CN1369008A - Intramolecularly cross-linked subtilisins with reduced immunogenicity - Google Patents

Abstract

The present invention describes subtilisin proteases having an intramolecular cross-link, wherein the intramolecular cross-link comprises a covalent linkage between an amino acid of a first residue of the protease and an amino acid of a second residue of the protease. The immunogenicity of the protease is reduced compared to the parent protease. Thus, this protease is suitable for use in several types of compositions including, but not limited to, laundry, dish cleaning, hard surface cleaning, skin care, hair care, beauty, oral care, and contact lens cleaning compositions.

Description

The subtilisin of the intramolecular crosslinking that immunogenicity reduces

Invention field

The present invention relates to change the subtilisin of structure, this proteolytic enzyme is used to composition, for example personal care composition, laundry composition, rigid surface cleaning compositions and meticulous fabric cleaning compositions.

Background of invention

Enzyme constitutes a maximum class natural protein.Class of enzymes wherein is can other proteinic proteolytic enzyme of catalytic hydrolysis.By natural and engineered protein enzyme are spiked in the cleaning compositions, especially with the laundry with having made that the ability of this protein hydrolysate is developed in those relevant cleaning compositions.

In the cleaning applications, the most frequently used proteolytic enzyme 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, Siezen, people such as Roland J. " the homology simulation and the protein engineering strategy of subtilisin, subtilisin sample serine stretch protein enzyme family ", protein engineering, Vol.4, No.7,719-737 page or leaf (1991).Unfortunately, the effect of wild-type protease in its natural surroundings often can not be optimized in the environment of non-natural cleaning compositions.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 this proteolytic enzyme in the non-natural wash environment.These methods comprise that the gene reconstitution of proteolytic enzyme and/or chemically modified are to strengthen thermostability and the oxidative stability of proteolytic enzyme under far different condition.

But, because the proteolytic enzyme of this modification is ectogenic for Mammals, so they are potential antigen.As antigen, these proteolytic enzyme can cause that mammiferous immunogenicity 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 developing the more efficient protein enzyme that is used for doing washing by genetically engineered and chemically modified, this proteolytic enzyme also not commercialization be used for personal care composition and meticulous fabric washing composition.The major cause of not adding these proteolytic enzyme in the products such as soap, gel, body lotion and shampoo is because human body sensitization can cause the problem of bad immunne response.Therefore, providing a kind of has the clean-up performance of proteolytic enzyme but does not excite the personal care composition of immunne response or meticulous fabric washing composition will have crucial meaning.

At present, by immobilization, granulation, the proteolytic enzyme that parcel or soluble chemistry are modified can be reduced to minimum level with the immunogenic response to proteolytic enzyme to avoid proteolytic enzyme to carry out airborne transmission.Though these methods have considered the human consumer and contacted with airborne proteolytic enzyme, but still exist the danger that tissue and final composition contact lastingly.

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 NovoNordisk 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.

The inventor has been surprised to find by described proteolytic enzyme is carried out the immunogenicity that intramolecular crosslinking can reduce described proteolytic enzyme.The proteolytic enzyme of also finding these intramolecular crosslinkings still keeps the activity of parent protease separately.In addition, the inventor has found the crosslinked specific residue of the most suitable participation in the described proteolytic enzyme, this be because, for example, the epitope regions of described relatively proteolytic enzyme (promptly, the position that described proteolytic enzyme is relevant with immunne response) or due to the locus of cleavage site (that is, the position of hydrolysis takes place described proteolytic enzyme in vivo).

The inventor has found to excite the immunogenic response that weakens but still has kept it as effectively and have a subtilisin of active proteolytic enzyme.Therefore, proteolytic enzyme of the present invention is suitable for using in the composition of several types, and said composition includes but not limited to, laundry, cleaning tableware, cleaning rigid surface, the composition of skin care, hair care, beauty treatment, mouth care and cleaning contact lens.

Summary of the invention

The present invention relates to have the subtilisin of intramolecular crosslinking, wherein intramolecular crosslinking comprises covalently bound between the amino acid of second residue of the amino acid of first residue of described proteolytic enzyme and described proteolytic enzyme.

Described proteolytic enzyme of the present invention has the immunogenicity that is lower than parent protease.Therefore, this proteolytic enzyme is applicable to the composition of several types, and said composition includes but not limited to, laundry, cleaning tableware, cleaning rigid surface, the composition of skin care, hair care, beauty treatment, mouth care and cleaning contact lens.

Detailed Description Of The Invention

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

The present invention can comprise any of the present invention must or optional components and/or limit the quantity of, perhaps by or substantially by any 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 present invention mentions the trade(brand)name of some materials, and described material includes, but are not limited to enzyme.The inventor does not want that the material with certain trade(brand)name limits.The equivalent of the material of being represented by trade(brand)name (for example, those source difference and title or catalogue (reference) number different materials) can be used to alternative and be used for proteolytic enzyme 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 insertion of amino-acid residue in the wild-type protein sequence.

The term " parent " that the present invention uses is meant such protein (wild-type or variant), and it is used to further modify and forms a kind of protease conjugates.

As described herein, term " wild-type " is meant a kind of protein by the microorganisms of not suddenling change, for example a kind of proteolytic enzyme or other enzyme.

As described herein, term " variant " is meant that its aminoacid sequence is different from the protein of the proteinic aminoacid sequence of its corresponding wild type.

As described herein, the molecular weight of all polymkeric substance is represented with weight-average molecular weight.

As described herein, when intramolecular crosslinking proteolytic enzyme of the present invention was not only limited to those proteolytic enzyme that contain subtilisin BPN ' and its variant, all amino acid numberings were 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.II, 7911-7925 page or leaf (1983).

As used in the present invention, wherein hyphen is used to define a zone of being made up of several amino-acid residues, and it represents that this zone comprises each residue between listed residue and the listed residue.For example, regional 70-84 represents by amino-acid residue 70,71,72,73,74,75,76,77,78,79,80,81,82,83 and 84 zones of forming.

Intramolecular crosslinking proteolytic enzyme of the present invention

The present invention relates to the subtilisin of intramolecular crosslinking, wherein intramolecular crosslinking comprises covalently bound between the amino acid of second residue of the amino acid of described proteinic first residue and described proteolytic enzyme.If be not limited to theory, the crosslinked degradation rate (that is, can increase stability) of proteolytic enzyme in the endosome compartment of antigen presenting cell that reduce that should produce certainly, what this conversely again can impede protein enzyme epi-position presents.

But the proteolytic enzyme of intramolecular crosslinking of the present invention is subtilisin sample proteolytic enzyme, its wild-type or variant.Term " subtilisin sample proteolytic enzyme " is meant that a kind of and sequence subtilisin BPN ' have at least 50%, the proteolytic enzyme of preferred 80% amino acid sequence homology as used in this article.Wild-type Bacillus subtilus sample proteolytic enzyme by, for example, basophilia genus bacillus, bacillus amyloliquefaciens, Bacillus amylosaccharicus, microorganisms such as Bacillus licheniformis, bacillus lentus and subtilis.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.7,719-737 page or leaf (1991).

But the proteolytic enzyme of the preferred intramolecular crosslinking of the present invention 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/SAlcalase ^(can be from Novo Industries, Copenhagen, Denmark buys), hope bacterium enzyme (Esperase) ^(Novo Industries), husky fragrant enzyme (Savinase) ^(NovoIndustries), Maxatase ^(can buy) from Genencor International Inc., Maxacal ^(Genencor International Inc.), Maxapem 15 ^(GenencorInternational Inc.), and the variant of above-mentioned enzyme.But the proteolytic enzyme of the particularly preferred intramolecular crosslinking of the present invention is proteolytic enzyme and the variant thereof that obtains from bacillus amyloliquefaciens.The most preferred proteolytic enzyme of the present invention is subtilisin BPN ' and variant thereof.

Be hereinafter referred to as " protease A ", the particularly preferred subtilisin BPN ' variant that uses among the present invention is disclosed in the U.S. Pat 5 that was presented to Venegas on July 9th, 1991,030, in 378, it is characterized in that in the aminoacid sequence of subtilisin BPN ', having following sudden change:

(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 ", that the variant of the preferred subtilisin BPN ' of another that uses in intramolecular crosslinking of the present invention is disclosed on January 7th, 1988 is disclosed, transfer the EP-B251 of Genencor international corporation, in 446, described variant is characterised in that the aminoacid sequence of wild-type subtilisin BPN ' has sudden change in following one or more sites: 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; Two or more sites in the perhaps above-listed site combine with one or more sudden changes that are selected from following site: Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222.

Other the preferred subtilisin BPN ' variant that is used for intramolecular crosslinking of 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 described variant is characterised in that the aminoacid sequence of wild-type subtilisin BPN ' has a sudden change on the Asn76 site, be combined with one or more sudden changes that are selected from the following site: Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leul35, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265 and Ala274.

Other the preferred subtilisin BPN ' variant that is used for intramolecular crosslinking of the present invention, hereinafter be called " proteolytic enzyme D ", the U.S. Pat 4 that on July 26th, 1988 was authorized people such as Estell is seen in its description, 760,025, described variant is characterised in that in wild-type subtilisin BPN ' aminoacid sequence to have the one or more sudden changes that are selected from the following amino acid sites: Asp32, Ser33, His64, Tyr104, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222.

The preferred proteolytic enzyme that is used for intramolecular crosslinking of the present invention 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.

The proteolytic enzyme of intramolecular crosslinking of the present invention comprises covalently bound between two amino acid of an above-mentioned proteolytic enzyme.These amino acid are called as the amino acid of first residue and the amino acid of second residue in the present invention independently.Term " first residue " and " second residue " should not be interpreted as being meant site 1 and the site 2 corresponding to subtilisin BPN ' separately as used herein.But these terms only are used to show a residue or another residue.For example, described first residue can be corresponding to the site 118 of subtilisin BPN ' corresponding to site 27 and described second residue of subtilisin BPN '.Certainly, scope of the present invention comprises also that certainly first residue is corresponding to the site 1 of subtilisin BPN ' or second residue site 2 corresponding to subtilisin BPN '.

In addition, expression needing should not be interpreted as the natural amino acid that is present in this site corresponding to the use of the term " corresponding to the site " of the specific site of subtilisin BPN '.But this description is intended to represent the site sequence number, rather than is present in the particular amino acid residue in this site.In fact, according to the present invention, halfcystine (non-natural is present among the subtilisin BPN ') is the amino acid of very preferred first residue or second residue.

According to the present invention, the intramolecular crosslinking of subtilisin comprises a kind of covalently bound between the amino acid of the amino acid of first residue of described proteolytic enzyme and second residue.This covalently bound connection portion that comprises first residue of bridge joint and second residue.Described connection portion can be an any structure, and by this structure, first residue can be by covalently bridged (connection) with second residue.Yet as used herein, the connection portion is not only that covalent linkage is as for example forming the only covalent linkage of a disulphide bridges.Be not limited to theory, the inventor has been found that this disulphide bridges for the proteolytic enzyme that a kind of intramolecular crosslinking is provided, and it is inoperative that the immunogenicity of this proteolytic enzyme is lower than parent's immunogenicity.

For example, the connection portion can be any small molecules, and promptly a kind of molecular weight is less than about 1600 greatly, preferably is less than about 800 greatly, more preferably is less than about 400 greatly, most preferably is less than about 300 molecule greatly.

Most preferred connection portion comprises the covalently bound one or more cysteine residues of those energy, the N-terminal connection portion of lysine residue and/or described proteolytic enzyme.For example, in a preferred embodiment of the invention, the amino acid of first residue contains an amino (for example, as a limiting examples, the amino acid of first residue is that the amino acid of Methionin (by natural existence or sudden change) or first residue is the aminoterminal of described proteolytic enzyme).In addition, in this preferred embodiment, the amino acid of second residue contains a sulfydryl, and for example, as a limiting examples, the amino acid of second residue is halfcystine (by natural existence or sudden change).For example, following non-limiting reagent can be used to form proteolytic enzyme covalently bound of intramolecular crosslinking of the present invention: 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] capronate; 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-iodoacetyl] 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.Hereinafter enumerated and used similar agents to prepare the limiting examples of intramolecular crosslinking proteolytic enzyme of the present invention.

Other example of connection portion is disclosed in the United States Patent (USP) of issuing August 29 nineteen ninety-five of authorizing Harris 5,446,090 with relevant chemistry; 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 the methoxyl group of the replacement of selectivity protein modification-poly-(ethylene glycol) derivative ", synthesising communication, Vol.22,2417-2424 page or leaf (1992); And people such as Ishii, " state of succinimide basic ring to the fluorescence of the aa-tropomyosin of pyrene maleimide mark and the influence of structural performance ", biophysics magazine, Vol.50,75-80 page or leaf (1986).Most preferred connection portion is (for example, alkyl) or the unsubstituted succinimide that replaces.

In a preferred embodiment of the invention, the amino acid of first residue (for example comprises an amino, as a limiting examples, the amino acid of first residue is the aminoterminal that the amino acid of Methionin (by natural existence or sudden change) or first residue comprises described proteolytic enzyme).Therefore preferably will be positioned at be not on first and second residue sites that lysine residue sports one or more other amino-acid residues so that for example the crosslinked of the lysine residue on first residue be optionally.For example, lysine residue is arranged on the site 43 of the first nearly epitope regions of subtilisin BPN ' of this paper definition.All lysine residues of other that exists in the proteolytic enzyme carry out can optionally making lysine residue and second residue on the site 43 crosslinked after the site selectivity sudden change.Perhaps, the amino acid of naturally occurring first residue can be sported Methionin (for example), then carry out the site selectivity sudden change of other all lysine residues of existing in the proteolytic enzyme, and make described lysine residue and second residue optionally crosslinked.

In this identical preferred embodiment, preferably the amino acid of second residue comprises a sulfydryl, and for example, as a non-limiting instance, the amino acid of second residue is halfcystine (by natural existence or sudden change).In this example, wherein to be present in one be not on the site of second residue to cysteine residues, preferably replaces halfcystine with another amino-acid residue on each site in these sites so that carry out optionally crosslinked between first residue and second residue.

In another preferred embodiment of the present invention, the amino acid of first and second residues comprises a sulfydryl separately.For example, as a non-limiting instance, the amino acid of first residue is that the amino acid of halfcystine (by natural existence or sudden change) and second residue is halfcystine (by natural existence or sudden change).In this example, wherein to be present in one be not on the site of first and second residues to cysteine residues, preferably the halfcystine on each site in these sites is substituted by another amino-acid residue so that carry out optionally crosslinked between first residue and second residue.

In another preferred embodiment of the present invention, the amino acid of first and second residues comprises an amino separately.For example, as a non-limiting instance, the amino acid of first residue can be that the amino acid of Methionin or aminoterminal (by natural existence or sudden change) and second residue is Methionin (by natural existence or sudden change).In this example, wherein to be present in one be not on the site of first and second residues to Methionin, preferably the Methionin on each site in these sites is substituted by another amino-acid residue so that carry out optionally crosslinked between first residue and second residue.

Therefore, in a preferred embodiment of the invention, the amino acid of first residue is Methionin or comprises aminoterminal and the amino acid of second residue is halfcystine.In another preferred embodiment of the present invention, the amino acid of first residue is that the amino acid of halfcystine and second residue is halfcystine.In another preferred embodiment of the present invention, the amino acid of first residue is that the amino acid of Methionin or aminoterminal and second residue is Methionin.

In a preferred embodiment of the invention, at least one in first and second residues is arranged in a definite zone.Have been found that subtilisin contains three epitope regions.Therefore, if be not limited to theory, it is crosslinked to affirm that one or more residue in these epitope regions has wherein participated in, owing to hindered presenting thereby having reduced immunogenicity of described epi-position.For example, wherein another residue of one or more residue in the epitope regions and proteolytic enzyme is crosslinked, needs to carry out the multidigit point cutting of described proteolytic enzyme before epi-position is presented.Have been found that the site 70-84 of first epitope regions corresponding to subtilisin BPN '; Second epitope regions is corresponding to the site 103-126 of subtilisin BPN '; And the 3rd epitope regions corresponding to the site 220-246 of 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 epitope regions of also finding some amino-acid residue of described proteolytic enzyme and described proteolytic enzyme in addition is closely adjacent.The inventor finds that the residue of first, second and the 3rd nearly epitope regions is closely adjacent with the 3rd epitope regions with first, second respectively.Therefore, if be not limited to theory, a residue be sure oing the one or more zones in first, second and the 3rd near epitope regions has participated in crosslinked, and immunogenic reduction is owing to having hindered presenting of epi-position.Have been found that the site 2,3,4,5,6,712,17,36,40,41,43,44,45,67,86,87,89,206,209,210,212,213,214,215 and 216 of the first nearly epitope regions corresponding to subtilisin BPN '; The second nearly epitope regions is corresponding to the site 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 the site 9,10,22,23,24,62,63,143,146,154 of the 3rd nearly epitope regions corresponding to subtilisin BPN ', 155,156,157,172,173,187,189,195,197,203,204,253,254,256,265,267,269,271,272 and 275.Referring to, for example Rubingh equals the interim U.S. Patent application 60/144979 of application on July 22nd, 1999, " protease conjugates with epitope regions of space protection ".

In addition, as the preferred embodiments of the invention, find that also subtilisin comprises first " cleavage site " zone and second " cleavage site " (that is, the position of hydrolysis takes place described proteolytic enzyme in vivo).If be not limited to theory, it is crosslinked to be sure of that one or more residues in these cleavage site zones have participated in, and the hydrolysis of proteolytic enzyme has been subjected to obstruction, thereby presenting of epi-position also correspondingly has been subjected to obstruction.Have been found that the site 156-165 of first cleavage site corresponding to subtilisin BPN ', 170,186,191-196 and 259-262 and second cleavage site are corresponding to the site 12-24,27 of subtilisin BPN ', 84-88,271 and 274.Referring to, for example Weisgerber equals the interim U.S. Patent application 60/144981 of application on July 22nd, 1999, " protease conjugates with cleavage site of space protection ".

Therefore, in a preferred embodiment of the invention, proteolytic enzyme of the present invention comprises a kind of intermolecular crosslinked, covalently bound between the amino acid of wherein intermolecular crosslinked first residue that comprises described proteolytic enzyme and the amino acid of second residue, wherein at least one residue is arranged in a zone that is selected from lower area:

(a) an aminoterminal zone corresponding with the site 1 of subtilisin BPN ';

(b) first epitope regions corresponding with the site 70-84 of subtilisin BPN ';

(c) second epitope regions corresponding with the site 103-126 of subtilisin BPN ';

(d) three epitope regions corresponding with the site 220-246 of subtilisin BPN ';

(e) with the site 156-165 of subtilisin BPN ', 170,186, the first cleavage site zone of 191-196 and 259-262 correspondence;

(f) with the site 12-24,27 of subtilisin BPN ', 84-88, the second cleavage site zone of 271 and 274 correspondences;

(g) with the site 2,3,4,5,6,7,12,17,36,40,41,43,44,45,67,86,87,89,206,209,210,212,213,214 of subtilisin BPN ', the first nearly epitope regions of 215 and 216 correspondences;

(h) with the site 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, the second nearly epitope regions of 141,144 and 145 correspondences; With

(i) with the site 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, the 3rd nearly epitope regions of 272 and 275 correspondences.

Wherein have only a residue in first residue and second residue to be arranged in an above-mentioned zone, other residue is selected from other any site of described proteolytic enzyme.

Preferably, at least one residue is positioned at and is selected from the aminoterminal zone, first epitope regions, and second epitope regions, the 3rd epitope regions is in the zone in first cleavage site zone and the second cleavage site zone.More preferably, at least one residue is positioned at and is selected from the aminoterminal zone, and first epitope regions is in the zone in second epitope regions and the 3rd epitope regions.Even more preferably, at least one residue is positioned at and is selected from first epitope regions, in the zone in second epitope regions and the 3rd epitope regions.Most preferably, at least one residue is positioned at first epitope regions.

In another the preferred embodiments of the invention, in first and second residues of described proteolytic enzyme each is positioned at and is selected from the aminoterminal zone, first epitope regions, second epitope regions, the 3rd epitope regions, the first cleavage site zone, the second cleavage site zone, the first nearly epitope regions, the second nearly epitope regions is in the zone in the 3rd nearly epitope regions.More preferably, first residue is positioned at corresponding to the site 1 of subtilisin BPN ' and second residue and is selected from first epitope regions, second epitope regions, the 3rd epitope regions, the first cleavage site zone, the second cleavage site zone, the first nearly epitope regions, the second nearly epitope regions is in the zone in the 3rd nearly epitope regions.

Wherein at least one residue is arranged in first epitope regions, and this zone is preferably corresponding to the site 75-83 of subtilisin BPN '.More preferably, this zone is corresponding to the site 78 of subtilisin BPN '.

Wherein at least one residue is arranged in second epitope regions, and this zone is preferably corresponding to the site 109,114 and 118 of subtilisin BPN '.It most preferably is site 118.

Wherein at least one residue is arranged in the 3rd epitope regions, and this zone is preferably corresponding to the site 240 of subtilisin BPN '.

Wherein at least one residue is arranged in the first cleavage site zone, and this zone is preferably corresponding to the site 158,159,160,161,162,163 of subtilisin BPN ', 164,165,170,186,191,192,193,194,196,259,260,261 and 262.More preferably, this zone is corresponding to the site 158,159,160,161,162,163,164,165,170,191,192,193,194,261 and 262 of subtilisin BPN '.Even more preferably, this zone is corresponding to the site 158,159,160,161,162,163,164,192,193,194,261 and 262 of subtilisin BPN '.Most preferably, this zone is corresponding to the site 160,161,162,163 and 261 of subtilisin BPN '.

Wherein at least one residue is arranged in the second cleavage site zone, and this zone is preferably corresponding to the site 13,14,15,16,18,19,20 and 21 of subtilisin BPN '.More preferably, this zone is corresponding to the site 14,15,16,18,19,20 and 21 of subtilisin BPN '.Most preferably, this zone is corresponding to the site 18,19,20 and 21 of subtilisin BPN '.

Wherein at least one residue is arranged in the first nearly epitope regions, and this zone is preferably corresponding to the site 2,3,4,5,6,7,12,17,40,41,43,67,86,87,89,206,209,214 and 215 of subtilisin BPN '.More preferably, this zone is corresponding to the site 2,3,4,5,17,40,41,43,67,86,87 and 214 of subtilisin BPN '.

Wherein at least one residue is arranged in the second nearly epitope regions, and this zone is preferably corresponding to the site 25,26,27,46,47 of subtilisin BPN ', 48,49,50,51,52,53,54,91,99,100,101,102,127,128,129,130,131,132,134,136,137,138,140,141,144 and 145.Most preferably, this zone is corresponding to the site 27,47,48,50,52,102,127,128,130,131,132,134,138 and 141 of subtilisin BPN '.

Wherein at least one residue is arranged in the 3rd nearly epitope regions, and this zone is preferably corresponding to the site 9,10,22,23 of subtilisin BPN ', 24,62,63,243,246,154,155,156,157,172,173,187,189,195,197,203,204,253,254,256,265,267,269,271,272 and 275.Most preferably, this zone is corresponding to the site 22,23,24,143,146,155,173,189,197,203,204,253,254,265 and 275 of subtilisin BPN '.

Below table 2 listed the limiting examples of the subtilisin of preferred intramolecular crosslinking, wherein, in each example, listed site sequence number is represented first and second residues respectively, that is, intermolecular crosslinkedly undertaken by these two residues.With disclosed herein consistent, the numbering of all residues is all corresponding to the numbering of subtilisin BPN '.

Table 2

First residue	Second residue
		27 (in the second cleavage site zones)	118 (in second epitope regions)
141 (in the second nearly epitope regions)	118 (in second epitope regions)
		114 (in the second nearly epitope regions)	118 (in second epitope regions)
25 (in the second nearly epitope regions)	118 (in second epitope regions)
		118 (in second epitope regions)	240 (in the 3rd epitope regions)
25 (in the second nearly epitope regions)	240 (in the 3rd epitope regions)
		275 (in the 3rd nearly epitope regions)	240 (in the 3rd epitope regions)
137 (in the second nearly epitope regions)	109 (in second epitope regions)
		????????????55	109 (in second epitope regions)
133 (in the second nearly epitope regions)	109 (in second epitope regions)
		1 (aminoterminal zone)	79 (first epitope regions)
1 (aminoterminal zone)	78 (first epitope regions)
		43 (in the first nearly epitope regions)	75 (in first epitope regions)
43 (in the first nearly epitope regions)	82 (in first epitope regions)
		12 (in the second cleavage site zones)	82 (in first epitope regions)
24 (in the second cleavage site zones)	82 (in first epitope regions)
		18 (in the second cleavage site zones)	82 (in first epitope regions)
4 (in the first nearly epitope regions)	82 (in first epitope regions)
		186 (in the first cleavage site zones)	158 (in the first cleavage site zones)
186 (in the first cleavage site zones)	160 (in the first cleavage site zones)
		262 (in the first cleavage site zones)	158 (in the first cleavage site zones)
262 (in the first cleavage site zones)	160 (in the first cleavage site zones)
		262 (in the first cleavage site zones)	161 (in the first cleavage site zones)
130 (in the second nearly epitope regions)	159 (in the first cleavage site zones)
		130 (in the second nearly epitope regions)	162 (in the first cleavage site zones)
78 (in first epitope regions)	212 (in the first nearly epitope regions)

Optional part

The proteolytic enzyme of intramolecular crosslinking of the present invention can comprise one or more other chemical structures in addition, the any residue that comprises (for example) and described proteolytic enzyme, especially do not participate in those residues of intramolecular crosslinking, covalently bound one or more small molecules, polypeptide and/or polymkeric substance (being defined as " extention " in this article).As used herein, term " small molecules " is meant a kind of molecular weight less than about 1600, preferably less than about 800, more preferably less than about 400 with most preferably less than about 300 molecule.As used herein, term " polypeptide " is meant a kind of molecule that contains two or more amino-acid residues.As used herein, term " polymer " " be meant a kind of two or more identical (preferably five or more a plurality of identical) monomeric molecule that contains.

Additional part can comprise the polypeptide portion of putting down in writing as in the following document, polymer moieties and connection portion: for example on June 13rd, 1996 is disclosed and transferred the WO 96/17929 of NovoNordisk by Olsen etc.; On December 19th, 1996 is disclosed and transferred the WO 96/40792 of Novo Nordisk by Olsen etc.; On August 21st, 1997 is disclosed and transferred the WO 97/30148 of Novo Nordisk by Bisgard-Frantzen etc.; On July 16th, 1998 is disclosed and transferred the WO 98/30682 of Novo Nordisk by Olsen etc.; On August 13rd, 1998 is disclosed and transferred the WO98/35026 of Novo Nordisk by Vov Der Osten etc.; On January 7th, 1999 is disclosed and transferred the WO 99/00849 of Novo Nordisk by Olsen etc.; On January 5th, 1999 promulgation and United States Patent (USP) 5856451 transfer NovoNordisk by Olsen etc.; On October 9th, 1997 is disclosed and transferred the WO 97/37007 of Genencor International Inc. by Bott etc.; On June 2nd, 1998 is by applications such as Weisgerber and U.S. Patent application 09/088912 that transfer P﹠G; On July 30th, 1997 is by applications such as Weisgerber and U.S. Patent application 08/903298 that transfer P﹠G; On July 22nd, 1999 is by the interim U.S. Patent application 60/144979 of applications such as Rubingh, " protease conjugates with epitope regions of space protection "; On July 22nd, 1999 is by the interim U.S. Patent application 60/144981 of applications such as Weisgerber, " protease conjugates with cleavage site of space protection ".

The preparation method

Suddenly change by nucleotide sequence and to prepare the described proteolytic enzyme part that in one or more epi-positions protection sites (or other any position of described part), has a replacement coding parent aminoacid sequence.These class methods are known in the art; A kind of limiting examples of this quadrat method is as described below:

The phagemid (pSS-5) that will contain wild-type subtilisin BPN ' gene changes intestinal bacteria dut-ung-bacterial strain CJ236 over to, and to people such as Yuckenberg, " use contains the DNA of uridylic and the external site-directed mutagenesis of phagemid carrier ", site-directed mutagenesis-a kind of practical approach, McPherson, M.J. write, behind the method improvement of describing in 27-48 page or leaf (1991) one literary compositions, with VCSM13 helper phage (people such as Kunkel, " do not need the quick of Phenotypic Selection; effective site-directed mutagenesis ", Enzymology method, Vol 154,367-382 page or leaf (1987)) preparation contains the single stranded DNA template of uridylic.By at Zoller and Smith, " carry out oligonucleotide-directed mutagenesis with M13 deutero-carrier: a kind of being used for produces the effective universal method of point mutation at any dna fragmentation ", nucleic acids research, Vol.10, disclosed method is improved and the primer site-directed mutagenesis that comes is used to prepare all mutant (providing as people such as Yuckenberg basically, above-mentioned) in the 6487-6500 page or leaf (1982).

With 380B dna synthesizer (Applied Biosystems Inc.) preparation oligonucleotide.The mutagenesis reaction product is changed in the intestinal bacteria MM294 bacterial strain (American type culture collection intestinal bacteria 33625).All sudden changes all confirm through dna sequencing, and change separated DNA over to Bacillus subtilus expression strain PG632 (people such as Saunders, " optimization of the signal sequence cleavage site of the 34-amino acid fragment of secretion human parathyroid hormone from Bacillus subtilus ", gene, Vol.102, people such as 277-282 page or leaf (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.160,15-21 page or leaf (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, soluble fractions hydrolysis in the fermention medium are (referring to " biology of genus bacillus: industrial application ", Doi, R.H. and M.McGloughlin, write (1992)).The pH of meat soup is constant in the fermentation operation process remains on 7.5.Add kantlex (50 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 variant by following step process fermentation culture.Cross bacillus subtilis cell in the 0.16 μ m filter membrane filtering nutrient solution by tangential flow.Then, cut off the acellular nutrient solution 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.By the cation-exchange chromatography of carrying out, and be further purified this proteolytic enzyme with the NaCl gradient elution with the S-sepharose.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's analytical chemistry such as (, Vol.99,316-320 page or leaf (1979)) DelMar.This assay method discharges the speed of p-Nitroaniline in the time of can measuring the synthetic substrate succinyl--Ala-Ala of described protease hydrolysis solubility-proline(Pro)-phenylalanine-p-Nitroaniline (sAAPF-pNA).Measure the xanchromatic speed that is produced by hydrolysis reaction at the 410nm place with spectrophotometer, this speed and organized enzyme concentration are proportional.In addition, determine total protein concentration with the absorbance measuring value at 280nm place.The ratio of organized enzyme/total protein provides the purity of described proteolytic enzyme, and is used to identify the fraction of collecting as stoste.

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

Using in the preparation as purpose, by Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size-exclusion column wash-out proenzyme liquid removing propylene glycol, and exchange buffering liquid.MES buffer exchange in the proenzyme liquid is become the KH of 0.01M ₂PO ₄Solution, pH5.5.

Provided the non-limiting example of the method for preparing a kind of intramolecular crosslinking proteolytic enzyme of the present invention below, wherein in each embodiment:

A kind of proteolytic enzyme shown in the symbolic representation as mentioned.

Embodiment 1

The tyrosine of preparation on the site 217 replaced by leucine and site 78 on the variant of the subtilisin BPN ' that replaced by halfcystine of Serine.In the damping fluid of pH5.5 with the dithiothreitol (DTT) (DTT of 5 liters of 5mM; From Sigma-Aldrich Co., St.Louis, MO buys) with this variant dialysis (100ml, concentration are approximately 2mg/mL) 4 hours.Transfer to this variant in the fresh buffer that does not contain DTT and dialysed about 16 hours.The concentration of this variant is by measuring in the absorbancy at 280nm place.The concentration (referring to Deaken etc., journal of biological chemistry, Vol.89,263 pages (1963)) of measuring mercaptan is with the concentration of the guaranteeing free mercaptan concentration greater than described proteolytic enzyme.Add N-(γ-dimaleoyl imino butyric acid base) succinimide ester (from Pierce Chemical, Rockford, IL buys) with the amount that surpasses 1.2 times of free mercaptan concentration.After five minutes, add the damping fluid of the 1M pH6.8 of about 20mL.After 30 minutes, add the damping fluid of the 1M pH5.5 of about 100mL.Then by the damping fluid of about 5 liters 0.01M pH5.5 this reaction mixture of dialysing.The proteolytic enzyme that has covalently bound intramolecular crosslinking between halfcystine by ion-exchange column purification point 78 on the throne and the aminoterminal.

Embodiment 2

The tyrosine of preparation on the site 217 is replaced by leucine, the l-asparagine on the site 240 replaced by halfcystine and site 118 on the variant of the subtilisin BPN ' that replaced by Methionin of l-asparagine.Described but other all lysine residues proteolytic enzyme are replaced by other amino acid beyond Methionin or the halfcystine.In the damping fluid of pH5.5 with the dithiothreitol (DTT) (DTT of 5 liters of 5mM; From Sigma-Aldrich Co., St.Louis, MO buys) with this variant dialysis (100ml, concentration are approximately 2mg/mL) 4 hours.Transfer to this variant in the fresh buffer that does not contain DTT and dialysed about 16 hours.The concentration of this variant is by measuring in the absorbancy at 280nm place.The concentration (referring to Deaken etc., journal of biological chemistry, Vol.89,263 pages (1963)) of measuring mercaptan is with the concentration of the guaranteeing free mercaptan concentration greater than described proteolytic enzyme.Add succinimido-4-(N-maleimide ylmethyl)-hexanaphthene-1-carboxyl (6-aminocaprolc acid ester) succinimide ester (from PierceChemical, Rockford, IL buys) with the amount that surpasses 1.2 times of free mercaptan concentration.After five minutes, add the damping fluid of the 1M pH6.8 of about 20mL.After 30 minutes, add the damping fluid of the 1M pH5.5 of about 100mL.Then by the damping fluid of about 5 liters 0.01M pH5.5 this reaction mixture of dialysing.The proteolytic enzyme that has covalently bound intramolecular crosslinking between halfcystine by ion-exchange column purification point 240 on the throne and the Methionin on the site 118.

Embodiment 3

N-(γ-maleimide butyric acid) succinimide ester that uses among the embodiment 1 is substituted by following any reagent place so that form covalently bound between the halfcystine in site 78 and aminoterminal: N-(α-dimaleoyl imino acetoxyl) succinimide ester; N-(β-dimaleoyl imino propionyloxy) succinimide ester, or-dimaleoyl imino benzoyl-N-hydroxyl succinimide ester.Each reagent is all from Pierce Chemical, Rockford, and IL buys.Perhaps, any reagent that comprises the maleimide amine moiety succinimide ester moiety similar with size can be synthesized out and replace N-(γ-dimaleoyl imino butyric acid base) succinimide ester (referring to Kalgutar etc., the medical chemistry magazine, Vol.39,1692-1703 page or leaf (1996) and the reference of being quoted thereof).

Embodiment 4

Succinimido-4-(N-maleimide ylmethyl)-hexanaphthene-1-carboxyl (6-aminocaprolc acid ester) succinimide ester that uses among the embodiment 2 is substituted by following any reagent place so that form covalently bound between the halfcystine in site 240 and the Methionin on the site 118: succinimido-4-(right-the dimaleoyl imino phenyl)-butyric ester, sulfosuccinimide base-4-(right-the dimaleoyl imino phenyl)-butyric ester, or N-[κ-dimaleoyl imino undecane acidic group] the sulfosuccinimide ester.Each reagent is all from PierceChemical, Rockford, and IL buys.Perhaps, any reagent that comprises the maleimide amine moiety succinimide ester moiety similar with size can be synthesized out and replace N-(γ-dimaleoyl imino butyric acid base) succinimide ester (referring to Kalgutar etc., the medical chemistry magazine, Vol.39,1692-1703 page or leaf (1996) and the reference of being quoted thereof).

Embodiment 5

The tyrosine of preparation on the site 217 replaced by leucine and site 78 on the variant of the subtilisin BPN ' that replaced by halfcystine of Serine.Described but other all lysine residues proteolytic enzyme are replaced by other amino acid beyond Methionin or the halfcystine.In the damping fluid of pH7-8 with the dithiothreitol (DTT) (DTT of 5 liters of 5mM; From Sigma-AldrichCo., St.Louis, MO buys) with this variant dialysis (100ml, concentration are approximately 2mg/mL) 4 hours.Transfer to this variant in the fresh buffer that does not contain DTT and dialysed about 4 hours.The concentration of this variant is by measuring in the absorbancy at 280nm place.The concentration (referring to Deaken etc., journal of biological chemistry, Vol.89,263 pages (1963)) of measuring mercaptan is with the concentration of the guaranteeing free mercaptan concentration greater than described proteolytic enzyme.Add succinimido [4-iodoacetyl] Aminobenzoate (from PierceChemical, Rockford, IL buys) with the amount that surpasses 1.2 times of free mercaptan concentration.After five minutes, add the damping fluid of the 1M pH6.8 of about 20mL.After 30 minutes, add the damping fluid of the 1M pH5.5 of about 100mL.Then by the damping fluid of about 5 liters 0.01M pH5.5 this reaction mixture of dialysing.The proteolytic enzyme that has covalently bound intramolecular crosslinking between halfcystine by ion-exchange column purification point 78 on the throne and the aminoterminal.

Embodiment 6

The tyrosine of preparation on the site 217 replaced by leucine and site 78 on the variant of the subtilisin BPN ' that replaced by halfcystine of Serine.All lysine residues of other of described proteolytic enzyme are replaced by other amino acid beyond Methionin or the halfcystine.In the damping fluid of pH5.5 with the dithiothreitol (DTT) (DTT of 5 liters of 5mM; From Sigma-AldrichCo., St.Louis, MO buys) with this variant dialysis (100ml, concentration are approximately 2mg/mL) 4 hours.Transfer to this variant in the fresh buffer that does not contain DTT and dialysed about 16 hours.The concentration of this variant is by measuring in the absorbancy at 280nm place.The concentration (referring to Deaken etc., journal of biological chemistry, Vol.89,263 pages (1963)) of measuring mercaptan is with the concentration of the guaranteeing free mercaptan concentration greater than described proteolytic enzyme.Add N-dimaleoyl imino [4 '-nitrine-2 ' oil of mirbane amino] butyric ester with the amount that surpasses 1.2 times of free mercaptan concentration.After five minutes, this reactant is exposed under the UV-light (320nm-350nm) and shone 10 minutes.Perhaps, this reactant is exposed to 10 flashes of light of standard camera shutter.Then by the damping fluid of about 2 liters 0.01M pH5.5 this reaction mixture of dialysing.The proteolytic enzyme that has covalently bound intramolecular crosslinking between halfcystine by ion-exchange column purification point 78 on the throne and the aminoterminal.

Embodiment 7

The GMAB that uses among the embodiment 6 is substituted by following any reagent place so that form covalently bound (all lysine residues of other of wherein said proteolytic enzyme are replaced by another amino acid) between the Methionin in site 78 and aminoterminal: N-5-azido--2-p-nitrobenzoic acid-base succinimide, N-succinimido-6-(4 '-azido--2 ' oil of mirbane amino) capronate, N-hydroxysulphosuccinimide base-4-triazobenzene manthanoate, sulfosuccinimide base (4-azido-salicyl amino) capronate, sulfosuccinimide base-7-azido--4-methylcoumarin-3-acetic ester, sulfosuccinimide base-6-(4 '-azido--2 ' oil of mirbane amino) capronate and sulfosuccinimide base-4-(right-the azido-phenyl) butyric ester.Each reagent is all from Pierce Chemical, Rockford, and IL buys.

Embodiment 8

The tyrosine of preparation on the site 217 is replaced by leucine, the Serine on the site 78 replaced by halfcystine and site 1 on the variant of the subtilisin BPN ' that replaced by halfcystine of L-Ala.Described but other all lysine residues proteolytic enzyme are replaced by other amino acid beyond Methionin or the halfcystine.In the damping fluid of pH5.5 with the dithiothreitol (DTT) (DTT of 5 liters of 5mM; From Sigma-Aldrich Co., St.Louis, MO buys) with this variant dialysis (100ml, concentration are approximately 2mg/mL) 4 hours.Transfer to this variant in the fresh buffer that does not contain DTT and dialysed about 16 hours.The concentration of this variant is by measuring in the absorbancy at 280nm place.The concentration (referring to Deaken etc., journal of biological chemistry, Vol.89,263 pages (1963)) of measuring mercaptan is with the concentration of the guaranteeing free mercaptan concentration greater than described proteolytic enzyme.With 10mL 1M (N-(2-hydroxyethyl) piperazine-N '-(2 ethane sulfonic aicd)) (HEPES)/NaOH pH of buffer 7.0 is added in the described protein enzyme solution.Add dimaleoyl imino hexane (from Pierce Chemical, Rockford, IL buys) with the amount that surpasses 1.2 times of free mercaptan concentration.After one hour, the damping fluid of the 0.01M pH5.5 by 20 volumes is with twice of this reaction mixture dialysis.The proteolytic enzyme that has the intramolecular crosslinking that its valency connects between halfcystine by ion-exchange column purification point 78 on the throne and the N-terminal halfcystine.

Analytical procedure

Measure and use following two kinds of methods, the proteolytic enzyme of intramolecular crosslinking of the present invention can be used to the mensuration and the immunogenic response of enzymic activity, and these two kinds of methods are known by those skilled in the art.Perhaps also can use other currently known methods in present technique field.

Protease activity

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

Use Scotch ^The #3750G band strips the human body skin chip from experimenter's shank repeatedly and be paved with chip substantially on belt.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 of 2mL 0.75mg/mL joined 0.01M KH ₂PO ₄, pH5.5 damping fluid in.The sodium laurate solution that in this solution, adds 1mL 2.5%, pH8.6.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 continuous gently mixing.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.Range estimation or the square band that relatively obtains from control enzyme with colorimeter and difference from colour intensity between the square band of described proteolytic enzyme acquisition.Compare with control enzyme square band, the colour intensity of the every square band of albumen a little less than, this explanation protease activities is higher.Painted collagen activity methods

50mL is contained 0.01M CaCl ₂And pH be that (collagen of azo pigment dipping, available from SigmaChemical Co., St.Louis MO) mixes for 8.6 0.1M tris damping fluid (three-methylol-aminomethane) and 0.5g azocoll.At 25 ℃ of these mixtures of following incubation, on oscillator plate, mix gently simultaneously.Filter this mixture of 2mL with 0.2 micron injection filter, read this mixture and after the absorbancy at 520nm place, the spectrophotometer adjustment is made zero.In remaining 48ml tris/azocoll mixture, add 1ppm control enzyme (for example, subtilisin BPN ') or testing protein enzyme.In time of 10 minutes altogether, filtered the solution that contains contrast/proteolytic enzyme of 2mL with 0.2 micron injection filter every 2 minutes.For the sample after every part of filtration, read the absorbancy at 520nm place immediately.The drawing result data are with respect to the curve of time.The slope of the contrast and the proteolytic enzyme of surveying is represented the relative reactivity of this sample.The big more expression activity of slope is high more.Survey protease activities (slope) can be expressed as the per-cent of contrast activity (slope).

Be used to measure test in the immunogenic mouse nose

Measure the immunogenicity potentiality of testing the proteolytic enzyme that to measure intramolecular crosslinking of the present invention in the immunogenic mouse nose 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 intramolecular crosslinking 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 the proteolytic enzyme and the subtilisin BPN ' of intramolecular crosslinking.

Composition of the present invention

The proteolytic enzyme of intramolecular crosslinking of the present invention can be used to be fit to use in any application of parent protease separately.Such example comprises cleaning compositions.Because the proteolytic enzyme of intramolecular crosslinking of the present invention can reach desirable weak immunogenic requirement, this proteolytic enzyme can also be used to once be benefited from use proteolytic enzyme in the minimum application.The example that this class is used comprises the application that the proteolytic enzyme of intramolecular crosslinking must closely contact with mammal skin, for example uses the application of personal care composition.

Cleaning compositions

Proteolytic enzyme of the present invention can be used to the meticulous fabric cleaning compositions that cleaning compositions includes, but not limited to laundry composition, rigid surface cleaning compositions, comprises dish washing compositions, and the automatic dishwasher detergent composition.

Cleaning compositions of the present invention contains one or more proteolytic enzyme of the present invention and a kind of cleaning compositions carrier of significant quantity.

" proteolytic enzyme of significant quantity " or similar statement that the present invention uses are meant the necessary amounts that reaches the proteolytic enzyme of the intramolecular crosslinking 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 proteolytic enzyme for example, the purposes of cleaning, the concrete composition of cleaning compositions, and the composition that 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%～about 1%, and one or more proteolytic enzyme of the present invention of 0.01%～about 0.1% most preferably from about.Several examples that described proteolytic enzyme can be used for the different cleaning composition have been discussed in more detail below.

Except that proteolytic enzyme 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 described proteolytic enzyme.The term " cleaning compositions material " that the present invention uses (for example is meant any selected required cleaning composition that is used for particular type and product form, liquid, particle, bar-shaped, sprays, bar, cream, gel) material, these materials are also compatible with the proteolytic enzyme that is used for composition.The concrete selection of cleaning compositions material can be by considering surfacing to be cleaned, easily carrying out at the required composition forms of the clean conditions in the use 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 proteolytic enzyme be reduced to such degree, and promptly described proteolytic enzyme is effective unlike desired under normal service condition.Hereinafter itemized concrete cleaning compositions material.

Proteolytic enzyme of the present invention can be used to expect in foam is abundant and cleaning effect the is good multiple cleanser compositions.Therefore, described proteolytic enzyme can use so that rigid surface sanitising agent, wash dishes composition, fabric cleaning composition of abundant preparation etc. are provided with various conventional ingredients.This based composition can be liquid, particle, form such as bar-shaped.This based composition can be mixed with " concentrating " sanitising agent, and this sanitising agent 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 is 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+1) 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.The present invention is alkyl alkoxy sulfate (AES) and alkyl alkoxy carboxylate salt (AEC) preferably.In addition, according to prescription teacher's needs, also be preferred 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-methyl glucose amide, its description are seen the U.S. Pat of authorizing people such as Connor 5,194,639 of promulgation on March 16th, 1993.

Can contain multiple other composition that is used for the washing composition cleaning compositions in the composition of the present invention, comprising, for example, other activeconstituents, carrier, water solubility promoter, 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 this 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 cleanser composition 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%.

Detergent composition of the present invention preferably is formulated in aqueous clean operation uses, and makes that 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 usage level of recommendation 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 compositions of the present invention and clean fabric composition, prescription Shi Keneng wishes to use the various washing assistants of about 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, prescription Shi Keneng wishes in this based composition to use various additional enzymes, 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 detergent applications.

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, n-nonanoic acid base 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 polyacrylic ester and polyaspartate, whitening agent, especially negatively charged ion whitening agent, suds suppressor, especially siloxanes and secondary alcohol, fabric softener, especially smectic clays etc. can 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%).

Variant of the present invention also can be used for the rigid surface cleaning compositions." rigid surface cleaning compositions " that the present invention uses be meant and be used to clean rigid surface, for example liquid the and granular cleanser compositions of floor, wall, bathroom tile etc.Rigid surface cleaning compositions of the present invention comprises one or more proteolytic enzyme of the present invention of significant quantity, protease 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 proteolytic enzyme, this class rigid surface cleaning compositions typically also comprises a kind of tensio-active agent and a kind of water miscible chelating washing assistant.But, at some special product, for example in the spray-type window sanitising agent, 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, preferably 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 to wanting clean Surface " full strength " to use 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 compositions

	Embodiment 7	Embodiment 8	Embodiment 9	Embodiment 10	Embodiment 11	Embodiment 12
							The proteolytic enzyme of 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％
Dodecyl (oxyethyl group) sodium sulfate	?????-	????2.20％	??????-	????2.20％	?????-	????2.20％
							Oxidizing dodecyl dimethyl amine	?????-	????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, dish washing compositions comprises one or more variants of the present invention." dish washing compositions " 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
					The proteolytic enzyme of embodiment 1	????0.05％	???0.50％	????0.02％	???0.40％
C 12-C 14The N-methyl glucose amide	????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 clean fabric composition

	Embodiment 17	Embodiment 18	Embodiment 19
				The proteolytic enzyme of 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

Proteolytic enzyme of the present invention is particularly suitable for personal care composition, for example retention type and rinsing type hair conditioner, shampoo, retention type and rinsing type control acne composition, face breast and skin-protecting agent, shower gels, soap, foam and clean agent of non-foam type, makeup, hand are used, face is used and body and function washing lotion, wetting Agent for Printing Inks, patch and facial mask, retain profile portion wetting Agent for Printing Inks, make up and cleaning liniment, oral care composition and contact lens care composition.Personal care composition of the present invention comprises one or more proteolytic enzyme of the present invention and personal care carrier.

For convenience of explanation, proteolytic enzyme 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 on December 17th, 1996 promulgation; The United States Patent (USP) 4,939,179 of authorizing people such as Cheney of July 3 nineteen ninety promulgation (hand with, face with and the body and function washing lotion); 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 compositions) 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 that on May 24th, 1988 issued again, 672 (contact lens cleaning compositions); And the United States Patent (USP) of authorizing Schafer 4,609,493 (contact lens cleaning compositions) of promulgation on September 2nd, 1986.

In order to further specify oral cleaning composition of the present invention, one or more proteolytic enzyme of the present invention that can add pharmaceutically acceptable amount in composition are used for removing the proteinic spot on tooth or the artificial tooth." oral cleaning composition " that the present invention uses is meant dentifrice, toothpaste, gutta-percha, tooth powder, mouth wash shua, oral spray, chewing-gum, chewing gum, lozenge, sachet, tablet, xanthan gel, prophylaxis pastes, dental procedure liquid etc.Preferably, described oral cleaning composition comprises one or more proteolytic enzyme of the present invention of about 0.0001%～about 20% (weight), more preferably from about 0.001%～about 10%, and also more preferably from about 0.01%～about 5%, and pharmaceutically acceptable carrier." pharmaceutically acceptable " 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., and 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 oral cleaning composition of the present invention are known by those skilled in the art of the present technique.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 compositions that is used for cleaning artificial tooth outside the oral cavity comprises one or more proteolytic enzyme of the present invention.One or more described proteolytic enzyme that comprise significant quantity in this class artificial tooth cleaning compositions, and artificial tooth cleaning carrier, the weight percentage of described proteolytic enzyme in composition preferably about 0.0001%～about 50%, more preferably about 0.001%～about 35%, also more preferably about 0.01%～about 20%.Various artificial tooth cleaning compositions forms, for example effervescent tablet etc. all by the present technique field known (referring to, for example U.S. Pat 5,055,305, Young), and are generally suitable for mixing one or more described proteolytic enzyme and are used to remove protein contaminants on the artificial tooth.

In another embodiment of the invention, comprise one or more proteolytic enzyme of the present invention in the contact lens cleaning compositions.One or more the described proteolytic enzyme and the contact lens cleaning carrier that comprise significant quantity in this class contact lens cleaning compositions, described proteolytic enzyme is shared weight percent preferably about 0.01%～50% in composition, more preferably about 0.01%～about 20%, also more preferably about 1%～about 5%.Various contact lens cleaning compositions forms, for example tablet, liquid etc. are all known by the present technique field, and are generally suitable for mixing one or more proteolytic enzyme of the present invention and are used to remove protein contaminants on the contact lens.

Embodiment 20-23

The contact lens cleaning soln

	Embodiment 20	Embodiment 21	Embodiment 22	Embodiment 23
					Proteolytic enzyme 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％	??????-	?????-	??????-
					Docosoic 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％
					The proteolytic enzyme of 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％
Docosoic 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％
					The proteolytic enzyme of 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 moisturizing compositions

	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％	-
The proteolytic enzyme of embodiment 4	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％
		The proteolytic enzyme of embodiment 1	????0.05％
Water	????91.0％

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

Sequence table＜110〉P﹠G＜120〉subtilopeptidase A＜130〉crosslinking protein enzyme＜140 of the intramolecular crosslinking that reduces of immunogenicity〉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?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?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 (12)

1. A subtilisin-like protease, characterized in that said protease has an intermolecular cross-link, wherein the intermolecular cross-link comprises the amino acid of the first residue of said protease and the second amino acid of said protease A connecting moiety between amino acids of residues. 2. The protease of claim 1, wherein at least one residue is located in a region selected from the following regions: (a) an amino-terminal region corresponding to site 1 of subtilisin BPN'; (b) a first epitope region corresponding to positions 70-84 of subtilisin BPN'; (c) a second epitope region corresponding to positions 103-126 of subtilisin BPN'; (d) a third epitope region corresponding to positions 220-246 of subtilisin BPN'; (e) first cleavage site regions corresponding to positions 156-165, 170, 186, 191-196 and 259-262 of subtilisin BPN'; (f) a second cleavage site region corresponding to positions 12-24, 27, 84-88, 271 and 274 of subtilisin BPN'; (g) with subtilisin BPN' at sites 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86, 87, 89, 206, The first near-epitope regions corresponding to 209, 210, 212, 213, 214, 215 and 216; (h) with subtilisin BPN' at sites 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, the second proximal epitope region corresponding to 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145; and (i) with subtilisin BPN' at sites 9, 10, 22, 23, 24, 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 correspond to the third proximal epitope region. 3. The protease of claim 2, wherein the first near-epitope region corresponds to sites 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43 of subtilisin BPN', 67, 86, 87, 89, 206, 209, 214 and 215; the second proximal epitope region corresponds to the subtilisin BPN' sites 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138 and 141; the third proximal epitope region corresponds to the subtilisin BPN' sites 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254, 265 and 275. 4. The protease of claim 3, wherein the second cleavage site region corresponds to positions 13-24 of subtilisin BPN'. 5. The protease of claim 4, wherein the first cleavage site region corresponds to positions 156-165, 170, 191-195, 261 and 262 of subtilisin BPN'. 6. The protease of claim 1, wherein the first epitope region corresponds to positions 75-83 of subtilisin BPN'. 7. The protease of claim 6, wherein the amino acid of the first residue is lysine or wherein the amino acid of the first residue corresponds to position 1 of subtilisin BPN'; and wherein the second residue The base amino acid is cysteine. 8. The protease of claim 6, wherein each residue in the first and second residues is located in an amino-terminal region, a first epitope region, a second epitope region, a third epitope region, the first cleavage site region, the second cleavage site region, the first near-epitope region, the second near-epitope region, and one of the third near-epitope region. 9. The protease of claim 8, wherein the first residue corresponds to site 1 of subtilisin BPN' and the second residue is located in a region selected from the first epitope, the second epitope, the second epitope In one of the three epitope regions, the first cleavage site region, the second cleavage site region, the first near-epitope region, the second near-epitope region, and the third near-epitope region. 10. The protease of claim 9, wherein the linking moiety comprises an alkyl succinimide; the second residue is located in the first epitope region; and wherein the second residue corresponds to subtilisin BPN ' at position 78. 11. A cleaning composition comprising a protease as claimed in claim 1 and a cleaning composition carrier. 12. A personal care composition comprising a protease according to claim 1 and a personal care carrier.