CN1317052A - Proteases fused with variants of streptomyces subtilisin proteases 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

The proteolytic enzyme that merges mutually with the variant of i (streptomyces) subtilisin inhibitor

The cross reference of related application

The application requires to declare in the United States Patent (USP) provisional application No.60/091 on July 7th, 1998, the rights and interests in 904.

Invention field

The present invention relates to following proteic fusion rotein: the variant of (1) proteolytic enzyme and (2) i (streptomyces) subtilisin inhibitor (SSI) and with SSI tool homology inhibitor (SSI sample inhibitor).These fusion roteins can be used for cleaning compositions and Personal hygiene composition.The invention still further relates to the cleaning compositions that contains fusion rotein of the present invention and the gene of Personal hygiene composition and this fusion rotein of encoding.

Background of invention

Enzyme has been formed the maximum naturally occurring albumen of a class.Class in the enzyme comprises other proteoclastic proteolytic enzyme of catalysis.The ability of this protolysate is used for albumen naturally occurring and that engineering produces is introduced cleaning compositions, especially is relevant to the cleaning compositions that clothes washing is used.In addition, although trial is few, other people also introduce the Personal hygiene composition with these proteolytic enzyme.Yet, during the expression of the storage of these compositions even proteolytic enzyme, these proteolytic enzyme often by self degraded or may degradation composition in other enzyme of existing.As the consequence of degraded, these cleanings and Personal hygiene composition are reaching limited in one's ability on the reinforced effects of expection.

Therefore, the inhibitor that adds protease activity to said composition will be useful with self hydrolysis of limit protein enzyme with to the degraded of other enzyme.The reversible proteinase inhibitor is provided, so that be used for cleaning or at cleaning ambient and when diluted at composition, proteolytic enzyme no longer is suppressed and can the protolysate spot, and this is very valuable.In addition, these inhibitor must enough be stablized to give full play to its inhibit feature.

People in this area are to experimentizing the stable protein proteinase inhibitor of enzyme in the cleaning compositions.Nature provides the protein proteinase inhibitor with modulin enzyme in vivo.But because these naturally occurring protein proteinase inhibitor tend to instability, their commercial uses in cleaning that contains proteolytic enzyme and Personal hygiene carrier vector have certain restriction.

The protein proteinase inhibitor typically is long peptide chain, and it can and suppress its activity with the combination of protease activities site.Homology according to the one-level aminoacid sequence, these inhibitor range usually several families (family's I is to family's IX) (see Laskowski etc., " ProteinInhibitors of Proteinases ", Annual Review of Biochemistry, Vol.49, pp.593-626 (1980)).These inhibitor comprise the inhibitor that is commonly referred to family's VI and the inhibitor of family's III, and family's VI inhibitor comprises eglin and barley chymotrypsin inhibitor, and family's III inhibitor comprises streptomyces subtilisin inhibitor (SSI) and plasminostretin.

These inhibitor tend in conjunction with specific proteolytic enzyme, and to a little less than other proteolytic enzyme combination.So just easily consider inhibitor at a specific proteases.For this reason, ability is discussed " proteolytic enzyme/peptide inhibitor to " usually.The right example of known protein enzyme/peptide inhibitor is subtilisin BPN '/SSI.Example is seen Mitsui etc., " CrystalStructure 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 etc., " Crystal Structure at 1.6 Resolution of theComplex of Subtilisin BPN ' with Streptomyces SubtilisinInhibitor ", Journal of Molecular Biology, Vol.178, pp.389-413 (1984).

SSI is stable under the situation that Subtilisin BPN ' exists, as long as the amount of this inhibitor is enough to the protease activity that suppresses all.But the inhibitor that it is believed that tool high protein enzyme affinity can not dissociate with it when diluted in wash environment.See WO 92/03529, Mikkelson etc., be appointed as Novo Nordisk A/S, on March 5th, 1992 published.

But if the binding constant of an inhibitor (Ki) allows some the enzyme tool activity in containing the right cleaning compositions of enzyme/inhibitor, inhibitor may be hydrolyzed with the enzyme in the composition.Therefore, find that can to keep suitable stable SSI variant or other inhibitor under the situation that proteolytic enzyme and cleaning and Personal hygiene composition exist be very valuable.In addition, these inhibitor preferably have preferred Ki for the specific proteases that need suppress.This Ki must allow composition lay up period in the end that wherein protease-producing is suppressed.But when diluted, this proteolytic enzyme and inhibitor must dissociate and not be subjected to the activity of arrestin enzyme with generation in wash environment for these cleanings and Personal hygiene composition.

But, the stability existing problems of these proteinase inhibitor.WO 98/13387, and Correa et al. is appointed as The Procter﹠amp; Gamble Co., on April 2nd, 1992 was published (corresponding to U.S. Patent Application Serial Number No.60/026,944), had disclosed the variant that enhanced stability can be provided.

And including the proteolytic enzyme that is used to clean with the Personal hygiene composition has himself distinctive problem at interior proteolytic enzyme finished product.For example, proteolytic enzyme production may be subjected to fermenting and purge process in from the restriction of hydrolysis.Unfortunately, in fermented liquid or purified mixture, add proteinase inhibitor and need buy and add excessive inhibitor.Because the hydrolysis of proteolytic enzyme may take place before adding inhibitor, being added with of inhibitor that therefore should the stage may be late.And in fact the interpolation of inhibitor may reduce the output of proteolytic enzyme.

As the example that adds at fermentation step, be appointed as Nagase﹠amp; Co., be published in the German Patent 2,131,451 on December 30th, 1971 and disclosed the process that is used to produce Sumizyme MP.Described according to it, this process need adds water miscible borate with as inhibitor.These borates filter the active output that has also therefore improved proteolytic enzyme according to its described enhancing.Yet, it is found that in fact the borate of certain level can postpone the production of enzyme.

Joergensen et al.; WO 93/13125; be appointed as Novo Nordisk A/S; be published on July 8th, 1993; disclosed a kind of process of in the fluid production medium, producing " susceptible inactivating protein "; this process prevents it at the production phase inactivation by " continuously and can protect " albumen with replying, and releasing is to proteic protection, and the recovery protein product is finished.This process is disclosed as and can be used for by making albumen can recover the protein yield that the ground inactivation obtains to improve.Yet this process may need to add the material of external source, and this material may be than costliness and inefficiency, and may need further interpolation process and this process of making restive.

Saunders et al., WO 98/13483, is appointed as The Procter﹠amp; GambleCo., be published on April 2nd, 1998, the needs by providing fusion rotein in vivo proteolytic enzyme to be suppressed have been provided.Hartman et al., WO 97/15670, is appointed as ArrisPharmaceutical Corp., is published on May 1st, 1997, mentioned the use of fusion rotein.These fusion roteins are by providing inhibitor/proteolytic enzyme to having practical value, and its cost savings and output improve.By producing stoichiometric inhibitor together with proteolytic enzyme, early stage degraded certainly can be lowered or eliminate the protein production stage.But proteinase inhibitor itself must enough be stablized to reach this purpose.

Being surprised to find SSI inhibitor, SSI sample inhibitor and their variant is being hydrolyzed corresponding to 63 of SSI and 64 places.Therefore, the present inventor provides inhibitor/proteolytic enzyme fusion rotein, and its inhibitor is SSI, SSI inhibitor and SSI sample inhibitor variant, and they are especially modified by an amino-acid residue replacement on 63 through modifying.This modification makes proteinase inhibitor obtain enhanced stability.The present inventor incorporates fusion rotein at this with these variants, has overcome the problem of proteolytic enzyme vivo degradation mentioned above with this.Therefore the present invention provides fusion rotein, and it contains higher proteolysis stability of tool and the proteolytic enzyme affinity of comparing low with parent's inhibitor, this can assist reduce proteolytic enzyme from hydrolysis.

Brief summary of the invention

Fusion rotein provided by the invention comprises:

(a) a proteolytic enzyme part;

(b) variant part, its variant part has the modified amino acid sequence of parent's aminoacid sequence, this modified amino acid sequence is replaced having monoamino-acid corresponding to 63 of SSI, and its parent's aminoacid sequence is selected from the colony that is made up of SSI, SSI sample inhibitor, SSI variant and SSI sample inhibitor variant; Fusion rotein also can according to circumstances contain

(c) a junction branch, the proteolytic enzyme part can link to each other by the connection portion covalency with variant part when the connection portion exists.

Preferred proteolytic enzyme partly comprises with SSI being the proteolytic enzyme of inhibitor.Such proteolytic enzyme for example comprises, originate from Bacillus alcalophilus, Bacillusamyloliquefaciens, Bacillus amylosaccharius, Bacilluslicheniformis, the proteolytic enzyme of Bacillus lentus and Bacillus subtilis.The invention still further relates to the gene and the cleaning compositions and the Personal hygiene composition that contain these fusion roteins of these fusion roteins of coding.

Detailed Description Of The Invention

At this basal component of the present invention is described.Here also comprise that plurality of optional is selected and non restrictive description preferred ingredients, they are useful on embodiment of the present invention.

The present invention can comprise any essential as described herein or selectable component, batching and/or restriction, or is formed or be made up of them substantially by them.

Unless indicate especially, all per-cent and ratio are all calculated by weight.Unless indicate especially, all per-cent all calculates on the basis of all compositions.

Be the trade(brand)name of material as mentioned herein, material comprises proteolytic enzyme and selectable component, but is not limited thereto.Do not wish materials limitations in material this present inventor titled with specific commodity.The material (for example) suitable with the material of quoting trade(brand)name can substitute the composition that is used in this paper.

All components, batching or composition levels all refer to the activity level of these components, batching or composition, do not comprise impurity, and as residual solvent or byproduct, they all may exist at merchandise resources.

At these all documents of quoting, comprise whole patents, patent application and printed publication, all be incorporated herein by reference in full.

Abbreviation used herein will be used as description amino acid.Table 1 provides the tabulation of abbreviation used herein.

Table 1

Amino acid	The trigram abbreviation	The single-letter abbreviation
			L-Ala	??????ala	???????A
Arginine	??????Arg	???????R
			L-asparagine	??????Asn	???????N
Aspartic acid	??????Asp	???????D
			Halfcystine	??????Cys	???????C
Glutamine	??????Gln	???????Q
			L-glutamic acid	??????Glu	???????E
Glycine	??????Gly	???????G
			Histidine	??????His	???????H
Isoleucine	??????Ile	???????I
			Leucine	??????Leu	???????L
Methionin	??????Lys	???????K
			Methionine(Met)	??????Met	???????M
Phenylalanine	??????Phe	???????F
			Proline(Pro)	??????Pro	???????P
Serine	??????Ser	???????S
			Threonine	??????Thr	???????T
Tryptophane	??????Trp	???????W
			Tyrosine	??????Tyr	???????Y
Xie Ansuan	??????Val	???????V

Definition

Term used herein " fusion rotein " has the meaning of its this area regulation, that is, two albumen are expressed on same amino acid chain, expresses typically under the control of a controlling element.For example, fusion rotein is used (example is seen Sambrooket al., Molecular Cloning:A Laboratory Manual, 2nd Ed., ColdSpring Harbor Press (1989)) recent years in numerous application.Business-like expression vector has been arranged recently to be used for producing target protein by integration technology.Discussion about the fusion rotein that contains SSI variant and proteolytic enzyme is also shown in the al. in Saunders et, U.S. Patent application No.60/026, and 947, it is corresponding to Saunders et al., and WO 98/13483, is appointed as TheProcter﹠amp; Gamble Co. is published on April 2nd, 1998.

Term used herein " sudden change " refers to the variation on the aminoacid sequence that gene order and these gene orders produced.Sudden change can be deletion, replacement or the interpolation of wild-type or parental array upper amino acid residue.

Term used herein " parent " refers at the wild-type of replacing corresponding to no amino acid on 63 of SSI or misfolded proteins enzyme, proteinase inhibitor, albumen or polypeptide (that is, the amino acid on 63 replace be abiogenous).One of these parents' example is known i (streptomyces) subtilisin inhibitor (SSI) (SEQ ID NO:1).SSI is at Ikenaka etc., " Amino Acid Sequence of an alkaline ProteinaseInhibitor (Streptomyces Subtilisin Inhibitor) fromStreptomyces albogriseoulus S-3253 ", Journal of Biochemistry, Vol.76, pp.1191-1209 has further description in (1974).Amino acid used herein numbering is from Ikenaka etc..The present inventor has also used a synthetic SSI gene that is designed to be rich in adenine and thymine, as the DNA of B.subtilis.By the construction process of expression plasmid, this synthetic gene is at four amino-acid residues of the extra coding of the N-terminal of polypeptide.This modified aminoacid sequence that comprises these four extra amino acid is represented with SEQ ID NO:2.

Term used herein " wild-type " refers to protein or the polypeptide by the organism generation of not sudden change, refers in particular to proteolytic enzyme or proteinase inhibitor at this.

Term used herein " variant " refers to albumen or polypeptide, refers in particular to proteinase inhibitor or proteolytic enzyme at this, and they have the aminoacid sequence that is different from parent protease inhibitor or parent protease respectively.

Fusion rotein of the present invention

The fusion rotein that the present inventor finds comprises: (a) proteolytic enzyme part (for easy, this paper also is called proteolytic enzyme); (b) variant part (be easy, this paper also is called variant); And (c) a junction branch that can comprise according to circumstances, the proteolytic enzyme part can link to each other by the connection portion covalency with variant part when the connection portion exists.Variant has the modified amino acid sequence of parent's aminoacid sequence, this modified amino acid sequence is replaced having monoamino-acid corresponding to 63 of SSI, and its parent's aminoacid sequence is selected from the colony that is made up of SSI, SSI sample inhibitor, SSI variant and SSI sample inhibitor variant.Fusion rotein of the present invention has utility value, particularly because the stability of its variant.

Do not attempt bound by theoryly, this fusion rotein minimizes or has avoided in the defective that may exist when being higher than other proteic speed expressing protein.This fusion rotein can allow the proteolytic enzyme and the inhibitor (variant) of synthetic specific molar weight simultaneously.For example, can construction of fusion protein double proteolytic enzyme and other analogues with inhibitor (variant) that equimolar amount is provided and proteolytic enzyme or variant molar weight.So just may in as far as possible early, prevent proteolytic enzyme from hydrolysis, for example, after just being translated in vivo.Fusion rotein can contain one or more identical or different proteolytic enzyme parts, as long as fusion rotein contains a proteolytic enzyme part and a variant part at least.Most preferred fusion rotein contains proteolytic enzyme part, a variant part, and one can be selected for use according to circumstances but is preferred connection portion.

Other proteolytic enzyme and/or inhibitor also are taken into account outside the fusion rotein, they are produced by same cell, they can with the same plasmid of antigen-4 fusion protein gene on, or coexisting as on the different plasmids of cell with it, or they are on a plasmid when antigen-4 fusion protein gene is on karyomit(e), and perhaps they are present in the cell chromosome.In addition, can produce a kind of proteolytic enzyme, it is the inhibitor that is specific to another kind of proteolytic enzyme, perhaps produces a kind of proteolytic enzyme, and it is the inhibitor that is specific to the proteolytic enzyme in this fusion rotein.And fusion rotein can be with one or more extra inhibitor coexpressions, and this inhibitor can be same with the variant part that comprises fusion rotein.The proteolytic enzyme part

As mentioned above, the present invention relates to the fusion rotein that contains proteolytic enzyme part (proteolytic enzyme).Therefore, the basal component of this fusion rotein is a proteolytic enzyme, and it can be suppressed by variant part of the present invention (variant).Proteolytic enzyme can be from animal, plant or microorganism, and wherein microorganism is comparatively preferred.Preferred proteolytic enzyme comprises that inhibitor is the proteolytic enzyme of SSI.These proteolytic enzyme comprise and originate from Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharius, Bacillus licheniformis, the proteolytic enzyme of Bacilluslentus and Bacillus subtilis.Preferably comprise subtilisin BPN, subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, Proteinase K and thermitase in these proteolytic enzyme, it comprises A/S Alcalase ^(Novo Industries, Copenhagen, Denmark), Esperase ^(Novo Industries), Savinase ^(Novo Industries), Maxatase ^(Gist-Brocades, Delft, Netherlands), Maxacal ^(Gist-Brocades), Maxapem 15 ^(Gist-Brocades) and variant above.Preferred proteolytic enzyme used herein comprises proteolytic enzyme and the variant thereof that derives from Bacillus amyloliquefaciens.Most preferred wild-type protease is subtilisin BPN '.

This paper is referred to as the variant useful as protease of the subtilisin BPN ' of " proteolytic enzyme monoid A " later on, they are disclosed in U.S. Patent number No.5,030,378, Venegas, on July 9th, 1991, it is characterized by the following subtilisin BPN ' aminoacid sequence (sequence is represented by SEQ ID NO:3) that suddenlys change of tool.

(a) 166 Gly is Asn, Ser, and Lys, Arg, His, Gln, Ala or Glu substitute; 169 Gly is Ser, substitute; 222 Met is Gln, Phe, His, Asn, Glu, Ala or Thr; Perhaps

(b) 160 Gly is substituted by Ala, and 222 Met is substituted by Ala.

This paper is referred to as other variants useful as protease in this article of the sublisin BPN ' of " proteolytic enzyme monoid B " later on, they are disclosed in european patent number EP-B-251,446, be appointed as Genencor International, Inc., deliver on January 7th, 1988, on December 28th, 1994 obtained the authorization, and it is characterized by the wild sublisin BPN ' aminoacid sequence in the sudden change of one or more following sites tool:

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, Tyr214, Gly215, and Ser221; Or tool sudden change on following two or more Sites Combination,

Asp32,Ser33,Tyr104,Ala152,Asn155,Glu156,Gly166,Gly169,Phe189,Tyr217,and?Met222.

Other in this article the sublisin BPN ' variant of useful as protease be referred to as " proteolytic enzyme monoid C " hereinafter, WO 95/10615 is seen in its description, be appointed as GenencorInternational Inc., be published in April 20 nineteen ninety-five, it is characterized by the wild sublisin BPN ' aminoacid sequence of tool sudden change, this sports the combination of Asn76 position with following one or more sudden changes

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 in this article the preferred subtilisin BPN ' variant of useful as protease be referred to as " proteolytic enzyme monoid D " hereinafter, U.S. Patent number 4 is seen in its description, 760,025, Estell, et al., on July 26th, 1988, be characterized as the wild subtilisin BPN ' aminoacid sequence of the one or more sudden changes of tool, its mutational site is selected from the Asp32 of colony that is made up of following site, Ser33, His64, Tyr104, Asn155, Glu156, Glu166, Gly169, Phe189, Tyr217 and Met222.

Preferred proteolytic enzyme used herein is selected from by Alcalase ^, the colony that forms of subtilisin BPN ', proteolytic enzyme monoid A, proteolytic enzyme monoid B, proteolytic enzyme monoid C, proteolytic enzyme monoid D.Most preferred proteolytic enzyme is selected from proteolytic enzyme monoid D.Variant part

Remove the proteolytic enzyme part, fusion rotein of the present invention contains a variant part (variant).This variant is the modified amino acid sequence with parent's aminoacid sequence, its modified amino acid sequence is replaced having an amino acid corresponding to 63 of i (streptomyces) subtilisin inhibitor (this paper is called SSI), and its parent's aminoacid sequence is selected from the colony that is made up of SSI, SSI sample inhibitor, SSI variant and SSI sample inhibitor variant.These variants can be merged in vivo with proteolytic enzyme.Preferred variant can be resisted the hydrolysis of its corresponding proteolytic enzyme part.

Replacing corresponding to the amino acid on 63 of SSI can be anyly can produce the more amino-acid residue of high stability than parent's aminoacid sequence.Replace corresponding to the amino acid on 63 of SSI and under most preferred situation, to be replaced by Isoleucine.A variant like this is represented as " L63I ".Having provided the original amino acid that appears in parent's aminoacid sequence in expression earlier during this variant, is thereafter the site number, and the 3rd what provide is amino acid after replacing.Like this, it is alternative by Isoleucine (I) that L63I refers to the leucine (L) that appears at natural inhibitor SSI the 63rd amino acid position (63).Position Number is corresponding with Ikenaka etal. (SEQ ID NO:1) above, and has ignored and be present in aminoterminal 4 the extra amino-acid residues of synthetic SSI (SEQ IDNO:1).All expressions in this way of other replacements that this paper is listed.

The variant of this paper is not limited to 63 SSI that are replaced.63 replacement also can be carried out on parent's aminoacid sequence (nucleotide sequences that comprises this aminoacid sequence of encoding certainly), and this variant as SSI variant, SSI sample inhibitor or SSI sample inhibitor of this parent.Preferred parent's aminoacid sequence comprises SSI and SSI variant.Most preferred parent's aminoacid sequence is the SSI variant.The example of the disclosure of SSI variant has Kojima etc., " Inhibition ofSubtilisin BPN ' by Reaction Site P1 Mutants of StreptomycesSubtilisin Inhibitor ", Journal of Molecular Biologv, Vol.109, pp.377-382 (1991); Tamura etc., " Mechanisms of TemporaryInhibition in Streptomyces Subtilisin Inhibitor Induced by anAmino Acid Substitution; Typtophan 86 Replaced by Histidine ", Bioehemistry, Vol.30, pp.5275-5286 (1991); JO 3099-099-A transfers Tsumura﹠amp; Co., be issued on September 12nd, 1989; Mikkelson etc., U.S. Patent number 5,674,833 transfers Novo Nordisk A/S, and on October 7th, 1997 authorized; WO 93/17086, Nielsen etc., transfer Novo Nordisk A/S, on September 2nd, 1993 published.The variant of other SSI is disclosed in U.S. Patent Application Serial Number No.60/026,944 WO 98/13387, Correa etc., transfer The Procter﹠amp; Gamble Co., on April 2nd, 1992 published, and these variants this paper is referred to as " inhibitor monoid A ".Preferred variant SSI (being used as parent's aminoacid sequence at this) is the variant among the inhibitor monoid A.Preferred SSI variant as parent's aminoacid sequence is classified following form 2-6 as at this.Equally, all Position Numbers are corresponding to as Ikenaka etc.Described SSI.

The non-limiting example of parent's aminoacid sequence that table 2 tool unit point is replaced

The parent 1	????D83C
		The parent 4	????M73D
The parent 34	????M73P

The non-limiting example of parent's aminoacid sequence that table 3 tool dibit point is replaced

The parent 2	????M73D+D83C
		The parent 3	????M73D+D83C
The parent 5	????M70Q+D83C
		The parent 29	????M73P+S98D
The parent 30	????M73P+S98E
		The parent 31	????M73P+S98A

The non-limiting example of parent's aminoacid sequence that replace in table 4 tool three sites

The parent 6	????M73P+D83C+S98A
		The parent 7	????M73P+Y75A+D83C
The parent 8	????M73P+D83C+S98V
		The parent 9	????M70Q+M73P+D83C
The parent 10	????M73P+V74A+D83C
		The parent 11	????M73P+M74F+D83C
The parent 12	????M70Q+D83C+S98A
		The parent 13	????G47D+M70Q+D83C
The parent 14	????G47D+D83C+S98A
		The parent 15	????G47D+M73P+D83C
The parent 16	????G47D+M73D+D83C
		The parent 27	????M73Q+D83C+S98D
The parent 28	????M73P+D83C+S98E

The non-limiting example of parent's aminoacid sequence that replace in table 5 tool four sites

The parent 17	?M70Q+M73P+V74F+D83C
		The parent 18	?M70Q+M73P+V74W+D83C
The parent 19	?M70Q+M73P+D83C+S98A
		The parent 20	?G47D+M73P+V74F+D83C
The parent 21	?G47D+M73P+V74W+D83C
		The parent 22	?G47D+M73P+D83C+S98A
The parent 32	?G47D+M73P+D83C+S98D
		The parent 33	?G47D+M73P+D83C+S98E

The non-limiting example of parent's aminoacid sequence that replace in table 6 tool five sites

The parent 23	?G47D+M70Q+M73P+V74F+D83C
		The parent 24	?G47D+M70Q+M73P+V74W+D83C
The parent 25	?G47D+M73P+V74F+D83C+S98A
		The parent 26	?G47D+M73P+V74W+D83C+S98A

Like this, the non-limiting example of variant of the present invention can be described to variant 1, variant 2 or the like, and for example, wherein variant 1 can be expressed as L63 ^*+ D83C, its " ^*" representative any non-SSI 63 original amino acid whose amino acid, variant 1-I can be expressed as L63I+D83C.The variant that is more preferably among them is listed in table 7, is all replaced by Isoleucine for 63 of these variants.

Table 7

The non-limiting example of preferred variants of the present 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 is continuous)

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

The preferred parent's aminoacid sequence of other of this paper is included in the variant that replacement is arranged on 62 that correspond to SSI.Replacement on 62 can be to remove the outer any amino acid of the amino acid (for SSI, the amino acid of natural appearance is L-Ala) of natural appearance among the parent.Preferred replacement on 62 is selected from Lys, Arg, Glu, Asp, Thr, Ser, Gln, Asn and Trp, more preferably replace and be selected from Lys, Arg, Glu, Asp, Thr, Ser, Gln and Asn, preferred replacement is selected from Lys, Arg, Glu and Asp, the replacement that is more preferably is selected from Lys and Arg, and most preferred is Lys.The preferred amino acids sequence also has a replacement on 62 except that listing in table replacing it among the 2-6.Such parent's example is named as parent X-A62 ^*, wherein X is corresponding to the parent who exemplifies in table 2-6.Like this, parent 6-A62 ^*Just corresponding to A62 ^*+ M73P+D83C+S98A.Similarly, parent 6-A62K is corresponding to A62K+M73P+D83C+S98A.Similarly, a variant exemplifying of this paper is variant 6-I-A62 ^*, it is corresponding to A62 ^*+ L63I+M73P+D83C+S98A.Like this, variant 6-I-A62K is corresponding to A62K+L63I+M73P+D83C+S98A.Table 8 has been listed other preferred variants of the present invention in this mode.

Table 8

The non-limiting example of other preferred variants of the present 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 *+L63U+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+L63U+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 11-1-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 *	????A52 *+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+IG47D+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 is useful on the variants that parent's aminoacid sequence of the present invention (its for SSI variant) is included in to be had single variant of replacing and two replacements are arranged on corresponding to SSI 98 on 98 and 62.Table 9 has been listed the preferred parent's aminoacid sequence in this classification.

Table 9

The non-limiting example of parent's aminoacid sequence

The parent 32	????A62K+S98Q
		The parent 33	????A62K+S98D
The parent 34	????A62K+S98E
		The parent 35	????A62R+S98Q
The parent 36	????A62R+S98D
		The parent 37	????A62R+S98E
The parent 38	??????S98A
		The parent 39	????A62K+S98A
The parent 40	????A62R+S98A
		The parent 41	??????S98Q
The parent 42	??????S98D
		The parent 43	??????S98E

The related example of variant of the present invention is listed in the table below 10.

Table 10

The non-limiting example of variant of the present 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 the binary form exist.Like this, not bound by theory, it also is possible that the binary SSI to the stabilization of excessive proteolytic enzyme tool enhanced resistance is provided.Preferred this stabilization binary SSI comprises two covalent attachment monomer together.This is in conjunction with can being that ester, acid amides, two sulphur or other appear at the key in amino acid or their side chain usually.Here " covalency dimerization " refers to the monomer that covalent attachment like this becomes binary with " covalence stablilityization ".More preferably dimerization is undertaken by disulfide linkage.Variant of the present invention has a mind to comprise the variant that exists with the binary form, and no matter they are with intramolecularly or intermolecular forces combination.

Other useful parent's aminoacid sequence comprises SSI sample inhibitor (being commonly referred to SSI sample (SIL) albumen) and SSI sample inhibitor variant herein.The visible Laskowski of related background information of SSI sample inhibitor etc., " Protein Inhibition of Proteases ", Annual Review of Biochemistry, Vol.49, pp.593-626 (1980).Preferred SSI sample inhibitor and the about same aminoacid sequence more than 50% of SSI tool, the same sequence of tool more than 65% more preferably, the same sequence of preferred tool more than 70%, wherein more preferably inhibitor can classify as family's III inhibitor.Middle Laskowski etc. sees above.Document.The example of such SSI sample inhibitor comprises SIL10 (sequence is provided in SEQ IDNO:4), SIL13 (SEQ ID NO:5) and SIL14 (SEQ ID NO:6), Terabe etc. is seen in further describing of they., " Three Novel Subtilisin-TrypsinInhibitors from Streptomyces:Primary Structures andInhibitor Properties ", Journal of Biochemistry, Vol.116 pp.1156-1163 (1994), such example also has SIL2 (sequence is provided in SEQ ID NO:9), SIL3 (SEQ ID NO:10) and SIL4 (SEQ ID NO:11), Taguchi etc. is seen in further describing of they., " Comparative Studies on the PrimaryStructure and Inhibitory Properties of Subtilisin-trypsinInhibitors from Streptomyces ", European Journal ofBiochemistry, Vol.220, pp.911-918 (1994).Two examples in addition of such SI sample inhibitor comprise STI1 (sequence is provided in SEQ ID NO:7) and STI2 (SEQ IDNO:8), it further describes visible Strickler etc., " Two Novel StreptomycesProtein Protease Inhibitors ", The Journal of BiologicalChemistry, Vol.267, No.5, pp.3236-3241 (1992).Another SSI sample inhibitor is known as plasminostreptin (sequence is provided in SEQ ID NO:12), it further describes visible Sugino etc., " plasminostreptin; a ProteinProtease Inhibitors Produced by Streptomycesantifibrinostreptin ", The Journal of Biological Chemistry, Vol.253, No.5, pp.1546-1555 (1978).Another SSI sample inhibitor is SLPI (sequence is provided in SEQ ID NO:13), it further describes visible Ueda etc., " 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 sample inhibitor is SAC I (sequence is provided in SEQ ID NO:14), it further describes visible Tanabe etc., " Primary Structure andReactive Site of Streptoverticillium Anticoagulant (SAC); aNovel Protein Protease Inhibitors Produced byStreptoverticillium cinnamoneum Subsp.cinnamoneum ", Journal of Biochemistry, Vol.115, pp.752-761 (1994).Another SSI sample inhibitor is SILI (sequence is provided in SEQ ID NO:15), it further describes visible Kojima etc., " Primary Structure and InhibitoryProperties of a Protease Inhibitor Produced by Streptomycescacaoi ", Biochemica et Biophysica Acta, Vol.1207, pp.120-125 (1994).The visible Taguchi of discussion of other SSI sample inhibitor etc., " HighFrequency of SSI-like Protease Inhibitors Among Streptomyces ", Bioscience, Biotechnology, and Biochemistry, Vol.57, pp.522-524 (1993), Taguchi etc., " Streptomyces SubtilisinInhibitors-Like Proteins Are Distributed Widely inStreptomycetes ", Applied and Environmental Microbiology, pp.4338-4341 (Dec.1993), and Suzuki etc., " Partial Amino AcidSequence of an alkaline Protease Inhibitor ", AgriculturalBiological Chemistry, Vol.45, pp.629-634 (1981).As known to a person skilled in the art, also have other SSI sample inhibitor to describe to some extent in the art.

The variant of SSI sample inhibitor also can be at this as parent's aminoacid sequence.These variants comprise that those are selected from SSI sample inhibitor mentioned above, and its aminoacid sequence contains one or more sudden changes.All replacements that illustration is crossed in the variant that this paper mentions all can be in the opposite position manufacturing of SSI sample inhibitor to provide parent's aminoacid sequence.The non-limitative example of other the SSI sample inhibitor variant that can be used as parent's aminoacid sequence is disclosed in Nielson etc., WO93/17086 transfers Novo Nrdisk A/S, is issued on September 2nd, 1993.As known to persons skilled in the art, 63 (examples) of the natural coding of the variant of SSI sample inhibitor, its variant or SSI may and not correspond to 63 of SSI.Therefore, as known in the art, may need sequence numbering is adjusted with 63 (examples) of location corresponding to SSI.The arrangement of sequence is found in reference that this paper quotes and other reference of this area.

The K that the preferred variant of the present invention is showed _iCan make this variant nearly all proteolytic enzyme of inhibition (preferably can suppress about 60% proteolytic enzyme, preferred suppress about 99%) in cleaning and Personal hygiene composition, and can dissociate with inhibitor after the dilution and/or during washing.Preferred inhibitors is to the K of proteolytic enzyme _iBe about 10 ^-12M to 10 ^-4M is more preferably 10 ^-10M to 10 ^-6M, most preferred 10 ^-8M to 10 ^-7M.Certainly, if the concentration of the size of washing plant or product changes, must adjust K in view of the above _iTo a useful K _iPrediction can be undertaken by those skilled in the art, and need not to consider such as in use dilution of composition, binding constant to the dependence of the temperature of employed relevant purging method, variant the similarly over-drastic tests of parameter such as stoichiometric ratio with proteolytic enzyme.The connection portion

Except that proteolytic enzyme part and variant part, this fusion rotein also can according to circumstances contain a junction branch.Preferred fusion protein contains a junction branch.This connection portion is a hydrolyzable amino acid chain, and it separates the proteolytic enzyme part with variant part, and its proteolytic enzyme part links to each other by the connection portion covalency with variant part.

Those skilled in the art can make up the connection portion to reach multiple various objectives.For example, the amino-acid residue of connection portion can be designed to the good substrates of hydrolysis.And this aminoacid sequence can design the separating after translation in order to accessory protein enzyme part and variant part, or in order to optimize variant part with respect to proteolytic enzyme partly combination or the position of avtive spot.

Preferred selectable connection portion is about 20 amino-acid residues or shorter.Preferred connection portion is easy to partly be cut by proteolytic enzyme.

When fusion rotein did not contain the connection portion, proteolytic enzyme part and variant part were directly covalently bound.

The further feature of fusion rotein

Because fusion rotein is finally coded by DNA in vivo, this DNA can be used to define the sequence of fusion rotein.The DNA of coding variant can be used for any amount of plasmid and/or expression system, comprise expression in vivo system and vivoexpression system, for example plant (is used for the comparatively preferred of biotechnology, comprise tobacco, the oleaginous seed crop, for example Semen Brassicae campestris, soybean and other similar things, cereal, for example corn, barley, oat, other vegetables, for example tomato, potato and similar plants) and the microbial expression system, the latter comprises fungi, for example yeast, and bacterium, for example Bacillus subtilus, intestinal bacteria and other similar bacterium.Preferred expression system is a microorganism, is more preferably natural bacteria, and most preferred is Bacillus subtilus or intestinal bacteria, and Bacillus subtilus is more better.

The DNA of coding variant can participate in activated plasmid or phage in cell, can also directly participate in the organic genome that is used to clone or express fusion rotein of the present invention.

Should understand, those skilled in the art will think that be used to the to encode DNA of a variant of the present invention can introduce with other variant of the present invention the same plasmid, phage or karyomit(e) under guidance of the present invention.In addition, this plasmid, phage or karyomit(e) can also the proteins encoded enzymes, and it comprises and contain inhibitor and/or proteolytic enzyme as its a part of fusion rotein no matter whether it is suppressed all can by fusion rotein of the present invention.

Those skilled in the art it is also understood that above-mentioned DNA also can expect and disclose the rna transcription basis of DNA.Those skilled in the art need not experiment just can know the RNA sequence by detecting dna sequence dna.

The invention still further relates to the gene of these fusion roteins of coding.

In a preferred embodiment of the invention, fusion rotein can with one or more proteinase inhibitor, preferably, another kind of proteinase inhibitor is coexpression in same expression system.Under the preferable case, the ease variants of interpolation is selected from SSI, SSI sample inhibitor, SSI variant and SSI sample inhibitor variant.Under the preferred situation, the proteinase inhibitor of interpolation is for carrying the variant of " variant part " defined herein independently, and this comprises preferred restriction.Under the most preferred situation, the proteinase inhibitor of interpolation is the variant identical with the variant part of fusion rotein.

Therefore, the present inventor provides expression system at this, and this system comprises the DNA of encoding fusion protein and can contain one or more extra proteinase inhibitor according to circumstances.Preferred expression system is the organism of living, and is more preferably natural bacteria.

Can expect that those skilled in the art can prepare the antibody of fusion rotein of the present invention.These antibody can known method prepare.

For example, fusion rotein of the present invention can be injected into suitable Mammals, as mouse, rabbit and other similar animal.Suitable scheme relates to the duplicate injection of immunogen in the presence of adjuvant, and injection is carried out according to the timetable that can improve the antibody production in the serum.The measurement of the titre of immune serum can be undertaken by the immunoassay step of standard in this area as antigen by albumen of the present invention.

Antiserum(antisera) can directly use, and spleen that can also be by collecting peripheral blood lymphocyte or immune animal also makes the antibody producing cell immortalization, uses the immune analysis method of standard to confirm suitable antibody produced cell thereupon, and obtains monoclonal antibody.

Polyclone or monoclonal antibody can be used to detect expression of the present invention by the detection method of standard.Like this, expectation can utilize these Antibody Preparation test kits to be used to measure expression level and other.

These antibody also can utilize the coupling method of standard to be coupled to the scintiscanning mark, for example technetium 99 or I-131, or be coupled to fluorescent mark.The antibody of mark also can be used for competitive analysis, and for example dynamic analysis is measured K with this _i

Fusion rotein of the present invention can also further contain " part " so that the function of needs to be provided, and for example cellulase is in conjunction with the territory, lipase, amylase and cellulase.

As known in the art, have accidental mistake in DNA and the aminoacid sequence.Therefore, those of ordinary skill in the art can find any mistake and suitably changes when repeating present inventor's work by this paper institute published content by routine techniques.

Method of manufacture and use thereof

Following example is not will the invention of being applied for be limited, but provides about how making and use guidance of the present invention to those skilled in the art.The guidance of these examples, other disclosure content of this paper and the out of Memory that those skilled in the art obtain have easily been arranged, and those skilled in the art can make and use the present invention.Succinct for composing a piece of writing, the repeated citing of this area and known art method all is removed, because they are predicted by those skilled in the art.

Variant part (variant) can suddenly change by the nucleotide sequences to coding parent aminoacid sequence and prepare, and has so just produced the variant of the aminoacid sequence of tool modified.These methods are known in the art, and are wherein a kind of as follows.

With a phagemid transformed into escherichia coli dut who contains with the corresponding gene of parent's aminoacid sequence ^-Ung ^-Bacterial strain CJ236 and utilize the VCSM13 helper phage to produce dna profiling (the Kunkel et al. that strand contains uridylic, " Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection ", Methodsin Enzymology, Vol.154, pp.367-382 (1987), Yuckenberget al. is seen in change, " Site Directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectors ", Mutagenesis-APractical Approach, McPherson, M.J.ed., pp.27-48 (1991)).The primer sites specific mutant can be by the method (Zoller of Zoller and Smith, M.J., and M.Smith, " Oligonucleotid-Directed Mutagenesis Using M13-Derived Vectors:An Efficient and General Procedure for theProduction of Point Mutations in any Fragment of DNA ", NucleicAcids Research, Vol.10, pp.6487-6500 (1982)) is used to produce all variants (document of Yuckenberg et al. as mentioned is described basically).

Use the 380B dna synthesizer to make oligonucleotide.The jump reaction product is transformed into coli strain MM294 (American Type Culture Collection E.coli33625).By dna sequencing confirm all sudden changes and will separate DNA be transformed into Bacillus subtilus expression strain PG632 (Sauders et al., " Optimization of theSiganal-Sequence Cleavage Site for secretion from Bacillussubtilis of a 34-amino acid Fragment of Human ParathyroidHormone ", Gene, Vol.102, pp.277-282 (1991) and Yang et al., " Cloning of the Neutral Protease Gene of Bacillus subtilis andthe Use of the Cloned Gene to Create an in vitro-DerivedDeletion Mutation ", Journal of Bacteriology, Vol.160, pp.15-21 (1984)).

The gene of coding variant can merge with proteolytic enzyme.The method of a standard is to introduce restricted point of contact in the appropriate location of each gene.Carry out restrictive diges-tion and connect the restriction enzyme fragment, for example, use the T4 ligase enzyme.According to the character of plasmid, with connecting mixture transformed into escherichia coli or subtilis.For example, can use the subtilisin that is carried by plasmid, this plasmid is at intestinal bacteria and the equal reproducible of subtilis and give the former amicillin resistance and latter's kalamycin resistance.Can utilize rite-directed mutagenesis (3 ' end) behind the DNA of coding Validase TSP Concentrate II carboxyl terminal amino-acid residue and then to introduce EcoR I site.Also can behind the DNA of coding terminator codon, (3 ' end) and then introduce BamH I site.Can make up inhibitor gene makes its dna sequence dna 5 ' at next-door neighbour's coding-terminal amino acid residue locate to have an EcoR I site.In addition, a BamH I site can be placed (3 ' end) behind the DNA of coding terminator codon.Can handle variant and subtilisin inhibitor gene with restriction enzyme EcoR I and BamH I earlier, handle with the T4 ligase enzyme subsequently.Can use and connect mixture transformed into escherichia coli MM294 to obtain amicillin resistance.Transform subtilis to obtain kalamycin resistance in case can from intestinal bacteria, be recovered to just available its of the plasmid of encoding fusion protein.

The subtilis that contains the purpose plasmid can be cultivated in substratum, this substratum contains 20g/l Tryptones, 20g/l yeast extract and 5g/l sodium-chlor and replenishes maltrinM100 (Grain Processing Corporation, Muscatine, IA) to 1.25%, the HEPES to 100mM of pH7.5, MnCl2 to 80 μ M and kantlex to 50 μ M.Culture was 37 ℃ of following incubations 24 hours.

Fusion rotein is secreted to substratum, therefrom it can be proposed.Any a series of chromatographic step comprises that ion-exchange and gel permeation chromatography all can use.

The characteristic of this fusion rotein

The detected protease activity of supernatant and the protease inhibitory activity that contain fusion rotein of the present invention.

But the Y217L variant of SSI arrestin enzyme, especially subtilisin BPN ' and subtilisin BPN '.In reference group, SSI is mixed with proteolytic enzyme, room temperature incubation 15 minutes.Press DelMar et al. immediately, Analytical Biochemistry, Vol.99, the method among the pp.316-320 (1979) is measured protease activity.Add 0.1MTris, pH8.6, the CaCl of 10mM ₂To volume be 990 μ l.N-succinyl--the Ala-Ala-Pro-Phe-that adds 10 μ l is right-and pyridine amine (20mg/ml) is with initial action.Measure speed of reaction according to the enhancing that 410nm absorbs down.

The culture supernatant that detects fusion rotein of the present invention in a like fashion suppresses the ability of the Y217L variant of subtilisin BPN '.Check up and return detected its on the culture and produce the ability of proteolytic enzyme.

The obvious shortage of inhibitor activity and protease activity is consistent with the fusion rotein that variant part and proteolytic enzyme by mutual cancellation (or basic cancellation) partly constitutes, and this is that the present invention is desirable.This is interpreted as Western trace result and supports that it shows that fusion rotein is made into.

For the needs of fusion rotein of the present invention being integrated with in cleaning or the Personal hygiene composition, its stability in the product environment is also tested.If the proteolytic enzyme of fusion rotein and inhibitor activity are stable, the level of protease activity is stable with respect to the time.Yet, if variant part by the inhibitor part institute hydrolysis of fusion rotein, protease activity can raise.The same liquid detergents composition of fusion rotein culture supernatant mixes mutually, and this detergent compositions is made according to following prescription:

Component	Weight percent
		?C 14-15Alkyl (oxyethyl group 2.25) sulfonic acid	?????18.0
?C 12-13Alkyl ethyl oxide compound	?????2.0
		?C 12-N-methyl glutamine	?????5.0
Citric acid	?????4.0
		Ethanol	?????3.5
Monoethanolamine	?????2.0
		1,2 propylene glycol	?????7.0
Sodium formiate	?????0.6
		Tetren ethyl oxide compound	?????1.18
?Soil?release?Polymer	?????0.25
		The siloxane foams inhibition	?????0.10
Whitening agent	?????0.10
		Water, NaOH	Add surplus to 100%

This composition account for population of samples long-pending 1/3rd.With the sample of 15 μ l 0.1M, Tris HCl and the 0.01MCaCl of pH8.6 with 975 μ l ₂Mix mutually.At room temperature this dilution of incubation is 30 minutes.Add substrate behind the incubation and calculate the amount of proteolytic enzyme.Detect the degraded of variant part by the increase of protease activity after several weeks.This degraded can be directly compares with the corresponding data such as the SSI of fusion rotein.

The K of variant _iCalculate as follows.With fusion rotein with 600 μ g/ml N-succinyl--Ala-Ala-Pro-Phe-right-pyridine amine mixes in the Tris solution of 990 μ l 50mM pH8.Follow the trail of 20 minutes hydrolysis rate.Process can observe stable speed in last 10 to 15 minutes.According to Goldstein, " The Mechanism of Enzyme-Inhibitor-Substrate Reactions ", Journal of GeneralPhysiology, Vol.27, equation among the pp.529-580 (1944) is compared and calculating K with the speed under the no variant situation with this speed _i

Cleaning compositions of the present invention

In another embodiment of the present invention, one or more of significant quantity fusion rotein of the present invention is contained in the cleaning compositions, and said composition can be used for cleaning must remove the peptide surfaces contaminated.These cleaning compositions comprise clean fabric composition, hard-surface cleaning compositions, comprise the light-duty cleaning compositions of dish cleaning compositions, and the detergent compositions of dish automatic rinser, but are not limited to these.

The cleaning compositions of this paper comprises one or more fusion roteins of the present invention and a cleaning compositions carrier of significant quantity.Under most preferred situation, such fusion rotein has a proteolytic enzyme part, a variant part and can choosing wantonly but comparatively preferred connection portion.In a preferred embodiment of the invention, cleaning compositions further contains one or more extra proteinase inhibitor except that fusion rotein at this.Under the preferable case, the ease variants of interpolation is selected from SSI, SSI sample inhibitor, SSI variant and SSI sample inhibitor variant.Under the preferred situation, the proteinase inhibitor of interpolation is for carrying the variant of " variant part " defined herein independently, and this comprises preferred restriction.Under the most preferred situation, the proteinase inhibitor of interpolation is the variant identical with the variant part of fusion rotein.

In this cleaning compositions, variant is 3: 1 to 1: 1 with the preferred molar ratio (variant is than proteolytic enzyme) (wherein fusion rotein variant part and any extra proteinase inhibitor sum are the variant mole number) of proteolytic enzyme in the cleaning compositions, preferred ratio is 3: 1 to 1.5: 1, and most preferred ratio is 2: 1.

Employed " fusion rotein of significant quantity " of this paper or other similar terms mean to obtaining the amount of the essential necessary fusion rotein of proteolytic activity of concrete cleaning compositions.The those of ordinary skill in field can more easily be determined this significant quantity on the basis of many factors, the example of these factors as, the concrete combination of used specific fusion proteins, cleaning applications, cleaning compositions, need liquid still dryness (as particle, column) composition and other similar factors.Preferred cleaning compositions contains one or more fusion roteins of the present invention of 0.0001% to 10%, and preferred cleaning compositions contains 0.001% to 1%, and most preferred cleaning compositions contains 0.01% to 0.1%.Several embodiment that may be applied to the multiple cleaning compositions of this fusion rotein will go through hereinafter.

Except that fusion rotein of the present invention, cleaning compositions of the present invention also comprises a cleaning compositions carrier, and it comprises the cleaning compositions material that one or more are compatible with fusion rotein.Term used herein " cleaning compositions material " refers to the material of any required cleaning composition that is selected to particular type and product form (as liquid state, particle, column, spraying, bar-shaped, pasty state, gel), and this material should be compatible with used fusion rotein in the composition.The desired form that is used for the composition of cleaning ambient during according to material to be cleaned and use can more easily be made concrete selection to the cleaning compositions material.Term used herein " compatible " means the cleaning compositions material can not reach the fusion rotein degree of the required usefulness of tool not when normal use that makes to the reduction of the inhibitor activity of fusion rotein and/or proteolytic activity.Hereinafter that detailed illustration is concrete cleaning compositions material.

Fusion rotein of the present invention can be used to the detergent compositions that multiple needs enrich foam and good cleaning action.Like this, this variant can be used to the hard surface cleaner, dish cleaning compositions, fabric laundry composition of fully preparation and other together with multiple traditional composition.These compositions can be liquid state, particle, column and other form.These compositions can " concentrate " the form preparation of stain remover, and they contain the tensio-active agent of weight percent 30% to 60%.

The cleaning compositions of this paper can preferably contain kinds of surface promoting agent (for example negatively charged ion, nonionic or zwitterionics) according to situation.It is 5% to 35% of composition that typically there is content in these tensio-active agents.

The non-limitative example of tensio-active agent used herein comprises traditional C ₁₁-C ₁₈Alkyl benzene sulphonate (ABS) and uncle and at random alkylsurfuric acid, C ₁₀-C ₁₈Secondary (2,3) alkylsurfuric acid, its molecular formula is CH ₃(CH ₂) _x(CHOSO ₃) ^-M ⁺CH ₃And CH ₃(CH ₂) _y(CHOSO ₃ ^-M ⁺) CH ₂CH ₃, x and wherein (y+1) for being at least 7 integer, more preferably then for being at least 9 integer, M is water miscible positively charged ion, sodium ion especially, non-limitative example also comprises C ₁₀-C ₁₈Alkyl alkoxy sulfuric acid (especially EO 1-5 oxyethyl group sulfuric acid), C ₁₀-C ₁₈Alkyl alkoxy carboxylic acid (especially EO 1-5 ethoxy carboxylate), C ₁₀-C ₁₈Alkyglycosides and their corresponding sulfuric acid polyglycosides, C ₁₂-C ₁₈α-sulfonated fatty acid ester, C ₁₂-C ₁₈Alkyl and alkylphenol alcoxylates (especially ethoxylate or ethoxy/third oxygen mixture), C ₁₂-C ₁₈Trimethyl-glycine and thetine (" sultaines "), C ₁₀-C ₁₈Amine oxide and other analogue.Alkyl alkoxy sulfuric acid (AES) and alkyl alkoxy carboxylic acid (AEC) are comparatively preferred herein.These tensio-active agents also are more preferably with being used in combination of amine oxide and/or trimethyl-glycine or Saltaine, and this depends on the requirement of formulator.Other useful conventional surfactant is listed with received text.Useful especially tensio-active agent comprises C ₁₀-C ₁₈N-methyl glucoside acid amides, it is disclosed in U.S. Patent number 5,194,639, Connor etc., registration on March 16th, 1993.

In the cleaning compositions of the present invention other component very widely comprises other active ingredient, carrier, hydrotrote, processing subsidiary, dyestuff or pigment and the solvent that is used for liquid preparation.Foamy additionally increases if desired, can be with such as C ₁₀-C ₁₆The such foam of alkylolamide (alkolamide) produces agent and adds composition, and typical content is about 1% to about 10%.C ₁₀-C ₁₄Monoethanolamine or diglycollic amide are that the such foam of a typical class produces agent.Such foam is produced agent with high foam cosurfactant, and it also is useful that for example above-mentioned amine oxide, trimethyl-glycine and thetine are used together.If desired, can add the magnesium salts of solubility to produce more foam, as MgCl ₂, MgSO ₄Or the like, its concentration is generally about 0.1% to about 2%.

The liquid detergents composition of this paper can be moisture or other solvent as carrier.Low-molecular-weight uncle or secondary alcohol are suitable for using, for example methyl alcohol, ethanol, propyl alcohol and Virahol.For the soluble surfaces promoting agent, single hydroxyl alcohol is comparatively preferred, but also can use many alcohol of containing about 2 to 6 carbon atoms and about 2 to 6 oh groups (for example 1, ammediol, 1,2 ethylidene glycol, glycerine and 1,2-propylene glycol).Composition can contain these carriers of about 5% to about 90%, typically contains about 10% to about 50%.

The detergent compositions of this paper is preferably prepared when cleaning operation in water, and the pH of water for cleaning is at 6.8 to 11.The finished product typically are mixed with in this scope.Be used for the technology that pH is controlled at the suggestion usage level is comprised use such as damping fluid, alkali and acid.These technologies are known by those skilled in the art.

In the process of preparation hard-surface cleaning compositions of the present invention and clean fabric composition, formulator may be ready to use multiple washing assistant, and its weight percent is about 5% to about 50%.Typical washing assistant comprises the succsinic acid, stratification silicate, phosphoric acid salt of the zeolite of 1-10 micron, many carboxylic acids such as citric acid and oxo and other.Other traditional washing assistant is listed in the prescription table look-up of standard.

Similarly, formulator may be willing to be intended in these compositions to use multiple additional enzyme, for example cellulase, lipase, amylase and proteolytic enzyme, and their content typically is weight percent about 0.001% to about 1%.Known multiple cleaning and fabric enzyme in the laundry stain remover field.

Multiple bleaching compounds, for example similar compound such as percarbonate, perborate can be used to these compositions, and their content typically is weight percent about 1% to about 15%.If desired, these compositions also can contain bleach activator, for example tetra-acetylated ethylene diamine, nonanoyl Oxybenzene sulfonic acid and other similar compounds, and it also is known in this area.It uses content typically to be weight percent about 1% to about 10%.

These compositions can also use the spot agent (soil release agent) of dissociating, oligomerization ester anionic particularly, sequestrant, particularly amino phosphonates do and inferior quadrol succinate, the clay remover, ethoxyquin tetracthylene pentamine particularly, dispersion agent, particularly polyacrylic ester and poly-asparagine (polyasparatate), brightener, negatively charged ion brightener particularly, froth suppressor, particularly siloxanes and secondary alcohol, fabric softener, smectic clays particularly, or the like similar substance, their content is weight percentage about 1% to about 35%.The prescription table look-up of standard and delivered the numerous detailed descriptions of patent relevant for these traditional materials.

Enzyme stabilizers also can be used for cleaning compositions.Such enzyme stabilizers comprises propylene glycol (preferred content is about 1% to about 10%), sodium formiate (preferred content is about 0.1% to about 1%) and calcium formiate (preferred content is about 0.1% to about 1%).

Other useful cleaning compositions comprises clay remover, dispersion agent, brightener, froth suppressor and fabric softener.

Fusion rotein of the present invention can be used for hard-surface cleaning compositions." hard-surface cleaning compositions " used herein is used to clean liquid state or the particulate state detergent compositions such as floor, wall, bathroom tile or the like crust.Hard-surface cleaning compositions typically contains tensio-active agent and water-soluble sequester washing assistant.Yet, in some specialty products such as window spray cleaner, do not use tensio-active agent sometimes, because they may be at glass surface residual film shape and/or mottled vestige.

But when tensio-active agent exists intrinsic energy be low to moderate this paper composition 0.1%, but said composition typically contains about 0.25% to about 10%, contain about 1% to about 5% more preferred.

This composition typically contains about 0.5% to about 50% decontamination lotion promoter, and more preferably content is 1% to about 10%.

More preferably the pH value is about 7 to 12.Regulate pH if desired, can use traditional pH regulator reagent, for example sodium hydroxide, yellow soda ash or hydrochloric acid.

Solvent can be contained in the composition.Useful solvent comprises glycol ether, for example diethylidene ethylene glycol ether, diethylidene ethylene glycol monobutyl ether, ethylidene ethylene glycol monobutyl ether, ethylidene ethylene glycol ether, propylidene ethylene glycol monobutyl ether, dipropylene ethylene glycol monobutyl ether, useful solvent also comprises glycol, for example 2,2,4-trimethylammonium-1,3-pentanediol and 2-ethyl-1,3 hexylene glycols, the example of solvent is not limited thereto.。The typical content of these solvents is for about 0.5% to about 15% during use, and preferred content is about 3% to about 11%.

In addition, when composition the surface is carried out " complete imitate " use after not to surface washing, this composition can use strong volatile solvent such as Virahol or ethanol with faster the evaporation from the surface of auxiliary said composition.Volatile solvent typical content in said composition is about 2% to about 12% during use.

Also be useful in the cleaning compositions that variant of the present invention comprises in the following stated: United States Patent (USP) provisional application No.60/079,477, Rubingh etc., declared on March 26th, 1998; United States Patent (USP) provisional application No.60/079,397, Rubingh etc., declared on March 26th, 1998; Application No. No.09/048,174, Weisgerber etc., declared on March 26th, 1998; Application No. No.09/088912 requires to have precedence over the provisional application No.60/079 of United States Patent (USP), 477, Weisgerber etc., declared on June 2nd, 1998.

Hard-surface cleaning compositions is illustrated by following examples.

Embodiment 1-6

Liquid hard-surface cleaning compositions

	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6
							It is variant part that fusion rotein comprises variant 6-I proteolytic enzyme, BPN 1The Y217L variant be the proteolytic enzyme part	?0.05％	?0.50％	??0.02％	??0.03％	??0.30％	??0.05％
???????EDTA	???-	???-	??2.90％	??2.90％	????-	????-
							Trisodium Citrate	???-	???-	????-	????-	??2.90％	??2.90％
?C 12Sodium alkyl benzene sulfonate	?1.95％	???-	??1.95％	????-	??1.95％	????-
							?C 12Sodium alkyl sulfate	???-	?2.20％	????-	??2.20％	????-	??2.20％
?C 12(oxyethyl group) sodium sulfate	???-	?2.20％	????-	??2.20％	????-	??2.20％
							?C 12The dimethyl amine oxide compound	???-	?0.50％	????-	??0.50％	????-	??0.50％
The isopropyl benzene sodium sulfonate	?1.30％	???-	??1.30％	????-	??1.30％	????-
							The hexyl carbitol	?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 prescriptions all transfer to pH7

In embodiment 1-6, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace above fusion rotein, and its result is similar substantially.

Embodiment 7-10

Liquid dish stain remover

	Example 7	Example 8	Example 9	Example 10
					It is variant part that fusion rotein comprises variant 7-I-A62K/ proteolytic enzyme, and the Y217L variant of BPN1 is the proteolytic enzyme part	?0.05％	?0.50％	?0.02％	?0.40％
????????C 12-C 14N-methyl glucoside acid amides	?0.90％	?0.90％	?0.90％	?0.90％
					??????????C 12Oxyethyl group (1) sulfuric acid	?12.0％	?12.0％	?12.0％	?12.0％
2-methyl undecanoic acid	?4.50％	?4.50％	?4.50％	?4.50％
					?????????C 12Oxyethyl group (2) carboxylic acid	?4.50％	?4.50％	?4.50％	?4.50％
?????????C 12Alcohol ethoxylate (4)	?3.00％	?3.00％	?3.00％	?3.00％
					????????????C 12Amine oxide	?3.00％	?3.00％	?3.00％	?3.00％
The isopropyl benzene sodium sulfonate	?2.00％	?2.00％	?2.00％	?2.00％
					Ethanol	?4.00％	?4.00％	?4.00％	?4.00％
????????????Mg 2+(as MgCl 2)	?0.20％	?0.20％	?0.20％	?0.20％
					????????????Ca 2+(as CaCl 2)	?0.40％	?0.40％	?0.40％	?0.40％
Water	?65.45％	?65％	?65.48％	?65.1％

All prescriptions all transfer to pH7.

In embodiment 7-10, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace above fusion rotein, and its result is similar substantially.

Embodiment 11-13

Liquid clean fabric composition

	Example 11	Example 12	Example 13
				It is variant part that fusion rotein comprises variant 2-I/ proteolytic enzyme, BPN 1The Y217L variant be the proteolytic enzyme part	???0.05％	???0.03％	???0.30％
?????????????C 12-C 14Sodium alkyl sulfate	???20.0％	???20.0％	???20.0％
				The 2-butyl is sad	???5.0％	???5.0％	???5.0％
Trisodium Citrate	???1.0％	???1.0％	???1.0％
				?????????????C 10Alcohol ethoxylate (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 embodiment 11-13, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace above fusion rotein, and its result is similar substantially.

Variant of the present invention also is applicable to the Personal hygiene composition, these compositions are selected from leave-on and the agent of rinse-off hair care, shampoo, leave-on and rinse-off acne composition, cleansing milk and nursing agent, body wash, perfumed soap, foaming and still facial cleansing agent, makeup, hand, face and body washing lotion and moistening agent, leave-on facial moisturizer, makeup and cleaning wipe, oral hygiene composition and contact lens health composition.Personal hygiene composition of the present invention contains one or more fusion roteins of the present invention and a kind of Personal hygiene carrier.Fusion rotein, comprise be preferably limited in, all descriptions of with good grounds cleaning compositions in this article.Under most preferred situation, such fusion rotein has a proteolytic enzyme part, a variant part and can choosing wantonly but comparatively preferred connection portion.In a preferred embodiment of the invention, cleaning compositions further contains one or more extra proteinase inhibitor except that fusion rotein at this.Under the preferable case, the ease variants of interpolation is selected from SSI, SSI sample inhibitor, SSI variant and SSI sample inhibitor variant.Under the preferred situation, the proteinase inhibitor of interpolation is for carrying the variant of " variant part " defined herein independently, and this comprises preferred restriction.Under the most preferred situation, the proteinase inhibitor of interpolation is the variant identical with the variant part of fusion rotein.

In this Personal hygiene composition, variant is 3: 1 to 1: 1 with the preferred molar ratio (variant is than proteolytic enzyme) (wherein fusion rotein variant part and any extra proteinase inhibitor sum are the variant mole number) of proteolytic enzyme, preferred ratio is 3: 1 to 1.5: 1, and most preferred ratio is 2: 1.

For ease of explanation, be suitable for below with reference to the described composition of document with fusion rotein of the present invention: U.S. Patent number 5,641,479, Linares et al. was issued June 24 (skin cleaner) in 1997; U.S. Patent number 5,599,549, Wivell et al. was issued February 4 (skin cleaner) in 1997; U.S. Patent number 5,585,104, Ha etal. was issued December 17 (skin cleaner) in 1996; U.S. Patent number 5,540,852, Kefauver et al. was issued July 30 (skin cleaner) in 1996; U.S. Patent number 5,510,050, Dunbar et al.. were issued April 23 (skin cleaner) in 1996; U.S. Patent number 5,612,324, Guang lin et al. was issued March 18 (anti-acne formulations) in 1997; U.S. Patent number 5,587,176, Warren etal. was issued December 24 (anti-acne formulations) in 1996; U.S. Patent number 5,549,888, Venkateswaran et al. was issued August 27 (anti-acne formulations) in 1996; U.S. Patent number 5,470,884, Corless et al. was issued November 28 nineteen ninety-five (anti-acne formulations); U.S. Patent number 5,650,384, Gordon et al. was issued July 22 (body wash) in 1997; U.S. Patent number 5,607,678, Mooreet al. was issued March 4 (body wash) in 1997; U.S. Patent number 5,624,666, Coffindaffer et al. issues on April 29th, 1997 (hair care agent and/or shampoo); U.S. Patent number 5,618,524, Bolich et al. issues on April 8th, 1997 (hair care agent and/or shampoo); U.S. Patent number 5,624,666, Coffindaffer etal. issues on April 29th, 1997 (hair care agent and/or shampoo); U.S. Patent number 5,612,301, Inman issues on March 18th, 1997 (hair care agent and/or shampoo); U.S. Patent number 5,573,709, Wells issues on November 12nd, 1996 (hair care agent and/or shampoo); U.S. Patent number 5,482,703, Pings issues on January 9th, 1996 (hair care agent and/or shampoo); U.S. Patent number Re.34,584, Grote et al. issues again on April 12nd, 1994 (hair care agent and/or shampoo); U.S. Patent number 5,641,493, Date et al. was issued June 24 (makeup) in 1997; U.S. Patent number 5,605,894, Blank et al. was issued February 25 (makeup) in 1997; U.S. Patent number 5,585,090, Yoshioka et al. was issued December 17 (makeup) in 1996; U.S. Patent number 4,939,179, Chenev et al. issues in July 3 nineteen ninety (hand, face and body washing lotion); U.S. Patent number 5,607,980, McAtee et al. issues on March 4th, 1997 (hand, face and body washing lotion); U.S. Patent number 4,045,364, Richet et al. was issued on August 30th, 1977 (makeup and cleaning wipe); European patent application, EP 0 619 074, and Touchet et al. is published on October 12nd, 1994 (makeup and cleaning wipe); U.S. Patent number 4,975,217, Brown-Skrobot et al. was issued December 4 nineteen ninety (makeup and cleaning wipe); U.S. Patent number 5,096,700, Seibel was issued March 17 (oral cleaning composition) in 1992; U.S. Patent number 5,028,414, Sampathkumar was issued July 2 (oral cleaning composition) in 1991; U.S. Patent number 5,028,415, Benedict et al. was issued July 2 (oral cleaning composition) in 1991; U.S. Patent number 5,028,415, Benedict et al. was issued July 2 (oral cleaning composition) in 1991; U.S. Patent number 4,863,627, Davies et al. was issued on September 5th, 1989 (contact lens cleaning soln); U.S. Patent number Re 32,672, Huth et al. was issued again on May 24th, 1988 (contact lens cleaning soln); U.S. Patent number 4,609,493, Schafer was issued on September 2nd, 1986 (contact lens cleaning soln).

In the Personal hygiene composition that the variant of invention comprises in the following stated also is useful: the provisional application No.60/079 of United States Patent (USP), and 477, Rubingh et al. was declared on March 26th, 1998; The provisional application No.60/079 of United States Patent (USP), 397, Rubingh etal. was declared on March 26th, 1998; Application No. No.09/048,174, Weisgerber et al. was declared on March 26th, 1998; Application No. No.09/088912 requires to have precedence over the provisional application No.60/079 of United States Patent (USP), and 477, Weisgerber et al. was declared on June 2nd, 1998.

For further specifying oral cleaning composition of the present invention, one or more fusion roteins of the present invention are contained in the composition that is used for removing from tooth or artificial tooth the Deproteinization spot." oral cleaning composition " used herein refers to analogues such as dentifrice, toothpaste, gutta-percha, tooth powder, washing liquid of oral cavity, mouth spray, buccal cavity gel, gum, lozenge, folliculus, tablet, xanthan gum, prophylaxis pastes, dental treatment solution.

The common content of Personal hygiene carrier component in the oral cavity cleaning component of typical oral cleaning composition is weight percentage about 50% to about 99.99%, and preferred content is about 65% to about 99.99%, and more preferably content is about 65% to about 99%.

The Personal hygiene carrier component and the optional components that can be contained in the oral cleaning composition of the present invention are known by those skilled in the art.The miscellaneous types of compositions, carrier component and the optional components that can be used for oral cleaning composition have been disclosed in the reference that this paper above quotes.

In another embodiment of the invention, be used for outside oral cavity denture care the artificial tooth cleaning compositions contain one or more variants of the present invention.These artificial tooth cleaning compositions one or more fusion roteins of the present invention and a kind of Personal hygiene carrier.The form of multiple artificial tooth cleaning compositions known in the art, (example is seen U.S. Patent number 5,055,305, and Young), they are applicable to that usually one or more are in order to remove the fusion rotein of Deproteinization spot from artificial tooth as effervescent tablet or the like.

In another embodiment of the invention, the contact lens cleaning compositions contains one or more variants of the present invention.These contact lens cleaning compositions one or more fusion roteins of the present invention and a kind of Personal hygiene carrier, the form of multiple contact lens cleaning compositions known in the art, as tablet or liquid or the like, they are applicable to that usually one or more are in order to remove the fusion rotein of Deproteinization spot from contact lens.

Embodiment 14-17

The contact lens cleaning compositions

	Example 14	Example 15	Example 16	Example 17
					It is variant part BPN that fusion rotein comprises variant 9-I/ proteolytic enzyme 1The Y217L variant be the proteolytic enzyme part	?0.01％	??0.5％	??0.1％	??2.0％
Glucose	?50.0％	??50.0％	??50.0％	??50.0％
					Nonionogenic tenside (poloxalkol)	?2.0％	??2.0％	??2.0％	??2.0％
Anion surfactant (polyoxyethylene-alkylphenylether sodium sulfricester)	?1.0％	??1.0％	??1.0％	??1.0％
					Sodium-chlor	?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 embodiment 14-17, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace above variant, and its result is similar substantially.

Embodiment 18-21

The body cleaning product

	Example 18	Example 19	Example 20	Example 21
					Water	???62.62％	????65.72％	???57.72％	????60.72％
The EDTA disodium	???0.2％	????0.2％	???0.2％	????0.2％
					Glycerine	???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％
					The Iauraphoacetate sodium salt	???6.0％	????6.0％	???6.0％	????6.0％
Tetradecanoic acid	???1.6％	????1.6％	???1.6％	????1.6％
					Bitter salt	???0.3％	????0.3％	???0.3％	????0.3％

(table) is continuous

Trihydroxy-stearic acid R-Glyceric acid	??0.5％	??0.5％	??0.5％	????0.5％
					PEG-6 is sad/the capric tri-glyceride	??3.0％	???-	???-	?????-
??Sucrose?polyesters?of ??cottonate?fatty?acid	??3.0％	???-	???-	?????-
					??Sucrose?polyesters?of ??behenate?fatty?acid	??3.0％	???-	??4.0％	?????-
Vaseline	???-	??4.0％	??8.0％	?????-
					Mineral oil	???-	???-	???-	????6.0％
The DMDM glycolylurea	?0.08％	?0.08％	?0.08％	????0.08％
					It is variant part that fusion rotein comprises variant 14-I/ proteolytic enzyme, BPN 1The Y217L variant be the proteolytic enzyme part	?0.1％	??2.0％	??2.0％	????5.0％
Citric acid	?1.40％	?1.40％	?1.40％	????1.40％

In embodiment 18-21, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace variant 14-I, and its result is similar substantially.

Embodiment 22-25

Clean product

	Example 22	Example 23	Example 24	Example 25
					Water	??66.52％	??65.17％	??68.47％	???68.72％
The EDTA disodium	???0.1％	???0.1％	???0.2％	????0.2％
					Citric acid	????-	????-	???1.4％	????1.4％
Sodium?Laureth-3?Sulfate	???3.0％	???3.5％	????-	?????-
					????Sodium?Laureth-4 ??????Carboxylate	???3.0％	???3.5％	????-	?????-
??????Laureth-12	???1.0％	???1.2％	????-	?????-
					???Polyquaternium10	????-	????-	???0.4％	????0.4％
???Polyquaternium25	???0.3％	???0.3％	????-	?????-
					Glycerine	???3.0％	???3.0％	???3.0％	????3.0％

(table) is continuous

????Sodium ????Lauroamphoacetate	???-	???-	??6.0％	?????6.0％
					Lauric acid	??6.0％	??6.0％	??3.0％	?????3.0％
Tetradecanoic acid	???-	???-	??3.0％	?????3.0％
					Bitter salt	??2.3％	??2.0％	??2.0％	?????2.0％
Trolamine	??4.0％	??4.0％	??4.0％	?????4.0％
					The trihydroxy-stearin	??0.5％	??0.5％	??0.5％	?????0.5％
????Sucrose?polyesters?of ????behenate?fatty?acid	??2.0％	??2.0％	???-	??????-
					????Sucrose?polyesters?of ????cottonate?fatty?acid	??3.0％	??2.0％	???-	??????-
PEG-6 is sad/the capric tri-glyceride	???-	???-	???-	?????2.0％
					Vaseline	???-	???-	??4.0％	??????-
Mineral oil	???-	???-	???-	?????2.0％
					The Cocamidopropyl trimethyl-glycine	??2.0％	??3.0％	??1.8％	?????1.8％
The lauryl dimethyl amine oxide	??1.0％	??1.2％	??1.2％	?????1.2％
					Dex pantoyl ester	??1.0％	?0.25％	?0.25％	??????-
The DMDM glycolylurea	?0.08％	?0.08％	?0.08％	?????0.08％
					It is variant part that fusion rotein comprises variant 24-I/ proteolytic enzyme, BPN 1The Y217L variant be the proteolytic enzyme part	??1.0％	??2.0％	??0.5％	?????0.5％
Spices	??0.2％	??0.2％	??0.2％	?????0.2％

In embodiment 22-25, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace above fusion rotein, and its result is similar substantially.

Embodiment 26-27

The leave-on skin moisturizing compositions

	Example 26	Example 27
			Glycerine	?????5.0％	?????-
Stearic acid	?????3.0％	?????-
			????C 11-13Isoparaffin	?????2.0％	?????-
Glycol stearate	?????1.5％	?????-
			Propenyl ethylene glycol	??????-	????3.0％
Mineral oil	?????1.0％	????10.0％
			Sesame oil	??????-	????7.0％
Vaseline	??????-	????1.8％
			Trolamine	?????0.7％	?????-
Cetyl acetate	?????0.65％	?????-
			Stearin	?????0.48％	????2.0％
The TEA stearate	??????-	????2.5％
			Hexadecanol	?????0.47％	?????-
Lanosterol	??????-	????1.8％
			DEA-phosphoric acid hexadecyl ester	?????0.25％	?????-
Para methyl paraben	?????0.2％	????0.2％
			????Propylparaben	?????0.12％	????0.1％
????Carbomet934	?????0.11％	?????-
			The EDTA disodium	?????0.1％	?????-
It is variant part that fusion rotein comprises variant 13-I/ proteolytic enzyme, BPN 1The Y217L variant be the proteolytic enzyme part	?????0.1％	????0.5％
			Water	?????84.32％	????71.1％

In embodiment 26-27, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace variant 13-I, and its result is similar substantially.

Embodiment 28

The clean wiping composition

Propyleneglycoles	????1.0％
		Texapon Special	????0.6％
Succsinic acid	????4.0％
		Sodium succinate	????3.2％
????Triclosan 	????0.15％
		It is variant part that fusion rotein comprises variant 20-I/ proteolytic enzyme, BPN 1The Y217L variant be the proteolytic enzyme part	????0.05％
Water	????91.0％

Above-mentioned composition is soaked into the woven absorbing sheet of a cellulose and/or polyester, and composition levels is 250% of this absorbing sheet weight.

In embodiment 28, at table 7, the preferred variants that this paper quoted from the variant of quoting again in 8,10 and other variants is used to replace above fusion rotein, and its result is similar substantially.

Sequence table＜110〉Saunders, Charles W.

Correa,Paul?E.

Sun,Yiping

Rubingh, Donn N,＜120〉streptomyces subtilisin occupies stabilized variants＜130 of white pure inhibitor〉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〉white light gray streptomycete (Streptomycas albogriseolus)＜400〉1Asp Ala Pro Ser Ala Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Val 15 10 15Gly Lys Gly Val Ser Ala Thr Thr Ala Ala Pro Glu Arg Ala Val THr

20??????????????????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?Leu?Leu?65??????????????????70??????????????????75??????????????????80Thr?Val?Asp?Gly?Val?Trp?Gln?Gly?Lys?Arg?Val?Ser?Tyr?Glu?Arg?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〉white light gray streptomycete (Streptomyces albogriseolus)＜400〉2Ala Gly Glu Phe Asp Ala Pro Ser Ala Leu Tyr Ala Pro Ser Ala Leu 15 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?Asp?65??????????????????70??????????????????75??????????????????80Pro?Val?Leu?Leu?Thr?Val?Asp?Gly?Val?Trp?Gln?Gly?Lys?Arg?Val?Ser

85??????????????????90??????????????????95Tyr?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 amyloliquefaciens (Bacillus amyloliquetaciens)＜400〉3Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu, 15 10 15His Ser Gln Gly Tyr 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?Val?Ala?Gly?Thr?Val?Ala?Ala?Leu?Asn?Asn?Ser?Ile?Gly?65??????????????????70??????????????????75??????????????????80Val?Leu?Gly?Val?Ala?Pro?Ser?Ala?Ser?Leu?Tyr?Ala?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?Gly?Val?Val?Val?Val?Ala?Ala?Ala?Gly?Asn?Glu?Gly?Thr?Ser?Gly145?????????????????150?????????????????155?????????????????160Ser?Ser?Ser?Thr?Val?Gly?Tyr?Pro?Gly?Lye?Tyr?Pro?Ser?Val?Ile?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

195?????????????????200?????????????????205Leu?Pro?Gly?Asn?Lys?Tyr?Gly?Ala?Tyr?Asn?Gly?Thr?Ser?Met?Ala?Ser

210?????????????????215?????????????????220Pro?His?Val?Ala?Gly?Ala?Ala?Ala?Leu?Ile?Leu?Ser?Lye?His?Pro?Asn225?????????????????230?????????????????235?????????????????240Trp?Thr?Asn?Thr?Gln?Val?Arg?Ser?Ser?Leu?Glu?Asn?Thr?Thr?Thr?Lys

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〉heat-resisting streptomycete (Streptomyces thermotolerans)＜400〉4Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly Glu Ser Ala 15 10 15Ile Ala Ala Thr Pro Glu Arg Ala Val Thr Leu Thr Cys Ala Pro Lys

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

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

50??????????????????55??????????????????60Cys?Thr?Lys?Gln?Tyr?Asp?Pro?Val?Val?Val?Thr?Val?Glu?Gly?Val?Trp?65??????????????????70??????????????????75??????????????????80Gln?Gly?Lys?Arg?Val?Ser?Tyr?Glu?Arg?Thr?Phe?Ser?Asn?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〉cadmium yellow streptomycetes (streptomyces galbus)＜400〉5Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Met Gly His Gly Glu, 15 10 15Ser Ala Ala Ala Val Ser Pro Ala Arg Ala Val Thr Leu Asn Cys Ala

20??????????????????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?Lys?Gln?Tyr?Ala?Pro?Val?Val?Ile?Thr?Val?Asp?Gly?65??????????????????70??????????????????75??????????????????80Val?Trp?Gln?Gly?Lys?Arg?Val?Ser?Tyr?Glu?Arg?Thr?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〉RRT＜213〉blue or green streptomycete far away (Streptomyces azureus)＜400〉6Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly Glu Gly Glu Ser Ala 15 10 15Ala Ala Ala Thr 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?Trp?65??????????????????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〉shallow Streptomyces glaucoviolaceus (Streptomyces lividans)＜400〉7Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly Glu 15 10 15Ser Ala Ala Thr 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?Vel?Asp?Gly?65??????????????????70??????????????????75??????????????????80Val?Trp?Gln?Gly?Arg?Arg?Leu?Ser?Tyr?Glu?Arg?Thr?Phe?Ala?Asn?Glu

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

100 105 110＜210〉8＜211〉110＜212〉PRT＜213〉long spore streptomycete (Streptomyces longisporus)＜400〉8Ala Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly 15 10 15Thr Ser Ala Ala Ala Ala Thr Pro Leu Arg Ala Val Thr Leu Asn Cys

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

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

50??????????????????55??????????????????60Gly?Val?Ile?Cys?Asn?Lys?Leu?Tyr?Asp?Pro?Val?Val?Val?Thr?Val?Asp?65??????????????????70??????????????????75??????????????????80Gly?Val?Trp?Gln?Gly?Lys?Arg?Val?Ser?Tyr?Glu?Arg?Thr?Phe?Gly?Asn

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

100 105 110＜210〉9＜211〉113＜212〉PRT＜213〉small streptomycete (Streptomyces parvulus)＜400〉9Thr Ala Pro Ala Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Ile 15 10 15Gly Gln Gly Glu Ser Ala Ala Ala Thr Ser Pro Leu Arg Ala Val Thr

20??????????????????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?Val?65??????????????????70??????????????????75??????????????????80Thr?Val?Asp?Gly?Val?Trp?Gln?Gly?Arg?Arg?Leu?Ser?Tyr?Glu?Arg?Thr

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

100 105 110Phe＜210〉10＜211〉107＜212〉PRT＜213〉streptomyces coelicolor (Streptomyces coelicolor)＜400〉10Tyr Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly Glu Ser Ala, 15 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?Trp?65??????????????????70??????????????????75??????????????????80Gln?Gly?Arg?Arg?Leu?Ser?Tyr?Glu?Arg?Thr?Phe?Ala?Asn?Glu?Cys?Val

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

100 105＜210〉11＜211〉116＜213〉grey violet ash streptomycete (Streptomyces lavendulae)＜400〉11Ala Pro Aap Ala Ala Pro Ala Ser Leu Tyr Ala Pro Ser Ala Leu Val, 15 10 15Leu Thr Ile Gly His Gly Gly Ala Ala Ala Thr Ala Thr Pro Glu Arg

20??????????????????25??????????????????30Ala?Val?Thr?Leu?Thr?Cys?Ala?Pro?Thr?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?Asp?Val?Trp?Cys?Asn?Lys?Leu?Tyr?Asp?Pro?65??????????????????70??????????????????75??????????????????80Val?Val?Val?Thr?Ala?Gln?Gly?Val?Trp?Gln?Gly?Gln?Arg?Val?Ser?Tyr

85??????????????????90??????????????????95Glu?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〉anti-fibrous strands mould (Streptomyces antifibrinolyticus)＜400〉the 112Gly Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Met Gly His Gly Asn 15 10 15Ser Ala Ala Thr Val Asn Pro Glu Arg Ala Val Thr Leu Asn Cys Ala that separate

20??????????????????25??????????????????30Pro?Thr?Ala?Ser?Gly?Thr?His?Pro?Ala?Ala?Leu?Gln?Ala?Cya?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?Gly?65??????????????????70??????????????????75??????????????????80Val?Trp?Gln?Gly?Lys?Arg?Val?Ser?Tyr?Glu?Arg?Thr?Phe?Ala?Asn?Glu

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

200 205＜210〉13＜211〉107＜212〉PRT＜213〉shallow Streptomyces glaucoviolaceus (Streptomyces lividans)＜400〉13Tyr Ala Pro Ser Ala leu Val Leu Thr Val Gly His Gly Glu Ser Ala 15 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?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?Trp?65??????????????????70??????????????????75??????????????????80Gln?Gly?Arg?Arg?Leu?Ser?Tyr?Glu?Arg?Thr?Phe?Ala?Asn?Glu?Cys?Val

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

100 105＜2l0〉14＜211〉110＜212〉PRT＜213〉Chinese cassia tree type streptoverticillums (Streptoverticillium cinnamoneum)＜400〉14Ser Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr Ile Gly Gln Gly Asp, 15 10 15Ser Ala Ala Ala Ala Gly Ile Gln Arg Ala Val Thr Leu Thr Cys Met

20??????????????????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?Glu?65??????????????????70??????????????????75??????????????????80Gly?Val?Trp?Glu?Gly?Arg?Arg?Val?Ser?Phe?Glu?Arg?Thr?Phe?Ala?Asn

85??????????????????90??????????????????95Pro?Cys?Glu?Leu?Lys?Ala?Gly?Lys?Gly?Thr?Val?Phe?Glu?Phe

100 105 110＜210〉15＜211〉110＜212〉PRT＜213〉cocoa streptomycetes (Streptomyces cacaoi)＜400〉15Ser Leu Tyr Ala Pro Ser Ala Val Val Ile Ser Lys Thr Gln Gly Ala, 15 10 15Ser Ala Asp Ala Pro Ala Gln Arg Ala Val Thr Leu Arg Cys Leu Pro

20??????????????????25??????????????????30Val?Gly?Gly?Asp?His?Pro?Ala?Pro?Glu?Lys?Ala?Cys?Ala?Ala?Leu?Arg

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?Gln?65??????????????????70??????????????????75??????????????????80Gly?Val?Trp?Asp?Gly?Arg?Arg?Ile?Asp?His?Ala?Gln?Thr?Phe?Ser?Asn

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

100?????????????????105?????????????????110

Claims (10)

1. a fusion rotein is characterized by:

(1) one proteolytic enzyme part, and

(2) one variant parts, wherein variant part has the modified amino acid sequence of parent's aminoacid sequence, this modified amino acid sequence is characterised in that it replaces having monoamino-acid corresponding to 63 of SSI, and its parent's aminoacid sequence is selected from the colony that is made up of SSI, SSI sample inhibitor, SSI variant and SSI sample inhibitor variant.

2. the fusion rotein of claim 1, it is further characterized in that a junction branch, wherein the proteolytic enzyme part links to each other with the connection portion covalency with variant part.

3. the fusion rotein of above any claim wherein replaces with Isoleucine at 63 amino acid corresponding to SSI.

4. the fusion rotein of above any claim, wherein parent's aminoacid sequence is selected from the colony that is made up of SSI and SSI variant.

5. the fusion rotein of above any claim, wherein the Ki of variant part performance makes variant part:

(1) arrestin enzyme part in containing the cleaning compositions of fusion rotein, and

(2) when dilution, partly dissociate with proteolytic enzyme.

6. the fusion rotein of above-mentioned any claim, it is selected from the colony that is made up of following:

(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；

(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+D33C；

(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)L63I+G47D+M73P+D83C+S98D；

(iii)L63I+G47D+M73P+D83C+S98E；and

(jjj)L63I+M73P.

7. the DNA of fusion rotein of any aforesaid right requirement encodes.

8. a composition, it contains fusion rotein and a kind of carrier of above any claim, and this carrier is selected from the colony that comprises a kind of cleaning compositions carrier and a kind of Personal hygiene combination carrier.

9. the composition of claim 8, it further contains proteinase inhibitor.

10. expression system, it contains the DNA of claim 7.