CN1315997A - Polypeptide-polymer conjugate with improved wash performance - Google Patents

Abstract

The present invention relates to a polypeptide-polymer conjugate having coupled one or more polymers covalently to the parent polypeptide, wherein the polymers is homo-polymers, graft, block, alternate, or ramdom co-polymers. The invention also relates to industrial compositions and products comprising a conjugate of the invention and to the use of said conjugate for improving the wash performance of industrial composition and products such as detergent compositions.

Description

Polypeptide-polymer conjugate with improved scourability

Invention field

The present invention relates to polypeptide-polymer conjugate, wherein polymkeric substance is homopolymer, graft copolymer, segmented copolymer, alternating copolymer or the random copolymers that is coupled at the polypeptide surface.The invention still further relates to the industry group compound that comprises conjugate of the present invention and product, polypeptide-polymer conjugate of the present invention and be used to improve the purposes of the scourability of industry group compound and product, also relate to the method for the scourability of improving polypeptide.

Background of invention

In detergent industry, enzyme is applied to cleaning formulation and has surpassed 30 years.The enzyme that is applied in these prescriptions comprises proteolytic enzyme, lipase, amylase, cellulase and other enzyme, or their miscellany.Commercial most important enzyme is a proteolytic enzyme.

The enzyme (as proteolytic enzyme) that the commerce that grows with each passing day is used is the variant that the wild-type protease of natural generation is transformed through protein engineering, as DURAZYM  (Novo Nordisk A/S), RELASE  (Novo Nordisk A/S), MAXAPEM  (Gist-BrocadesN.V.), PURAFECT  (Genencor International, Inc.).

Yet, though disclose many useful enzyme variants in the document, still need new improved enzyme or enzyme variants, be used for many industrial uses.

Because polypeptide may cause that the immunne response of non-expectation--is determined by method of application--that normally IgG and/or IgE reply, developed the technology that reduces immunne response in nearest 30 years.

WO 97/24421 and WO 97/24427 disclose by being covalently bound on the activated polymkeric substance and enzyme have been carried out immobilization.Use of the immobilization of activated polymkeric substance, especially demonstrate the improved antigenicity characteristic of zymoprotein one or more enzymes.The contriver claims, can realize by the structural modification of enzyme improving, and not influence the performance of enzyme in detergent solution.

Though immobilization might avoid forming airborne substances, in the operating process of immobilization step, this method still exists formation dust or aerocolloidal risk.

Another kind of technology is a coupling technology, wherein many polymerizable moleculars and desired polypeptides coupling.When this technology of use, immunity system is difficult to discern the epi-position of being responsible for forming antibody on the polypeptide surface, reduces immunne response thus.

Learn the polypeptide (being medicine) of effect for direct introducing human recycle system to produce special physiological, typical potential immunne response is that IgG and/or IgM reply, and the polypeptide (being industrial polypeptide) that sucks by respiratory system may cause that IgE replys (being transformation reactions).

Explain that a kind of theory that immunne response reduces is to be responsible for causing the epi-position of the immunne response that antibody forms on the polymerizable molecular maskable polypeptide surface.Another kind of theoretical (or being the part factor at least) is that conjugate is huge more, and the immunne response of generation is fallen lowly more.

Be generally used for the polypeptide coupling be homopolymer with the polymkeric substance that forms conjugate, promptly form, as oxyethane (EO), polyoxyethylene glycol (PEG) or propylene oxide (PO), polypropylene glycol (PPG) by a kind of repeating unit.Carbohydrate such as dextran, also uses.

U.S. Patent number 4,179,337 relate to the polypeptide of non-immunogenic, such as with polyoxyethylene glycol (PEG) or polypropylene glycol (PPG) link coupled enzyme and peptide hormone.

WO 96/17929 (Novo Nordisk A/S) relates to and polymerizable molecular (particularly polyoxyethylene glycol (PEG)) the modified polypeptide conjugate of link coupled.

The surprising discovery of the present inventor is now compared with the polypeptide of unmodified, and polymkeric substance-polypeptide conjugate has improved scourability.

Summary of the invention

Aspect first, the present invention relates to have the polypeptide-polymer conjugate of improved scourability.

The present inventor finds that if the homopolymer of molecular weight 0.1-60 kDa and parent's polypeptide of molecular weight 4-100 kDa are carried out coupling, then compare with the scourability of parent's polypeptide, the scourability of this polypeptide can be improved.

The present inventor also finds, if graft copolymer, segmented copolymer, alternating copolymer or random copolymers (are had general formula: EOxPOy (I), x=1-99% wherein, y=1-99%, and x+y=100%) and the parent's polypeptide that is used for industrial use carry out covalent coupling, then compare with parent's polypeptide, its scourability can be improved.

In both of these case, to compare with parent enzyme, it is breathed allergy and also may reduce.In the later case, even compare with the corresponding conjugate of coupling PEG or other homopolymer, it is breathed allergy and has reduction.

Aspect second, the present invention relates to the composition that in comprising the Industrial products of conjugate of the present invention, uses.

Aspect the 3rd, the present invention relates to use conjugate to improve scourability.

Aspect in the end the present invention relates to improve the method for polypeptide scourability.The industry polypeptide

The polypeptide that is used for industrial application has enzymatic and/or antimicrobial acivity usually.Industry polypeptide (opposite with pharmaceutical polypeptide) is not intended to introduce the human recycle system.

Therefore, the industrial polypeptide (such as enzyme) that uses as the activeconstituents of industry group compound and/or product (by hereinafter definition) (such as a clothing and a bowl dish washing composition, be used for handling composition and personal care product's (comprising makeup) of textiles), unlikely directly contact with human or animal's the recycle system because not with these polypeptide product of these polypeptide (or comprise) injection (or similarly) in blood flow.

Therefore, in the situation of industrial polypeptide, potential risk is to suck the respiratory allergies (being that IgE replys) that polypeptide causes by respiratory pathways.

In content of the present invention, " industrial polypeptide " is defined as the polypeptide that is not intended to introduce the people and/or the animal body recycle system, comprises peptide, protein and/or enzyme.

The example of these polypeptide comprises having the hereinafter polypeptide of the enzymic activity of definition.

Yet, if one or more polymkeric substance and enzyme are carried out coupling, to compare with parent enzyme, the performance of this enzyme will keep roughly the same or reduce.

The catalytic performance of enzyme depends on that enzyme and substrate are in many factors such as avtive spot contact.If polymkeric substance and enzyme are carried out coupling, polymkeric substance will be distributed in the surface of enzyme with random fashion usually.And, polymkeric substance will with near the enzyme coupling avtive spot of this enzyme, cause sterically hindered.Therefore predict that the performance of enzyme will affect adversely.

The surprising discovery of the present inventor, by with the puting together of polymkeric substance, the performance of enzyme may strengthen.

Detailed Description Of The Invention

The present surprising polypeptide-polymer conjugate that successfully provides of the present inventor, wherein the catalytic performance of polypeptide is improved.

The present invention relates to be fit to industrial application and mix polypeptide-polymer conjugate in the Industrial products as activeconstituents.Conjugate of the present invention also may have the breathing allergy of reduction.

Term " polypeptide-polymer conjugate " in content of the present invention, refers to that one or more polymkeric substance combine with polypeptid covalence.

Term " allergy of reduction ", in content of the present invention, refer to after sucking modified polypeptide of the present invention, compare with corresponding parent's polypeptide, the amount of the IgE that can cause abnormal state of its generation (in human body, the molecule for having comparable performance in particular animals) has reduced.Also can use term " breathing allergy ".Term " improved scourability " in content of the present invention, refers to compare with the Δ reflectance value (delta reflectance) of the tester that cleans with parent enzyme (non-conjugate), and the Δ reflectance value of the test material that cleans with conjugate has increased.

When term " improved scourability " when using (wherein can not obtain the Δ reflectance value) as skin care products, this term refers to that the cleaning effect when using parent enzyme (non-conjugate) compares, its cleaning effect is improved.

The present inventor has been found that then scourability can be improved if parent's unmodified polypeptide and homopolymer, graft copolymer, segmented copolymer, alternating copolymer or random copolymers are carried out coupling.Compare with corresponding parent's unmodified polypeptide, the potential allergenicity that the suction polypeptide causes is replied also and may be reduced.

In one aspect, the present invention relates to conjugate, wherein parent's polypeptide can with molecular weight 100-10,000 Da, preferred 100-5,000 Da, more preferably 100-2,000 Da, particularly 100-1, the polymerizable molecular coupling of 000 Da.

Because known weak point/tendency that light polymerizable molecular suppresses the polypeptide functionally active is less, so will lack/light polymerizable molecular and desired polypeptides coupling are favourable.For example, with coupling the corresponding enzyme of big/reaggregation molecule compare, the easier arrival of substrate and molecular weight ranges meet the avtive spot of the polymerizable molecular link coupled enzyme of embodiment of the present invention, because polymerizable molecular is sterically hindered more not remarkable at this moment.And, the distortion minimum of polypeptide structure, with contain big/compare with polypeptide link coupled conjugate than the reaggregation molecule, the polypeptide-polymer conjugate that contains less/light polymerizable molecular has improved stability, because it is less to push polypeptide structure the weight of different directions to.

Use another benefit of little polymerizable molecular to be, they are more cheap, because polymkeric substance is sold by kilogram.This has reduced the cost of production conjugate of the present invention.

In addition, compare with immobilized enzyme (covalently bound with the multivalence of the activated polymer with a plurality of reactive groups as enzyme), conjugate of the present invention shows that single polymer molecule is covalently bound to protein surface.This has been avoided the crosslinked of enzyme, causes more mean deviation increase of the distribution of catalyzer in applicating medium.This may cause the proteinic performance of conjugate per unit of the present invention better than immobilized enzyme.

As everyone knows, polymkeric substance can adopt different conformation/forms, and this main (but not being) depends on molecular structure, solvent (being water), temperature and concentration (S.Forster and M.Antonietti herein, Adv.Mater., 1998,10 (3), 195-217).Those conformation/forms comprise micella, thin slice, orderly right cylinder or the bicontinuous structure of different shape.The molecular conformation of multipolymer in aqueous medium, as solvation random coil, abduction curl, shaft-like polymkeric substance, supercoil and vesicle be well-known (Watersoluble polymers, M.J.Comstock compiles, ACS SymposiumSeries, 1991).

Therefore, be not subjected to the restriction of any theory, we believe be subjected to better shielding when conformational energy that graft copolymer, segmented copolymer, alternating copolymer or random copolymers that polypeptide surface connects are taked makes surface ratio with more hydrophilic homopolymer in water.And the synergistic effect that the formation supramolecular structure causes may reduce the getatability on polypeptide surface.And more lipophilic multipolymer (comparing with the PEG homopolymer) strengthens with the repulsion of antibody, may also play a role.

In addition, graft copolymer, segmented copolymer, alternating copolymer or random copolymers more inflexible structure (comparing) with homopolymer may make antibody more be difficult to (by inflexible polymkeric substance more and the conformation taked) to arrive and be responsible for forming the epi-position that causes the IgE that metamorphosis replys on the polypeptide surface.

The hydrophobicity of polymkeric substance is considered to that also polypeptide-polymer conjugate potential allergenicity is had influence.

By suitable selective polymer and molecular structure, can cover better, more desirably shield the lip-deep epi-position of polypeptide.And, by adjusting the characteristic of the polymkeric substance that connects, can optimize the characteristic of different ingredients (as washing composition).

In yet another aspect, the present invention relates to the polypeptide-polymer conjugate that one or more polymkeric substance and parent's polypeptide have carried out covalent coupling, wherein polymer characterization is general formula: EOxPOy

(Ⅰ)

X=1-99% wherein, y=1-99%, and x+y=100%.

Described polymkeric substance preferably is characterized by logical formula I, x=10-90% wherein, y=10-90%, x+y=100%.

In a preferred embodiment of the invention, polymkeric substance is made up of ethylene oxide unit and propylene oxide units, its ratio (EO unit: the PO unit) be 10: 90,20: 80,30: 70,40: 60,50: 50,60: 40,70: 30,80: 20 and 90: 10.

In preferred embodiments, the molecular weight of described polymkeric substance is 100 to 100, and 000Da is preferred 100 to 50,000Da, particularly 100 to 10,000Da.

In a more preferred embodiment, the molecular weight of described polymkeric substance is 100 to 12,000Da, more preferably 300 to 3,000Da.

In embodiments of the invention, polymkeric substance is Synthetic rubber, isoprene-styrene, hydrogenated, block, diblock, triblock copolymer, multi-block polymer.Logical formula I is understood to include the polymkeric substance that wherein EO unit and PO unit are placed independently of one another.Allergenicity is estimated

Can check by sucking, and compare the effect of using parent's polypeptide in the tracheae and the effect of using the corresponding modified polypeptide of the present invention and estimate allergenicity.

Existing several interior animal model of body that the polypeptide allergenicity is estimated that is used for.Some such model provides suitable foundation for the danger evaluation among the mankind.The model that is fit to comprises guinea pig model and mouse model.The function of institute's inductive irritant reaction in the animal of sensitization before these models manage the respiratory allergen is expressed as.According to these simulations, with the allergen declared by introducing animal in the tracheae.

The suitable strain of cavy (Dunkin Hartley strain) can be because of allergic response produce IgE antibody, and this point is with human different.Yet, they produce the IgG antibody 1A of another kind of type and IgG1B (referring to, for example, Prent φ, ATLA, 19, P.8-14,1991), these antibody have caused and the polypeptide (comprising enzyme) that is sucked is produced allergenicity have replied.Therefore, when using Dunkin Hartley animal model, the relative quantity of IgG1A and IgG1B can be represented level of allergenicity.

The rat strain that is suitable for tracheae interior contact polypeptide such as enzyme is a Brown Norway strain.Brown Norway rat produces IgE as allergic response.

In cavy and mouse, estimate the allergenic more details of respiratory by (1996) such as Kimber, basis and applied toxicology, 33, P.1-10 describe.

Other animal (as rabbit etc.) also can be used for similar research.Polymerizable molecular

Be coupled to polymerizable molecular on the polypeptide and can be the polymerizable molecular of any suitable molecular weight by the present invention's definition, comprise natural and the synthetic homopolymer [as polyvalent alcohol (promptly poly--OH), polyamine (i.e. poly--NH ₂) and poly carboxylic acid (promptly poly--COOH)] and heteropolymer, promptly comprise the polymkeric substance of one or more different coupling group (as hydroxyl and amido).

The example of the polymerizable molecular that is fit to comprises the polymerizable molecular that is selected from the group that contains following each quasi-molecule: polyalkylene oxide (PAO), as polyalkylene glycol (PAG), comprise polyoxyethylene glycol (PEG), methoxy poly (ethylene glycol) (mPEG) and polypropylene glycol, PEG-glycidyl ether (Epox-PEG), PEG-oxygen carbonylic imidazole (CDI-PEG), the PEG of branch, star-like PEGs, polyvinyl alcohol (PVA), polycarboxylate, poly-(V-Pyrol RC), poly-D, L-amino acid, ethene-copolymer-maleic anhydride, vinylbenzene-oxysuccinic acid anhydride copolymer, dextran, comprise Sensor Chip CM 5, heparin, the homologous albumin, Mierocrystalline cellulose, comprise methylcellulose gum, carboxymethyl cellulose, ethyl cellulose, Natvosol, carboxyethyl cellulose and hydroxypropylcellulose, the hydrolysate of chitosan, starch (as hydroxyethylamyle and hydroxypropylated starch), glycogen, agarose and derivative thereof, guar gum, amylopectin, inulin, xanthan gum (xanthangum), carrageenin (carrageenan), pectin, alginic acid hydrolysate and biological polymer, polyoxyethylene ester, comprise stearate, PEG8 stearate (Myrj 45) for example, PEG40 stearate (Myrj 52), PEG50 stearate (Myrj 53), PEG100 stearate (Myrj 59) and polyoxyethylene 25 propylene glycol stearates, Soxylat A 25-7, comprise 2 ethyl ethers, 2 amyl ethers, 2 cetyl ether, 2 stearyl ether, 2 vaccenic acid ethers, 3 hexyl ether, 3 Octyl Ether, 3 decyl ethers, 3 lauryl ethers, 3 myristyl ether, 3 cetyl ether, 3 stearyl ether, 4 heptyl ethers, 4 Octyl Ether, 4 decyl ethers, 4 lauryl ethers, 4 myristyl ether, 4 cetyl ether, 4 stearyl ether, 5 hexyl ether, 5 Octyl Ether, 5 decyl ethers, 5 lauryl ethers, 5 myristyl ether, 5 cetyl ether, 5 stearyl ether, 6 decyl ethers, 6 lauryl ethers, 6 myristyl ether, 6 cetyl ether, 6 stearyl ether, 7 decyl ethers, 7 lauryl ethers, 7 myristyl ether, 7 stearyl ether, 8 decyl ethers, 8 lauryl ethers, 8 myristyl ether, 8 cetyl ether, 8 stearyl ether, 9 lauryl ethers, 10 lauryl ethers, 10 tridecyl ethers, 10 cetyl ether, 10 stearyl ether, 10 oleyl ethers, 20 cetyl ether, 20 isocetyl ethers, 20 stearyl ether, 20 oleyl ethers, 21 stearyl ether, 23 lauryl ethers, 100 stearyl ether and polyoxyethylene sorbitol acid anhydride class, comprise mono-laurate, monoleate, monopalmitate, monostearate, trioleate, tristearate.

Preferred polymerizable molecular is avirulent polymerizable molecular, as polyoxyethylene glycol (PEG), also requires these molecule covalent couplings relatively simple to the chemical process on the linking group of enzyme surface.

Generally it seems, polyalkylene oxide (PAO) is (as polyethylene oxide, as PEG) be preferred polymerizable molecular, because these polymerizable moleculars compare with polysaccharide (as dextran, amylopectin etc.), the crosslinked active group (not catering to the need) of the energy that they had is seldom.

Also can be graft copolymer, segmented copolymer, alternating copolymer or random copolymers with polypeptide link coupled polymkeric substance with following formula:

EO _xPO _y(Ⅰ)

X=1-99% wherein, y=1-99%, and x+y=100%.

In the preferred embodiment of the invention, polymkeric substance is made up of ethylene oxide unit and propylene oxide units, its ratio (EO unit: the PO unit) be 10: 90,20: 80,30: 70,40: 60,50: 50,60: 40,70: 30,80: 20 or 90: 10.

In preferred embodiments, the molecular weight of described polymkeric substance is 100 to 100, and 000Da is preferred 100 to 50,000Da, particularly 100 to 10,000Da.

In embodiments of the invention, polymkeric substance is Synthetic rubber, isoprene-styrene, hydrogenated, block, diblock, triblock copolymer, multi-block polymer.

Can be used for carrying out the concrete multipolymer of link coupled with the polypeptide surface for example has: poly-(ethylene glycol-altogether-propylene glycol); Poly-(ethylene glycol-altogether-propylene glycol) single-butyl ether; Poly-(ethylene glycol-altogether-propylene glycol) monomethyl ether.

Preferred polymkeric substance is the nontoxic polymer of for example being made up of PEG and PPG multipolymer.Preferred those are covalently coupled to the polymkeric substance that the lip-deep linking group of enzyme needs simple relatively chemical process.

The embodiment that can be used for being coupled to the lip-deep concrete block polymer of polypeptide is: poly-(propylene glycol)-block-poly-(ethylene glycol)-block-poly-(propylene glycol); Poly-(ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol); Poly-(propylene glycol)-block-poly-(ethylene glycol)-block-poly-(propylene glycol) single-butyl ether; Poly-(ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol) single-butyl ether; Poly-(propylene glycol) block-poly-(ethylene glycol)-block-poly-(propylene glycol) monomethyl ether; Poly-(ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol) monomethyl ether.

Preferred block polymer is the block polymer with following general formula: H (OCH ₂CH ₂-) _x[OCH (CH ₃) CH ₂-] _y(OCH ₂CH ₂-) _zOH, its molecular-weight average (M _n) be 1,100, ethylene glycol content is 10wt%, M _n=1,900 and 50wt%, M _n=2,000 and 10wt%, M _n=2,800 and 10wt%, M _n=2,800 and 15wt%, M _n=2,900 and 40wt%, M _n=4,400 and 30wt%, M _n=5,800 and 30wt%, M _n=8,400 and 80wt%.

Other preferred block polymer is the block polymer with following general formula: H[-OCH (CH ₃) CH ₂-] _x(OCH ₂CH ₂-) _y[OCH (CH ₃) CH ₂-] _zOH, its molecular-weight average (M _n) be 2,000 and ethylene glycol content be 50wt%, M _n=2,700 and 40wt%, and M _n=3,300 and 10wt%.

The example of concrete block polymer is p7120:Pluronics, is purchased from BASF (Germany), and Tergitol is purchased from Union Carbide (USA), and Synperonic is purchased from Fluka (Switzerland).

The example that can be used for being coupled to the concrete multipolymer on polypeptide surface is: poly-(ethylene glycol-altogether--propylene glycol), particularly poly-(ethylene glycol-altogether-propylene glycol), its molecular-weight average M _nBe 2,500 and ethylene glycol content be 75wt%, molecular-weight average M _nBe 12,000 and ethylene glycol content be 75wt%; Poly-(ethylene glycol-altogether-propylene glycol) single-butyl ether, particularly its M _nBe 970 and 50wt% ethylene glycol, M arranged _nBe 1,700 and 50wt% ethylene glycol and M arranged _nBe 3,900 and 50wt% ethylene glycol, M arranged _nBe 1,700 and 50wt% ethylene glycol and M arranged _nBe 3,900 and poly-(ethylene glycol-altogether-propylene glycol) single-butyl ether of 50wt% ethylene glycol arranged; Poly-(ethylene glycol-altogether-propylene glycol) monomethyl ether.

Preferred polymkeric substance is the nontoxic polymer of for example being made up of PEG and PPG multipolymer.Preferred those are covalently coupled to the polymkeric substance that the lip-deep linking group of enzyme needs simple relatively chemical process.

The example of concrete EO-oligopolymer is: glycol ether, diglycol monotertiary methyl ether, triglycol, triglycol monomethyl ether, Tetraglycol 99, Tetraglycol 99 monomethyl ether, five glycol, five glycol monomethyl ether, hexaethylene glycol, hexaethylene glycol monomethyl ether, seven glycol, seven glycol monomethyl ether, the perhaps linear nonbranched C2-C14 monoalky lether of an ethylene glycol and glycol oligomer and 2-7 ethylene oxide unit.

Graft copolymer, segmented copolymer, alternating copolymer or random copolymers can be star-like or the branching form.The preparation of suitable polymkeric substance

The polymkeric substance that is connected with parent's polypeptide surface can adopt and well known to a person skilled in the art the technology preparation.And then various polymkeric substance can be purchased from each following company: BASF (Germany), Union Carbide (USA), Aldrich, Shearwater, Sigma (USA) etc.The activation of polymkeric substance

If will not have activity, just must use appropriate methodology to make its activation with polypeptide bonded polymkeric substance.According to the present invention, block polymer or multipolymer also can be by joint and polypeptide couplings.The joint that is fit to is known those of skill in the art.

The method and the principles of chemistry that are used for the activated polymerization molecule and are used for conjugated protein have a detailed description in the literature.

The method that generally is used to activate insoluble polymer comprises: activate functional group with following compounds: cyanogen bromide, Periodic acid, glutaraldehyde, diepoxides (biepoxides), Epicholorohydrin, divinylsulfone, carbodiimide, alkylsulfonyl halogenide, three chlorotriazines etc. are (referring to R.F.Taylor, (1991), " protein is fixed; basis and application ", Marcel Dekker, N.Y.; S.S.Wong, (1992), " protein is puted together and crosslinked chemistry ", CRC press, Boca Raton; G.T.Hermanson etc., (1993), " fixed affinity ligands technology ", press of institute, N.Y.).The some of them method relates to the activation of insoluble polymer, but also is applicable to the activation of soluble polymer (as Periodic acid, three chlorotriazines, alkylsulfonyl halogenide, divinylsulfone, carbodiimide etc.).Is linking group selected on amino, hydroxyl, sulfydryl, carboxyl, aldehyde or sulfhedryl and the protein selecting activation with puting together the functional group that must consider in the chemical process on the polymkeric substance, activate and put together chemical process and generally include: the ⅰ) activation of polymkeric substance, ⅱ) put together, ⅲ) sealing of remaining activity group.

Below concise and to the point many suitable polymer activation methods are described.Yet, should be appreciated that also and can use other method.

Polymerizable molecular is coupled on the free acid group of polypeptide can by means of imide and, for example amino-PEG or diazanyl-PEG (Pollak etc., (1976), J.Amr.Chem.Soc., 98,289-291) or diazo acid ester/acid amides (Wong etc., (1992), " protein is puted together and cross-linking chemistry ", CRC press) carry out.

With polymerizable molecular be coupled to hydroxyl normally very the difficulty because this must carry out in water.And common hydrolysis reaction is than more preponderating with the reaction of hydroxyl.

Polymerizable molecular is coupled to the free sulfhydryl base can be reached with special groups (as maleimide or positive pyridyl disulfide).Vinyl sulphone (United States Patent (USP) no.5,414,135, (1995), Snow etc.) also has preference for sulfhedryl, but does not resemble other compound of mentioning selective.

Can be by the reached arginine residues in the group target polypeptide chain that comprises two contiguous carbonyls.

It is also useful to relate to the technology that the PEG with the electrophilicity activatable is coupled on the lysine amino.The many conventional leavings group of alcohol forms the amine key.For example, can use alkylsulfonate, as tresylates (Nilsson etc., (1984), Enzymology method, 104 volumes, Jacoby W.B. compiles, press of institute: Orlando, P.56-66; Nilsson etc., (1987), Enzymology method, 135 volumes, Mosbach K. compiles, press of institute: Orlando, P.65-79; Scouten etc., (1987), Enzymology method, 135 volumes, Mosbach K. compiles, press of institute: Orlando, 1987, P.79-84; Crossland etc., (1971), J.Amr.Chem.Soc.1971,93, pp.4217-4219), mesylate (Harris, 1985, the same; Harris etc., (1984), J.Polym.Sci.Polym.Chem.Ed.22, pp 341-352), arylsulphonate (as tosylate) and p-nitrophenyl sulfonate.

Organic SULPHURYL CHLORIDE (for example Tresyl Chloride) can change into good leavings group (sulfonate) with the hydroxyl in many polymkeric substance (for example PEG) effectively, when with polypeptide in nucleophilic group (as amino) when reacting, this leavings group makes can form stable key between polymkeric substance and polypeptide.Except high puting together the productive rate will be arranged, reaction conditions also requires usually relatively gentleer (neutrality or slight alkalinity pH value seldom or are not destroyed to avoid sex change and activity), and satisfies the nondestructive condition of polypeptide needs.

Tosylate is stronger than mesylate reactivity, and resolve into more astatically PEG, diox and sulfonic acid (Zalipsky, (1995), the bioconjugates chemistry, 6,150-165).Epoxide also can be used to produce the amine key, but the above-mentioned group of specific activity is much weak.

With carbonyl chloride PEG being changed into chloro-formic ester will produce and lysine amino formic acid ester bond.This transformation can be carried out by multiple version, as using N-hydroxy-succinamide (United States Patent (USP) no.5,122,614, (1992); Zalipsky etc., (1992), biotechnology applications and biological chemistry, 15, P.100-114; Monfardini etc., (1995), bioconjugates chemistry, 6,62-69), with imidazoles (Allen etc., (1991), Carbohydr.Res., 213, pp 309-319), with p-NP, DMAP (EP 632 082 A1, (1993), Looze, alternative chlorine such as Y.).Usually by being reacted, chloro-formic ester and required leavings group prepare derivative.All these groups all produce the amino-formate bond with peptide.

In addition, can use isocyanate and isothiocyanate to produce urea and thiocarbamide respectively.

Use aforesaid identical leavings group and cyclic imid thrones, can obtain acid amides (United States Patent (USP) no.5,349,001, (1994), Greenwald etc.) from PEG acid.The reactive behavior of these compounds is very high, but hydrolysis is accelerated.

Also can use from the PEG succinate of succinyl oxide prepared in reaction.Formed thus ester group make conjugate to hydrolysis more responsive many (United States Patent (USP) no.5,122,614, (1992), Zalipsky).This group can activate with N-hydroxy-succinamide.

In addition, can introduce a special joint.That the most ancient is cyanuryl chloride (Abuchowski etc., (1977), journal of biological chemistry, 252,3578-3581; United States Patent (USP) no.4,179,337, (1979), Dayis etc.; Shafer etc., (1986), J.Polym.Sci.Polym.Chem.Ed., 24,375-378).Polymkeric substance can also be by pyrimidine ring and polypeptide coupling (referring to US 4,144,128, US4,195,128 and US4,298,395).

PEG is coupled on the aromatic amine, and diazotization subsequently will produce very active diazonium salt, its in position can with reactive polypeptide.Azlactone derivative by making PEG (United States Patent (USP) no.5,321,095, (1994), and Greenwald, therefore R.B.) reaction introduces another amido linkage, also can form amido linkage.

Because some peptides do not comprise many Methionins, therefore, will be connected to more than a PEG might be favourable on the identical Methionin.This can be by for example using 1, and 3-diamino-2-propyl alcohol carries out.

Also PEG can be connected to by amino-formate bond on the amino of enzyme (WO 95/11924, Greenwald etc.).Lysine residue also can be used as main chain.

Be used to prepare the functionalized generality general introduction of the polymer activation of relevant conjugate and PEG referring to following document: Zaplisky, S., bioconjugates chemistry, 1995,6,150-165, Hermanson, G.T., Academic Press, San Diego, 1996, and S.Herman, G.Hooftman, E.Schacht, bioactivation and compatible polymeric magazine (Journal of Bioactive and Compatible polymers), the 10th volume, 1995,145-187.The position of link coupled block polymer or multipolymer

In fact, all ionogens (as the amino of lysine residue) all are positioned at the surface (referring to for example Thomas E.Creighton, (1993), " protein ", W.H.Freeman and Company, New York) of peptide molecule.Therefore, easy to reach linking group (promptly amino) number equals the number that lysine residue in the polypeptide primary structure adds the N-terminal amino group on the surface of polypeptide.

According to the present invention, can there be 1-100 polymerizable molecular, preferred 4-50 polymerizable molecular, a 5-35 polymerizable molecular to be coupled on the purpose parent polypeptide.Parent's polypeptide

Can be at parent's polypeptide, normally molecular weight is on parent's polypeptide basis of 4-100kDa, preferred 15-60kDa, utilizes any suitable technique known in the art to prepare modified polypeptide of the present invention.

Term " parent " polypeptide is meant any not link coupled polypeptide (promptly wanting modified polypeptides).This polypeptide preferably can be microbe-derived, and as bacterium, filamentous fungus or yeast source, perhaps it can derive from plant.

Parent's polypeptide can be naturally occurring (or wild-type) polypeptide or its variant.

When selecting parent's polypeptide, preferably use polypeptide with a plurality of linking groups.

In the preferred embodiment of the invention, polymerizable molecular is distributed widely on the surface of parent's polypeptide.For enzyme, near preferred not coupling of zone block polymer or the multipolymer avtive spot.

In the context of the present invention, " be distributed widely in " and be meant that its residing position can make and be coupled to the different piece that polymerizable molecular on the polypeptide linking group covers the polypeptide surface (preferably whole surface or approach whole surface), to guarantee that discernible relevant epi-position is covered, thus not by immune antibody recognition, thereby obtain hypoallergenic enzyme.It is believed that: interactional surface-area is approximately 500 between polypeptide and the antibody ²(26 * 19 ) (referring to Sheriff etc., (1987), NAS's journal, 84 volumes, p.8075).

For enzyme,, preferably polymkeric substance is not coupled in the short range of avtive spot in order to ensure the loss minimum of enzymatic activity.Usually it seems: with distance avtive spot 5 , preferably not have the polymkeric substance coupling in the scope of 10 be good.

And, according to the present invention, can by the known epi-position place of immune system recognition or approach said epi-position coupling the polypeptide of polymkeric substance also be regarded as very beneficial.If the position of epi-position is unknown, then many polymkeric substance are coupled to the polypeptide surface can and linking group on be rather favourable.Preferably said linking group is distributed widely in the surface of polypeptide with the distance that is fit to from avtive spot.

According to the present invention, preferably meet the parent polypeptide of above-mentioned relevant coupling polymer in the lip-deep distribution requirement of polypeptide.

For enzyme, especially preferably do not have or have only only a few polymkeric substance (be 0-2) be coupled to apart from the avtive spot 0-5 scope, the enzyme in the 0-10 scope preferably.Enzymic activity

Parent enzyme can have any activity that becomes known for industry group compound as described below and product.Related enzymic activity comprises oxydo-reductase (E.C.1, " enzyme nomenclature, (1992), publishing company of institute), as laccase and superoxide-dismutase (SOD); Lytic enzyme E.C.3 comprises proteolytic enzyme, serine protease particularly, and as subtilisin, and lipolytic enzyme; Transferring enzyme, (E.C.2), as trans-glutaminases (TG enzyme); Isomerase (E.C.5), as protein disulfide isomerase (Protein Disulfide Isomerase, PDI).Parent protease

Parent protease comprises according to biological chemistry and (the International Union of Biochemistry and MolecularBiology of molecular biology League of Nations, IUBMB) recommendation (Recommendations, 1992) classification is arranged in the proteolytic enzyme of enzyme classification numbering E.C.3.4 group.

Example comprises that classification is positioned at the proteolytic enzyme under the following enzyme classification numbering (E.C.):

3.4.11 (being so-called aminopeptidase) comprises 3.4.11.5 (prolyl aminopeptidase), 3.4.11.9 (X-pro aminopeptidase), 3.4.11.10 (bacterium LAP), 3.4.11.12 (thermophilic aminopeptidase), 3.4.11.15 (lysyl aminopeptidase), 3.4.11.17 (tryptophyl aminopeptidase), 3.4.11.18 (Peptidase MSTN);

3.4.21 (being so-called Serine endopeptidase) comprises 3.4.21.1 (Quimotrase), 3.4.21.4 (trypsinase), 3.4.21.19 (glutamy endopeptidase), 3.4.21.25 (Cucumisin (cucumisin)), 3.4.21.32 (brachyurin), 3.4.21.48 (Saccharomyces mycetin (cerevisin)) and 3.4.21.62 (subtilisin);

3.4.22 (being so-called halfcystine endopeptidase) comprises 3.4.22.2 (papoid), 3.4.22.3 (ficin (ficain)), 3.4.22.6 (Disken), 3.4.22.7 (asclepain), 3.4.22.14 (Actinidin (actinidain)), 3.4.22.30 (papaya proteolytic enzyme (caricain)) and 3.4.22.31 (bromeline (ananain));

3.4.23 (being so-called aspartic acid endopeptidase) comprises 3.4.23.1 (pepsin A), 3.4.23.18 (aspergillus asparagus fern acyl proteolytic enzyme I (aspergillopepsin I)), 3.4.23.20 (mould asparagus fern acyl proteolytic enzyme (penicillopepsin)) and 3.4.23.25 (yeast asparagus fern acyl proteolytic enzyme (saccharopepsin)); With

3.4.24 (being so-called Zinc metalloproteinase) comprises 3.4.24.28 (tetaine (Bacillolysin)).

The example of relevant subtilisin comprises subtilisin BPN ', subtilisin amylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147, thermitase, aqualysin, genus bacillus PB92 proteolytic enzyme, Proteinase K, proteolytic enzyme TW7 and proteolytic enzyme TW3.

The particular example of the commercial proteolytic enzyme that these obtain easily comprises Esperase , Alcalase , Neutrase , Durazym , Savinase , Pyrase , Pancreatic Trypsin NOVO (PTN), Bio-Feed Pro, Clear-LensPro, Everlase , Kanase , Relase , V8Proteinase  (all enzymes can be obtained by Novo Nordisk A/S).

The example of other commercial proteolytic enzyme comprises Maxatase , Maxacal , Maxapem , Opticlean , Properase  and the Purafect  that Genencor International sells.

Be appreciated that ease variants also is considered as parent protease.EP 130.756 (Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), people such as Thomas, (1985), Nature, 318,375-376, people such as Thomas, (1987), J.Mol.Biol., 193,803-813, people such as Russel, (1987), Nature, 328,496-500, WO88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Novo Nordisk A/S), WO 91/00345 (Novo Nordisk A/S), EP525 610 (Solvay), with the example of having described these ease variants among the WO 94/02618 (Gist-Brocades N.V.).

Also should mention disclosed C component among EP 482,879 B1 (Shionogi).

Protease activities can be as " Methods of Enzymatic Analysis ", the third edition, and 1984, Verlag Chemie, Weinheim, that describes in the 5th volume measures.

The desirable protein lytic enzyme comprises the proteolytic enzyme that is selected from down group: have above-mentioned characteristic (be number, the linking group of linking group the position, or the like) acid aspartate protease, L-Cysteine HCL Anhydrous, serine protease (such as subtilisin) or metalloprotease.

The particular example that contains the suitable parent protease of suitable number linking group is listed in following table 1: table 1:

Enzyme	The quantity of linking group	Molecular weight kDa	Reference
				????PD498	????13	????29	????SEQ?ID?NO:2 ????WO?93/24623
????Savinase	????6	????27	People such as von der Osten, (1993), Journal of Biotechnology, 28,55+
				Proteinase K	????9	????29	People such as Gunkel, (1989), Eur.J. Biochem., 179,185-194
Proteolytic enzyme R	????5	????29	People such as Samal, (1990), Mol. Microbiol., 4,1789-1792
				Proteolytic enzyme T	????14	????29	People such as Samal, (1989), Gene, 85, p.329-333
Subtilisin DY	????13	????27	People such as Betzel, (1993), Arch. Biophys., 302 (2), 499-502
				????Lion?Y	????15	????46	????SEQ?ID?NO:4 ????JP?04197182-A
				????Jal?6	????5	????28	????WO?92/17576
Thermolysin	????12	????34	People such as Titani, (1972), Nature New Biol., 238,35-37 and SEQ ID NO:5
				Alcalase  (the Carlsberg variant of natural subtilisin)	????10	????27	People such as von der Osten, (1993), Journal of Biotechnology, 28,55+

Subtilisin PD498 molecular weight is 29kDa, and by SEQ ID NO:2 as can be seen, has 12 to be positioned at Methionin group and 1 N-terminal amino that the enzyme surface can supply polymkeric substance to connect.As mentioned above, preferred enzyme has the Methionin that extensively distributes on the surface.PD498 does not have lysine residue in the distance of distance avtive spot 0-10 dust, make it to be particularly suitable for modified forms.And wherein lysine residue is distributed widely in the surface (promptly away from avtive spot) of this enzyme.

Subtilisin DY molecular weight is 27kDa, and 12 amino (being lysine residue) and 1 N-terminal amino (seeing SEQ ID NO:3) that are positioned at the enzyme surface are arranged.

Parent protease Lion Y molecular weight is 46kDa, and 14 amino (being lysine residue) and 1 N-terminal amino (seeing SEQ ID NO:4) that are positioned at the enzyme surface are arranged.

Neutral metal proteolytic enzyme thermophilic bacteria protein enzyme molecular weight is about 34kDa, and 11 amino (being lysine residue) and 1 N-terminal amino (seeing SEQ IDNO:5) that are positioned at the enzyme surface are arranged.Parent lipase

Parent lipase comprises the lipase that is arranged in enzyme classification numbering E.C.3.1.1 (carboxylic ester hydrolase) group according to recommendation (1992) classification of biological chemistry and molecular biology League of Nations (IUBMB).

Example comprises that classification is positioned at the lipase under the following enzyme classification numbering:

3.1.1 (being so-called carboxylic ester hydrolase) comprises (3.1.1.3) triacylglycerol lipase and (3.1.1.4) Phospholipase A2.

The example of lipase comprises by following microorganism deutero-lipase.Listed patent publications is quoted as a reference herein:

Humicola is as H.brevispora, H.lanuginosa, H.brevisvar.thermoidea and H.insolens (US4,810,414);

Rhodopseudomonas, as Pseudomonas fragi, Pseudomonas stutzeri, pseudomonas cepacia and Pseudomonas fluorescens (WO 89/04361), plant pseudomonas, gladiolus pseudomonas (U.S. Patent number 4,950,417 (Solvay enzymes)), (WO 88/09367 for Pseudomonas alcaligenes, pseudomonas pseudoalcaligenes (EP 218 272) or pseudomonas mendocina; US 5,389, and 536);

Fusarium is as sharp sickle spore (EP 130,064) or F.solani pisi (WO90/09446);

Mucor (being also referred to as Rhizomucor (Rhizomucor)) is as rice black wool mould (EP238 023);

Chromobacterium (particularly thickness look bacillus);

Aspergillus (particularly aspergillus niger);

Mycocandida is as C.cylindracea (be also referred to as and wrinkle candiyeast), C.antarctica (WO 88/02775) or C.Antarctica lipase A or B (WO94/01541 and WO 89/02916);

Geotrichum, as geotrichum candidum (people such as Schimada, (1989), J.Biochem., 106,383-388);

Penicillium, as penicillium cammenberti (people such as Yamaguchi, (1991), Gene, 103,61-67);

Rhizopus, as R.delemar (people such as Hass, (1991), Gene, 109,107-113), snow-white head mold (people such as Kugimiya, (1992), Biosci.Biotech.Biochem., 56,716-719) or Rhizopus oryzae;

Bacillus, as subtilis (people such as Dartois, (1993), Biochemica et Biophysica acta, 1131,253-260), bacstearothermophilus (JP 64/7744992) or bacillus pumilus (WO 91/16422).

The particular example of the commercial lipase that obtains comprises Lipolase , Lipolase  Ultra, Lipozyme , Palatase , Novozym  435, Lecitase  (can be obtained by Novo Nordisk A/S) easily.

The example of other lipase is Lumafast , the pseudomonas mendocina lipase of Genencor Int.Inc.; Lipomax , the pseudomonas pseudoalcaligenes lipase of Gist-Brocades/Genencor Int.Inc.; The fusarium solanae lipase (at) of Unilever; The genus bacillus lipase of Solvay enzymes.Can obtain other lipase by other company.

Be appreciated that lipase Variant also can be considered parent enzyme.As having described these examples among WO 93/01285 and the WO95/22615.

The activity of lipase can be as " Methods of Enzymatic Analysis ", the third edition, 1984, Verlag Chemie, Weinhein describes in the 4th volume, or disclosed mensuration among AF 95/5 GB (can ask for to Novo Nordisk A/S).

The fat hydrolase of expectation comprises Humicola lanuginosa lipase, as disclosed lipase among EP258 068 and the EP 305 216; Rhizomucor miehei lipase is as disclosed among the EP238 023; The mould fat hydrolase of colter (WO 96/13578); Lipase from candida sp is such as C.antarctica lipase, as C.antarctica lipase A or the B that describes among the EP 214 761; Pseudomonas lipase, such as the Pseudomonas alcaligenes of describing among the EP 218 272 and pseudomonas pseudoalcaligenes lipase, as disclosed pseudomonas cepacia lipase among the EP331376, disclosed pseudomonas lipase among the WO 95/14783; Genus bacillus lipase, as bacillus subtilis lipase (people such as Dartois, (1993), Biochemica et Biophysica acta, 1131,253-260), bacstearothermophilus lipase (JP 64/744992) and bacillus pumilus lipase (WO91/16422).The fat hydrolase of other kind comprises at, as by Humicolainsolens, pseudomonas mendocina (WO 88/09367) or Fusariumsolani pisi (as describing among the WO 90/09446) deutero-at.Parent's oxydo-reductase

Parent's oxydo-reductase comprises the oxydo-reductase that is arranged in enzyme classification numbering E.C.1 (oxydo-reductase) group according to recommendation (1992) classification of biological chemistry and molecular biology League of Nations (IUBMB).

Example comprises that classification is positioned at the oxydo-reductase under the following enzyme classification numbering:

Glyceraldehyde-3 phosphate dehydrogenase NAD+ (1.1.1.8), glyceraldehyde-3 phosphate dehydrogenase NAD (P)+(1.1.1.94), phosphoric acid 3-glycerine 1-desaturase NADP (1.1.1.94), glucose oxidase (1.1.3.4), hexose oxidase (1.1.3.5), catechol-oxydase (1.1.3.14), bilirubin oxidase (1.3.3.5), alanine dehydrogenase (1.4.1.1), glutamate dehydrogenase (1.4.1.2), glutamate dehydrogenase NAD (P)+(1.4.1.3), glutamate dehydrogenase NADP+ (1.4.1.4), L-amino acid dehydrogenase (1.4.1.5), serine dehydrogenase (1.4.1.7), valine dehydrogenase NADP+ (1.4.1.8), leucine dehydrogenase (1.4.1.9), glycine dehydrogenase (1.4.1.10), L-amino-acid oxidase (1.4.3.2), D-amino-acid oxidase (1.4.3.3), L-L-GLOD (1.4.3.11), protein-Methionin 6-oxydase (1.4.3.1 3), L-lysyl oxidase (1.4.3.14), L-aspartate oxidase (1.4.3.16), D-amino acid dehydrogenase (1.4.99.1), protein disulfide reductase enzyme (1.6.4.4), thioredoxin reductase (1.6.4.5), protein disulfide reductase enzyme (gsh) (1.8.4.2), laccase (1.10.3.2), catalase (1.11.1.6), peroxidase (1.11.1.7), lipoxidase (1.13.11.12), superoxide-dismutase (1.15.1.1).

Described glucose oxidase can be derived by aspergillus niger and be obtained.

Described laccase can be derived by Polyporus pinsitus, Myceliophtorathermophila, Coprinus cinereus, dry thread Pyrenomycetes, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera and be obtained.

Bilirubin oxidase can deriving obtains by myrothecium verrucaria (Myrothechecium verrucaria).

Peroxidase can by (as) soybean, horseradish or Coprinus cinereus derive and obtain.

The protein disulfide reductase enzyme can be any protein disulfide reductase enzyme of mentioning in the DK number of patent application 768/93,265/94 and 264/94 (Novo Nordisk A/S) (herein quoting as a reference), comprises the protein disulfide reductase enzyme of Niu Qiyuan, by aspergillus oryzae or aspergillus niger deutero-protein disulfide reductase enzyme and intestinal bacteria deutero-DsbA or DsbC.

The particular example of the commercial oxydo-reductase that obtains comprises Gluzyme  (can be obtained by Novo Nordisk A/S) easily.Yet, can obtain other oxydo-reductase by other company.

Be appreciated that the redox enzyme variants also can be considered parent enzyme.

The activity of oxydo-reductase can be as " Methods of EnzymaticAnalysis ", the third edition, and 1984, Verlag Chemie, Weinheim, that describes in the 3rd volume measures.

The laccase of expectation comprises from disclosed laccase among the WO 96/00290 of Novo Nordisk and the WO95/33836.

Other oxydo-reductase comprises catalase, glucose oxidase, peroxidase, haloperoxidase (haloperoxidase), superoxide-dismutase and lipoxidase.Parent's carbohydrase

Parent's carbohydrase can be defined as the enzyme of all sugar chains that can decompose (particularly) five and six-membered ring structure (as starch), promptly is positioned at enzyme under the enzyme classification numbering E.C.3.2 (Glycosylase) according to the recommendation (1992) of biological chemistry and molecular biology League of Nations (IUBMB) classification.Also comprise in the carbohydrase group of the present invention and carbohydrate (as six-membered ring structure, such as D-glucose) can be isomerizated into as five-membered ring structure, as the enzyme of D-fructose.

Example comprises that classification is positioned at the carbohydrase under the following enzyme classification numbering:

α-Dian Fenmei (3.2.1.1), beta-amylase (3.2.1.2), dextran 1,4-alpha-glucosidase (3.2.1.3), cellulase (3.2.1.4), inscribe-1,3 (4)-beta-glucanases (3.2.1.6), inscribe-1,4-beta-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase (3.2.1.14), polygalacturonase (3.2.1.15), N,O-Diacetylmuramidase (3.2.1.17), beta-glucosidase enzyme (3.2.1.21), alpha-galactosidase (3.2.1.22), beta-galactosidase enzymes (3.2.1.23), amylo-1:4,1:6-transglucosidase, 6-Polyglucosidase (3.2.1.33), xylan 1,4-xylobiase (3.2.1.37), dextran inscribe-1,3-β-D-Polyglucosidase (3.2.1.39), Schardinger dextrin inscribe-1,6-Polyglucosidase (3.2.1.41), sucrose alpha-glucosidase (3.2.1.48), dextran inscribe-1,3-alpha-glucosidase (3.2.1.59), dextran 1,4-beta-glucosidase enzyme (3.2.1.74), dextran inscribe-1,6-beta-glucosidase enzyme (3.2.1.75), arabinan inscribe-1,5-α-arabinofuranosidase/xylosidase (3.2.1.99), Sumylact L (3.2.1.108), chitoanase (chitonanase, 3.2.1.132), and xylose isomerase (5.3.1.5).

The example of relevant carbohydrase comprises by Trichoderma harzianum deutero-α-1,3-dextranase; By paecilomyces bacterial strain deutero-α-1,6-dextranase; By subtilis deutero-beta-glucanase; By Humicola insolens deutero-beta-glucanase; By aspergillus niger deutero-beta-glucanase; By Trichoderma bacterial strain deutero-beta-glucanase; Bacterial strain deutero-beta-glucanase by Oerskovia xanthineolytica; Circumscribed-1 by the aspergillus niger deutero-, 4-α-D-Polyglucosidase (glucoamylase); By subtilis deutero-α-Dian Fenmei; By bacillus amyloliquefaciens deutero-α-Dian Fenmei; By bacstearothermophilus deutero-α-Dian Fenmei; By aspergillus oryzae deutero-α-Dian Fenmei; By non-pathogenic microorganism deutero-α-Dian Fenmei; By aspergillus niger deutero-alpha-galactosidase; By Humicola insolens deutero-pentosanase, zytase, cellobiase, cellulase, hemicellulase; By Trichoderma reesei deutero-cellulase; By non-pathogenic moulds deutero-cellulase; By aspergillus niger deutero-polygalacturonase, cellulase, arabinase (arabinase), hemicellulase; By thin mould deutero-dextranase; By non-pathogenic moulds deutero-endoglucanase; By Bacillus acidopullyticus deutero-Starch debranching enzyme; By Kluyveromyces fragilis deutero-beta-galactosidase enzymes; By Trichoderma reesei deutero-zytase.

The particular example of the commercial carbohydrase that obtains comprises Alpha-Gal  easily, Bio-Feed  Alpha, Bio-Feed  Beta, Bio-Feed  Plus, Novozyme  188, Carezyme , Celluclast , Cellusoft , Ceremyl , Citrozym , Denimax , Dezyme , Dextrozyme , Finizym , Fungamyl , Gamanase , Glucanex , Lactozym , Maltogenase , Pentopan , Pectinex , Promozyme , Pulpzyme , Novamyl , Termamyl , AMG (amyloglucosidase Novo), Maltogenase , Sweetzyme , Aquazym , Natalase  (can obtain) by Novo Nordi sk A/S.Can obtain other carbohydrase by other company.

Should be appreciated that the carbohydrase variant also is considered as parent enzyme.

The activity of carbohydrase can be as " Methods of Enzymatic Analysis ", the third edition, and 1984, Verlag Chemie, Weinheim, that describes in the 4th volume measures.The parent transfer enzyme

The parent transfer enzyme comprises according to the recommendation (1992) of biological chemistry and molecular biology League of Nations (IUBMB) classification and is positioned at transferring enzyme under the enzyme classification numbering E.C.2.

The parent transfer enzyme can be any transferring enzyme in the following transferring enzyme subgroup: the transferring enzyme (E.C.2.1) that shifts a carbon-based group; Shift the transferring enzyme (E.C.2.2) of aldehyde radical; Acyltransferase (E.C.2.3); Transglucosylase (E.C.2.4); The alkyl of transfer except that methyl or the transferring enzyme of aromatic base (E.C.2.5); Shift the transferring enzyme (E.C.2.6) of nitrogen-containing group.

In preferred embodiments, the parent transfer enzyme is trans-glutaminases (transglutaminase) E.C.2.3.2.13 (protein-glutamine m-glutamyl transferase).

Trans-glutaminases be can the catalyzing acyl shift reaction enzyme, wherein the γ of peptide bonded glutamine residue-Carboxylamide group is an acry radical donor.Primary amino group in the multiple compound can be used as acyl acceptor and plays a role, and forms single γ-acid amides that replaces of peptide bonded L-glutamic acid subsequently.If the epsilon-amino of the lysine residue in the peptide chain is as acyl acceptor, transferring enzyme will form intramolecularly or intermolecular gamma-glutamyl-ε-lysyl is crosslinked so.

The example of trans-glutaminases has been described in the unsettled DK number of patent application 990/94 (Novo Nordisk A/S).

Parent's trans-glutaminases can be people, animal (as ox) or microorganism origin.

The example of these parent's trans-glutaminasess is the trans-glutaminasess by animal derived, FX III a; By bull suede bubble bacterium deutero-microbial transglutaminase (people such as Klein, Journal of Bacteriology, 174,2599-2605); Kind by streptomyces, the kind that comprises lilac grey streptomycete, streptomyces lydicus (claiming Lebanon streptomycete in the past) and streptoverticillium, comprise Streptomyces mobaraensis, streptoverticillum, grey yellowish pink streptoverticillium deutero-trans-glutaminases (people such as Motoki, US 5,156,956; People such as Andou, US5,252,469; People such as Kaempfer, Journal of GeneralMicrobiology, 137,1831-1892; People such as Ochi, InternationalJournal of Sytematic Bacteriology, 44,285-292; People such as Andou, US 5,252, and 469; People such as Williams, Journal of GeneralMicrobiology, 129,1743-1813).

Should be appreciated that the transferring enzyme variant also is considered as parent enzyme.

The activity of trans-glutaminases can be as " Methods of EnzymaticAnalysis ", the third edition, and 1984, Verlag Chemie, Weinheim, that describes in the 1-10 volume measures.

Suitable transferring enzyme comprises disclosed any trans-glutaminases among WO 96/06931 (Novo Nordisk A/S) and the WO96/22366 (Novo Nordisk A/S).Parent's phytase

Parent's phytase comprises according to the recommendation (1992) of biological chemistry and molecular biology League of Nations (IUBMB) classification and is positioned at enzyme under the enzyme classification numbering E.C.3.1.3 (orthophosphoric ester monohydrolase).

Phytase is the enzyme that is changed into the microorganisms of inositol and inorganic phosphorus by the catalysis phytinic acid.

The microorganism that produces phytase comprises bacterium, such as subtilis, bacillus natto and pseudomonas; Yeast is such as yeast saccharomyces cerevisiae; And fungi, such as other kind of aspergillus niger, Fructus Fici aspergillus, Aspergillus awamori, aspergillus oryzae, terreus or Aspergillus nidulans and each Aspergillus.

The example of parent's phytase comprises that classification is positioned at the phytase under the following enzyme classification numbering: 3-phytase (3.1.3.8) and 6-phytase (3.1.3.26).

The activity of phytase can be as " Methods of Enzymatic Analysis ", the third edition, 1984, Verlag Chemie, Weinheim, that describes in the 1-10 volume measures, and perhaps can measure according to the method for describing among the EP-A1-0 420 358 embodiment 2A.Isomerase

Parent's isomerase comprises according to the recommendation (1992) of biological chemistry and molecular biology League of Nations (IUBMB) classification and is positioned at enzyme under the enzyme classification numbering E.C.5.

The example of parent's isomerase is a protein disulfide isomerase.Suitable protein disulfide isomerase comprises the PDI that describes among the WO 95/01425 (Novo Nordisk A/S), but is not limited to these.The industry group compound

Another aspect of the invention relates to and contains " the industry group compound " with the modified polypeptide that improves scourability.

In the context of the invention, " industry group compound " refers to not plan to import the composition of the recycle system.In other words, refer to the composition of not planning through intracutaneous, intravenously or subcutaneous administration.

As mentioned above, for polypeptide, the industrial application of enzyme for example, subject matter are may be because of respiratory system sucks, and cause the reaction of respiratory allergic effect as contact in the tracheae or in the nose.

The example of " industry group compound " is a used polypeptide in composition or product such as the washing composition, particularly enzyme and antimicrobial polypeptide, described washing composition comprises that laundry is used and a bowl dish washing composition, household cleaning product, agrochemicals, personal care product such as skin care products, comprise beauty treatment and shower product, oral and transdermal drug, processing/machining textile compositions for use, hard-surface cleaning compositions etc., particularly including in the present invention be skin care products and washing composition.

Skin care products

Among the present invention, all personal care products that " skin care products " are contained body skin clean, nursing and/or improved looks used also comprise other products, and as hair care product, they in use may contact skin or respiratory system.The corresponding product that is used for animal is also included within the scope of the invention.

The object lesson of related skin care products of the present invention has soap, makeup, cleansing cream, clean lotion for skin care, clean skin milk, soap frost (cream soap), whitening powder, soap powder, cake soap, transparent soap, nail varnish sanitising agent, shampoo, face cream, hair conditioner etc.

Be suitable for the enzymic activity of skin care

The conjugate that skin care compositions of the present invention comprises washing of the present invention or cleaning effect improves to some extent, allergenicity decreases and become known for all compositions of skin care compositions.

Known many enzymic activitys can be used for skin care compositions.

Proteolytic enzyme

Proteolytic enzyme is the effective constituent in the clean skin product.Proteolytic enzyme can be removed the upper strata of the dead keratinocyte of skin, makes skin look more glossy, more pure and fresh thus.And proteolytic enzyme also can improve the slipperiness of skin.

Proteolytic enzyme can be used for toiletry, bath in a tub and shower product, comprises shampoo, amendment, lotion, emulsifiable paste, soap-cake, perfumed soap and liquid soap.

Lipase

The effective constituent that lipase can be used as in clean skin product and the anti-acne products is used for cosmetic use, to remove excess skin lipid, also can be used as skin care effective constituent and is used for bath in a tub and shower product (as emulsifiable paste and lotion).

Lipase also can be used for hair cleaning product (for example shampoo), to remove sebum and other fatty substance from hair surface effectively.

Oxydo-reductase

The most frequently used oxydo-reductase that is used for personal care applications is can guarantee to produce H with substrate (as glucose) ₂O ₂Oxydase (normally notatin), H then ₂O ₂Can initial peroxidase (normally lactoperoxidase) to for example SCN ^-Or I ^-Oxidation, form antimicrobial reagent (SCNO ^-Or I ₂).Known this enzymatic mixture is natural for example to be present in the milk and saliva.

Commercial, always as antimicrobial system, in these products, it also can be used in combination with amyloglucosidase peroxidase in dental care products (collutory, tooth powder, chewing gum), to produce glucose.Known these systems also are used for cosmetic product, are beneficial to preserve.

The oxydo-reductase Another application is to use oxydase, peroxidase and laccase to carry out oxidative coloration of hair (referring to WO 96/00290 or the WO95/33836 of for example Novo Nordisk).

The known free radical that forms on skin (with hair) surface is relevant with the weathering process (damage of hair) of skin.

Free radical meeting intensity of activation causes the chain reaction of adipose membrane, collagen and cytoclasis.

The application of free-radical scavengers (as superoxide-dismutase) in makeup be well-known (R.L.Goldemberg, DCI, Nov.93, P.48-52).

Protein disulfide isomerase (PDI) also is a kind of oxydo-reductase.It can be used for hair-waving (make the disulfide bond reduction of hair and reoxidize) and repair damaged hair (wherein damaging mainly is that reduction has taken place original disulfide linkage).

Change paddy oxanamide enzyme

The skin care compositions that is used for skin, hair or nail comprises: (a) amino functional activeconstituents, (b) the crosslinked trans-glutaminases of catalytic active component and skin, hair or nail, and (c) at United States Patent (USP) NO.5, known carrier in 490,980.

The make-up composition that is suitable for mammal skin, hair or nail comprises: (a) amount is enough to form at least a keratinocyte (corneocyte) envelope protein of protective layer on said skin, hair or nail; (b) amount is enough to make the trans-glutaminases that forms covalent linkage between the keratinocyte protein of the outer exposed in keratinocyte envelope protein and said skin, hair or horny layer of nail; (c) amount is enough to activate the calcium ion of trans-glutaminases; And (d) acceptable carrier in the makeup, wherein said composition comprises and has the biphase emulsifying agent, wherein said keratinocyte envelope protein matter be included in one in this two-phase mutually in, and trans-glutaminases be included in another mutually in (referring to United States Patent (USP) NO.5,525,336).

JP 3083908 has described a kind of skin cosmetics material, wherein contains the trans-glutaminases with the water-soluble substances modified.This modification material is one or more in polyoxyethylene glycol, ethylene glycol, propylene glycol, glycerine, polyvinyl alcohol, glucose, sucrose, Lalgine, carboxymethyl cellulose, starch and the hydroxypropylcellulose for example.This modification is by for example importing active group and finishing with the enzyme bonding.Provide so a kind of relatively gentle to skin, fade in time and spoiled degree is less and have good treatment coarse skin, moisture retention and the material that beautifies the conditioning skin performance.

Skin care products of the present invention

A third aspect of the present invention relates to skin care products, and it comprises skin care compositions of the present invention.Term " skin care products " as above defines.

Skin care products of the present invention can comprise the modifying enzyme of the present invention of significant quantity.This well known to a person skilled in the art significant quantity be generally final skin care products greater than 0-5%.

Skin care products involved in the present invention include but not limited to following product: soap, makeup, cleansing cream, clean lotion for skin care, clean skin milk, soap frost, soap powder, cake soap, transparent soap, nail varnish sanitising agent, shampoo, face cream, hair conditioner etc.Conventional skin care products prescription

Term " composition that is used for skin care products " is meant and comprises all the components that becomes known for the skin care products prescription.The example of this composition can (be edited by Wilfried Umbach referring to " makeup and toiletry ", publish Britain, (1991) by Ellis Horwood company limited) and " tensio-active agents in the consumer's goods " (edit by J.Falbe, publish (1987) by Spring-Verlag).

Below non-limit listed some directiveness prescriptions.These preparations have been summarized important skin care products prescription involved in the present invention.

About 0.5 dyestuff of perfumed soap composition example % tensio-active agent soap (sodium salt) 83-87 sequestrant edetate 0.1-0.3 consistency modifiers sodium-chlor＜0.1 optical whitening agent＜0.1 antioxidant 2, two (1, the 1-the dimethyl ethyl)-4-first 0.1-0.3 of 6-

Base phenol (BHT) whitening agent titanium dioxide 0.1-0.3 spices 1.0-2.0 protease enzyme/lipase 0-5 moisture difference

Synthetic detergent composition example % tensio-active agent lauryl sulfate 30-50

The list of lauryl sulfosuccinate 1-12 fatting agent fatty alcohol 10-20 binder glycerine/distearyl ester 0-10 filler starch 0-10 activating agent salicylic acid 0-1 dyestuff＜0.2 spices 0-2 protease enzyme/lipase 0-5 moisture difference

Foam bath and shower

Foam bath shower composition example % % tensio-active agent lauryl ether sulfate 10-20 10-12

Cocamidopropyl propyl amide dimethyl betaine 2-4 2-4

Ethoxylated fatty acid 0.5-2-fatting agent Fatty Alcohol(C12-C14 and C12-C18) 0.5-3

Ethoxylized fatty alcohol 0.5-5 0-4 protease enzyme/lipase 0-5 0-5

The quaternized hydroxypropyl cellulose of bubble bath shower composition example % % foam stabiliser Marlamid 0.2-2 0-4 conditioner-0-0.5 thickener sodium chloride 0-3 0-3 pearling agent ethylene glycol stearate 0-2-activating agent plant extracts 0-1 0-1 anticorrisive agent 5-bromo-5-nitro-1,3-diox 0.1 0.1 dyestuff 0.1-0.2 0.1 spices 0.3-3 0.3-2 protease enzyme/lipase 0-5 0-5 moisture difference difference

Shampoo composition example % tensio-active agent lauryl ether sulfate 12-16

Fatty acid distribution of coconut oil acyl aminopropyl dimethyl betaine 2-5

Fatty acid polyglycol ester 0-2 suds booster fatty acid ethanol amide 0.5-2.5 amendment quaternized hydroxyethylcellulos 0.4-1

The lanolin alcohol 0.2-1 additive dandruff removing agent 0-1 anticorrisive agent 5-bromo-5-nitro-1 of protein hydrolysate 0.2-1 fatting agent ethoxylation, 3-diox 0.1-0.3 pearling agent ethylene glycol stearate 0-2 dyestuff＜0.1pH conditioning agent acid/alkali 0.1-1 spices 0.3-0.5 protease enzyme/lipase 0-5 moisture difference

Hair conditioner and hair conditioner

Hair conditioner hair conditioner composition example % % tensio-active agent fatty alcohol polyglycol ether 0.1-1.5-

0.2?????????2.5

Palmityl trimethyl ammonium chloride 0.5-1-

The list of dimethyl benzyl stearyl chlorination ammonium-0.5-1 fatting agent glycerine/two spermaceti acid/stearate 0.5-1.5-

1.5 2.5 consistency modifiers Fatty Alcohol(C12-C14 and C12-C18) 1-2.5 2.5-

3.5 thickening material methylhydroxypropylcellulose 0.3-0.4-

0.6 0.8 amendment quaternized hydroxyethylcellulos 0.1-0.3-

0.3 0.4 sanitas p-Hydroxybenzoate 0.1-0.1-

0.3 0.3 dyestuff＜0.1＜0.1pH conditioning agent acid/alkali 0.1-1 0.1-1 spices 0.2-0.2-

0.5 0.5 protease enzyme/lipase 0-5 0-5 moisture difference difference washing composition disclosure

Detergent composition of the present invention for example can be made hand washing and machine washing of clothes detergent composition, comprise laundry additive composition and be suitable for the pretreated composition of dirty fabric, the fabric softening compositions that rinsing is added is suitable for normal domestic use hard surface cleaning operation (comprising that microbial film removes) and the bowl dishwashing is washed the composition of operation.

In microbial film removes, should mention that the alginic acid lyase is a kind of preferred enzyme (referring to JPl012728 A K.K.GUNZE and TANABE SEIYAKU GO, it is incorporated herein by reference).

Detergent composition of the present invention comprises conjugate of the present invention and tensio-active agent.Can randomly comprise washing assistant in addition, other enzyme, froth suppressor, softener, dye transfer inhibitor, and other composition that uses traditionally in the washing composition, as soil-suspending agent, soil release agent, white dyes, abrasive material, sterilant, discoloration inhibitor, tinting material and/or coating or the spices that do not coat.

Detergent composition of the present invention can be liquid, paste, gel, strip or granular form.PH (recording in the aqueous solution of working concentration) is normally neutral or alkaline, in the pH7-11 scope.Granular composition of the present invention also can be " a fine and close form ", and promptly they have granulated detergent relative higher density, the i.e. 550-950g/l than routine.

Press composition weight meter, generally with 0.00001-2%, preferred 0.0001-1%, more preferably 0.001-0.5%, most preferably the amount of 0.01-0.2% zymoprotein is incorporated into enzyme conjugate of the present invention or other optional enzyme of mixing in the detergent composition in the detergent composition of the present invention.But the dosage of enzyme depends on the allergenicity of enzyme and the scourability of improvement, that is, low allergenicity can adopt high dosage, has improved scourability and then can adopt low dosage.Surfactant system

Surfactant system can comprise nonionogenic tenside, anion surfactant, cats product, amphoterics and/or zwitterionics.Surfactant system preferably is made up of anion surfactant, or the mixture of anion surfactant and nonionogenic tenside, accounts for 50-100% as anion surfactant, and nonionogenic tenside accounts for 0-50%.Also can contain cats product, amphoterics, zwitterionics and semi-polarity tensio-active agent in the laundry detergent composition, and nonionogenic tenside and/or anion surfactant but not above-mentioned those.Tensio-active agent generally exists with the amount of 0.1-60% weight.Introduce some examples of tensio-active agent below.Nonionogenic tenside:

Tensio-active agent can comprise polyalkylene oxide (as the polyoxyethylene) condenses of alkylphenol.These alkyl can comprise about 6-14 carbon atom, can be the straight or branched configurations.The amount that oxyethane exists is equivalent to every mole of about 2-25 mole of alkylphenol.

Tensio-active agent also can comprise the condenses of primary and secondary fatty alcohol and about 1-25 moles of ethylene oxide.The alkyl chain of this fatty alcohol can be a straight or branched, and contains 8-22 the carbon atom of having an appointment usually.

In addition, nonionogenic tenside can comprise condensation product, alkyl polysaccharide and its mixture of polyoxyethylene condenses, primary and secondary fatty alcohol and about 1-25 moles of ethylene oxide of alkylphenol.The C that most preferably has 3-15 oxyethyl group ₈-C ₁₄Alkylphenol ethoxylate and C with 2-10 oxyethyl group ₈-C ₁₈(preferred average out to C ₁₀) alcohol ethoxylate and its mixture.Anion surfactant:

Suitable anion surfactant comprises formula RO (A) _mSO ₃The water-soluble salt of M or the such alkyl alkoxylated suifate of acid, wherein R is unsubstituted C ₁₀-C ₂₄Alkyl or have C ₁₀-C ₂₄The hydroxyalkyl of moieties, preferred C ₁₂-C ₂₀Alkyl or hydroxyalkyl, more preferably C ₁₂-C ₁₈Alkyl or hydroxyalkyl, A are oxyethyl group or propoxy-unit, and m is greater than 0, be generally about 0.5-6,0.5-3 more preferably from about, M is H or positively charged ion, this positively charged ion can be: for example metallic cation (as sodium, potassium, lithium, calcium, magnesium etc.), ammonium or replace ammonium cation.Estimate to use alkyl ethoxylated sulfate and alkyl propoxylated sulphates herein.The object lesson that replaces ammonium cation comprises: methyl, dimethyl, trimethyl ammonium positively charged ion and quaternary ammonium cation such as tetramethyl-ammonium and lupetidine positively charged ion and from alkylamine such as ethamine, diethylamine, triethylamine, its those positively charged ions of mixture deutero-etc.

Other suitable anion surfactant comprises formula ROSO ₃The water-soluble salt of M or the such alkyl sulfate surfactant of acid, wherein R C preferably ₁₀-C ₂₄Alkyl preferably has C ₁₀-C ₂₀The alkyl of moieties or hydroxyalkyl, more preferably C ₁₂-C ₁₈Alkyl or hydroxyalkyl, M are H or positively charged ion (as sodium, potassium, lithium ion), perhaps ammonium or substituted ammonium ion.

Other anion surfactant comprise soap salt (comprise, for example sodium, potassium, ammonium and the ammonium that replaces such as list, two and triethanolamine salt), C ₈-C ₂₂Uncle or secondary paraffin sulfonate, C ₈-C ₂₄The sulfonation poly carboxylic acid of alkene sulfonate, the preparation of the sulfonation by the alkaline earth metal citrate pyrolysis product.Alkylbenzene sulfonate is proper, particularly linear (straight chain) alkylbenzene sulfonate (LAS), and wherein this alkyl preferably contains 10-18 carbon atom.

Usually contain the 1-40% that has an appointment, preferred this analog anion surfactants of about 3-20% in the laundry detergent composition.Builder system:

Composition of the present invention also can comprise builder system.Any conventional builder system all is applicable to herein, and it comprises alumino-silicate materials, silicate, multi-carboxylate and lipid acid, such as the material of edetate (EDTA), metal ion chelation agent aminopolyphosphonic acid salt for example.Also can use phosphate builders herein.

Suitable washing assistant can be an inorganic ion exchange material, normally the alumino-silicate materials of inorganic hydration, the synthetic zeolite of hydration, for example zeolite A, X, B, HS or the MAP of hydration more specifically.

The amount of detergent builder compound salt is generally the 5-80% of composition weight.The preferred amounts that is used for the washing assistant of liquid washing agent is 5-30%.Other detersive enzyme activity:

Except the conjugate of the present invention that contains the tool specific activity, detergent composition of the present invention also can contain other enzymic activity (for example also being the form of the above-mentioned enzyme conjugate of the present invention) that cleaning performance and/or fabric nursing benefit are provided, for example proteolytic enzyme, lipase, at, amylase, cellulase, peroxidase, haloperoxidase, oxydase (for example laccase).

The concrete enzyme example of considering be " enzymic activity " listed those in saving in the above.SYNTHETIC OPTICAL WHITNER:

Also can contain SYNTHETIC OPTICAL WHITNER in the detergent composition (particularly under the situation of granulated detergent), as oxygen type SYNTHETIC OPTICAL WHITNER or halogen-type SYNTHETIC OPTICAL WHITNER.Oxygen bleaching agent can be the hydrogen peroxide releasing agent, and for example perborate (as PB1 or PB4) or percarbonate perhaps can be percarboxylic acids, and its particle diameter can be the 400-800 micron.When having oxygen type bleaching compounds, the amount of its existence generally is about 1-25%.

The hydrogen peroxide releasing agent can be used with bleach-activating agent; this bleach-activating agent is: for example tetra acetyl ethylene diamine (TAED), nonanoly acyloxy benzene sulfonate (NOBS), 3,5-trimethyl acetyl oxygen base benzene sulfonate (ISONOBS) or penta-acetyl glucose (PAG).

The halogen-type SYNTHETIC OPTICAL WHITNER can be a hypohalite SYNTHETIC OPTICAL WHITNER for example, as TCCA (Trichloroisocyanuric acid), and the sodium of dichloroisocyanuric acid and sylvite, and N-chloro and N-bromo alkane sulphonamide.Such material adds with the 0.5-10wt% of the finished product weight, the amount of preferred 1-5wt% usually.Textile application proteolytic enzyme

Proteolytic enzyme is used for silk is come unstuck and the sand washing processing.Lipase

In the arrangement process of textiles, lipase is used to remove the lipid material (referring to for example WO93/13256 of Novo Nordisk A/S) that contains hydrophobicity ester (as Witepsol W-S 55).Oxydo-reductase

In the bleach clean-up of textiles, catalase can be used for removing unnecessary hydrogen peroxide.Carbohydrase

In the arrangement of jean clothes, cellulolytic enzyme is widely used, so that the local change of fabric colourity (enzyme promoted " granite-wash ").

Cellulolytic enzyme also can be used in the biopolishing processing.Biopolishing is the special processing that the sand washing surface is carried out, and can improve the character of fabric feeling and outward appearance aspect, but the fabric wetting properties is incurred loss.Can use the described method of WO93/20278 and carry out biopolishing.

In the fabrication processes of textiles, silk thread can be subjected to no small mechanical force usually.For avoiding fracture, usually use gel-like substance (slurry) to they starchings.The most generally the sizing agent of Shi Yonging is natural or the starch of modified forms.Only after having removed the slurry on the fabric (being so-called destarch), just finished peculiar and persistent arrangement.The destarch process of the fabric of crossing with the slurry sizing that contains starch or treated starch preferably utilizes amylolytic enzyme to promote.Material and method material enzyme: the proteolytic enzyme of the subtilisin type shown in the PD498:WO 93/24623.The sequence of PD498 shows in SEQ ID NO.1 and 2.Subtilisin DY: separate from the genus bacillus mutation, be shown in SEQ ID NO:3 subtilisin type proteolytic enzyme (Betzel etc. (1993), biophysics archives (Archives of Biophysics), 302 volumes, 2, P.499-502).Savinase ^Savinase variant R247K (adopt the BPN numbering, the arginine at 247 places is replaced by Methionin in the position).ELISA reagent: the anti-rabbit Ig of the pig of horseradish peroxidase-labeled (Dako, DK, P217, extent of dilution 1: 1000).Rat anti-mouse IgE (Serotec MCA419; Extent of dilution 1: 100).Mouse anti rat IgE (Serotec MCA193; Extent of dilution 1: 200).Biotin labeled mouse anti rat IgG1 monoclonal antibody (Zymed03-9140; Extent of dilution 1: 1000) biotin labeled rat anti-mouse IgG1 monoclonal antibody (Serotec MCA336B; Extent of dilution 1: 2000) Streptavidin-horseradish peroxidase (Kirkegard and Perry14-30-00; Extent of dilution 1: 1000).Damping fluid and solution :-PBS (pH7.2 (1 liter)) NaCl 8.00g KCl 0.20g K ₂HPO ₄1.04g KH ₂PO ₄0.32g-lavation buffer solution PBS, 0.05% (v/v) Tween20-sealing damping fluid PBS, 2% (wt/v) skim-milk-dilution buffer liquid PBS, 0.05% (v/v) Tween20,0.5% (wt/v) skim-milk-citrate buffer (0.1M, pH5.0-5.2 (1 liter)) Trisodium Citrate 20.60g citric acid 6.30g-stop bath (DMG damping fluid)-Sodium Tetraborate, borax (Sigma)-3,3-dimethylated pentanedioic acid (Sigma)-CaCl ₂(Sigma)-and Tween20: polyoxyethylene sorbitan mono-laurate (Merck catalog number (Cat.No.) 822184)-N-hydroxy-succinamide (Fluka art.56480)-carbonyl chloride (Fluka art.79380)-lactose (Merck 7656)-PMSF (phenyl methyl sulfonic acid fluoride) Sigma-succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline (Suc-AAPF-pNP) Sigma NO.S-7388, molecular weight 624.6g/ mole.-mPEG (Fluka) proteolytic enzyme model detergent 95 is the household detergent prescription: 25% STP (Na ₅P ₃O ₁₀) 25% Na ₂SO ₄10% Na ₂CO ₃20% LAS (Nansa 80S), 5% NI (Dobanol 25-7), 5% Na ₂Si ₂O ₅0.5% carboxymethyl cellulose (CMC), 9.5% water EMPA116: the blood on cotton, milk, prepared Chinese ink EMPA117: the blood on PE/BO, milk, the painted substrate of prepared Chinese ink: OPD: O-Phenylene Diamine, (Kementec catalog number (Cat.No.) 4260) experimental animal: Brown Norway rat (derives from Charles River, DE) equipment: XCEL II (Novex) ELISA readout instrument (UVmax, Molecular Devices) HPLC (Waters) PFLC (Pharmacia) Superdex-75 post, Mono-Q, MonoS (available from Pharmacia, SW.) SLT: available from the Fotometer of SLT LabInstruments.Size exclusion chromatography instrument (Spherogel TSK-G2000 SW).The size exclusion chromatography instrument (Superdex 200, Pharmacia, and SW) the Amicon sulculus adopts the Filtron UltrasetteMiniwash RobotJ﹠amp of Omega 10K film; M Tidas MMS/16 photometer has CLX 75X xenon lamp and fiber eyeglass method: (IT) stimulates in the tracheae of Brown Norway rat

The disposable syringe that use has 21/2 inchage metal probe carries out using molecule in the tracheae.This probe inserts in the tracheae of rat into 1cm under epiglottis greatly, places the molecular solution of 0.1ml.

Experimental animal is 10 every group a Brown Norway rat (BN).Weight is tested about 450 grams of weight when finishing greater than 200 grams when the experiment beginning.Determine the ELISA method of IgE antibody relative concentration in the Brown Norway rat

Use thriple decker sandwich ELISA (three layer sandwish ELISA) to determine the relative concentration of specific IgE serum antibody.

1) wraps by elisa plate with 10mg mouse anti rat IgE damping fluid 1.50 μ l/ holes.Under 4 ℃, be incubated overnight.

2) turned letter plate and with the sealing damping fluid at room temperature sealed at least 1/2 hour.200 μ l/ holes.Slightly shake.With lavation buffer solution wash plate 3 times.

3) with the rat blood serum incubation, from undiluted serum, and with 2 times of extent of dilution serial dilutions.Stay some holes only to handle (blank) with damping fluid 4.50 μ l/ holes.At room temperature incubation is 30 minutes.Slightly shake.Wash plate is 3 times in lavation buffer solution.

4) in dilution buffer liquid, enzyme is diluted to suitable protein concn.50 μ l/ holes are incubation 30 minutes at room temperature.Slightly shake.Wash plate is 3 times in lavation buffer solution.

5) dilute specific polyclonal antienzyme antiserum(antisera) serum (pIg) to be used to detect binding antibody with dilution buffer liquid.50 μ l/ holes are incubation 30 minutes at room temperature.Slightly shake.Wash plate is 3 times in lavation buffer solution.

6) the anti-pIg antibody of puting together with dilution buffer liquid dilution horseradish peroxidase.50 μ l/ holes are incubation 30 minutes at room temperature.Slightly shake.With lavation buffer solution wash plate 3 times.

7) in substrate buffer solution, mix 0.6mg ODP/ml+0.4 μ l H ₂O ₂/ ml.Use immediately after preparing this solution.Incubation 10 minutes.50 μ l/ holes.

8) termination reaction: add stop bath 50 μ l/ holes.

9) at 492 nm to dull and stereotyped reading, with the reading of 620nm as reference.Data also show with the Lotus computed in software.Determining molecular weight

Use the sds page (Novex) of 4-20% gradient, carry out proteinic electrophoretic separation by standard method.Protein dyes detection by silver.Mark-12 with respect to Novex ^The mobility of wide range of molecular weights standard is measured molecular weight.Protease activity is analyzed with Suc-Ala-Ala-Pro-Phe-pNa:

Key between proteolytic enzyme cutting peptide and the p-Nitroaniline is created in the visible yellow color that 405nm absorbs.Damping fluid: for example Britton and Robinson pH of buffer 8.3 substrates: 100mg suc-AAPF-pNa is dissolved in the 1ml dimethyl sulfoxide (DMSO) (DMSO).This solution of 100ml is diluted to 10ml with Britton and Robinson damping fluid.Analyze

Mix substrate and protein enzyme solution, monitoring is as the absorbancy of the function of time and ABS405nm/min under 405nm.Should controlled temperature (between 20-50 ℃, depending on proteolytic enzyme).This measurement be protease activity in the sample.

Embodiment

Embodiment 1

With poly-(the ethylene glycol)-block of N-succinimdyl carbonate activation-poly-(propylene glycol)-block-poly-(ethylene glycol) 1.900 (50wt% second

Glycol)

To gather (ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol) 1.900 (50wt% ethylene glycol) (from ALDRICH) and be dissolved in toluene (5ml/g polymkeric substance).Under normal pressure, steam about 20% with the azeotropic drying reactant.Solution is cooled to 20 ℃, adds the carbonyl chloride (1.93M, 7mol/mol polymkeric substance) in the toluene.Then, mixture is at room temperature stirred spend the night.Solvent removed in vacuo and excessive carbonyl chloride obtain a kind of buttery intermediate double (carbonochloridic acid ester).

Toluene (anhydrous 4ml/g polymkeric substance) is added oil is dissolved again.Add N-hydroxy-succinamide (NHS) (2.4mol/mol polymkeric substance), mixture is cooled off with ice bath.Drip triethylamine (2.2mol/mol polymkeric substance) down at 0 ℃ again.Can observe immediately and triethylamine hydrochloride precipitation (Et occur ₃N.HCl).Mixture at room temperature stirred spend the night.Adopt glass filter (G5) that mixture is filtered, remove Et ₃N.HCl.Under reduced pressure filtrate is evaporated to driedly, obtains the oily matter of 97% (mol/mol).NMR shows greater than 90% and is activated, and have less than 8o/o (mol/mol) not in conjunction with NHS.1H-NMR (the 400MHz) (CDCl of poly-(ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol) 1.900 pairs of (succinimdyl carbonate) (50wt% ethylene glycol) ₃) δ: 1.15bs (and I=330, PPG-CH ₃), 2.69s (the unreacted NHS of I=1.7), 2.83s (I=41, succinimide), 3.41m (I=110, the CH-CH among the PPG ₂), 3.55m (I=220, the CH-CH among the PPG ₂), 3.61m (I=440 main peak), 4.46t (I=19, the CH among the PEG ₂-O-CO-).

Embodiment 2

Activate with the N-succinimdyl carbonate

Poly-(ethylene glycol)-altogether-(propylene glycol)-single-butyl ether 970 (about 50wt% ethylene glycol)

To gather by (ethylene glycol)-be total to-(propylene glycol) single-butyl ether 970 (about 50wt% ethylene glycol) (from ALDRICH) is dissolved in the toluene (4ml/g polymkeric substance).Distillation removes about 25% with the azeotropic drying reactant under normal pressure.Solution is cooled to 0 ℃, adds the carbonyl chloride (1.93M, 5mol/mol polymkeric substance) in the toluene.Then, mixture was at room temperature stirred 21 hours.Solvent removed in vacuo and excessive carbonyl chloride obtain a kind of buttery intermediate carbonochloridic acid ester.

Toluene (anhydrous 2ml/g polymkeric substance) is added oil is dissolved again.At room temperature, add N-hydroxy-succinamide (NHS) (1.2mol/mol polymkeric substance), drip triethylamine (1.1mol/mol polymkeric substance) down at 0 ℃ again.Can observe immediately and triethylamine hydrochloride precipitation (Et occur ₃N.HCl).Mixture at room temperature stirred spend the night.Adopt sintered glass filter (G5) that mixture is filtered, remove undissolved Et ₃N.HCl.Under reduced pressure filtrate is evaporated to driedly, obtains the oily matter of 89% (mol/mol).NMR shows＞72% activation, and have＜50/0 (mol/mol) not in conjunction with NHS. ¹H-NMR (400MHz, CDCl ₃) δ: 0.91t (I=1000-CH ₃Butyl), 1.15bs (I=8744, in the propylene glycol-CH ₃), 1.39m (I=1320 CH ₃-CH ₂-CH ₂-butyl), 1.55m (I=656-CH ₂-O-butyl), 2.68s (the unreacted NHS of I=60.8), 2.83 s (I=963.2, succinimide), 3.40m (I=3059, the CH-CH in the propylene glycol ₂), 3.55m (I=2678, the CH-CH in the propylene glycol ₂), 3.61m (the I=1764 main peak, in the ethylene glycol-CH ₂-CH ₂-), 4.46m (CH ₂-O-CO-).

Embodiment 3

With N-succinimdyl carbonate activation mPEG 350

MPEG 350 is suspended in (4ml/g mPEG) in the toluene, and distillation removes about 20% with the azeotropic drying reactant under normal pressure.When solution is cooled to 20 ℃, add the carbonyl chloride (1.93M 1.5mol/mol mPEG) in the toluene, mixture at room temperature stirs and spends the night.The mixture reduction vaporization, the intermediate product chloro-formic ester of gained is an oily.

After evaporation, add methylene dichloride and toluene (1: 2, anhydrous 4ml/g mPEG) to dissolve this water white oil again.Add N-hydroxy-succinamide (NHS) (1.5mol/mol mPEG) with solid form, add 0 ℃ of triethylamine (1.1mol/mol mPEG) then, can be observed triethylamine hydrochloride (Et ₃N.HCl) precipitate immediately.Spend the night stirring under the mixture room temperature.With sintered glass filter (G5) filtering mixt to remove Et ₃N.HCl.Filtrate evaporated under reduced pressure to dry, is obtained the oil of 98% (mol/mol).NMR shows have 85-95% to be activated and less than 10o/o (mol/mol) HNEt ₃Cl.MPEG 350 succinimdyl carbonate (CDCl ₃) ¹H-NMR (400MHz) (CDCl ₃) δ is: 1.42t (I=1.4, HNEt ₃CH among the Cl ₃), 2.68s (the unreacted NHS of I=3.4), 2.84s (I=6.2 succinimide), 3.10dq (I=1.0, HNEt ₃CH among the Cl ₂), 3.38s (I=5.8, the CH among the OMe ₃), 3.64bs (I=50 main peak), 4.47t (I=3.0, the CH among the PEG ₂).

Embodiment 4

With N-succinimdyl carbonate activated PEG 300

PEG 300 is dissolved in the toluene (13ml/g mPEG).Distillation removes about 25% with the azeotropic drying reactant under normal pressure.When solution is cooled to 20 ℃, add the carbonyl chloride (1.93M 3.8mol/mol PEG) in the toluene, mixture at room temperature stirred 20 hours.With the mixture reduction vaporization, the intermediate product of gained two (chloro-formic ester) is an oily.

After evaporation, add dry toluene (10ml/g PEG) to dissolve this water white oil again.Add N-hydroxy-succinamide (NHS) (3.0mol/mol mPEG) with solid form, add triethylamine (2.4mol/mol mPEG) down at 0 ℃ then.Can be observed triethylamine hydrochloride (Et ₃N.HCl) precipitate immediately.Spend the night stirring under the mixture room temperature.With sintered glass filter (G5) filtering mixt to remove Et ₃N.HCl.Filtrate evaporated under reduced pressure to dry, is obtained the oil of 90% (mol/mol).NMR shows to have greater than 55% and is activated and less than 5o/o (mol/mol) HNEt ₃Cl. ¹H-NMR (400MHz, CDCl ₃) (I=1.8 is at NEt for δ: 1.11t ₃In CH ₃), 2.69s (I=1.39, unreacted NHS), 2.84s (I=20.0 succinimide), 3.64bs (I=113 main peak), 4.44m (I=10.0, the CH in PEG ₂).

Embodiment 5

With N-succinimdyl carbonate activation mPEG 550

MPEG 550 is dissolved in the toluene (9ml/g mPEG).Distillation removes about 10% with the azeotropic drying reactant under normal pressure.When solution is cooled to 20 ℃, add the carbonyl chloride (1.93M 1.5mol/mol mPEG) in the toluene, mixture at room temperature stirs and spends the night.With the mixture reduction vaporization, the intermediate product chloro-formic ester of gained is an oily.

After evaporation, add dry toluene (4ml/g PEG) and anhydrous methylene chloride (3ml/gPEG) to dissolve this water white oil again.Add N-hydroxy-succinamide (NHS) (1.2mol/mol mPEG) with solid form, add triethylamine (1.2mol/molmPEG) down at 0 ℃ then.Can be observed triethylamine hydrochloride (Et ₃N.HCl) precipitate immediately.Spend the night stirring under the mixture room temperature.With sintered glass filter (G5) filtering mixt to remove Et ₃N.HCl.Filtrate evaporated under reduced pressure to dry, is obtained the viscous oil of 89% (mol/mol).NMR shows to have greater than 77% and is activated and less than 2o/o (mol/mol) HNEt ₃Cl.MPEG 550 succinimdyl carbonates ¹H-NMR (400MHz) (CDCl ₃) δ: 1.41t (I=4.2, HNEt ₃CH among the Cl ₃), 2.69s (the unreacted NHS of I=24.4), 2.84s (I=81 succinimide), 3.10dq (I=3.7, HNEt ₃CH among the Cl ₂), the 3.38s (CH among the I=97OMe ₃), 3.64bs (I=1250 main peak), 4.44m (I=41, the CH among the in PEG ₂).

Embodiment 6

PD498 proteolytic enzyme and activatory mPEG 350 put together

In the final volume of 6ml, in 50mM Sodium Tetraborate (pH9.7), with 62mg PD498 with 20mg (about 200 μ l) with N-succinimdyl carbonate activatory mPEG350 (according to embodiment 1 preparation) incubation.Stir by magnetic, at room temperature react.Reaction times is 2 hours.Is 6.0 to come termination reaction by adding the 0.5M succsinic acid to whole pH.The molecular weight of gained derivative approximately is 33kDa, is equivalent to every mole of PD498 and has connected about 11 moles of mPEG.

With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 7

Subtilisin DY proteolytic enzyme and activatory mPEG 350 put together

Subtilisin DY is puted together through N-succinimdyl carbonate activatory mPEG350 with method identical described in use and the embodiment 2.

It will be apparent to those skilled in the art that, with reference to aforementioned disclosure, can make many modifications and variations and do not depart from its spirit and scope in enforcement of the present invention, therefore, it is the defined content of following claim that scope of the present invention is interpreted as.

Embodiment 8

Savinase variant R247K and activatory mPEG-350 put together

Savinase variant N-succinimdyl carbonate activatory mPEG 350 with 16mg in the Sodium Tetraborate (pH9.5) of 50mM of 21mg is carried out incubation, and final volume is approximately 2ml.Adopt magnetic to stir and at room temperature reacts, and be 9.0-9.5 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.By using 50mM Sodium Tetraborate, 5mM succsinic acid, 1mM CaCl ₂, (Pharmacia carries out size exclusion chromatography, on SW) and handles and remove excessive reagent pH6.0 equilibrated Superdex 75 HiLoad posts.With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 9

Savinase variant R247K and activatory mPEG-550 put together

Savinase variant N-succinimdyl carbonate activatory mPEG 550 with 25mg in the Sodium Tetraborate (pH9.5) of 50mM of 21mg is carried out incubation, and final volume is approximately 2ml.Adopt magnetic to stir and at room temperature reacts, and be 9.0-9.5 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.By using 50mM Sodium Tetraborate, 5mM succsinic acid, 1mM CaCl ₂, (Pharmacia carries out size exclusion chromatography, on SW) and handles and remove excessive reagent pH6.0 equilibrated Superdex 75 HiLoad posts.With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 10

Savinase variant R247K and activatory be two-the puting together of PEG-300

With the Savinase variant of 21mg N-succinimdyl carbonate activatory in the Sodium Tetraborate (pH9.5) of 50mM with 14mg two-PEG 300 carries out incubation, final volume is approximately 2ml.Adopt magnetic to stir and at room temperature reacts, and be 9.0-9.5 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.By using 50mM Sodium Tetraborate, 5mM succsinic acid, 1mM CaCl ₂, (Pharmacia carries out size exclusion chromatography, on SW) and handles and remove excessive reagent pH6.0 equilibrated Superdex 75 HiLoad posts.With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 11

Savinase and activatory be two-the puting together of PEG-200

With the Savinase variant of 827mg N-succinimdyl carbonate activatory in the Sodium Tetraborate (pH9) of 50mM with 420mg two-PEG200 carries out incubation, final volume is approximately 30ml.Adopt magnetic to stir and at room temperature reacts, and be 8.5-9.0 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.Adopt Filtron Ultrasette, cutoff value is that 10kD carries out ultrafiltration to remove excessive reagent.With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 12

Savinase and activatory be two-the puting together of PEG-300

With the Savinase variant of 827mg N-succinimdyl carbonate activatory in the Sodium Tetraborate (pH9) of 50mM with 610mg two-PEG300 carries out incubation, final volume is approximately 30ml.Adopt magnetic to stir and at room temperature reacts, and be 8.5-9.0 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.Adopt Filtron Ultrasette, cutoff value is that 10kD carries out ultrafiltration to remove excessive reagent.With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 13

Savinase and activatory be two-the puting together of PEG-600

With the Savinase variant of 827mg N-succinimdyl carbonate activatory in the Sodium Tetraborate (pH9) of 50mM with 1000mg two-PEG600 carries out incubation, final volume is approximately 100ml.Adopt magnetic to stir and at room temperature reacts, and be 8.5-9.0 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.Adopt Filtron Ultrasette, cutoff value is that 10kD carries out ultrafiltration to remove excessive reagent.With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 14

Savinase and activatory PEG 1000 put together

Savinase N-succinimdyl carbonate activatory PEG 1000 with 2.8g in the Sodium Tetraborate (pH9) of 50mM of 2g is carried out incubation, and final volume is approximately 200ml.Adopt magnetic to stir and at room temperature reacts, and be 8.5-9.0 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.

Adopt Filtron Ultrasette to carry out ultrafiltration to remove excessive reagent.

With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 15

Savinase and activatory PEG 2000 put together

Savinase N-succinimdyl carbonate activatory PEG 2000 with 7.8g in the Sodium Tetraborate (pH9) of 50 mM of 2g is carried out incubation, and final volume is approximately 200ml.Adopt magnetic to stir and at room temperature reacts, and be 8.5-9.0 to keep the pH value by adding 0.5M NaOH.Reaction times is 2 hours.Adding 1M HCl is 6.0 to make to react and stop to final pH value.

Adopt Filtron Ultrasette to carry out ultrafiltration to remove excessive reagent.

With parent enzyme relatively, to the remaining activity of peptide substrates (succinyl--alanyl-alanyl-prolyl-phenylalanyl p-Nitroaniline) near 100%.

Embodiment 16Savinase and poly-(ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol) 1900

Puting together of two (succinimdyl carbonates) (50wt% ethylene glycol)

To be adjusted to pH9.0 in the 148mg Savinase in the 3ml damping fluid with 0.5N NaOH, in enzyme, add this activatory block polymer of 450mg.Then, under magnetic stirs with reaction mixture incubation at room temperature, and with 0.5N NaOH maintenance pH9.0.After 2 hours, use the 0.5M succsinic acid with pH regulator to 6.0.Reaction mixture is carried out purifying through gel-filtration on Superdex 200 posts.Compare with parent enzyme, conjugate is 130% to the remaining activity of DMC.

Embodiment 17 Savinase and poly-(ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol) 2900

Puting together of two (succinimdyl carbonates) (40wt% ethylene glycol)

To be adjusted to pH9.0 in the 148mg Savinase in the 3ml damping fluid with 0.5N NaOH, in enzyme, add this activatory block polymer of 700mg.Then, under magnetic stirs with reaction mixture incubation at room temperature, and with 0.5N NaOH maintenance pH9.0.After 2 hours, use the 0.5M succsinic acid with pH regulator to 6.5.Reaction mixture is carried out purifying through gel-filtration on Superdex 200 posts.Compare with parent enzyme, conjugate is 84% to the remaining activity of DMC.

Embodiment 18 Savinase and poly-(ethylene glycol)-block-poly-(propylene glycol)-block-poly-(ethylene glycol) 8400

Puting together of two (succinimdyl carbonates) (80wt% ethylene glycol)

To be adjusted to pH9.0 in the 148mg Savinase in the 4ml damping fluid with 0.5N NaOH, this activatory block polymer of 2000mg will be dissolved among the 6ml 1mM HCl, add in the enzyme then.Under magnetic stirs with reaction mixture incubation at room temperature, and with 0.5N NaOH maintenance pH9.0.After 2 hours, use the 0.5M succsinic acid with pH regulator to 6.5.Reaction mixture is carried out purifying through gel-filtration on Superdex 200 posts.Compare with parent enzyme, conjugate is 103% to the remaining activity of DMC.

Embodiment 19 Savinase and poly-(ethylene glycol)-block-poly-(propylene glycol)-altogether-poly-(ethylene glycol) 12000

Puting together of two (succinimdyl carbonates) (75wt% ethylene glycol)

To be adjusted to pH9.0 in the 148mg Savinase in the 4ml damping fluid with 0.5N NaOH, this activatory multipolymer of 2800mg will be added in the enzyme.Under magnetic stirs with reaction mixture incubation at room temperature, and with 0.5N NaOH maintenance pH9.0.After 2 hours, use the 0.5M succsinic acid with pH regulator to 6.0.Reaction mixture carries out purifying through gel-filtration on Superdex 200 posts.Compare with parent enzyme, conjugate is 140% to the remaining activity of DMC.

Embodiment 20 Savinase and poly-(ethylene glycol)-block-poly-(propylene glycol)-altogether-poly-(ethylene glycol) 970

Puting together of two (succinimdyl carbonates) (50wt% ethylene glycol)

To be adjusted to pH9.0 in the 148mg Savinase in the 4ml damping fluid with 0.5N NaOH, this activatory multipolymer of 340mg will be added in the enzyme.Under magnetic stirs with reaction mixture incubation at room temperature, and with 0.5N NaOH maintenance pH9.0.After 2 hours, use the 0.5M succsinic acid with pH regulator to 6.0.Reaction mixture carries out purifying through gel-filtration on the Superdex200 post.Compare with parent enzyme, conjugate is 124% to the remaining activity of DMC.

Embodiment 21

The PD498 conjugate of little mPEG polymkeric substance is to Brown Norway

(IT) experiment in the tracheae of rat

PD498 diluted sample to 0.75 μ g protein/ml with known protein matter concentration (optical density(OD) and amino acid sequence analysis by derivative are measured).

Dilute sample is divided into the 1.5ml component to be used for each immunization.These components are stored under-20 the stable condition standby.When research beginning and end, analyze.A new component is all taken in each immunity and analysis.

As described in above-mentioned embodiment, the enzyme conjugate is puted together N-succinimide activatory mPEG350,550,750.Compare with corresponding parent enzyme.

Detect following sample:

Group 1:PD498 (not link coupled parent enzyme-contrast)

Group 2:PD498-SPEG 750

Group 3:PD498-SPEG 550

Group 4:PD498-SPEG 350

With 0.9% (wt/vol) NaCl solution, 100 μ l (control group) or above-mentioned PD498 protein diluent 100 μ l immune rats, weekly, totally 15 times.

Every group has 10 Brown Norway rats.Every once the immunity after a week but before next immunity from eye blood sampling (2ml), by blood coagulation and the centrifugal serum that obtains.

Detect special IgE level with the above-mentioned ELISA test that is specific to rat IgE, measure 1/2 extent of dilution titre of the undiluted liquid of serum, measure the optical density(OD) of 492/620nm.

The IT result of experiment is shown in the following table, shows when research finishes total optical density value of observed per 100 μ l serum in the Brown Norway rat of corresponding PD498 derivative processing.

PD498 conjugate experimental result is shown in the following table 2: table 2:

Immune time	Unmodified	PEG350	?PEG550	?PEG750	????NaCl
						????0	?0.3(0.6)	0.3(0.6)	0.3(0.6)	0.3(0.6)	0.3(0.6)
????15	?5.3(1.6)	2.7(1.2)	1.6(0.6)	1.5(1.4)	0.3(0.6)

Value in the bracket: the standard error of institute's lining average.

As can be seen from Table 2, compare with the rat that is exposed to the unmodified parent enzyme in the tracheae, be exposed to coupling in the tracheae its specific IgE of rat of PD498 conjugate level of replying of little polymkeric substance reduce, so allergenicity decreases.

Embodiment 22

(IT) experiment in the Brown Norway rat tracheae of subtilisin DY conjugate

Repeat embodiment 5 described Brown Norway rat IT researchs, subtilisin DY-SPEG750 conjugate and corresponding parent's subtilisin DY (seeing SEQ IDNO:3) are done one relatively.

Subtilisin DY-PEG750 experimental result is shown in the following table 3: table 3:

Immune time	Unmodified	PEG750	?NaCl
				????0	?0.3(0.6)	0.3(0.6)	0.3(0.6)
????15	?7.2(2.0)	1.9(0.4)	0.3(0.6)

Value in the bracket: the standard error of institute's lining average.

As can be seen from Table 3, compare with the rat that is exposed to the unmodified parent enzyme in the tracheae, be exposed to coupling in the tracheae its specific IgE of rat of subtilisin DY conjugate level of replying of 750Da polymkeric substance reduce, so allergenicity decreases.

Embodiment 23

The skin care formulation that contains the PD498-SPEG conjugate

Preparation contains the following skin care formulation of conjugate of the present invention: skin-care liquid (making 100g) oil phase: whiteruss 35g hexadecanol 5gTween80 7g water: single propylene glycol (MPG) 10g0.4% citrate buffer solution ^*PH5.8 42.9gmethyl paraben 0.1gPD498-SPEG550 ^*10mg (in zymoprotein)

Separately mix oil phase and water, and be heated to 80 ℃.Slowly pour oil phase into aqueous phase while stirring, mixture is cooled to about 35 ℃, add the PD498-SPEG550 conjugate, skin-care liquid is cooled off fast. ^*0.4% citric acid monohydrate compound, pH transfers to 5.9 ^*Usually provide with the preparation that is added with MPG.Should regulate the MPG of aqueous phase according to the amount of MPG in the zymin.Gel (making 100g) MPG 20g ^*H ₂The about 100g citric acid of O 0.4g ^*Carbapol 940 1gPD498-SPEG350 10mg (in zymoprotein)

Mix each composition by above order, before adding carbapol, regulate pH to 5.6.Add carbapol re-adjustment pH afterwards. ^*Regulate according to the amount in the zymin. ^**pH5.6

Embodiment 24

The scourability table 4 of PEG-Savinase and EOPO-Sayinase: experiment is provided with number 1

Washing composition	Washing composition 95 types, 3.0g/l
			The water hardness	????6°dH(2∶1?Ca/Mg)
Enzyme (concentration is benchmark in the absorptiometry value under the 280nm)	Protease Savinase Savinase-PEG1000bis Savinase-PEG2000bis Savinase-PEG4000bis Savinase-PEG6000bis Savinase-PEG10000bis	Concentration 8.1 * 10 -4M ????1.1×10 -4M ????1.2×10 -4M ????1.4×10 -4M ????1.1×10 -4M ????1.1×10 -4M
			Washing time	15 minutes
Temperature	????15℃
			Enzyme concn	????10nM
Experimental technique	Miniwash repeats for robot-3 time
			Sample (Swatch)/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA117

Table 5: experiment is provided with numbers 2

Washing composition	The Tide powdered detergent, 1.0g/l
			The water hardness	????6°dH(2∶1?Ca/Mg)
Enzyme (concentration is benchmark in the absorptiometry value under the 280nm)	Protease Savinase Savinase-PEG1000bis Savinase-PEG2000bis Savinase-PEG4000bis Savinase-PEG6000bis Savinase-PEG10000bis	Concentration 8.1 * 10 -4M ????1.1×10 -4M ????1.2×10 -4M ????1.4×10 -4M ????1.1×10 -4M ????1.1×10 -4M???
			Washing time	10 minutes
Temperature	????25℃
			Enzyme concn	????0；3；6；9；15；25nM
Experimental technique	Miniwash repeats for robot-3 time
			Sample (Swatch)/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA117

Table 6: experiment is provided with numbers 3

Washing composition	The Tide powdered detergent, 1.0g/l
			The water hardness	????6°dH(2∶1?Ca/Mg)
Enzyme (concentration is benchmark in the absorptiometry value under the 280nm)	Proteolytic enzyme Savinase Savinase-PEG1000bis Savinase-PEG2000bis	Concentration 8.1 * 10 -4M ????1.1×10 -4M ????1.2×10 -4M??
			Washing time	10 minutes
Temperature	????25℃
			Enzyme concn	????0；3；6；9；15；25nM
Experimental technique	Miniwash repeats for robot-3 time
			Sample (Swatch)/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA117

Table 7: experiment is provided with numbers 4

Washing composition	Washing composition 95 types, 3.0g/l
			The water hardness	6 ° of dH (EMPA117), 18 ° of dH (grass), (2: 1 Ca/Mg)
Enzyme (concentration is benchmark in the absorptiometry value under the 280nm)	Protease Savinase Savinase-PEG200bis Savinase-PEG300bis Savinase-PEG600bis Savinase-PEG1000bis Savinase-PEG1000bis PS174-PEG1000bis	Concentration 6.8 * 10 -4M ?1.7mg/ml~6.4×10 -5M ?1.6mg/ml~5.8×10 -5M ?6.5mg/ml~2.4×10 -4M ?9.6mg/ml~3.6×10 -4M ?3.4mg/ml~1.3×10 -4M ?1.9mg/ml~6.9×10 -5M
			Washing time	15 minutes
Temperature	????15℃
			Enzyme concn	????10nM
Experimental technique	Miniwash repeats for robot-3 time
			Sample (Swatch)/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA117

Table 8: experiment is provided with 5

Washing composition	????Omo?Color,4g/l
				The water hardness	????18°dH(EMPA116)
Enzyme	Proteolytic enzyme Savinase Savinase-PEG300bis Savinase-EO50PO50	Tension and relaxation (Remission) 25.8 26.2 27.2	Δ tension and relaxation 4.3 4.8 5.8
				Washing time	20 minutes
Temperature	????30℃
				Enzyme concn	????2.5nM
Experimental technique	Miniwash repeats for robot-3 time
				Sample/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA116

Table 9: experiment is provided with 6

Washing composition	????Omo?Color,4g/l
				The water hardness	????18°dH(EMPA116)
Enzyme	Proteolytic enzyme Savinase Savinase-EO50PO50	Tension and relaxation 28.2 28.4	Δ tension and relaxation 6.7 7.0
				Washing time	20 minutes
Temperature	????30℃
				Enzyme concn	????5.0nM
Experimental technique	Miniwash repeats for robot-3 time
				Sample/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA116

Table 10: experiment is provided with 7

Washing composition	????Wisk?HDP,1g/l
				The water hardness	????6°dH(EMPA117)
Enzyme	Proteolytic enzyme Savinase Savinase-PEG300bis Savinase-mPEG350 Savinase-EO50PO50	Tension and relaxation 13.7 14.6 14.4 14.1	Δ tension and relaxation 2.7 3.7 3.4 3.1
				Washing time	10 minutes
Temperature	????25℃
				Enzyme concn	????5.0nM
Experimental technique	Miniwash repeats for robot-3 time
				Sample/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA116

Table 11: experiment is provided with 8

Washing composition	????Wisk?HDP,1g/l
				The water hardness	????6°dH(EMPA117)
Enzyme	Proteolytic enzyme Savinase Savinase-PEG300bis Savinase-mPEG350 Savinase-EO50PO50	Tension and relaxation 15.0 16.1 15.8 16.0	Δ tension and relaxation 4.5 5.6 5.3 5.5
				Washing time	10 minutes
Temperature	????25℃
				Enzyme concn	????10.0nM
Experimental technique	Miniwash repeats for robot-3 time
				Sample/volume	3 * 6cm experiment material, the 50ml detergent solution
Experiment material	????EMPA117

The pH value of detergent solution is adjusted to 10.5 with HCl/NaOH.By in deionized water, adding CaCl ₂And MgCl ₂The adjusting water hardness (referring to, tensio-active agent-theory, technology and application in the consumer's goods, springer Verlag 1986).The pH value of detergent solution is adjusted to 10.5 through adding HCl usually.

By the proteolytic enzyme inactivations that in microwave oven, detergent solution existed in the commercial powder washing composition 85 ℃ of following heating 5 minutes.

The reflectance of experiment material (reflectance) is measured and is carried out under 460nm, adopts J﹠amp; M Tidas MMS/16 photometer, it has CLX 75W xenon lamp and fibre optics eyeglass.Each pieces of fabric is measured together with other pieces of fabric (identical setting) as a setting individually.

(Student-Newman-Keuls) compared in the t-check that utilizes SAS 6.12 softwares that experimental data is carried out variance analysis and 95% significance.

The scourability of different Savinase  variants by Δ reflectance relatively (DeltaReflectance, DR) value is estimated:

DR=R _{Proteolytic enzyme}-R _Blank

DR: Δ reflectance

R _{Proteolytic enzyme}: the reflectance of using the experiment material of the proteolytic enzyme washing of puting together.

R _Blank: with the reflectance of the experiment material of unconjugated proteolytic enzyme washing.The result

Capitalization indicates is checking (SNK, the statistics grouping in hurdle a=0.05) based on t-.If two in identical group (identical letter), then they can not separate statistically.Table 12: average reflection degree value and Statistical information

Experiment number 1	Reflectance
		Blind experiment (Blind)	????9.5??F
????Savinase	????14.3?B
		????Savinase-PEG1000bis	????14.7?A
????Savinase-PEG2000bis	????14.5?B
		Root-mean-square deviation (Root MSE)	????0.2
Square root (R-square)	????0.99

Table 13: average reflection degree value

Experiment numbers 2	????Savinase	????PEG1000	????PEG2000
				Blind experiment	????10.7	????11.1	????10.8
????3nM	????14.2	????15.3	????14.0
				????6nM	????14.2	????15.8	????15.0
????9nM	????15.4	????16.9	????15.3
				????15nM	????16.3	????17.4	????16.3
????25nM	????16.6	????17.6	????17.6

Table 14: average reflection degree value

Experiment numbers 3	????EMPA117
				????Savinase	????PEG1000	????PEG2000
	Blind experiment	????11.5	????12.5				????12.0
????3nM				????13.8	????15.1	????15.5
	????6nM	????14.7	????16.3				????15.8
????9nM				????15.1	????17.1	????16.6
	????15nM	????16.0	????18.3				????17.2
????25nM				????16.4	????18.3	????18.3

Table 15: average reflection degree value

Experiment numbers 4	????EMPA117
		Blind experiment	????9.8??D
????Savinase	????14.9?AB
		????Savinase-PEG200bis	????15.2?AB
????Savinase-PEG300bis	????15.3?AB
		????Savinase-PEG1000bis	????15.5?A
Savinase variant R247K-PEG1000bis	????15.2?AB
		Root-mean-square deviation	????0.4
Square root	????0.96

As can be seen from the above table, compare with unconjugated Savinase, PEG-Savinase and EOPO-Savinase have the scourability of improvement.

Sequence table＜110〉NOVO NORDISK A/S＜120〉have the polypeptide-polymer conjugate of improved scourability＜130〉5625; HkBk＜140〉＜141＜160〉5＜170〉PatentIn Ver.2.1＜210〉1＜211〉840＜212〉DNA＜213〉Bacillus sp.PD498; NCIMB No.40484＜400〉1tggtcaccga atgaccctta ctattctgct taccagtatg gaccacaaaa cacctcaacc 60cctgctgcct gggatgtaac ccgtggaagc agcactcaaa cggtggcggt ccttgattcc 120ggagtggatt ataaccaccc tgatcttgca agaaaagtaa taaaagggta cgactttatc 180gacagggaca ataacccaat ggatcttaac ggacatggta cccattgtgc cggtactgtt 240gctgctgata cgaacaatgg aattggcgta gccggtatgg caccagatac gaagatcctt 300gccgtacggg tccttgatgc caatggaagt ggctcacttg acagcattgc ctcaggtatc 360cgctatgctg ctgatcaagg ggcaaaggta ctcaacctct cccttggttg cgaatgcaac 420tccacaactc ttaagagtgc cgtcgactat gcatggaaca aaggagctgt agtcgttgct 480gctgcaggga atgacaatgt atcccgtaca ttccaaccag cttcttaccc taatgccatt 540gcagtaggtg ccattgactc caatgatcga aaagcatcat tctccaatta cggaacgtgg 600gtggatgtca ctgctccagg tgtgaacata gcatcaaccg ttccgaataa tggctactcc 660tacatgtctg gtacgtccat ggcatcccct cacgtggccg gtttggctgc tttgttggca 720agtcaaggta agaataacgt acaaatccgc caggccattg agcaaaccgc cgataagatc 780tctggcactg gaacaaactt caagtatggt aaaatcaact caaacaaagc tgtaagatac 840＜210〉2＜211〉280＜212〉PRT＜213〉Bacillus sp.PD498,NCIMB No.40484＜400〉2Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln 1 5 10 15Asn Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr

20??????????????????25??????????????????30Gln?Thr?Val?Ala?Val?Leu?Asp?Ser?Gly?Val?Asp?Tyr?Asn?His?Pro?Asp

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

50??????????????????55??????????????????60Asn?Pro?Met?Asp?Leu?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Thr?Val?65??????????????????70??????????????????75??????????????????80Ala?Ala?Asp?Thr?Asn?Asn?Gly?Ile?Gly?Val?Ala?Gly?Met?Ala?Pro?Asp

85??????????????????90??????????????????95Thr?Lys?Ile?Leu?Ala?Val?Arg?Val?Leu?Asp?Ala?Asn?Gly?Ser?Gly?Ser

100?????????????????105?????????????????110Leu?Asp?Ser?Ile?Ala?Ser?Gly?Ile?Arg?Tyr?Ala?Ala?Asp?Gln?Gly?Ala

115?????????????????120?????????????????125Lys?Val?Leu?Asn?Leu?Ser?Leu?Gly?Cys?Glu?Cys?Asn?Ser?Thr?Thr?Leu

130?????????????????135?????????????????140Lys?Ser?Ala?Val?Asp?Tyr?Ala?Trp?Asn?Lys?Gly?Ala?Val?Val?Val?Ala145?????????????????150?????????????????155?????????????????160Ala?Ala?Gly?Asn?Asp?Asn?Val?Ser?Arg?Thr?Phe?Gln?Pro?Ala?Ser?Tyr

165?????????????????170?????????????????175Pro?Asn?Ala?Ile?Ala?Val?Gly?Ala?Ile?Asp?Ser?Asn?Asp?Arg?Lys?Ala

180?????????????????185?????????????????190Ser?Phe?Ser?Asn?Tyr?Gly?Thr?Trp?Val?Asp?Val?Thr?Ala?Pro?Gly?Val

195?????????????????200?????????????????205Asn?Ile?Ala?Ser?Thr?Val?Pro?Asn?Asn?Gly?Tyr?Ser?Tyr?Met?Ser?Gly

210?????????????????215?????????????????220Thr?Ser?Met?Ala?Ser?Pro?His?Val?Ala?Gly?Leu?Ala?Ala?Leu?Leu?Ala225?????????????????230?????????????????235?????????????????240Ser?Gln?Gly?Lys?Asn?Asn?Val?Gln?Ile?Arg?Gln?Ala?Ile?Glu?Gln?Thr

245?????????????????250?????????????????255Ala?Asp?Lys?Ile?Ser?Gly?Thr?Gly?Thr?Asn?Phe?Lys?Tyr?Gly?Lys?Ile

260?????????????????265?????????????????270Asn?Ser?Asn?Lys?Ala?Val?Arg?Tyr

275?????????????????280<210>3<211>274<212>PRT<213>Bacillus?sp.variant<400>3Ala?Gln?Thr?Val?Pro?Tyr?Gly?Ile?Pro?Leu?Ile?Lys?Ala?Asp?Lys?Val??1???????????????5??????????????????10?????????????????15Gln?Ala?Gln?Gly?Tyr?Lys?Gly?Ala?Asn?Val?Lys?Val?Gly?Ile?Ile?Asp

20??????????????????25??????????????????30Thr?Gly?Ile?Ala?(Ala/Ser)?Ser?His?Thr?Asp?Leu?Lys?Val?Val?Gly?Gly?Ala

35???????????????????????40??????????????????45Ser?Phe?Val?Ser?Gly?Glu?Ser?Tyr?Asn?Thr?Asp?Gly?Asn?Gly?His?Gly

50??????????????????55??????????????????60Thr?His?Val?Ala?Gly?Thr?Val?Ala?Ala?Leu?Asp?Asn?Thr?Thr?Gly?Val?65??????????????????70??????????????????75??????????????????80Leu?Gly?Val?Ala?Pro?Asn?Val?Ser?Leu?Tyr?Ala?Ile?Lys?Val?Leu?Asn

85??????????????????90??????????????????95Ser?Ser?Gly?Ser?Gly?Thr?Tyr?Ser?Ala?Ile?Val?Ser?Gly?Ile?Glu?Trp

100?????????????????105?????????????????110Ala?Thr?Gln?Asn?Gly?Leu?Asp?Val?Ile?Asn?Met?Ser?Leu?Gly?Gly?Pro

115?????????????????120?????????????????125Ser?Gly?Ser?Thr?Ala?Leu?Lys?Gln?Ala?Val?Asp?Lys?Ala?Tyr?Ala?Ser

130?????????????????135?????????????????140Gly?Ile?Val?Val?Val?Ala?Ala?Ala?Gly?Asn?Ser?Gly?Ser?Ser?Gly?ser145?????????????????150?????????????????155?????????????????160Gln?Asn?Thr?Ile?Gly?Tyr?Pro?Ala?Lys?Tyr?Asp?Ser?Val?Ile?Ala?Val

165?????????????????170?????????????????175Gly?Ala?Val?Asp?Ser?Asn?Lys?Asn?Arg?Ala?Ser?Phe?Ser?Ser?Val?Gly

180?????????????????185?????????????????190(Ala/Ser)?Glu?Leu?Glu?Val?Met?Ala?Pro?Gly?Val?Ser?Val?Tyr?Ser?Thr?Tyr

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

210?????????????????215?????????????????220His?Val?Ala?Gly?Ala?Ala?Ala?Leu?Ile?Leu?Ser?Lys?Tyr?Pro?Thr?Leu225?????????????????230?????????????????235?????????????????240Ser?Ala?Ser?Gln?Val?Arg?Asn?Arg?Leu?Ser?Ser?Thr?Ala?Thr?Asn?Leu

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

260?????????????????265?????????????????270Ala?Gln?<210>4<211>433<212>PRT<213>Bacillus?sp.Y<400>4Asn?Asp?Val?Ala?Arg?Gly?Ile?Val?Lys?Ala?Asp?Val?Ala?Gln?Asn?Asn??1???????????????5??????????????????10??????????????????15Tyr?Gly?Leu?Tyr?Gly?Gln?Gly?Gln?Leu?Val?Ala?Val?Ala?Asp?Thr?Gly

20??????????????????25??????????????????30Leu?Asp?Thr?Gly?Arg?Asn?Asp?Ser?Ser?Met?His?Glu?Ala?Phe?Arg?Gly

35??????????????????40??????????????????45Lys?Ile?Thr?Ala?Leu?Tyr?Ala?Leu?Gly?Arg?Thr?Asn?Asn?Ala?Ser?Asp

50??????????????????55??????????????????60Pro?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Ser?Val?Leu?Gly?Asn?Ala?65??????????????????70??????????????????75??????????????????80Leu?Asn?Lys?Gly?Met?Ala?Pro?Gln?Ala?Asn?Leu?Val?Phe?Gln?Ser?Ile

85??????????????????90??????????????????95Met?Asp?Ser?Ser?Gly?Gly?Leu?Gly?Gly?Leu?pro?Ser?Asn?Leu?Asn?Thr

100?????????????????105?????????????????110Leu?Phe?Ser?Gln?Ala?Trp?Asn?Ala?Gly?Ala?Arg?Ile?His?Thr?Asn?Ser

115?????????????????120?????????????????125Trp?Gly?Ala?Pro?Val?Asn?Gly?Ala?Tyr?Thr?Ala?Asn?Ser?Arg?Gln?Val

130?????????????????135?????????????????140Asp?Glu?Tyr?Val?Arg?Asn?Asn?Asp?Met?Thr?Val?Leu?Phe?Ala?Ala?Gly145?????????????????150?????????????????155?????????????????160Asn?Glu?Gly?Pro?Asn?Ser?Gly?Thr?Ile?Ser?Ala?Pro?Gly?Thr?Ala?Lys

165?????????????????170?????????????????175Asn?Ala?Ile?Thr?Val?Gly?Ala?Thr?Glu?Asn?Tyr?Arg?Pro?Ser?Phe?Gly

180?????????????????185?????????????????190Ser?Ile?Ala?Asp?Asn?Pro?Asn?His?Ile?Ala?Gln?Phe?Ser?Ser?Arg?Gly

195?????????????????200?????????????????205Ala?Thr?Arg?Asp?Gly?Arg?Ile?Lys?Pro?Asp?Val?Thr?Ala?Pro?Gly?Thr

210?????????????????215?????????????????220Phe?Ile?Leu?Ser?Ala?Arg?Ser?Ser?Leu?Ala?Pro?Asp?Ser?Ser?Phe?Trp225?????????????????230?????????????????235?????????????????240Ala?Asn?Tyr?Asn?Ser?Lys?Tyr?Ala?Tyr?Met?Gly?Gly?Thr?Ser?Met?Ala

245?????????????????250?????????????????255Thr?Pro?Ile?Val?Ala?Gly?Asn?Val?Ala?Gln?Leu?Arg?Glu?His?Phe?Ile

260?????????????????265?????????????????270Lys?Asn?Arg?Gly?Ile?Thr?Pro?Lys?Pro?Ser?Leu?Ile?Lys?Ala?Ala?Leu

275?????????????????280?????????????????285Ile?Ala?Gly?Ala?Thr?Asp?Val?Gly?Leu?Gly?Tyr?Pro?Ser?Gly?Asp?Gln

290?????????????????295?????????????????300Gly?Trp?Gly?Arg?Val?Thr?Leu?Asp?Lys?Ser?Leu?Asn?Val?Ala?Tyr?Val305?????????????????310?????????????????315?????????????????320Asn?Glu?Ala?Thr?Ala?Leu?Ala?Thr?Gly?Gln?Lys?Ala?Thr?Tyr?Sar?Phe

325?????????????????330?????????????????335Gln?Ala?Gln?Ala?Gly?Lys?Pro?Leu?Lys?Tle?Ser?Leu?Val?Trp?Thr?Asp

340?????????????????345?????????????????350Ala?Pro?Gly?Ser?Thr?Thr?Ala?Ser?Tyr?Thr?Leu?Val?Asn?Asp?Leu?Asp

355?????????????????360?????????????????365Leu?Val?Ile?Thr?Ala?Pro?Asn?Gly?Gln?Lys?Tyr?Val?Gly?Asn?Asp?Phe

370?????????????????375?????????????????380Ser?Tyr?Pro?Tyr?Asp?Asn?Asn?Trp?Asp?Gly?Arg?Asn?Asn?Val?Glu?Asn385?????????????????390?????????????????395?????????????????400Val?Phe?Ile?Asn?Ala?Pro?Gln?Ser?Gly?Thr?Tyr?Ile?Ile?Glu?Val?Gln

405?????????????????410?????????????????415Ala?Tyr?Asn?Val?Pro?Ser?Gly?Pro?Gln?Arg?Phe?Ser?Leu?Ala?Ile?Val

420?????????????????425?????????????????430His<210>5<211>316<212>PRT<213>Bacillus?thermoproteolycicus<400>5Ile?Thr?Gly?Thr?Ser?Thr?Val?Gly?Val?Gly?Arg?Gly?Val?Leu?Gly?Asp??1???????????????5??????????????????10??????????????????15Gln?Lys?Asn?Ile?Asn?Thr?Thr?Tyr?Ser?Thr?Tyr?Tyr?Tyr?Leu?Gln?Asp

20??????????????????25??????????????????30Asn?Thr?Arg?Gly?Asp?Gly?Ile?Phe?Thr?Tyr?Asp?Ala?Lys?Tyr?Arg?Thr

35??????????????????40??????????????????45Thr?Leu?Pro?Gly?Ser?Leu?Trp?Ala?Asp?Ala?Asp?Asn?Gln?Phe?Phe?Ala

50??????????????????55??????????????????60Ser?Tyr?Asp?Ala?Pro?Ala?Val?Asp?Ala?His?Tyr?Tyr?Ala?Gly?Val?Thr?65??????????????????70??????????????????75??????????????????80Tyr?Asp?Tyr?Tyr?Lys?Asn?Val?His?Asn?Arg?Leu?Ser?Tyr?Asp?Gly?Asn

85??????????????????90??????????????????95Asn?Ala?Ala?Ile?Arg?Ser?Ser?Val?His?Tyr?Ser?Gln?GLy?Tyr?Asn?Asn

100?????????????????105?????????????????110Ala?Phe?Trp?Asn?Gly?Ser?Glu?Met?Val?Tyr?Gly?Asp?Gly?Asp?Gly?Gln

115?????????????????120?????????????????125Thr?Phe?Ile?Pro?Leu?Ser?Gly?Gly?Ile?Asp?Val?Val?Ala?His?Glu?Leu

130?????????????????135?????????????????140Thr?His?Ala?Val?Thr?Asp?Tyr?Thr?Ala?Gly?Leu?Ile?Tyr?Gln?Asn?Glu145?????????????????150?????????????????155?????????????????160Ser?Gly?Ala?Ile?Asn?Glu?Ala?Ile?Ser?Asp?Ile?Phe?Gly?Thr?Leu?Val

165?????????????????170?????????????????175Glu?Phe?Tyr?Ala?Asn?Lys?Asn?Pro?Asp?Trp?Glu?Ile?Gly?Glu?Asp?Val

180?????????????????185?????????????????190Tyr?Thr?Pro?Gly?Ile?Ser?Gly?Asp?Ser?Leu?Arg?Ser?Met?Ser?Asp?Pro

195?????????????????200?????????????????205Ala?Lys?Tyr?Gly?Asp?Pro?Asp?His?Tyr?Ser?Lys?Arg?Tyr?Thr?Gly?Thr

210?????????????????215?????????????????220Gln?Asp?Asn?Gly?Gly?Val?His?Ile?Asn?Ser?Gly?Ile?Ile?Asn?Lys?Ala225?????????????????230?????????????????235?????????????????240Ala?Tyr?Leu?Ile?Ser?Gln?Gly?Gly?Thr?His?Tyr?Gly?Val?Ser?Val?Val

245?????????????????250?????????????????255Gly?Ile?Gly?Arg?Asp?Lys?Leu?Gly?Lys?Ile?Phe?Tyr?Arg?Ala?Leu?Thr

260?????????????????265?????????????????270Gln?Tyr?Leu?Thr?Pro?Thr?Ser?Asn?Phe?ser?Gln?Leu?Arg?Ala?Ala?Ala

275?????????????????280?????????????????285Val?Gln?Ser?Ala?Thr?Asp?Leu?Tyr?Gly?Ser?Thr?Ser?Gln?Glu?Val?Ala

290?????????????????295?????????????????300Ser?Val?Lys?Gln?Ala?Phe?Asp?Ala?Val?Gly?Val?Lys305?????????????????310?????????????????315

Claims (25)

1. one kind has the polypeptide-polymer conjugate that improves scourability.

2. according to the conjugate of claim 1, wherein, described conjugate has the allergenicity of reduction.

3. according to the conjugate of claim 1 or 2, wherein, described polypeptide is a kind of enzyme.

4. according to each conjugate of aforementioned claim, wherein, the molecular weight of parent's polypeptide is 4 kDa to 100 kDa, it is conjugated on the homopolymer, and the molecular weight of described homopolymer is 0.1 kDa to 60 kDa, preferred 100 Da to 10,000 Da, particularly 100 Da to 2,000 Da.

5. according to the conjugate of claim 4, wherein, parent's polypeptide, particularly enzyme, its molecular weight are 15 to 60 kDa.

6. according to each conjugate of claim 1 to 5, wherein, 1 to 100 polymerizable molecular, preferred 4 to 50, more preferably 5 to 35 polymerizable moleculars are covalently coupled on the parent enzyme.

7. according to each conjugate of claim 1 to 6, wherein, polymerizable molecular is selected from natural or synthetic homopolymer and heteropolymer.

8. according to the conjugate of claim 7, wherein, polymerizable molecular is selected from and comprises the synthesized polymer molecule, comprises in the group of the PEG of branch, star-like PEG, PEG ether and PEG ester.

9. according to the conjugate of aforementioned each claim, wherein, polymerizable molecular is selected from and comprises in the group of PEG that molecular weight is 100 to 6,000 Da, preferred 100 to 2,000 Da.

10. according to the conjugate of claim 7, wherein, polymerizable molecular is selected from and contains in the group that comprises following natural polymerization molecule: dextran, comprise carboxymethyl-dextran, and Mierocrystalline cellulose, the hydrolysate of methylcellulose gum, carboxymethyl cellulose, ethyl cellulose, Natvosol, hydroxypropylcellulose and chitosan for example, starch, for example hydroxyethyl-starch, hydroxypropyl-starch, glycogen, agarose, guar gum, inulin, amylopectin, xanthan gum, carrageenin, pectin and alginic acid.

11. according to the conjugate of claim 1 to 3, wherein, polypeptide-polymer conjugate be on this polypeptide surface coupling graft copolymer, segmented copolymer, alternating copolymer or random copolymers.

12. according to the conjugate of claim 11, wherein, polypeptide-polymer conjugate has one or more polymkeric substance to be covalently coupled on parent's polypeptide, wherein, polymkeric substance is characterized by following general formula:

EO _xPO _y????(Ⅰ)

Wherein, x=1-99%, y=1-99%, and x+y=100%.

13. according to the conjugate of claim 12, wherein, the molecular weight of polymkeric substance is 100 to 100,000 Da, particularly 100 to 50,000 Da, especially 100 to 10,000 Da.

14. according to the conjugate of claim 12 or 13, wherein, the molecular weight of parent's polypeptide is 1 to 100 kDa, particularly a kind of enzyme, its molecular weight are 15 to 60 kDa.

15. according to each conjugate of claim 12 to 14, wherein, block polymer or multipolymer comprise ethylene oxide unit (EO) and propylene oxide units (PO), and its mol ratio is 10: 90 or 20: 80 or 30: 70 or 40: 60 or 50: 50 or 60: 40 or 70: 30 or 80: 20 or 90: 10.

16. according to each conjugate of claim 12 to 15, wherein, 1 to 100 polymerizable molecular is arranged, preferred 4 to 50, especially 5 to 35 polymerizable moleculars are covalently coupled on the parent enzyme.

17. according to claim 4 to 10 or 11 to 16 each conjugates, wherein, described polypeptide is microbe-derived, as bacterium, filamentous fungus or yeast source, perhaps derives from plant.

18. conjugate according to claim 17, wherein, described polypeptide is a kind of enzyme that is selected from down group: lytic enzyme, comprise proteolytic enzyme, comprise serine protease, for example subtilisin and metalloprotease, or carbohydrase, or lipase, or oxydo-reductase, for example laccase and haloperoxidase or superoxide-dismutase.

19. conjugate 18 according to claim 18, wherein, parent protease is selected from: PD498, Sayinase , Sayinase variant R247K, Proteinase K, proteolytic enzyme R, Thermitase, subtilisin DY, Lion Y, Alcaiase , proteolytic enzyme T and JA16.

20. according to the conjugate of claim 18, wherein, parent's carbohydrase is selected from: Novamyl , Fungamyl , Natalase  and Termamyl .

21. an industry group compound, it comprises the conjugate of claim 1 to 20.

22. according to the industry group compound of claim 21, it is a kind of washing composition, as laundry detergent composition, bowl dish detergent composition or hard-surface cleaning compositions.

23. each the purposes of conjugate in improving claim 21 or 22 each the scourabilities of industry group compound of claim 1 to 20.

24. according to the purposes of claim 23, it is used for reducing the breathing allergenicity.

25. method of improving the polypeptide scourability, comprise claim 4,6 to 10 each defined homopolymer are coupled on claim 4,5,17 to 20 each the parent's polypeptide, and/or the block polymer of claim 11,12,13,15,16 each definition or multipolymer are coupled on claim 14,17 to 20 each the parent's polypeptide.