene can be used for subtilisin carried in a plasmid that replicates both in E. coli and in B. subtilis, and that confers resistance to ampicillin to the first bacterium and resistance to kanamycin to the second bacterium. Oligonucleotide-directed mutagenesis can be used to place an EcoR \ site immediately after the DNA (3 'to) that codes for the carboxyl-terminal amino acid residue of subtilisin. A BamYW site can also be placed immediately after the DNA (3 'to) that codes for the stop codon. The gene for the inhibitor can be constructed such that there is an EcoRI site just 5 'to and adjacent to the DNA sequence encoding the N-terminal amino acid residue. further, a BamYW site can be placed after the DNA (3 'to) that codes for the stop codon. The genes for the inhibitor and subtilisin can be treated with restriction enzymes EcoRI and BamYW and subsequently treated with the T4 DNA ligase. The ligand mixture can be used to transform E. coli MM294 cells to exhibit resistance to ampicillin. Once the plasmid encoding the fusion protein is recovered from E. coli, that plasmid can be used to transform to β. Subtilis to be resistant to kanamycin. Bacillus subtilis cells containing the plasmid of interest are cultured 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% malnorm M100 (Grain Processing Corporation, Muscatine, IA), 100 mM HEPES, pH 7.5, 80 μM of MnCl2 and 50 μM of kanamycin. The cultures are incubated for 24 hours at 37 ° C. The fusion protein is secreted in the culture medium, from which it can be isolated. Any of a number of chromatographic steps can be used, including ion exchange and gel filtration chromatography.

Characterization of the fusion proteins of the present invention The fermenon supernatants containing a fusion protein of the present invention are evaluated for their protease activity and for their protease inhibition activity. SSI inhibits, inter alia, subtilisin BPN 'and a variant Y217L of subtilisin BPN'. In the control, SSI is mixed with protease and incubated for 15 minutes at room temperature. The protease activity is then measured using the method of DelMar et al., Analvtical Biochemistry. Vol. 99, pp. 316-320, (1979). A solution of 0.1 M Tris, pH 8.6, 10 mM CaCl2 is added to bring the volume to 990 μl. 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.

Culture supernatants of a fusion protein of the present invention are tested for their ability to inhibit the Y217L derivative of subtilisin BPN 'in a similar manner. Culture supernatants are also tested for their ability to produce protease. The lack of activity of significant inhibition and protease activity is consistent with a fusion protein that is prepared in which the variant part and the protease part each deny (or substantially deny) the activity of the other, such as is desired in the present invention. This interpreon can be reinforced by the results of the Western Blot analysis indicating that the fusion protein has been prepared. Because it is desired to incorporate a fusion protein of the present invention into the compositions for cleaning or for personal care, the stability in the environment of the product is also evaluated. If the protease and inhibitor activities are stable, the level of protease activity is constant over time. However, if the variant part is hydrolyzed by the protease part in the fusion protein, the protease activity will increase. The culture supernatants of the fusion protein are mixed with 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 directly compared to that of, for example, SSI in a fusion protein. The K i of a fusion protein is determined as follows. The fusion protein and 600 μg / ml of succinyl-Ala-Ala-Pro-Fe-p-nitroanilide are mixed in 990 μl of a 50 mM Tris solution, pH8. It is observed a constant speed is observed during the last ten to fifteen minutes. This velocity, compared to velocity using only the protease, is used to calculate the Ki according to Goldstein's equations "The Mechanism of Enzyme-Inhibitor-Substrate Reactions", Journal of General Physiology. 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 fusion proteins of the present invention is included in cleaning compositions useful for cleaning a variety of surfaces that require stain removal. of peptide. Such cleaning compositions include, but are not limited to, fabric cleaning compositions, hard surface cleaning compositions, light duty cleaning compositions including tableware 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. In more preferred way, such fusion protein has a part of protease, a part of variant and optionally, but preferably, a linking part. In a preferred embodiment of the present invention, the cleaning compositions of the present invention also comprise, in addition to the fusion protein, one or more additional protease inhibitors. Preferably, the additional protease inhibitor is a variant of the protease inhibitors selected from SSI, SSI-type inhibitors, SSI variants and SSL-type inhibitors. Most preferably, the additional protease inhibitor is a variant which has, independently, the same definition as for the "variant parts" discussed in the present invention, including the preferred limitations. Most preferably, the additional protease inhibitor is the same variant as the variant part of the fusion protein. In the cleaning compositions of the present invention, the preferred molar ratio of the variant to the protease (variant to protease ratio) (in which the variant part of the fusion protein and any of the additional protease inhibitors represent in collectively the molar amount of the variant), is from about 3: 1 to about 1.5: 1, and most preferred is about 2: 1. As used in the present invention, "effective amount of fusion protein" or the like, refers to the amount of fusion protein that is needed to achieve the necessary proteolytic activity in the specific cleaning composition. Such effective amounts can be readily determined by the person skilled in the art and are based on many factors, such as the particular fusion protein used, the cleaning application, the specific composition of the cleaning composition, whether a liquid or dry composition (for example granulate or stick), and the like. Preferably, the cleaning compositions comprise from about 0.0001% to about 10%, more preferably from about 0.001% to about 1% and more preferably from about 0.01% to about 0.1% of one or more of the fusion proteins of the present invention. Subsequently, several examples of various cleaning compositions in which the fusion proteins can be used are discussed in greater detail. In addition to the fusion proteins of the present invention, the present cleaning compositions also comprise a vehicle for cleaning composition comprising one or more of the materials for cleaning composition compatible wthe fusion protein. 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 (eg, liquid, granulate, stick, for spray, stick, paste, gel, etc.) whose materials are also compatible wthe fusion protein 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 and / or the proteolytic activity of the fusion protein to such an extent that the protein of Fusion 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 fusion proteins of the present invention can be used in a variety of detergent compositions in which high foam formation and good cleaning activity is desired. Thus, the fusion proteins can be used with various conventional ingredients to provide completely 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 alkylbenzene sulphonates and the primary and random alkylsulfates of conventional Cn-Cβ8, the secondary (2,3) alkyl sulfates of C ?0-C18 of the formulas CH3 (CH2) (CHOS? 3"M +) CH3 and CH3 (CH2) and (CHOS? 3" M +) CH2CH3 in which xy (y + 1) are integers of at least about 7, preferably at least about 9 and M is a cation conferring solubility in water, especially sodium, the C 1 -C 18 alkylalkoxy sulfates (especially the ethoxysulfates with EO 1-5), the C 1 -C 8 alkylalkoxycarboxylates (especially the ethoxycarboxylates with EO 1) -5), the alkyl polyglycosides of C-io-Ciß and their corresponding sulphated polyglucosides, the C 12 -C 18 fatty acid esters of α-sulfonated, alkylalkoxylates and C 12 -C 8 alkylphenolalkoxylates (especially the ethoxylates and ethoxy / mixed propoxy) , betaines and sulfobetaines (sultaines) of Ci2-C? 8, the amine oxides of C? o-C? 8 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, Connors et al., Issued March 16, 1993. A wide variety of other useful ingredients in the cleaning compositions of the present invention include, for example, other active ingredients, vehicles, hydrotropes, processing aids, colorants. or pigments and solvents for liquid formulations. If a further increase in foaming is desired, foaming enhancers such as the C 10 -Ci 6 alkanolamides can be incorporated into the compositions, typically at levels of from about 1% to about 10%. C10-C14 monoethanol- and diethanolamides illustrate a typical class of such foam enhancers. The use of such foam enhancers with high foam forming auxiliary surfactants is also advantageous. If desired, soluble magnesium salts such as MgC, MgSO4 and the like can be added, typically at levels from about 0.1% to about 2%, to provide additional foam formation. The liquid detergent compositions of the present invention may contain water and other solvents as carriers. Suitable primary and secondary low molecular weight alcohols exemplified by methanol, ethanol, propanol, e / so-propanol. Monohydric alcohols are preferred for solubilizing 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) can also be used. , ethylene glycol, glycerin, and 1-2 propanediol. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of said vehicles. The detergent compositions of the present invention will preferably be formulated such 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 in this range. Techniques for controlling pH at recommended levels of use include the use of, for example, pH, alkali and acid regulators. Such techniques are well known to those in the art. When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, 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, layered silicates, phosphates and the like. Other conventional detergency builders are listed on standard forms. Likewise, the formulator may wish to use various 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 bleach compounds, such as percarbonates, perborates and the like, can be used in such compositions, typically at levels of 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. Usage levels typically range from about 1% to about 10% by weight. In said compositions, dirt-releasing agents may be used, especially those of the anionic oligoester type, chelating agents, especially the aminophosphonates and ethylenediamindisuccinates, clay soil removal agents, especially ethoxylated tetraethylenepentamine, dispersing agents, especially polyacrylates and polyaspartates, brighteners, especially anionic brighteners, suds suppressors, especially silicones and secondary alcohols, fabric softeners, especially smectite clays, and the like, all 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 cleaning composition materials include clay soiling removal agents, dispersing agents, brightening agents, suds suppressors and fabric softeners. The fusion proteins of the present invention are useful in compositions for cleaning hard surfaces. As used in the present invention "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 hydrosoluble sequestering detergent builder. However, in certain specialized products such as aerosol window cleaners, surfactants are sometimes not used since they can produce a film and / or fluted residue on the surface of the glass. The surfactant component, when present, may constitute as low as 0.1% of the compositions of the present invention, but typically the compositions will contain from about 0.25% to about 10%, preferably about 1% up to about 5% surfactant. Typically the compositions will contain about 0. 5% to about 50% of a detergent builder, preferably from about 1% to about 10%.

Preferably, the pH should be in the range of about 7 to about 12. Conventional agents can be used to adjust the pH, such as sodium hydroxide, sodium carbonate or hydrochloric acid if 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-hexanediol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, preferably from about 3% to about 11%. Additionally, highly volatile solvents such as / so-propanol or ethanol can be used in the compositions of the present invention to facilitate the composition to evaporate from surfaces faster, when the surface is not rinsed after applying the composition " to total concentration "to the surface. When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions. The variants of the present invention are also useful to be included in the cleaning compositions described in the following documents: provisional patent application E.U.A. Serial No. 60 / 079,477Í Rubingh et al., Filed March 26, 1998; provisional patent application E.U.A. Serial No. 60 / 079,397, Rubingh et al., Filed March 26, 1998; patent application E.U.A. Serial No. 09 / 048,174, Weisgerber et al., Filed March 26, 1998; and patent application E.U.A. Serial No. 09/088912, which claims priority to patent application E.U.A. Serial 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 LEGIBLE COMPOSITIONS FOR CLEANING HARD SURFACES All the formulas are adjusted to pH 7. In Examples 1-6, the variants mentioned in Tables 7, 8 and 10, and the preferred variants mentioned in the present invention, among others, are replaced by the protein of previous fusion, with substantially similar results.

EXAMPLES 7-10 Dishwashing Liquid 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 substituted by the above fusion protein, with substantially similar results.

EXAMPLES 11-13 Lightweight 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 replaced by the above fusion protein, with substantially similar results.

Compositions for personal care The fusion proteins of the present invention are also suitable for use in personal care compositions selected from, for example, hair conditioners that are left in and rinsed, shampoos, acne compositions that they are left on the skin and are rinsed, facial creams and conditioners, bath gels, soaps, facial cleansers that make and foam and without foam, cosmetics, lotions and moisturizers for hands, face and body, facial moisturizers that are left in the skin, cosmetic and cleaning towels, oral care compositions, and contact lens care compositions. The compositions of the present invention for personal care comprise one or more fusion proteins of the present invention and a vehicle for personal care. The fusion proteins of the present invention, including the preferred limitations, are described in the present invention with respect to cleaning compositions. Preferably, such a fusion protein has a part of protease, a part of variant, and in optional form, but preferred, a linking part. In a preferred embodiment of the present invention, the personal care compositions of the present invention also comprise, in addition to the fusion protein, one or more additional protease inhibitors. Preferably, the additional protease inhibitor is a variant of the protease inhibitors selected from SSI, SSI type inhibitors, SSI variants and SSI type inhibitors. More preferred, the additional protease inhibitor is a variant that carries, independently, the same definition as that of the "variant part" discussed in the present invention, including preferred limitations. Most preferably, the additional protease inhibitor is the same variant as the variant part of the fusion protein. In the personal care compositions of the present invention, the preferred molar ratio of variant to protease (variant to protease ratio) (in which the variant portion of the fusion protein and any of the additional protease inhibitors represent collective, the molar amount of the variant) is from about 3: 1 to about 1: 1, more preferably from about 3: 1 to about 1.5: 1, and more preferably 2: 1. To polish them, The fusion proteins of the present invention are suitable to be included in the compositions described in the following references: EUA Patent No. 5,641, 479, Linares et al., Issued June 24, 1997 (cleansers for the skin); Patent E.U.A. No. 5,599,549, Wivell et al .; Issued on February 4, 1997; (cleansers for the skin); Patent E.U.A. No. 5,585,104; Ha et al .; issued December 17, 1996; (skin cleansers); Patent E.U.A. No. 5,540,852; Kefauver et al .; Issued on July 30, 1996; (cleansers for the skin); Patent E.U.A. No. 5,510,050; Dunbar et al .; Issued on April 23, 1996; (cleansers for the skin); Patent E.U.A. No. 5,612,324, Guang Ling et al .; Issued on March 18, 1997; (anti-acne preparations); Patent E.U.A. No. 5,587,176; Warren et al .; Issued on December 24, 1996; (anti-acne preparations); Patent E.U.A. No. 5,549,888; Venkateswaran Issued on August 27, 1996; (anti-acne preparations); Patent E.U.A. No. 5,470,884; Corless et al .; Issued on November 28, 1995; (anti-acne preparations); Patent E.U.A. No. 5,650,384; Gordon et al .; issued on July 22, 1997; (gels for bath); Patent E.U.A. No. 5,607,678; Moore et al .; Issued on March 4, 1997; (gels for bath); Patent E.U.A. No. 5,624,666; Coffindaffer et al .; issued on April 29, 1997; (conditioners and / or shampoos for hair); Patent E.U.A. No. 5,618,524; Bolich et al .: issued on April 8, 1997; (conditioners and / or shampoos for hair); Patent E.U.A. No. 5,573,709; Wells, issued November 12, 1996; (conditioners and / or shampoos for hair); Patent E.U.A. No. 5,482,703; Pines: issued on January 9, 1996; (conditioners and / or shampoos for hair); Patent E.U.A. No. Re. 34,584; Grote et al .; reissued on April 12, 1994; (conditioners and / or shampoos for hair); Patent E.U.A. No.5,641, 493; Date et al .; Issued on June 24, 1997; (cosmetics); Patent E.U.A. No. 5,605,894; Blank et al .; Issued on February 25, 1997; (cosmetics); Patent E.U.A. No. 5,585,090, Yoshioka et al .; Issued on Dber 17, 1996; (cosmetics); Patent E.U.A. No. 4,939,179; Chenev et al .: issued July 3, 1990; (lotions for hands, face and / or body); Patent E.U.A. No. 5,607,908; Mc Atee et al .: issued March 4, 1997; (lotions for hands, face and / or body); Patent E.U.A. No. 4, 045,364; Richter et al .; Issued on August 30, 1977; (cosmetic and cleaning towels); European Patent Application EP 0 619 074, Touchet et al .: published on October 12, 1994; (cosmetic and cleaning towels); Patent E.U.A. Do not. 4,975,217; Brown-Skrobot et al .: issued December 4, 1990; (cosmetic and cleaning towels); Patent E.U.A. No. 5,096,700; Seibel Issued on March 17, 1992; (compositions for oral cleansing); Patent E.U.A. No. 5,028,414, Sampathkumar. Issued on July 2, 1991; (compositions for oral cleansing); Patent E.U.A. No. 5,028,415, Benedict et al .; Issued on July 2, 1991; (compositions for oral cleansing); Patent E.U.A. No. 4,863,627; Davies et al .; September 5, 1989; (cleaning solutions for contact lenses); Patent E.U.A. No. Re 32,672; Huth et al .: reissued on May 24, 1988; (cleaning solutions for contact lenses); and Patent E.U.A. No. 4,609,493, Schaffer; issued September 2, 1986 (cleaning solutions for contact lenses). The fusion proteins of the present invention are also useful for inclusion in the personal care compositions described in the following documents: U.S. Patent Application. Provisional, Series No., 60 / 079,475; Rubingh et al: filed on March 26, 1998; patent application E.U. Provisional Series No. 60 / 079,397; Rubingh et al; filed on March 26, 1998; patent application E.U. Provisional Series No. 09 / 048,174; Weisgerber 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, Weisgerber et al; filed June 2, 1998. To further illustrate the oral cleansing compositions of the present invention, one or more fusion proteins of the present invention are included in compositions useful for removing protein spots from teeth or dentures. As used in the present invention, "oral cleansing compositions" refer to dentifrices, toothpastes, dental gels, dental powders, mouth rinses, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels , pastes for prophylaxis, solutions for dental treatments and the like. 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 the references cited above in the present invention. 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 of the fusion proteins of the present invention 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, e.g., U.S. Patent No. 5,055,305, Young). and are generally suitable for the incorporation of one or more fusion proteins 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 of the fusion proteins and a personal care vehicle. 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 fusion proteins of the present invention to remove protein spots. of contact lenses.

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 substituted by the above fusion protein, 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, among others, are substituted by the above fusion protein with substantially similar results.

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 substituted by the above fusion protein, with substantially similar results.

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 the above fusion protein with substantially similar results.

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 we 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 the above fusion protein with substantially similar results.

LIST OF SEQUENCES < 110 > Saunders, Charles W. < 120 > Proteases fused with subtilisin inhibitor variants of Streptomyces < 130 > Proteases fused with variants < 140 > < 141 > < 150 60/091, 904 < 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 1 5 10 15 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 35 40 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 > 1 17 < 212 > PRT < 213 > Streptomyces albogriseolus < 400 > 2 Wing Gly Glu Phe Asp Wing Pro Being Wing Leu Tyr Wing Pro being Wing Leu 1 5 10 15 Val Leu Thr Val Gly Lys Gly Val Ser Ala Thr Thr Ala Ala Pro Glu 25 30 Arg Ala Val Thr Leu Thr Cys Ala Pro Gly Pro Ser Gly Thr His Pro 35 40 45 Ala Ala Gly Ser 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 65 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 < 21 1 > 275 < 212 > PRT < 213 > Bacillus amyloliquefaciens < 400 > 3 'Ala Gln Ser Val Pro Tyr Gly Val Ser Glp 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 20 25 30 Ser Gly He Asp Ser Ser His Pro Asp Leu Lys Val Wing Ala Gly Gly Wing 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Wing Gly Thr Val Wing 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 Being Gly Gln Tyr Being Trp He He Asn Gly He Glu 100 105 110 Trp Wing Wing Wing Asn Asn Met Asp Val He Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Wing Wing Leu Lys Wing Wing Val Asp Lys Wing Val Wing 130 13S 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 Wing 165 170 175 Val Gly Ala Val Asp Ser As Asn Gln Arg Ala 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 Gly Wing Wing Ala Leu He Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 2S5 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu He Asn Val Gln Wing 260 265 270 Ala Ala i Glp 275 < 210 > 4 < 211 > 107 < 212 > PRT < 213 > Streptomyces thermotolerans < 400 > 4 Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly Glu Ser Ala 1 5 10 15 He Ala Ala Thr Pro Glu Arg Ala Val Thr Leu Thr Cys Ala Pro Lys 20 25 30 Wing Wing Gly Thr His Pro Wing Wing Gly Wing Wing Cys Wing Wing Glu Leu Arg 35 40 45 Gly Val Gly Gly Asp Phe Asp Wing Leu Thr Wing Arg Asp Gly Val Met 50 55 60 Cys Thr Lys Gln Tyr Asp Pro Val Val Val Thr 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 is Be Ala Ala Ala Ala Ser Ala Ala Ala Ala Thu Leu Asn Cys Ala 20 25 30 Pro Be Ala Be Gly Thr His Pro Ala Pro Ala Ala Ala Ala Ala Glu 35 40 45 Leu Arg Ala Ala Gly Gly Asp Leu Asp Ala Leu Ala Gly Pro Wing Asp 50 55 SO Thr Val Cys Thr Lys Gln Tyr Wing Pro Val Val He Thr Val Asp Gly 55 70 75 80 Val Trp Gln Gly Lys Arg Val, Ser Tyr Glu Arg Thr Phe Wing Asn Gly 85 90 95 Cys Val Lys Asn Wing Ser Gly Ser Ser Val Phe Wing Phe 100 105 < 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 Thr Pro Wing Glu Arg Wing Val Thr Leu Thr Cys Wing Pro Arg 20 25 30 Pro Ser Gly Thr His Pro Val Wing Gly Wing Wing Cys Wing Glu Leu Arg 35 40 45 Gly Val Gly Gly Asp Val His Wing Leu Thr Wing Thr Asp Gly Val Met 50 55 60 Cys Thr Lys Gln Tyr Asp Pro Val Val Val 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 15 Be Ala Ala Thr Ala Ala Pro Leu Arg Ala Val Thr Leu Thr Cys Ala 20 25 30 Pro Thr Wing Ser Gly Thr His Pro Wing Wing Wing Wing Wing Cys Wing Glu 35 40 45 Leu Arg Wing Wing His Gly Asp Pro Wing Wing Leu Wing Wing Glu Asp Ser 50 55 60 Val Met Cys Thr Arg Glu Tyr Wing Pro Val Val Val Thr Val Asp Gly 6S 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 Alaf Ser Leu Tyr Ala Pro Ser Ala Leu Val Le? ~ Thí7va? Gly His Gly 1 5 10 15 Thr Ser Ala Ala Ala Ala Thr Pro Leu Arg Ala Val Thr Leu Asn Cys 20 25 30 Wing Pro Thr Wing Being Gly Thr His Pro Wing Pro Wing Leu Wing Cys Wing 35 40 45 Asp Leu Arg Gly Val Gly Gly Asp He Asp Wing Leu Lys Wing Arg Asp 50 ss so Gly Val He Cys Asn Lys Leu Tyr Asp 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 100 105 110 < 210 > 9 < 211 > 113 < 212 > PRT < 213 > Streptomyces parvulus < 400 > 9 Thr Ala Pro Ala Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr He 1 5 10 15 Gly Gln Gly Glu Be Wing Wing Wing Thr Ser Pro Leu Arg Wing Val Thr 20 25 30 Leu Thr Cys Ala Pro Lys Ala 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 Met 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 Ala Asn Glu Cys Val Lys Asn Ala Gly Ser Ala Ser Val Phe Thr 100 105 110 Phe < 210 > 10 < 211 > 107 < 212 > PRT < 213 > Streptomyces coelicolor < 400 > 10 Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly Glu Ser 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 Asp Ala Ala Cys Ala Glu Leu Arg 35 40 45 - | Q Ala Ala His Gly Asp Pro Be Wing Leu Wing Wing Asp Asp Wing Val Met 50 55 60 Cys Thr Arg Glu Tyr Wing 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 Val Phe Thr Phe 100 105 < 210 > 11 < 211 > 116 < 212 > PRT < 213 > Streptomyces lavendulae < 400 > 11 Ala Pro Asp Ala Ala Pro Ala Ser Leu Tyr Ala Pro Ser Ala 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 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 Ala Phe 115 < 210 > 12 < 211 > 109 < 212 > PRT < 213 > Streptomyces antifibrinolyticus < 400 > 12 Gly Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Met Gly His Gly Asn 1 5 10 15 Be Ala Ala Thr Val Asn Pro Glu Arg Ala Val Thr Leu Asn Cys Ala 20 25 30 Pro Thr Wing Ser Gly Thr His Pro Wing Wing Ala Leu Gln Wing Cys Wing Glu 35 40 45 Leu Arg Gly Wing Gly Gly Asp Phe Asp Wing Leu Thr Val Arg Gly Asp 50 55 60 Val Wing Cy3 Thr Lys Glp Phe Asp Pro Val Val Val Thr Val Asp Gly 65 70 75 80 Val Trp Gln Gly Lys Arg Val Ser Tyr Glu Arg Thr Phe Wing Asn Glu 85 90 95 Cys Val Lys Asn Ser Tyr Gly Met Thr Val Phe Thr Phe 100 105 < 210 > 13 < 211 > 107 < 212 > PRT < 213 > Streptomyces lividans < 400 > 13 Tyr Ala Pro Be Wing Read Val Leu Thr Val Gly His Gly Glu Be Ala 1 5 10 1S Ala Thr Ala Ala Pro Leu Arg Ala Val Thr L * eu Thr Cys Ala Pro Thr 20 25 30 Ala Ser Gly Thr His Pro Ala Ala Ala Ala Ala Cys Ala Glu Leu Arg 35 40 45 H Ala Ala 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 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 Val Phe Thr Phe 100 105 < 210 > 14 < 211 > 110 < 212 > PRT < 213 > Streptomyces cinnamoneum < 400 > 14 Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr He Gly Gln Gly Asp 1 5 10 15 Be Ala Ala Ala Ala Gly He Gln Arg Ala Val Thr Leu Thr Cys Met 20 25 30 Pro Lys Wing Asp Gly Thr His Pro Asn Thr Arg 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 75 80 Gly Val Trp Glu Gly Arg Arg Val Ser Phe Glu Arg Thr Phe Ala Asn 85 90 95 Pro Cys Glu Leu Lys Wing Gly Lys Gly Thr Val Phe Glu Phe 100 105 110 < 210 > 15 < 211 > 110 < 212 > PRT < 213 > Streptomyces cacaoi Be Wing Asp Wing Pro Wing * Gln Arg Wing Val Thr Leu Arg Cys Leu Pro 25 30 Val Gly Gly Asp His Pro Pro Wing Glu Lys Wing Cys Ala Wing Leu Arg 40 45 Glu Wing Gly Gly Asp Pro Wing Wing Leu Pro Arg Tyr Val Glu Asp Thr 50 55 60 Gly Arg Val Cys Thr Arg Glu Tyr Arg Pro Val Thr Val Ser Val 61a _65 70 75 ~ 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 ios no

Claims (10)

NOVELTY OF THE INVENTION CLAIMS

1. - A fusion protein characterized by: (a) a part of protease; and (b) a variant part, in which the variant part has a modified amino acid sequence of a progenitor amino acid sequence, in which the modified amino acid sequence is characterized by an amino acid substitution at position 63 corresponding to SSI , and in which the progenitor amino acid sequence is selected from the group consisting of SSI, SSI type inhibitors, SSI variants, and SSI type inhibitor variants.

2. A fusion protein according to claim 1, further characterized by a linker part in which the protease part and the variant part are linked covalently by the linking part.

3. A fusion protein according to any of the preceding claims, further characterized in that the substitution of the amino acid at position 63 corresponding to SSI is with solucin.

4. A fusion protein according to any of the preceding claims, further characterized in that the progenitor amino acid sequence is selected from the group consisting of SSI and SSI variants.

5. - A fusion protein according to any of the preceding claims further characterized in that it has a Ki, such that the variant part: a) inhibits a protease in a composition comprising the fusion protein; and b) dissociates from the protease part after dilution.

6. The fusion protein according to any of the preceding claims, further characterized in that the variant part is 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 + D83C; (g) L63I + M70Q + M73P + V74W + D83C; (h) L63I + M70Q + M73P + D83C + S98A; (i) L63I + G47D + M73P + V74F + D83C; 0) 63I + G47D + M73P + V74W + D83C; (k) L63I + G47D + M73P + D83C + S98A; (I) L63I + G47D + M70Q + M73P + V74F + D83C; (m) L63I + G47D + M70Q + M73P + V74F + 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; (ce) 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 + 73P + V74W + D83C; (hh) A62 * + L63I + M70Q + M73P + D83C + S98A; (ii) A62 * + L63I + G47D + M73P + V74F + D83C; Gj) A62 * + L63I + G47D + M73P + V74W + D83C; (kk) A62 * + L63I + G47D + M73P + D83C + S98A; (II) 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; (ccc) 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) L63l + G47D + M73P + D83C + S98D; (iii) L63I + G47D + M73P + D83C + S98E; (jjj) L63I + M73P.

7. The DNA coding for a fusion protein according to any of the preceding claims.

8. A composition comprising a fusion protein according to any of the preceding claims and a vehicle selected from the group consisting of a vehicle for cleaning composition and a vehicle for composition for personal care.

9. A composition according to claim 8, further characterized in that it also comprises a protease inhibitor.

10. An expression system comprising the DNA according to claim 7.