CN1372593A - Protease conjugates having sterically protected epitope regions - Google Patents

Abstract

The present disclosure relates to subtilisin protease conjugate comprising a protease moiety and one or more addition moieties. Each addition moiety is covalently attached to an epitope protection position of the protease moiety. The protease conjugates have decreased immunogenicity relative to a parent protease. The present disclosure further relates to cleaning and personal care compositions comprising the protease conjugates.

Description

Protease conjugates with epitope regions of space protection

Invention field

The present invention relates to the subtilisin of chemically modified, this proteolytic enzyme can be used for composition, for example, personal care composition, laundry composition is in hard-surface cleaning compositions and the meticulous fabric cleaning combination.

Background of invention

Enzyme is the natural generation protein of a maximum class.A class wherein is can other proteinic proteolytic enzyme of catalytic hydrolysis.Typically, by natural generation and engineered protein enzyme are spiked in the cleaning combination, made that the ability of this protein hydrolysate is developed in those cleaning combinations of the usefulness of especially doing washing.

In the cleaning field, the most frequently used is serine protease.Most of serine proteases are that bacterium generates, and sub-fraction is wherein generated by other microorganism, for example fungi.Referring to, people such as Siezen " the homology simulation and the protein engineering strategy of subtilisin, subtilisin sample serine stretch protein enzyme family ", protein engineering, Vol.4, No.7, pp.719-737 (1991).Regrettably, the effect of wild-type protease in its natural surroundings often can not be optimized in the non-natural environment of cleaning combination.Specifically, property of protease, for example, thermostability, pH stability, oxidative stability and substrate specificity etc. all may not necessarily reach best user mode outside this proteolytic enzyme natural surroundings.

Now change the wild-type amino acid sequence of serine protease with several method, to strengthen the effect of proteolytic enzyme in the non-natural wash environment.These methods comprise that the hereditary reconstruct of proteolytic enzyme and/or chemically modified are to strengthen thermostability and the oxidative stability of proteolytic enzyme under far different condition.

But, because this proteinoid enzyme is ectogenic for Mammals, so they are potential antigen.As antigen, these proteolytic enzyme can cause that mammiferous immunogen and/or allergen reply (being referred to as immunogenic response among the present invention).

In addition, though obtained significant effect in the lasting research of the more efficient protein enzyme that is used for doing washing by hereditary reconstruct and chemically modified exploitation, this proteinoid enzyme is not also at commercial personal care composition and the meticulous fabric washing composition of being used for.The major cause of not adding proteolytic enzyme in such as products such as soap, gel, body lotion, shampoo and meticulous fabric washing composition is because above-mentioned human body sensitization can cause the problem of bad immunogenic response.Therefore, provide a kind of clean-up performance to make the immunogenic response that is excited reduce to minimum personal care composition or meticulous fabric washing composition simultaneously again and will have crucial meaning with proteolytic enzyme.

At present, can make immunogenic response reduce to minimum with following method: proteolytic enzyme immobilization, granulation, bag quilt or the dissolving of chemically modified is airborne to avoid it to become to proteolytic enzyme.Though these methods have been considered contacting of human consumer and airborne transmission proteolytic enzyme, contact the danger that contacts with dust that contains proteolytic enzyme or aerosol with workman in process of production lastingly but still exist tissue and final composition.

Also proposed can reach the immunogenic purpose that reduces proteolytic enzyme on the proteolytic enzyme by polymkeric substance is connected to.For example, referring to the United States Patent (USP) 4179337 of authorizing Davis etc. of promulgation in, on December 18th, 1979; On January 5th, 1999 promulgation and United States Patent (USP) 5856451 transfer Novo Nordisk by Olsen etc.; On January 7th, 1999 is disclosed and transferred the WO 99/00489 of NovoNordisk by Olsen etc.; On July 16th, 1998 is disclosed and transferred the WO 98/30682 of Novo Nordisk by Olsen etc.; Disclosed with on August 13rd, 1998, the WO 98/35026 of Von Der Osten etc.Yet these propose not show the importance that polymkeric substance is connected to the specific amino acids zone of described proteolytic enzyme in order to reduce immunogenic response most effectively.

Have been found that recently subtilisin comprises three epitope regions and finds one or more polymkeric substance, polypeptide, or other group should be connected the one or more upward to realize that the immunogenic significance of this proteolytic enzyme reduces of these zones.For example referring to U.S. Patent application 09/088912, Weisgerber etc. transfer P﹠G, and June 2 in 1998 submitted.

The inventor has been found that near the space protection one or more epitope regions of this proteolytic enzyme is an immunogenic alternative mechanism of presenting and reduce this proteolytic enzyme that prevents or hinder epi-position.Therefore, the inventor provides the subtilisin of chemically modified, wherein said modification be with close zone, one or more epitope regions space on.Therefore, the inventor has found to excite the immunogenic response that weakens but still has kept their active subtilisins as effective and active protease.Therefore, protease conjugates of the present invention is suitable for including but not limited in the composition of several types, laundry, cleaning tableware, cleaning rigid surface, skin care, hair care, beauty treatment, mouth care and the composition that cleans contact lens.

Summary of the invention

The present invention relates to protease conjugates, this conjugate comprises a proteolytic enzyme part and one or more extention, and wherein each extention all links to each other with the epi-position protective position covalency of described proteolytic enzyme part, wherein:

(a) the epi-position protective position in first epitope regions is selected from corresponding to 1,2,3,4,5,6,7,12,17,36,40,41,43,44,45,67,86,87,89,206,209,210,212,213,214,215 and 216 of subtilisin BPN ';

(b) the epi-position protective position in second epitope regions is selected from corresponding to 25,26,27,46,47,48,49,50,51,52,53 of subtilisin BPN ', 54,91,99,100,101,102,127,128,129,130,131,132,133,134,136,137,138,140,141,144 and 145; With

(c) the epi-position protective position in the 3rd epitope regions is selected from corresponding to 9,10,22,23,24,62,63,143,146,154 of subtilisin BPN ', 155,156,157,172,173,187,189,195,197,203,204,253,254,256,265,267,269,271,272 and 275; And wherein extention has following array structure independently of one another:

Wherein said X does not exist or is a junction branch; R ₁Do not exist or be selected from first polypeptide and first polymkeric substance; R ₂Do not exist or be selected from second polypeptide and second polymkeric substance; Wherein said X, R ₁And R ₂In have at least one not exist.

Protease conjugates of the present invention has the immunogenicity of reduction for parent protease.Therefore, such protease conjugates is suitable for the composition of several types, includes but not limited to: laundry composition, clean tableware, cleaning rigid surface, skin care, hair care, beauty treatment, mouth care and cleaning contact lens composition.

Detailed Description Of The Invention

Main ingredient of the present invention is as described below.Also comprise non restrictive description in addition to the various optional and preferred ingredient that is used for embodiment of the present invention.

The present invention can comprise any one of the present invention must or optional components and/or limit the quantity of, perhaps by or substantially by any one of the present invention must or optional components and/or the institute that limits the quantity of form.

Unless otherwise indicated, all per-cents and ratio all calculate with weight.Unless otherwise indicated, all per-cents all calculate according to total composition.

All components or composition content are reference with the active matter content of this component or composition all, and despumation, for example, may be present in residual solvent or by product in the commercial source product.

Mentioned all documents among the present invention comprise that all patents, patent application and printed publication all are incorporated herein by reference in full at this.

The material of commodity in use name includes, but are not limited to enzyme among the present invention.The inventor does not plan to be subjected to the restriction of a certain trade(brand)name material in the present invention.The equivalent of a certain trade(brand)name material (for example, title or catalogue (reference) number different material that obtains from difference source) can be used for substituting and being used for protease conjugates of the present invention and composition.

The present invention uses abbreviation to describe amino acid.The a series of abbreviations that provide the present invention to use in the table 1:

Table I

The abbreviation of amino acid trigram abbreviation single-letter

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

The term " sudden change " that the present invention uses is meant gene order and/or by the variation in the aminoacid sequence of this gene order generation.Sudden change comprises disappearance, replacement and the interpolation of amino-acid residue in the wild-type protein sequence.

The term " parent " that the present invention uses is meant a kind of protein (wild-type or variant) that can be used for further modifying to form protease conjugates of the present invention.

The term " wild-type " that the present invention uses is meant the protein that is produced by naturally occurring organism, for example proteolytic enzyme or other enzyme.

The term " variant " that the present invention uses is meant that its aminoacid sequence is different from the protein of the proteinic aminoacid sequence of corresponding wild type.

All polymericular weights that the present invention uses are all represented with weight-average molecular weight.

Although conjugate of the present invention is not only limited to those conjugates that contains subtilisin BPN ' and variant thereof, all amino acid numberings are all with reference to the aminoacid sequence of the subtilisin BPN ' that is represented by SEQ ID NO:1.The aminoacid sequence of subtilisin BPN ' further describe people such as seeing Wells, nucleic acids research, Vol.11, pp.7911-7925 (1983).

Protease conjugates of the present invention

Protease conjugates of the present invention is the compound that comprises proteolytic enzyme part and one or more extentions, and wherein said proteolytic enzyme part and extention link to each other by covalently bound (that is covalent linkage).The proteolytic enzyme part

Proteolytic enzyme of the present invention partly is subtilisin sample proteolytic enzyme, wild-type or its variant.The term " subtilisin sample proteolytic enzyme " that the present invention uses is meant that the sequence with subtilisin BPN ' has at least 50%, the proteolytic enzyme of preferred 80% amino acid sequence homology.Wild-type subtilisin sample proteolytic enzyme by, for example, basophilia genus bacillus, bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, bacillus lentus and subtilis microorganisms.Discussion about subtilisin sample serine protease and homology thereof can be referring to people such as Siezen, " the homology simulation and the protein engineering strategy of subtilisin; subtilisin sample serine stretch protein enzyme family ", protein engineering, Vol.4, No.7, pp.719-737 (1991).

The preferred proteolytic enzyme that uses among the present invention partly comprises, for example, the proteolytic enzyme that from bacillus amyloliquefaciens, Bacillus licheniformis and subtilis, obtains, subtilisin BPN, subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, Proteinase K and aluminothermy enzyme comprise A/S Alcalase ^(can be from Novo Industries, Copenhagen, Denmark buys), hope bacterium enzyme (Esperase) ^(Novo Industries), husky fragrant enzyme (Savinase) ^(Novo Industries), Maxatase ^(can buy) from Genencor International Inc., Maxacal ^(GenencorInternational Inc.), Maxapem 15 ^(Genencor International Inc.), and the variant of above-mentioned enzyme.The particularly preferred proteolytic enzyme that uses among the present invention partly comprises proteolytic enzyme and the variant thereof that obtains from bacillus amyloliquefaciens.Most preferred proteolytic enzyme partly is subtilisin BPN ' and variant thereof among the present invention.

Be hereinafter referred to as the particularly preferred subtilisin BPN ' variant that uses among the present invention of " protease A " and be disclosed in the United States Patent (USP) 5 that was presented to Venegas on July 9th, 1991,030,378, above-mentioned variant is characterised in that to have following sudden change in the aminoacid sequence of subtilisin BPN ':

(a) Gly on the 166th is selected from following amino-acid residue and is replaced: Asn, Ser,

Lys, Arg, His, Gln, Ala and Glu; Gly on the 169th is by Ser

Replace; And the Met on the 222nd is selected from following amino-acid residue replacement:

Gln, Phe, His, Asn, Glu, Ala and Thr; Perhaps

B) Gly on the 160th is replaced by Ala, and the Met on the 222nd is replaced by Ala.

Hereinafter be called " proteolytic enzyme B ", be disclosed in the EP-B251 of on January 7th, the 1988 disclosed Genencor of transferring international corporation in the present invention as another preferred subtilisin BPN ' variant of parent's aminoacid sequence, in 446, above-mentioned variant is characterised in that in the aminoacid sequence of wild-type subtilisin BPN ' to have sudden change in following one or more positions: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67, Ser87, Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Met124, Gly127, Gly128, Pro129, Leu 135, Lys170, Tyr171, Pro172, Asp197, Met199, Ser204, Lys2l3, Tyr214, Gly215, and Ser221; Perhaps two places in last column position or many places be selected from following locational one or more sudden changes and combine: Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222.

The preferred subtilisin BPN ' variant of other that uses among the present invention hereinafter is called " proteolytic enzyme C ", and the WO 95/10615 of the disclosed Genencor of transferring international corporation on April 20 nineteen ninety-five is seen in its description, and above-mentioned variant is characterised in that wild-type subtilisin BPN ' aminoacid sequence has a sudden change on the Asn76 position, be combined with and be selected from following locational one or more sudden changes: Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glul95, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265 and Ala274.

Be used for other preferred subtilisin BPN ' variant of the present invention, hereinafter be called " proteolytic enzyme D ", the United States Patent (USP) 4 of authorizing people such as Estell on July 26th, 1988 is seen in its description, 760,025, above-mentioned variant is characterised in that to have the one or more sudden changes that are selected from the following amino acid position: Asp32 in wild-type subtilisin BPN ' aminoacid sequence, Ser33, His64, Tyr104, Asn155, Glul56, Gly166, Gly169, Phe189, Tyr217 and Met222.

Preferred proteolytic enzyme of the present invention partly is selected from: subtilisin BPN ', and protease A, proteolytic enzyme B, proteolytic enzyme C, and proteolytic enzyme D, wherein proteolytic enzyme D is most preferably.

If be not limited to theory, proteolytic enzyme of the present invention partly has three epitope regions: first epitope regions, second epitope regions and the 3rd epitope regions.First epitope regions appears at the site 70-84 corresponding to subtilisin BPN '; Second epitope regions appears at the site 103-126 corresponding to subtilisin BPN '; And the 3rd epitope regions appear at site 220-246 corresponding to subtilisin BPN '.Referring to, for example, on June 2nd, 1998 is by applications such as Weisgerber and U.S. Patent application 09/088,912 that transfer P﹠G; On July 22nd, 1999 is by the common unsettled U.S. Patent application 60/144991 of applications such as Rubingh, " serine protease variants that has aminoacid replacement and disappearance at epitope regions "; With the common unsettled U.S. Patent application 60/144980 of on July 22nd, 1999 by applications such as Sikorski, " serine protease variants that has aminoacid replacement at epitope regions ".

The inventor be surprised to find with at least one above-mentioned epitope regions space on close epi-position protective position.Find that also by one or more extentions are linked to each other with amino acid covalency on the epi-position protective position of proteolytic enzyme part these epi-positions being protected exempts from hydrolysis, and thereby avoid epi-position to expose.

The epi-position protective position that is suitable for carrying out with extention covalent modification depends on that people expect the epi-position of protecting.More preferably, the epi-position protective position at least one extention and first epitope regions is covalently bound.

Have been found that the epi-position protective position in first epitope regions is corresponding to 1,2,3,4,5,6,7,12,17,36,40,41,43,44,45,67,86,87,89,206,209,210,212,213,214,215 and 216 of subtilisin BPN '.More preferably, the epi-position protective position in first epitope regions is corresponding to 1,2,3,4,5,17,40,41,43,67,86,87 and 214 of subtilisin BPN '.

Found further that the epi-position protective position in second epitope regions is corresponding to 25,26,27,46,47,48 of subtilisin BPN '; 49,50,51,52,53; 54,91,99,100,101; 102,127,128,129,130; 131,132,133,134,136; 137,138,140,141,144 and 145.Preferably, the epi-position protective position in second epitope regions is corresponding to 27,47,48,50,52,102,127,128,130,131,132,134,138 and 141 of subtilisin BPN '.

Found further that the epi-position protective position in the 3rd epitope regions is selected from corresponding to 9,10,22,23,24 of subtilisin BPN '; 62,63,143,146,154; 155,156,157,172,173; 187,189,195,197,203; 204,253,254,256,265; 267,269,271,272 and 275.Preferably, the epi-position protective position in the 3rd epitope regions is corresponding to 22,23,24,143,146,155,173,189,197,203,204,253,254,265 and 275 of subtilisin BPN '.

In a preferred embodiment of the invention, proteolytic enzyme partly comprises the sequence of the modification of parent's aminoacid sequence.Parent's aminoacid sequence can be any in the above-mentioned proteolytic enzyme, has preferred limiting factor same as described above.In this embodiment, parent's aminoacid sequence is substituted the amino acid replacement to generate the suitable proteolytic enzyme part that is connected with one or more extentions of the present invention on one or more parent's amino-acid residues.According to the present invention, described replacement should be carried out on one or more epi-position protective position.The epi-position protective position, and preferred limiting factor is as mentioned above.

In order to realize that best one or more extentions are connected with the selectivity of described proteolytic enzyme part on one or more epi-position protective position, described replacement should be carried out with (parent's aminoacid sequence the is exclusive) substituted amino acid that is not present in parent's aminoacid sequence.In this respect, any parent's aminoacid sequence exclusive substituted amino acid can use.For example; because cysteine residues is not present in the wild-type amino acid sequence of subtilisin BPN ', be suitable for the present invention so on one or more epi-position protective position of subtilisin BPN ', replace with one or more cysteine residues.When wherein cysteine residues is present on the position outside the epi-position protective position of parent's aminoacid sequence; preferably replace in above-mentioned each position, to guarantee on the epi-position protective position and one or more extention selectivity couplings with another kind of amino-acid residue.Halfcystine is to be used for the most preferred substituted amino acid that replaces on one or more epi-position protective position.

Other preferred substituted amino acid comprises Methionin.When wherein said substituted amino acid is Methionin; the locational lysine residue that preferably will be present in outside parent's aminoacid sequence epi-position protective position is mutated into another kind of amino-acid residue, so that make the functionalization of one or more lysine residues on this epi-position protective position have selectivity.For example, lysine residue is present on the 170th of subtilisin BPN ', and this position is as the defined epi-position protective position of the present invention.The site selectivity sudden change that comes across all other lysine residues in subtilisin BPN ' sequence can be carried out, then with the lysine residue on the 170th of the extention selectivity functionization.In addition, can make the arbitrary locational amino-acid residue of described epi-position protective position sport Methionin (for example), use extention selectivity functionization on those positions subsequently.Extention

Protease conjugates of the present invention comprises one or more extentions, and wherein each described extention is all covalently bound with 1 amino-acid residue on aforesaid epi-position protective position.Described extention can be any chemical structure.Preferably, this extention spatially hinders the epi-position protective position that is attached thereto, or as defined any other the epi-position protective position of the present invention.The limiting examples of extention includes, but not limited to molecular weight and is lower than approximately 1600, preferably is lower than approximately 800, more preferably less than about 400, and most preferably is lower than about 300 molecule; Polypeptide; And polymkeric substance.Term " polypeptide " used among the present invention is meant the molecule that comprises two or more amino-acid residues.Used term " polymer " among the present invention " be meant any molecule that comprises two or more identical (preferred 5 or a plurality of identical) monomeric unit.

Preferably, described extention has following structure: Wherein said X does not exist or is a connection portion; R ₁Do not exist or be selected from first polypeptide and first polymkeric substance; R ₂Do not exist or be selected from second polypeptide and second polymkeric substance; Wherein X, R ₁And R ₂In at least one does not exist.

Preferably, comprise 1～about 15 in the described protease conjugates, more preferably from about 2～about 10,1～about 5 extentions most preferably from about.

Wherein said R ₁And R ₂When independently being polypeptide portion or polymer moieties separately, R ₁And R ₂Can be identical or different.Preferably, wherein said R ₁When being polypeptide portion, R ₂Do not exist or for polypeptide portion, and most preferably do not exist.Most preferably, R wherein ₁When being polypeptide portion, R ₂Do not exist or for identical polypeptide portion, and most preferably do not exist.Preferably, wherein said R ₁When being polymer moieties, R ₂Do not exist or for polymer moieties.Most preferably, wherein said R ₁When being polymer moieties, R ₂Do not exist or for identical polymer moieties.R wherein ₁And R ₂In at least one when being described first polymkeric substance and second polymkeric substance respectively, X does not preferably exist.Polypeptide portion

Polypeptide portion of the present invention comprises the polypeptide portion that contains 2 or a plurality of amino-acid residues.Preferred polypeptide portion is selected from the protein that comprises enzyme.Preferred enzyme comprises proteolytic enzyme, cellulase, lipase, amylase, peroxidase, microperoxisome, hemicellulase, zytase, Phospholipid hydrolase, esterase, at, polygalacturonase, M-Zyme, reductase enzyme (comprises, for example, the NADH reductase enzyme), oxydase, phenol oxidase, lipoxygenase, ligninase, Starch debranching enzyme, tannase, the xylan polysulfate enzyme, malanases, beta-glucanase, arabinofuranosidase/xylosidase, Unidasa, chondrosulphatase, laccase, transferring enzyme, isomerase (comprises, for example, glucose isomerase and xylose isomerase), lyase, ligase enzyme, the enzyme in synthetic enzyme and fruit source (comprise, for example, papoid).More preferably comprise the enzyme in proteolytic enzyme, cellulase, amylase, lipase and fruit source as the enzyme of polypeptide portion, wherein proteolytic enzyme is more preferred.

Comprise lipase as the example of the lipase of polypeptide portion: the mould genus of humic, Rhodopseudomonas, fusarium, Mucor, chromobacterium, Aspergillus, Candida, geotrichum, Penicillium, Rhizopus and bacillus from following microorganism.

The example of commercialization lipase comprises Lipolase ^, Lipolase Ultra ^, Lipozyme ^, Palatase ^, Novozym435 ^, and Lecitase ^(all these can be from Novo NordiskA/S, Copenhagen, Denmark buys), Lumafast ^(can be available from Genencor, Int., Rochester, NY), and Lipomax ^(Genencor, Int.).

Proteolytic enzyme example as polypeptide portion comprises serine protease, Quimotrase and elastoser type enzyme.Most preferably comprise serine protease as the proteolytic enzyme of polypeptide portion, as the present invention above in the discussion of " proteolytic enzyme part " defined.

Most preferably, when wherein said polypeptide portion was serine protease, described polypeptide portion comprised the definition of the above-mentioned proteolytic enzyme part of the present invention independently.Preferably; as mentioned above; the aminoacid sequence of described polypeptide portion is to the sequence after parent's amino acid sequence modifications, being modified at (this parent's aminoacid sequence can be called " second " parent aminoacid sequence) on one or more aforesaid epi-position protective position wherein.In this case, one of described connection portion (wherein said proteolytic enzyme part does not exist) or proteolytic enzyme part (wherein said connection portion does not exist) are covalently bound to polypeptide portion by 1 substituted amino acid on 1 the epi-position protective position that is present in polypeptide portion.When wherein said polypeptide portion is serine protease, equal preferred, preferred, and most preferred epi-position protective position group is applicable to parent's aminoacid sequence of proteolytic enzyme part and their correspondence as mentioned above.

Most preferably, when wherein said polypeptide portion was serine protease, described polypeptide portion and described proteolytic enzyme partly were to be equal to part.In this case, described polypeptide portion preferably links to each other by disulphide bridges with described proteolytic enzyme part, and wherein X does not exist, and most preferably, R ₂Do not exist.Polymer moieties

Extention of the present invention can comprise polymer moieties.Used term " polymer " part among the present invention ", be meant any molecule that comprises two or more identical (preferred 5 or a plurality of identical) monomeric unit.The example of suitable polymers part comprises the hydrolysate of polyalkylene oxides, polyalcohols, polyvinyl alcohol, polycarbonate, polyvinylpyrrolidone, polyamino acid, Mierocrystalline cellulose, dextran, starch, glycogen, agarose, guar gum, amylopectin, inulin, xanthan gum, carrageenin, pectin, biological polymer, alginic acid hydrolysate and chitosan.Preferred polyalkylene oxides comprises polyoxyethylene glycol, methoxy poly (ethylene glycol) and polypropylene glycol.Preferred dextran comprises Sensor Chip CM 5.Preferred Mierocrystalline cellulose comprises methylcellulose gum, carboxymethyl cellulose, ethyl cellulose, Natvosol, carboxyethyl cellulose and hydroxypropylcellulose.Preferred starch comprises hydroxyethylamyle and hydroxypropylated starch.Preferred polymkeric substance is a polyalkylene oxides.Most preferred polymer moieties is a polyoxyethylene glycol.

R wherein ₁And R ₂When independently being polymer moieties separately, preferably, R ₁And R ₂Set molecular weight (that is R, ₁Molecular weight add R ₂Molecular weight) for about 0.2kD (kilodalton)～about 40kD, more preferably about 0.5kD～about 40kD, more preferably about 0.5kD～about 20kD, and most preferably be about 1kD～about 10kD.

R wherein ₁And R ₂When respectively doing for oneself polymer moieties, preferably, R ₁And R ₂The molecular weight that has 0.1kD～about 20kD independently of one another, more preferably molecular weight is 0.25kD～about 20kD, more preferably about 0.5kD～about 10kD, and most preferably be about 0.5kD～5kD.

R wherein ₁And R ₂When respectively doing for oneself polymer moieties, preferably, R ₁And R ₂The ratio of molecular weight is about 1: 10～about 10: 1, more preferably about 1: 5～about 5: 1, and most preferably be about 1: 3～about 3: 1.

R wherein ₁Be polymer moieties and R ₂When not existing, preferably, R ₁Molecular weight be about 0.1kD～about 40kD, more preferably for about 0.5kD～about 40kD, more preferably about 0.5kD～about 20kD, and most preferably be about 1kD～about 10kD.The connection portion

The X that the present invention uses can not exist or a junction branch; this connection portion and one or more polypeptide portion or one or more polymer moieties or both are covalently bound, and simultaneously also with an epi-position protective position of proteolytic enzyme part on amino-acid residue covalently bound.Described connection portion generally can be any small molecules, that is, molecular weight is less than about 1600, preferably less than about 800, is more preferably less than approximately 400, is more preferably less than about 300 molecule.Most preferred connection portion comprise those can with cysteine residues or the covalently bound connection portion of lysine residue, most preferably cysteine residues.

The example of connection portion and relevant chemistry are disclosed in the United States Patent (USP) of authorizing Harris 5,446,090 of promulgation on August 29 nineteen ninety-five; The United States Patent (USP) of authorizing Merrill 55,171,264 of promulgation on December 15th, 1992; The United States Patent (USP) 5,162,430 of authorizing people such as Rhee of on November 10th, 1992 promulgation; The United States Patent (USP) 5,153,265 of authorizing people such as Shadle of on October 6th, 1992 promulgation; The United States Patent (USP) of authorizing Zalipsky 5,122,614 of promulgation on June 16th, 1992; People such as Goodson, " the fixed point Pegylation of recombinant interleukin-2 on its glycosylation site ", biotechnology, Vol.8, No.4, pp.343-346 (1990); Kogan, " being applicable to the synthetic of methoxyl group-poly-(ethylene glycol) derivative after the replacement of selectivity protein modification ", synthesising communication, Vol.22, pp.2417-2424 (1992); And people such as Ishii, " succinimide ring status to the fluorescence of the α α-tropomyosin of pyrene maleimide mark and the influence of structural performance ", biophysics magazine, Vol.50, pp.75-80 (1986).Most preferred connection portion is (for example, alkyl) or the unsubstituted succinimide that replaces.

As other example, following non-limiting reagent can be used to form described connection portion: N-[α-dimaleoyl imino acetoxyl] succinimide ester; N-5-azido--2-p-nitrobenzoic acid-base succinimide; The dimaleoyl imino hexane; N-[β-dimaleoyl imino propionyloxy] succinimide ester; Two [2-(succinimido oxygen carbonyl oxygen base)-ethyl] sulfone; Two [sulfosuccinimide base] suberate; 1,5-two fluoro-2,4-dinitrobenzene; Dimethyl hexanedioyl imido-ester 2HCl; Dimethyl-g two imide acid esters 2HCl; Dimethyl-octa two imide acid esters 2HCl; Pentanedioic acid two succinimide esters; Disuccinimidyl suberate; Between-dimaleoyl imino benzoyl-N-hydroxyl succinimide ester; N-hydroxyl succinimido-4-azido-Whitfield's ointment; N-succinimido-6-[4 '-azido--2 ' oil of mirbane amino] acid esters; 2,3-dibromo-propionic acid N-hydroxyl succinimide ester; 4-[maleimide ylmethyl] hexanaphthene-1-carboxylic acid succinimide ester; 4-(right-the dimaleoyl imino tolyl) butyric acid succinimide ester; Succinimido-6-[(β-dimaleoyl imino propionamido) capronate]; Two [2-(sulfosuccinimide base oxygen carbonyl oxygen base)-ethyl] sulfone; N-[γ-dimaleoyl imino butyric acid base)] sulfosuccinimide ester; N-hydroxyl sulfosuccinimide base-4-triazobenzene manthanoate; N-[κ-dimaleoyl imino undecane acidic group] the sulfosuccinimide ester; Between-dimaleoyl imino benzoyloxy-N-hydroxyl sulfosuccinimide ester; Sulfosuccinimide base [4-azidosalicylic acid base amino] capronate; Sulfosuccinimide base-7-nitrine-4-methylcoumarin-3-acetic ester; Sulfosuccinimide base-6-[4 '-nitrine-2 '-nitrophenyl amino] capronate; Sulfosuccinimide base-4-[is to azidophenyl] butyric ester; Sulfosuccinimide base [4-sulphur ethanoyl] Aminobenzoate; Sulfosuccinimide base-4-[N-maleimide ylmethyl] hexanaphthene-1-carboxylicesters; With sulfosuccinimide base-4-[right-the dimaleoyl imino phenyl]-butyric ester.Each of these reagent is all from Pierce chemical company, Rockford, and IL buys.

Optional part

Protease conjugates of the present invention can comprise one or more other chemical structures in addition; comprise (for example) and other residue of illustrational proteolytic enzyme or do not having the one or more small molecules that are connected on the epi-position protective position of extention even in the present invention not, polypeptide and/or polymkeric substance (being called as " replenishing part " in this article).Additional part can comprise aforesaid polypeptide portion, polymer moieties, and connection portion.In addition, for example, molecular weight is about 100Da～about 5000Da, be preferably about 100Da～about 2000Da, more preferably be about 100Da～about 1000Da, more preferably for about 100Da～about 750Da, and be most preferably one or more polymkeric substance (most preferably polyoxyethylene glycol) of about 300Da can be covalently bound to the proteolytic enzyme of the present invention residue except that the cited residue of the present invention partly.Use technology of the present invention and well known in the art (comprise by connection portion) as described in the present invention, on any position of this proteolytic enzyme part, such polymer moieties can be directly connected to proteolytic enzyme of the present invention partly on.The limiting examples of the polymer conjugate of this optional type is narration to some extent in disclosed WO 99/00849 on January 7th, 1999 (Olsen etc., Novo NordiskA/S).

The preparation method

The described proteolytic enzyme part that has replacement on one or more epi-position protective position (or any other position of proteolytic enzyme part) is suddenlyd change by the nucleotide sequence to coding parent aminoacid sequence and is prepared from.These class methods are known in the art; A kind of limiting examples of such method is as described below:

To contain wild-type subtilisin BPN ' gene (Mitchison, C. and J.A.Wells, " protein engineering of the middle disulfide linkage of subtilisin BPN ' ", biological chemistry, Vol.28, pp.4807-4815 (1989)) phagemid (pSS-5) is transformed into intestinal bacteria dut-ung-bacterial strain CJ236, and to people such as Yuckenberg, " use contains the DNA of uridylic and the external site-directed mutagenesis of phagemid carrier ", the method of site-directed mutagenesis-a kind of practicality, McPherson, M.J. writes, behind the method improvement of describing in pp.27-48 (1991) one literary compositions, with the VCSM13 helper phage (people such as Kunkel, " and do not need Phenotypic Selection fast; effective site-directed mutagenesis ", Enzymology method, Vol 154, pp.367-382 (1987)) preparation contains the single stranded DNA template of uridylic.From Zoller, M.J. and M.Smith, " carry out oligonucleotide-directed mutagenesis with M13 deutero-carrier: a kind of effective universal method that is used for producing point mutation " at arbitrary dna fragmentation, nucleic acids research, Vol.10, disclosed method is improved and the primer site-directed mutagenesis that comes prepares all mutant (basically as people such as Yuckenberg, above-mentioned) in pp.6487-6500 (1982) one literary compositions.

With 380B dna synthesizer (Applied Biosystems Inc.) preparation oligonucleotide.The mutagenesis reaction product is transformed into intestinal bacteria MM294 bacterial strain (American type culture collection intestinal bacteria 33625).All sudden changes all confirm through dna sequencing, and separated DNA is transformed into Bacillus subtilus expression strain PG632 (people such as Saunders, " optimization of the signal sequence cleavage site of secretion human parathyroid hormone 34-amino acid fragment from Bacillus subtilus ", gene, Vol.102, people such as pp.277-282 (1991) and Yang, " application in setting up external deutero-deletion mutantion of the clone of bacillus subtilis neutral proteinase gene and this clone gene ", the bacteriology magazine, Vol.160, pp.15-21 (1984).

Ferment as follows.The bacillus subtilis cell (PG632) that will contain the target protein enzyme with one liter of LB meat soup that contains 10g/L glucose is cultured to mid-log phase, and be inoculated into cumulative volume be 9 liters Biostat C fermentor tank (Braun Biotech, Inc., Allentown, PA) in.The starch (Maltrin M-250), defoamer, damping fluid and the trace mineral that contain yeast extract, caseic hydrolysate, water-soluble portion hydrolysis in the fermention medium are (referring to " genus bacillus biology: industrial application ", Doi, R.H. and M.McGloughlin, write (1992)).The pH of meat soup is constant in the fermenting process remains on 7.5.Add kantlex (500 μ g/mL) the mutagenesis plasmid is carried out the microbiotic selection.Cell is cultivated 18 hours down until A at 37 ℃ ₆₀₀Be about 60, the results product.

Obtain pure proteolytic enzyme by following step process fermenting broth.With 0.16 μ m filter membrane by the bacillus subtilis cell in the tangential flow filtering meat soup.Then, cut off the acellular meat soup of filter membrane ultrafiltration and concentration with 8,000 molecular weight.With the MES damping fluid (2-(N-morpholino) ethyl sulfonic acid) after concentrating the pH value is transferred to 5.5.Be further purified this proteolytic enzyme with the S-sepharose by cation-exchange chromatography, and use the NaCl gradient elution.Referring to Scopes, R.K. " protein purification principle and operation ", Springer-Verlag, New York (1984)

Measure the activated protein enzyme concn in the fraction of collecting during the gradient elution with pNA assay method people such as (, analytical chemistry, Vol.99, pp.316-320 (1979)) DelMar.This assay method discharges the speed of p-Nitroaniline in the time of can measuring the synthetic substrate succinyl--Ala-Ala of this protease hydrolysis solubility-proline(Pro)-phenylalanine-p-Nitroaniline (sAAPF-pNA).Measure at the 410nm place by hydrolysis reaction generation xanchromatic speed with spectrophotometer, the concentration of this speed and active protease part is proportional.In addition, determine total protein concentration with the absorbance measuring value at 280nm place.The ratio of active protease/total protein provides proteolytic enzyme purity, and can be used to identify the fraction in the stoste of collecting.

The autolysis of proteolytic enzyme in the storage process such as adds at the propylene glycol of weight in the mixing fraction that obtains from chromatography column.After purge process is finished, detect the purity of proteolytic enzyme stoste with SDS-PAGE (SDS-PAGE), with II type trypsin inhibitor-T: turkey egg white (Sigma Chemical Company, St.Louis Missouri) measures absolute enzyme concn by the avtive spot titration method.

In for the preparation of using, by Sephadex-G25 (Pharmacia, Piscataway, NeW Jersey) size-exclusion column eluted protein proenzyme liquid removing propylene glycol, and exchange buffering liquid.MES buffer exchange in the proenzyme liquid is become 0.01M KH ₂PO ₄Solution, pH 5.5.

Can generate protease conjugates with the proteolytic enzyme that makes and one or more extention functionalization.Preferred activation extention precursor is to strengthen itself and the reactivity of proteolytic enzyme part precursor (protease conjugates that extention precursor and the generation of proteolytic enzyme part precursors reaction partly are made up of described extention and described proteolytic enzyme).This class activation method is known in the art.Provide protease conjugates preparation method's non-limiting example below.

Embodiment 1

According to such scheme; (wherein " P " expression deducts the proteolytic enzyme part that is replaced the sulfydryl that generates by halfcystine will to contain the proteolytic enzyme of cysteine residues and a polymer moieties coupling with following method on 1 epi-position protective position; n is polyalkylene glycol monomer number of repeating units (for example, a n=77)).

Replace the tyrosine on the 217th and replace the variant that Serine on the 161st prepares subtilisin BPN ' with leucine with halfcystine.The concentration of this variant in phosphate buffered saline buffer (pH5.5) reaches about 2mg/mL.Then, with rare sodium hydroxide solution pH is risen to 7.5.With the ratio of 25: 1 activated polymers, above-mentioned variant is mixed with monomethyl polyoxyethylene glycol maleimide with variant.After mixing 1 hour at ambient temperature, the pH of mixture is transferred to 5.5, and cut off ultra-fine filter with molecular weight and remove by filter excessive polymkeric substance with dilute phosphoric acid.The protease conjugates that contains purifying in the enriched material.

Embodiment 2

According to such scheme; the proteolytic enzyme part and a polymer moieties coupling (wherein " P " expression deducts the proteolytic enzyme part that is replaced the sulfydryl that generates by halfcystine, and n is the abundant number (for example n=77) of polyalkylene glycol monomer repeating unit) that will on 1 epi-position protective position, contain cysteine residues with following method.

Replace the tyrosine on the 217th and replace the variant that phenylalanine on the 261st prepares subtilisin BPN ' with leucine with halfcystine.The concentration of this variant in phosphate buffered saline buffer (pH 5.5) reaches about 2mg/mL.Then, with rare sodium hydroxide solution pH is risen to 7.5.With the ratio of 25: 1 activated polymers, above-mentioned variant is mixed with the dimethyl polyethylene glycol maleimide with variant.After mixing 1 hour at ambient temperature, the pH of mixture is transferred to 5.5, and cut off ultra-fine filter with molecular weight and remove by filter excessive polymkeric substance with dilute phosphoric acid.The protease conjugates that contains purifying in the enriched material.

Embodiment 3

The polymkeric substance of succinimide protection is coupled to optionally (wherein " MPEG " and " PEGM " is coordinator on the Methionin on one or more epi-position protective position; represent the monomethyl polyoxyethylene glycol, wherein " P " representative deducts the proteolytic enzyme part of illustrated Methionin amido):

Embodiment 4

The polymkeric substance of carbodiimide protection is coupled to optionally (wherein " MPEG " and " PEGM " is coordinator on the Methionin on one or more epi-position protective position; represent the monomethyl polyoxyethylene glycol; " P " representative deducts the proteolytic enzyme part of illustrated Methionin amido; X and X ' are for containing the side chain of carbodiimide part, for example alkyl):

Embodiment 5

The proteolytic enzyme part and alkyl maleimide coupling (wherein " P " expression deducts the proteolytic enzyme part that is replaced the sulfydryl that generates by halfcystine, and " R " is alkyl) that will on 1 epi-position protective position, contain cysteine residues with following method.Among this embodiment, R ₁And R ₂Not existing separately, is derived by alkyl maleimide in the connection portion.

Replace the tyrosine on the 217th and replace the variant that Serine on the 163rd prepares subtilisin BPN ' with leucine with halfcystine.Use 0.01M KH ₂PO ₄Damping fluid (pH 7) preparation 20mL concentration is about the solution of the above-mentioned variant of 1mg/mL.In this solution, add 1.5 normal alkyl maleimides.Slightly mixed this solution at ambient temperature 1 hour.From above-mentioned solution, obtain the protease conjugates of generation with standard method.

Embodiment 6

The proteolytic enzyme that contains cysteine residues with following method on 1 epi-position protective position partly forms dimer (wherein " P " expression deducts the proteolytic enzyme part that halfcystine replaces the sulfydryl that generates).Among this embodiment, described proteolytic enzyme part and polypeptide portion are (and X do not exist) that is equal to.

Replace the tyrosine on the 217th and replace the variant that Serine on the 163rd prepares subtilisin BPN ' with leucine with halfcystine.Use 0.01M KH ₂PO ₄Damping fluid (pH8.6) preparation 20mL concentration is about the solution of the above-mentioned variant of 1mg/mL.At ambient temperature, in solution, be blown into oxygen lightly about 1 hour, generate desirable protein enzyme conjugate dimer.From above-mentioned solution, obtain the protease conjugates of generation with standard method.

Analytical procedure

Can test the enzymatic activity and the immunogenic response of protease conjugates of the present invention with following method, these two kinds of methods all are well known by persons skilled in the art.Other method well known in the art also can substitute use.

The protease conjugates activity

Can measure the protease activity of protease conjugates of the present invention with method well known in the art.Two kinds of such methods are described below: the skin scrapings activity methods

With Scotch  #3750G band, strip the human body skin chip from experimenter's shank repeatedly and on belt, be paved with chip substantially.Then, belt is cut into 1 square inch square, put aside.In 10mm * 35mm Petri dish, control enzyme (for example, subtilisin BPN ') or the testing protein enzyme conjugate of 2mL 0.75mg/mL joined 0.01MKH ₂PO ₄In pH 5.5 damping fluids.In this solution, add 1mL 2.5% sodium laurate pH 8.6 solution.Solution is placed mixing gently on the platform vibrator.The above-mentioned square band that makes was soaked in the solution (chip is towards last) 10 minutes under constantly mixing gently.Then, with tap water gently the rinsing square be with 15 seconds.(3mL, available from Sigma Chemical Co., St.Louis MO) inhales and moves on in the clean Petri dish with the blue dye liquor of Stevenel.Square band with rinsing under mixing gently placed dye liquor (chip is towards last) 3 minutes.From dye liquor, take out the square band, rinsing continuously in two beakers that fill 300mL distilled water, each 15 seconds.Dry air should the square band.Vision or the square band that relatively obtains from control enzyme with colorimeter and difference from colour intensity between the square band of protease conjugates acquisition.Compare with control enzyme square band, the colour intensity of protease conjugates square band a little less than, this explanation protease conjugates active higher.Painted collagen activity methods

50m is contained 0.01M CaCl ₂0.1M tris damping fluid (three-methylol-aminomethane) (pH 8.6) and 0.5g azocoll (collagen of azo pigment dipping, available from Sigma Chemical Co., St.Louis MO) mixes.25 ℃ of these mixtures of following incubation mix on oscillator plate simultaneously gently.Filter this mixture of 2mL with 0.2 micron injection filter, read the absorbancy of this mixture spectrophotometric setter adjustment is made zero at the 520nm place.In remaining 48mltris/azocoll mixture, add 1ppm control enzyme (for example, subtilisin BPN ') or testing protein enzyme conjugate.In time of 10 minutes altogether, filtered the solution that 2mL contains contrast/protease conjugates with 0.2 micron injection filter every 2 minutes.For the sample after every part of filtration, read the absorbancy at 520nm place immediately.Drawing result is with respect to the curve of time.What the slope of contrast and conjugate to be measured showed is the relative reactivity of this sample.The big more expression activity of slope is high more.Testing protein enzyme conjugate activity (slope) can be expressed as the per-cent of contrast activity (slope).

Be used to measure test in the immunogenic mouse nose

Measure in the immunogenic mouse nose immunogenicity potentiality that test can be measured protease conjugates of the present invention with means known in the art or by following being used to of this paper.This test is similar to Robinson etc., " mouse is to the specific antibody responsing reaction of subtilisin Carlsberg (Alcalase): expose the development of model in a kind of nose ", basis and applied toxicology (Fundamental and Applied Toxicology), Vol.34,15-24 page or leaf (1996) and Robinson etc., " utilize and test the allergen effectiveness that (MINT) measures detergent enzyme in the mouse nose: " with the comparison of (GPIT) test in the guinea pig trachea, the toxicology science, Vol.43, the assay method of describing in the 39-46 page or leaf (1998), these two kinds of assay methods can be used for replacing test hereinafter described.

Weight is approximately 18 to the about 20 BDF1 female mices that restrain, and (Charles River laboratory, Portage MI) are used to this test.Before the administration these mouse are isolated a week.These mouse are closed in the cage that is placed with wood chip pad grass, and these cages are placed in the room of controlling moisture (30-70%) and temperature (67-77), carry out light and shade alternative circulation in 12 hours in this room.The arbitrarily edible Purina of these mouse ^Mouse food (Purina Mills, Richmond, IN) and water.

With potential antigen to be measured (as the Bacillus subtilus BPN ' of positive control or a kind of proteolytic enzyme of intermolecular cross-linking of the present invention) 5 mouse in a group are carried out administration.Before the administration, every mouse is anaesthetized by the mixture of intraperitoneal (i.p.) injection Ketaset (88.8mg/kg) and Rompun (6.67mg/kg).Dopey mouse is held in the palm, and the back of the body has dissolved damping fluid (the 0.01M KH of proteolytic enzyme down with 5mL ₂PO ₄, pH5.5) carry out intranasal administration.When every group of dosage is identical, can test at different dosage.Outside the nostril of every mouse and by mouse, suck in the place for the drug solns quilt gently.The 3rd, 10,17 and 24 days repeat administrations.

Collected serum sample at the 29th day.By enzyme-specific IgG 1 antibody in the antigen capture ELISA method mensuration mice serum.Utilize the ED of standard ₅₀Value can compare the immunogenicity of protease conjugates and subtilisin BPN '.

Composition of the present invention

Protease conjugates of the present invention can be used for being fit to use arbitrary situation of parent protease separately.Such example comprises cleaning compositions.Because the desirable immunogenicity that weakens of protease conjugates of the present invention, this protease conjugates can also be used for once from using the benefited minimum situation of proteolytic enzyme.The example of this class purposes comprises the situation that protease conjugates must closely contact with mammal skin (particularly human body skin), for example wherein uses personal care composition.

Cleaning compositions

Described protease conjugates can be used for cleaning compositions and includes, but not limited to laundry composition, rigid surface cleaning combination, meticulous fabric cleaning combination and comprise the wash dishes composition, and the automatic dishwasher detergent composition.

Cleaning compositions of the present invention comprises one or more protease conjugates of the present invention and the cleaning compositions carrier of significant quantity.

" protease conjugates of significant quantity " or similar statement that the present invention uses are meant the amount that is enough to reach the protease conjugates of desirable proteins hydrolytic activity in the specific cleaning compositions.Described significant quantity can easily be determined by those of ordinary skills, and depend on multiple factor, the concrete kind of used protease conjugates for example, the purposes of cleaning, the concrete composition of cleaning compositions, and what need to use is liquid or dried (for example, particulate state, bar-shaped) composition, etc.Preferably, comprise in the described cleaning compositions about 0.0001%～about 10%, more preferably from about 0.001%～1%, and one or more protease conjugates of the present invention of 0.01%～about 0.1% most preferably from about.Several examples that protease conjugates can be used for wherein different cleaning composition have been discussed in more detail below.

Except that protease conjugates of the present invention, cleaning compositions of the present invention also comprises the cleaning compositions carrier that wherein contains one or more cleaning compositions materials compatible with protease conjugates.The term " cleaning compositions material " that the present invention uses (for example is meant any selected required composition that is used for particular type and product form, liquid, particle, piece, sprays, rod, cream, gel) material, these materials also are compatible with the protease conjugates that is used for composition.The concrete selection of cleaning compositions material can be by considering surfacing to be cleaned, easily carrying out for the required composition forms of in use the cleaning condition use of washing composition (for example, by).Term used herein " compatible " is meant that the cleaning compositions material can not make the proteolytic activity of protease conjugates be reduced to such degree, and promptly proteolytic enzyme is effective unlike desired under normal service condition.Hereinafter illustrated in greater detail concrete cleaning compositions material.

Protease conjugates of the present invention can be used for expecting in foam is abundant and cleaning effect the is good multiple detergent composition.Therefore, protease conjugates can use with various conventional ingredients, and rigid surface clean-out system, wash dishes composition, fabric cleaning composition of abundant preparation etc. are provided.This based composition can liquid, particle, form such as bar-shaped use.This based composition can be mixed with " concentrating " washing composition, wherein contains up to about 30%～about 60% (weight) tensio-active agent.

Cleaning compositions of the present invention can be randomly, and preferably, comprise various tensio-active agents (for example, negatively charged ion, nonionic or zwitterionics).Typically, the content of this class tensio-active agent in composition about 5%～about 35%.

The limiting examples that is used for tensio-active agent of the present invention comprises conventional C ₁₁-C ₁₈Alkylbenzene sulfonate and uncle and random alkyl-sulphate; General formula CH ₃(CH ₂) _X(CHOSO ₃) ^-M ⁺) CH ₃And CH ₃(CH ₂) _y(CHOSO ₃ ^-M ⁺) CH ₂CH ₃C ₁₀-C ₁₈Secondary (2,3) alkyl-sulphate, wherein x and (y+l) be to be at least about 7 integer preferably be at least about 9, and M is a kind of water miscible positively charged ion, particularly sodium; C ₁₀-C ₁₈Alkyl alkoxy sulfate (particularly EO 1-5 ethoxy sulfate); C ₁₀-C ₁₈Alkyl alkoxy carboxylate salt (particularly EO 1-5 ethoxy carboxylate); C ₁₀-C ₁₈Alkyl polyglucoside and corresponding sulfation polyglucoside thereof; C ₁₂-C ₁₈α-sulfonated fatty acid ester; C ₁₂-C ₁₈Alkyl and alkyl phenolic alkoxy thing (particularly ethoxylate and blended oxyethyl group/propoxy-); C ₁₂-C ₁₈Trimethyl-glycine and sultaine; C ₁₀-C ₁₈Amine oxide etc.Preferred alkyl alkoxy sulfate of the present invention (AES) and alkyl alkoxy carboxylate salt (AEC).In addition, also preferably according to prescription teacher's hope with this class tensio-active agent and amine oxide and/or trimethyl-glycine or the coupling of sultaine tensio-active agent.The useful tensio-active agent of other routine is listed in the standard textbook.Useful especially tensio-active agent comprises C ₁₀-C ₁₈N-methylglucosamine, its description are seen the United States Patent (USP) 5,194,639 of authorizing people such as Connor of on March 16th, 1993 promulgation.

Can also comprise multiple other composition that can in the washing composition cleaning compositions, bring into play effect in the composition of the present invention, comprising, for example, other activeconstituents, carrier, hydrotropic agent, processing aid, dyestuff or colorant and the solvent that is used for liquid preparation.If wish the extra foam that increases, then can in composition, add C ₁₀-C ₁₆Suds boosters such as alkylolamide, typically add-on is about 1%～about 10%.C ₁₀-C ₁₄One glycollic amide and diglycollic amide are represented such suds booster of a quasi-representative.With this class suds booster and efficient foaming cosurfactant, for example above-mentioned amine oxide, trimethyl-glycine and sultaine coupling also are favourable.If desired, can add MgCl ₂, MgSO ₄Etc. the solubility magnesium salts, typically about 0.1%～about 2%, so that extra foam to be provided.

Liquid detergent compositions of the present invention can comprise water and other solvent as carrier.The example of low-molecular-weight uncle or secondary alcohol has methyl alcohol, ethanol, propyl alcohol and Virahol, and they all are suitable.Preferably dissolve tensio-active agent, but the polyvalent alcohol (for example, 1, ammediol, ethylene glycol, glycerol and 1,2-propylene glycol) that contains have an appointment 2～about 6 carbon atoms and about 2～about 6 hydroxyls can use also with monohydroxy-alcohol.That described composition can comprise is about 5%～and about 90%, this class carrier of typically about 10%～about 50%.

Preferably be mixed with detergent composition so that in aqueous cleaning operation between the usage period, the pH value of bath water is about 6.8～about 11.Therefore, typically finished product is prepared in this scope.The technology that the pH value is controlled at the recommendation usage level comprises, for example use of damping fluid, alkali and acid.This class technology is well known to those skilled in the art.

When preparation rigid surface cleaning combination of the present invention and fabric cleaning composition, the prescription teacher can wish to use about washing assistant of 5%～about 50% (weight).Typical washing assistant comprises 1-10 micron zeolite, polycarboxylate for example Citrate trianion and oxo disuccinate, stacked silicate, phosphoric acid salt etc.Other conventional washing assistant is seen the standard recipe handbook.

Equally, the prescription teacher can use various other enzymes in this based composition, for example cellulase, lipase, amylase and proteolytic enzyme, and typical amounts is about 0.001%～about 1% (weight).Various detergency enzymes and enzyme fabric care are all known by the detergent for washing clothes field.

Various bleaching compounds, for example percarbonate, perborate etc. also can be used for this based composition, and typical amounts is about 1%～about 15% (weight).If desired, this based composition can also comprise bleach-activating agent, for example tetraacetyl ethylene diamine, nonanoyl hydroxy benzene sulfonate etc., and these also are known in the art.Typical amounts is about 1%～about 10% (weight).

Stain remover, especially negatively charged ion oligomer ester type, chelating, especially phosphoro-amidate and quadrol disuccinate, clay soil remover, especially ethoxylation tetren, dispersion agent, especially polyacrylate and polyaspartic acid salts, whitening agent, especially negatively charged ion whitening agent, suds suppressor, especially siloxanes and secondary alcohol, fabric softener, especially smectic clays etc. may be used in this based composition, and consumption is about 1%～35% (weight).All comprised the detailed description of enriching of this class conventional material in standard recipe handbook and the disclosed patent.

Enzyme stabilizers also can be used for cleaning compositions.This fermentoid stablizer comprises propylene glycol (being preferably about 1%～about 10%), sodium formiate (being preferably about 0.1%～about 1%) and calcium formiate (being preferably about 0.1%～about 1%).

Protease conjugates of the present invention also can be used for the rigid surface cleaning combination." rigid surface cleaning combination " that the present invention uses be meant and be used to clean rigid surface, for example liquid the and granular detergent composition of floor, wall, bathroom tile etc.Rigid surface cleaning combination of the present invention comprises one or more protease conjugates of the present invention of significant quantity, protease conjugates content in the preferred composition is about 0.001%～about 10%, more preferably from about 0.01%～and about 5%, 0.05%～about 1% (weight) more preferably from about also.Except that containing one or more protease conjugates, this class rigid surface cleaning combination typically also comprises tensio-active agent and water miscible chelating washing assistant.But, at some special product, for example in the spray-type window clean-out system, not using described tensio-active agent sometimes, this is because they may produce film like and/or mottled vestiges at glass surface.

When containing surface active agent composition, its content in the present composition can be low to moderate 0.1%, still, typically, in the said composition content of tensio-active agent be about 0.25%～about 10%, more preferably from about 1%～about 5%.

Typically, comprise about detergent auxiliary of 0.5%～about 50% in the described composition, preferred about 1%～about 10%.

Preferred pH scope should be about 7～12.Regulate pH if desired, pH regulator agent commonly used, for example sodium hydroxide, yellow soda ash or hydrochloric acid can use.

Solvent also can be included in the described composition.Useful solvent includes, but not limited to for example diglycol monotertiary hexyl ether of glycol ether, the diglycol monotertiary butyl ether, ethylene glycol monobutyl ether, ethylene glycol mono hexyl ether, propylene glycol single-butyl ether, the dipropylene glycol single-butyl ether, and glycol for example 2,2,4-trimethylammonium-1,3-pentanediol and 2-ethyl-1, the 3-hexylene glycol.In use, the typical amounts of this kind solvent be about 0.5%～about 15%, more preferably from about 3%～about 11%.

In addition, when not cleaning this surface after the surface that will clean " full strength " uses composition of the present invention, can use the high volatile volatile solvent in said composition, for example Virahol or ethanol are to accelerate the evaporation that composition is gone up on the surface.During use, the typical amounts of volatile solvent in composition is about 2%～about 12%.

Embodiment 7-12

Liquid rigid surface cleaning combination

	Embodiment 7	Embodiment 8	Embodiment 9	Embodiment 10	Embodiment 11	Embodiment 12
							Protease conjugates among the embodiment 3	??0.05％	??0.50％	??0.02％	??0.03％	??0.30％	??0.05％
EDTA	??-	??-	??2.90％	??2.90％	??-	??-
							Trisodium Citrate	??-	??-	??-	??-	??2.90％	??2.90％
Sodium dodecylbenzene sulfonate	??1.95％	??-	??1.95％	??-	??1.95％	??-
							Sodium lauryl sulphate	??-	??2.20％	??-	??2.20％	??-	??2.20％
C 12(oxyethyl group) sodium sulfate	??-	??2.20％	??-	??2.20％	??-	??2.20％
							C 12The dimethylamine oxide compound	??-	??0.50％	??-	??0.50％	??-	??0.50％
Cumene sodium sulfonate	??1.30％	??-	??1.30％	??-	??1.30％	??-
							The hexyl Trivalin SF	??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 another embodiment of the invention, the wash dishes composition comprises one or more protease conjugates of the present invention." the wash dishes composition " that the present invention uses is meant the composition of the form of ownership that is used to clean tableware, includes but not limited to granular and liquid form.

Embodiment 13-16

Liquid tableware detergent

	Embodiment 13	Embodiment 14	Embodiment 15	Embodiment 16
					Protease conjugates among the embodiment 1	??0.05％	??0.50％	??0.02％	??0.40％
C 12-C 14The N-methylglucosamine	??0.90％	??0.90％	??0.90％	??0.90％
					C 12Oxyethyl group (1) vitriol	??12.0％	??12.0％	??12.0％	??12.0％
2-methyl undecanoic acid	??4.50％	??4.50％	??4.50％	??4.50％
					C 12Oxyethyl group (2) carboxylate salt	??4.50％	??4.50％	??4.50％	??4.50％
C 12Fatty alcohol ethoxylate (4)	??3.00％	??3.00％	??3.00％	??3.00％
					C 12Amine oxide	??3.00％	??3.00％	??3.00％	??3.00％
Cumene 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.

Embodiment 17-19

Liquid fabric cleaning composition

	Embodiment 17	Embodiment 18	Embodiment 19
				Protease conjugates among the embodiment 4	????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 10Fatty alcohol ethoxylate (3)	????13.0％	????13.0％	????13.0％
Monoethanolamine MEA BASF	????2.50％	????2.50％	????2.50％
				Water/propylene glycol/ethanol (100: 1: 1)	????58.45％	????58.47％	????58.20％

Personal care composition

Protease conjugates of the present invention is particularly suitable for personal care composition, for example retention type and the agent of rinsing type hair care, shampoo, retention type and rinsing type control acne composition, Cleansing Foam and amendment, shower gels, soap, foam and clean agent of non-foam type, makeup, hand with, face with and body and function softener, wetting Agent for Printing Inks, stick agent and facial mask, retain profile portion heat preserving agent, make up and cleaning liniment, oral care composition, catamenials and contact lens care composition.Personal care composition of the present invention comprises one or more protease conjugates of the present invention and personal care carrier.

For convenience of explanation, protease conjugates of the present invention is fit to be included in the composition described in the following reference: the United States Patent (USP) 5,641,479 (skin cleaner) of authorizing people such as Linares of on June 24th, 1997 promulgation; The United States Patent (USP) 5,599,549 (skin cleaner) of authorizing people such as Wivell of on February 4th, 1997 promulgation; The United States Patent (USP) 5,585,104 (skin cleaner) of authorizing people such as Ha of on December 17th, 1996 promulgation; The United States Patent (USP) 5,540,852 (skin cleaner) of authorizing people such as Kefauver of on July 30th, 1996 promulgation; The United States Patent (USP) 5,510,050 (skin cleaner) of authorizing people such as Dunbar of on April 23rd, 1996 promulgation; The United States Patent (USP) 5,612,324 (anti-acne preparation) of authorizing people such as Guang Lin of on March 18th, 1997 promulgation; The United States Patent (USP) 5,587,176 (anti-acne preparation) of authorizing people such as Warren of on December 24th, 1996 promulgation; The United States Patent (USP) of authorizing Venkateswaran 5,549,888 (anti-acne preparation) of promulgation on August 27th, 1996; The United States Patent (USP) 5,470,884 (anti-acne preparation) of authorizing people such as Corless of November 28 nineteen ninety-five promulgation; The United States Patent (USP) 5,650,384 (shower gels) of authorizing people such as Gordon of on July 22nd, 1997 promulgation; The United States Patent (USP) 5,607,678 (shower gels) of authorizing people such as Moore of on March 4th, 1997 promulgation; The United States Patent (USP) 5,624,666 (hair conditioner and/or shampoo) of authorizing people such as Coffindaffer of on April 29th, 1997 promulgation; The United States Patent (USP) 5,618,524 (hair conditioner and/or shampoo) of authorizing people such as Bolich of on April 8th, 1997 promulgation; The United States Patent (USP) of authorizing Inman 5,612,301 of promulgation on March 18th, 1997, (hair conditioner and/or shampoo); The United States Patent (USP) of authorizing Wells 5,573,709 (hair conditioner and/or shampoo) of promulgation on November 12nd, 1996; The United States Patent (USP) of authorizing Pings 5,482,703 (hair conditioner and/or shampoo) of promulgation on January 9th, 1996; The United States Patent (USP) Re.34 that authorizes people such as Grote that on April 12nd, 1994 issued again, 584 (hair conditioner and/or shampoos); The United States Patent (USP) 5,641,493 (makeup) of authorizing people such as Date of on June 24th, 1997 promulgation; The United States Patent (USP) 5,605,894 (makeup) of authorizing people such as Blank of on February 25th, 1997 promulgation; The United States Patent (USP) 5,585,090 (makeup) of authorizing people such as Yoshioka of order promulgation December 17 in 1996; The United States Patent (USP) 4,949,179 of authorizing people such as Cheney of July 3 nineteen ninety promulgation (hand with, face with and the body and function softener); The United States Patent (USP) 5,607,980 of authorizing people such as McAtee of on March 4th, 1997 promulgation (hand with, face with and the body and function softener); The United States Patent (USP) 4,045,364 (cosmetic or Clean-liniment) of authorizing people such as Richter of on August 30th, 1977 promulgation; October in 1994 people such as disclosed Touchet on the 12nd european patent application, EP 0 619 074 (cosmetic or Clean-liniment); The United States Patent (USP) 4,975,217 (cosmetic or Clean-liniment) of authorizing people such as Brown-Skrobot of December 4 nineteen ninety promulgation; The United States Patent (USP) of authorizing Seibel 5,096,700 (oral cleaning composition) of promulgation on March 17th, 1992; The United States Patent (USP) of authorizing Sampathkumar 5,028,414 (oral cleaning composition) of promulgation on July 2nd, 1991; The United States Patent (USP) 5,028,415 (oral cleaning composition) of authorizing people such as Benedict of on July 2nd, 1991 promulgation; The United States Patent (USP) 4,863,627 (contact lens cleaning combination) of authorizing people such as Davies of on September 5th, 1989 promulgation; The United States Patent (USP) Re.32 that authorizes people such as Huth of on May 24th, 1988 promulgation, 672 (contact lens cleaning combinations); And the United States Patent (USP) of authorizing Schafer 4,609,493 (contact lens cleaning combination) of promulgation on September 2nd, 1986.

In order to further specify oral cleaning composition of the present invention, be used for removing albumen spot on tooth or the artificial tooth but can in composition, add one or more protease conjugates of the present invention of receiving amount on the medicine." oral cleaning composition " that the present invention uses is meant dentifrice, toothpaste, tooth powder, mouth wash shua, oral spray, chewing gum, lozenge, sachet, tablet, xanthan gel, prophylaxis pastes, dental procedure solution etc.Preferably, described oral cleaning composition comprises one or more protease conjugates of the present invention of about 0.0001%～about 20% (weight), more preferably from about 0.001%～about 10%, also more preferably from about 0.01%～about 5%, and pharmaceutically acceptable carrier." acceptable on the pharmacology " that the present invention uses is meant that medicine, medicament or the inert fraction of this term description are suitable for contacting with zootic tissue with the people and not having excessive toxicity, uncompatibility, unstable, hormesis, anaphylaxis etc., has rational effect/risk ratio.

Typically, the pharmaceutically acceptable oral cavity cleaning carrier components of described oral cleaning composition account for usually composition weight about 50%～about 99.99%, preferred about 65%～about 99.99%, more preferably from about 65%～about 99%.

The pharmaceutically acceptable carrier components and the optional member that can join in the oral cavity cleaning component of oral cleaning composition of the present invention are well-known for those skilled in the art.Carrier components that uses in diversified types of compositions, the oral cleaning composition and optional member are all open in the above-mentioned reference of the present invention.

In another embodiment of the invention, the artificial tooth cleaning combination that is used for denture care outside the oral cavity comprises one or more protease conjugates of the present invention.One or more protease conjugates and the artificial tooth that comprise significant quantity in this class artificial tooth cleaning combination clean carrier, the weight percentage of described protease conjugates in composition preferably about 0.0001%～about 50%, more preferably from about 0.001%～and about 35%, also more preferably from about 0.01%～about 20%.Various artificial tooth cleaning combination forms, for example effervescent tablet etc. all be known in the art (referring to, for example United States Patent (USP) 5,055,305, Young), and are generally suitable for mixing one or more protease conjugates and are used to remove albumen spot on the artificial tooth.

In another embodiment of the invention, comprise one or more protease conjugates of the present invention in the contact lens cleaning combination.One or more protease conjugates and the contact lens that comprise significant quantity in this class contact lens cleaning combination clean carrier, described protease conjugates is shared weight percent preferably about 0.01%～50% in composition, more preferably from about 0.01%～and about 20%, also more preferably from about 1%～about 5%.Various contact lens cleaning combination forms, for example tablet, liquid etc. all are known in the art, and are generally suitable for mixing one or more protease conjugates and are used to remove albumen spot on the contact lens.

Embodiment 20-23

The contact lens cleaning combination

	Embodiment 20	Embodiment 21	Embodiment 22	Embodiment 23
					Protease conjugates among the embodiment 5	????0.01％	????0.5％	????0.1％	????2.0％
Glucose	????50.0％	????50.0％	????50.0％	????50.0％
					Nonionic surface active agent (polyoxyethylene-polyoxypropylene multipolymer)	????2.0％	????2.0％	????2.0％	????2.0％
Aniorfic surfactant (polyoxyethylene-alkyl phenyl ether sodium sulfovinate)	????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％

Embodiment 24-27

The bathing product

	Embodiment 24	Embodiment 25	Embodiment 26	Embodiment 27
					Water	????62.62％	????65.72％	????57.72％	????60.72％
The EDTA disodium	????0.2％	????0.2％	????0.2％	????0.2％
					Glycerol	????3.0％	????3.0％	????3.0％	????3.0％
Polyquatenium?10	????0.4％	????0.4％	????0.4％	????0.4％
					Zetesol NL	????12.0％	????12.0％	????12.0％	????12.0％
Coconut oleoyl amine MEA	????2.8％	????2.8％	????2.8％	????2.8％
					Lauroyl both sexes sodium acetate	????6.0％	????6.0％	????6.0％	????6.0％
Tetradecanoic acid	????1.6％	????1.6％	????1.6％	????1.6％
					Magnesium sulfate 7 hydrate	????0.3％	????0.3％	????0.3％	????0.3％
The trihydroxy-tristearin	????0.5％	????0.5％	????0.5％	????0.5％
					The PEG-6 caprylic/capric triglyceride	????3.0％	????-	????-	????-
Cottonseed acid lipid acid sucrose polyfatty acid esters	????3.0％	????-	????-	????-
					Mountain Yu acid lipid acid sucrose polyfatty acid esters	????3.0％	????-	????4.0％	????-
Vaseline	????-	????4.0％	????8.0％	????-
					Mineral oil	????-	????-	????-	????6.0％
The DMDM glycolylurea	????0.08％	????0.08％	????0.08％	????0.08％
					Protease conjugates among the embodiment 6	????0.1％	????2.0％	????2.0％	????5.0％
Citric acid	????1.40％	????1.40％	????1.40％	????1.40％

Embodiment 28-31

Face wash products

	Embodiment 28	Embodiment 29	Embodiment 30	Embodiment 31
					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％
Lauryl ether-3 sodium sulfate	????3.0％	????3.5％	????-	????-
					Lauryl ether-4 carboxylic acid sodium	????3.0％	????3.5％	????-	????-
Lauryl ether-12	????1.0％	????1.2％	????-	????-
					Polyquaternium?10	????-	????-	????0.4％	????0.4％
Polyquaternium?25	????0.3％	????0.3％	????-	????-
					Glycerine	????3.0％	????3.0％	????3.0％	????3.0％
Lauroyl both sexes sodium acetate	????-	????-	????6.0％	????6.0％
					Lauric acid	????6.0％	????6.0％	????3.0％	????3.0％
Tetradecanoic acid	????-	????-	????3.0％	????3.0％
					Magnesium sulfate 7 hydrate	????2.3％	????2.0％	????2.0％	????2.0％
Trolamine	????4.0％	????4.0％	????4.0％	????4.0％
					The trihydroxy-tristearin	????0.5％	????0.5％	????0.5％	????0.5％
Mountain Yu acid lipid acid sucrose polyfatty acid esters	????2.0％	????2.0％	????-	????-
					Cottonseed acid lipid acid sucrose polyfatty acid esters	????3.0％	????2.0％	????-	????-
The PEG-6 caprylic/capric triglyceride	????-	????-	????-	????2.0％
					Vaseline	????-	????-	????4.0％	????-
Mineral oil	????-	????-	????-	????2.0％
					Cocoamidopropyl	????2.0％	????3.0％	????1.8％	????1.8％
Lauryl dimethylamine oxide	????1.0％	????1.2％	????1.2％	????1.2％
					D-panthenol	????1.0％	????0.25％	????0.25％	????-
The DMDM glycolylurea	????0.08％	????0.08％	????0.08％	????0.08％
					Protease conjugates among the embodiment 2	????1.0％	????2.0％	????0.5％	????0.5％
Daily spices	????0.2％	????0.2％	????0.2％	????0.2％

Embodiment 32-33

Retention type skin insulation composition

	Embodiment 32	Embodiment 33
			Glycerine	????5.0％	????-
Stearic acid	????3.0％	????-
			C 11-13Isoparaffin	????2.0％	????-
Ethylene glycol stearate	????1.5％	????-
			Propylene glycol	????-	????3.0％
Mineral oil	????1.0％	????10.0％
			Sesame oil	????-	????7.0％
Vaseline	????-	????1.8％
			Trolamine	????0.7％	????-
The acetate cetyl	????0.65％	????-
			Stearin	????0.48％	????2.0％
The TEA stearate	????-	????2.5％
			Hexadecanol	????0.47％	????-
Wool wax alcohol	????-	????1.8％
			DEA-phosphoric acid cetyl	????0.25％	????-
Methyl p-hydroxybenzoate	????0.2％	????0.2％
			Propylparaben	????0.12％	????0.1％
Carbomer?934	????0.11％	????-
			The EDTA disodium	????0.1％	????-
Protease conjugates among the embodiment 14	????0.1％	????0.5％
			Water	????84.32％	????71.1％

Embodiment 34

The wipe cleaner composition

Propylene glycol	????1.0％
		Texapon Special	????0.6％
Succsinic acid	????4.0％
		Sodium succinate	????3.2％
Triclosan 	????0.15％
		Protease conjugates among the embodiment 1	????0.05％
Water	????91.0％

Above-mentioned composition is impregnated on the woven absorption layer of being made up of Mierocrystalline cellulose and/or polyester, and in absorption layer weight, composition is about 250%.

Sequence table＜110〉P﹠G＜120〉have the protease conjugates of the epitope regions of space protection＜130〉epi-position protections＜140〉PCT/US00/＜141〉2000-07-11＜160〉1＜170〉PatentIn Ver.2.0＜210〉1＜211〉275＜212〉PRT＜213〉bacillus amyloliquefaciens＜400〉1Ala 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?Lys?Tyr?Pro?Ser?Val?Ile?Ala

165?????????????????170?????????????????175Val?Gly?Ala?Val?Asp?Ser?Ser?Asn?Gln?Ar9?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?Lys?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

Claims (14)

1. protease conjugates; it is characterized in that described conjugate comprises a proteolytic enzyme part and one or more extention; wherein said proteolytic enzyme partly comprises first epitope regions; second epitope regions and the 3rd epitope regions; wherein each extention all links to each other with the epi-position protective position covalency of described proteolytic enzyme part, wherein:

(a) the epi-position protective position in first epitope regions is selected from corresponding to 1,2,3,4,5,6,7,12,17,36,40,41,43,44,45,67,86,87,89,206,209,210,212,213,214,215 and 216 of subtilisin BPN ';

(b) the epi-position protective position in second epitope regions is selected from corresponding to 25,26,27,46,47,48,49,50,51,52,53 of subtilisin BPN ', 54,91,99,100,101,102,127,128,129,130,131,132,133,134,136,137,138,140,141,144 and 145; With

(c) the epi-position protective position in the 3rd epitope regions is selected from corresponding to 9,10,22,23,24,62,63,143,146,154 of subtilisin BPN '; 155,156,157,172,173,187,189,195,197,203; 204,253,254,256,265,267,269,271,272 and 275.

2. according to the protease conjugates of claim 1, wherein each extention has following array structure independently:

Wherein X does not exist or is a junction branch; R ₁Do not exist or be selected from first polypeptide and first polymkeric substance; R ₂Do not exist or be selected from second polypeptide and second polymkeric substance; Wherein X, R ₁And R ₂In have at least one not exist.

3. according to the protease conjugates of claim 2; wherein said proteolytic enzyme partly has the aminoacid sequence of the modification of parent's aminoacid sequence, and the aminoacid sequence of wherein said modification is included on one or more epi-position protective position and is replaced by a substituted amino acid and wherein each extention and a substituted amino acid are covalently bound.

4. according to the protease conjugates of claim 3, wherein said substituted amino acid is a halfcystine.

5. according to the protease conjugates of claim 4, wherein said parent's aminoacid sequence is selected from subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, Proteinase K, aluminothermy enzyme, protease A, proteolytic enzyme B, proteolytic enzyme C, with proteolytic enzyme D, and their variant.

6. according to the protease conjugates of claim 5, wherein:

(a) the epi-position protective position in first epitope regions is selected from corresponding to 1,2,3,4,5,6,7,12,17,40,41,67,86,87,89,206,209,214 and 215 of subtilisin BPN ';

(b) the epi-position protective position in second epitope regions is selected from corresponding to 27,47,48,50,52,102,127,128,130,131,132,134,138 and 141 of subtilisin BPN '; With

(c) the epi-position protective position in the 3rd epitope regions is selected from corresponding to 22,23,24,143,146,155,173,189,197,203,204,253,254,265 and 275 of subtilisin BPN '.

7. according to the protease conjugates of claim 6, R wherein ₁And R ₂Do not exist.

8. according to the protease conjugates of claim 6, R wherein ₁For being selected from subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, Proteinase K, aluminothermy enzyme, protease A, proteolytic enzyme B, proteolytic enzyme C and proteolytic enzyme D, and first polypeptide of their variant.

9. protease conjugates according to Claim 8, wherein said first polypeptide is selected from the following position at it and links to each other with described connection portion or described proteolytic enzyme part covalency: corresponding to 1,2,3,4,5,6,7 of subtilisin BPN ', 9,10,12,17,22,23,24,25,26,27,36,40,41,43,44,45,46,47,48,49,50,51,52,53,54,62,63,67,86,87,89,91,99,100,101,102,127,128,129,130,131,132,133,134,136,137,138,140,141,143,144,145,146,154,155,156,157,172,173,187,189,195,197,203,204,206,209,210,212,213,214,215,216,253,254,256,265,267,269,271,272 and 275.

10. according to the protease conjugates of claim 9, wherein X does not exist and wherein said proteolytic enzyme part and first polypeptide link to each other by the disulphide bridges covalency.

11. according to the protease conjugates of claim 6, wherein R ₂Do not exist, first polymkeric substance is a polyoxyethylene glycol.

12. according to the protease conjugates of claim 11, wherein at least one extention be selected from first epitope regions, the epi-position protective position covalency in the epitope regions of second epitope regions and the 3rd epitope regions links to each other.

13. a cleaning compositions, described composition comprise the protease conjugates described in the claim 1 and a kind of cleaning compositions carrier.

14. a compositions for use in personal care, described composition comprise the protease conjugates described in the claim 1 and a kind of use in personal care carrier.