CN1253585A - Coupled enzyme for skin care - Google Patents

Abstract

The present invention relates to modified enzymes suitable for skin care having from 4 to 70 polymeric molecules, with a molecule weight from 1 to 35 kDa, coupled covalently to the surface of parent enzymes having a molecule weight from 15 to 100 kDa. Further the invention is directed towards skin care compositions and products comprising such modified enzymes and finally the use of said modified enzyme for reducing the sensitisation potential of skin care products.

Description

A kind of coupled enzyme that is used for skin care

Invention field

The present invention relates to coupled enzyme, comprise said coupled enzyme and become known for the purposes that the skin care compositions of all compositions in the skin care compositions, the skin care products that comprise skin care compositions of the present invention and said coupled enzyme are used to improve the stability of enzyme and/or reduce the sensitizability of enzyme.

Background of invention

Just like having a bath and shower from ancients.This does not also change up till now.For most of people of today, have a bath and shower be a daily life part that is used to keep good health health and obtains pleasant smell.Some people carrying out once salubrious shower in the morning or having a bath as important and essential mental experience, does not have this content yet, and these people just can't regain consciousness.

On the consumption market, can find many products that are used for body care and keep good health health, for example be used to clean product with moistening all parts of health.Some such products comprise the coupled enzyme as effective constituent.

The enzyme that is used for skin care

For a long time, know, handle skin with the enzyme of vegetables and fruit (as cucumber, tomato, Radix Dauci Sativae, banana etc.) form and have useful latent effect.

Yet, before the seventies in 20th century, enzyme is not introduced commercial skin care products as yet, in part because limited about the knowledge of enzyme, but also because it is believed that the stability of enzyme can not be satisfactory, and in skin care products, have some disadvantageous character.For example, it is found that cellulase can change the lotion that comprises carboxymethyl cellulose and the viscosity of emulsifiable paste; The emulsifiable paste that lipase can cause comprising fatty acid ester changes; Find that proteolytic enzyme can make protein component destruction and cause viscosity to reduce.

In addition, at that time, enzyme expensive also hindered the application of enzyme in this personal care product.

People's skin

Which floor people's skin be made of.The fibrous protein Keratin sulfate is contained in the superiors' (epidermis), and its function is to serve as the protective layer of isolation environment.The skin of epidermis is in an organized way dead and form by the cell of the granulosa that is arranged in its below.Discharge many enzymes in the granulosa, their cellular materials of checkmating change into Keratin sulfate.

Corium is connected with epidermis by basement membrane, and by the recycle system skin and health rest part is coupled together.Corium has blood vessel, nerve fiber and lymphatic vessel, and to comprise mainly be by collegen filament and limited amount elastin and the fibrous fibrous network of reticulin.

The coupled enzyme that is used for the personal care product

As mentioned above, the stability of some enzyme can not be satisfactory, and (mode that depends on contact) may cause immunne response under certain condition, and normally IgG and/or IgE reply.

Generally all recognize that when polypeptide (as enzyme) and polymerizable molecular coupling, the stability of polypeptide can be improved today, immunne response can reduce.

The technology that is used to connect polypeptide and polymerizable molecular is known in this area.

One of optimal commercial technology is as far back as early stage just describe (United States Patent (USP) no.4,179, the 337) seventies in 20th century.Said patent relates to the non-immunogenic polypeptide, as with polyoxyethylene glycol (PEG) or polypropylene glycol link coupled enzyme and peptide hormone.Wherein at least 15% polypeptide physiologically active is maintained.

English Patent no.1,183,257 (Crook etc.) have described the chemistry that enzyme is connected with polysaccharide by triazine ring.

In addition, the technology that is used to keep the enzymatic activity of enzyme-polymer conjugates also is known in this area.

WO 93/15189 (Veronese etc.) relates to a kind of by keeping the method through polyethyleneglycol modified hydrolase of proteolysis on the inhibitor that proteolytic ferment is connected to macromoleization.This binding substances is intended to medical applications.

Have been found that: polypeptide chain is received to be reduced polypeptide active on the polymerizable molecular usually or disturb polypeptide and its substrate interaction.EP 183 503 (Beecham Group PLC) discloses a kind of development of above-mentioned notion, and binding substances promptly is provided, and described binding substances contains the protein that is connected to the pharmaceutically useful at least a water-soluble polymers by means of the reversible linking group.

EP471,125 (Kanebo) disclose to comprise by triazine ring and have been coupled on the polysaccharide skin care products with the parent protease (bacillus protein enzyme Esperase ) that improves thermotolerance and storage stability.The coupling technology that is utilized has description at above-mentioned English Patent no.1 among 183,257 (Crook etc.).

JP 3083908 has described a kind of skin cosmetics material, contain in this material from the cavy liver, with the trans-glutaminases of one or more water-soluble substanceses (as PEG, starch, Mierocrystalline cellulose etc.) modified.This modification is coupled on the said enzyme by the activation polymerizable molecular and with it to be carried out.It is said that said composition is relatively gentleer to skin.

General knowledge general introduction based on prior art

Be used for the technology that one or more polymerizable moleculars are coupled on the peptide molecule is well known in the art.And, known that the immunne response of the enzyme-polymer conjugates of this modified reduces, stability improves.

Summary of the invention

The purpose of this invention is to provide the improved coupled enzyme binding substances that is suitable in skin care products, using.

Inventor of the present invention finds: when use has the active coupled enzyme that is suitable for skin care, to enzyme and polymer molecule certain requirement must be arranged, with the stability of acquisition raising and the sensitizability of reduction, and still kept significant residual enzymatic activity simultaneously.

Inventor of the present invention finds: with the number of enzyme surface link coupled polymerizable molecular and weight must with the weight and/or the surface-area balance of enzyme.And the position of coupling polymerization molecule is also very important.

First aspect of the present invention relates to a kind of coupled enzyme, and wherein molecular weight is that 4-70 the polymerizable molecular of 1-35kDa is the surface of the parent enzyme of 15-100kDa to molecular weight by covalent coupling.

At the parent enzyme molecular weight is under the situation of 15-35kDa, the polymerizable molecular of 4-20 covalent coupling should covalent coupling to the surface of said parent enzyme.

If the molecular weight of parent enzyme in the scope of 35-60kDa, then is coupled to the individual polymerizable molecular of 7-40 (preferably 10-30) on the surface of said parent enzyme.

Similarly, if parent enzyme has the molecular weight of 60-80kDa, then the individual polymerizable molecular of 10-50 (preferably 13-40) is coupled on the surface of said parent enzyme.

The individual polymerizable molecular of 15-70 (preferably 18-60) is coupled on the surface of the parent enzyme with 80-100kDa molecular weight.

Usually, polymerizable molecular is coupled to the lip-deep amino (NH of enzyme ₂) and N-terminal amino on, yet polymerizable molecular also can be coupled to the amino acid whose carboxyl (COOH) that is in the surface in the enzyme chain.

Preferred linking group is lysine residue and the amino that is positioned at N-terminal.

The carboxylic acid linking group can be the carboxylic acid group of aspartic acid or L-glutamic acid and the COOH group of C-terminal.

In this application, the number of " linking group " is the number that the amino number of lysine residue adds N-terminal amino in the polypeptide chain.

Parent enzyme of the present invention can be a lytic enzyme, comprises proteolytic enzyme (especially subtilisin) or lipase, or oxydo-reductase (comprising laccase and superoxide-dismutase).

Second aspect of the present invention relates to the skin care compositions that comprises coupled enzyme of the present invention and be used for the composition of skin care products.

The 3rd aspect of the present invention relates to the skin care products that comprise skin care compositions of the present invention.

Compare with corresponding skin care products (comprising parent enzyme), the stability of skin care products of the present invention increases, and sensitizability decreases.

Term " sensitizability reduction " is meant " allergenicity reduction " in the context of the present invention, this refers to when sucking coupled enzyme of the present invention, the amount of the IgE (in the mankind, being the molecule with similar effect in specific animal) that produces, can cause allergic state reduces than corresponding parent enzyme.

In the context of the present invention, " skin care products " comprise all personal care product and the other products that are used for the body skin cleaning, nurse and/or beautify, as hair care product, these products may contact with skin or respiratory system during use.The present invention also comprises the corresponding product that is used for animal.

Specific examples according to skin care products involved in the present invention is a soap, makeup, cold cream, skin-care gel, skin care milk, clean lotion for skin care, cleansing cream, cleaning lotion, clean skin milk, cold cream, cold soap, cosmetic base-material (make-up base), the cleaning breast, facial mask, contain the zinc astringent, T district essence (T zone essence), hand cream, face powder (essencepowder), whitening powder, soap powder, cake soap, transparent soap, lipstick, lipstick, nutrient substance, foundation cream, face powder, eye shadow powder, foundation cream, the nail varnish sanitising agent, hair tonic, hairdressing liquid, hair-cream, hair gel, mayonnaise agent, hair setting goods, hair dye, hair coloring agent, scalp moisturizing agent (scalp treatment), shampoo, balm, hair conditioner, hair jelly, suntan oil (sun oil), sunscreen products, shaving foam and gel, shaving cream, baby oil, the acne care article, antiperspirant, wormer, deodorant etc.

Allergenicity is estimated

Can check by sucking, and compare the effect of using parent enzyme in the tracheae and the effect of using the corresponding coupled enzyme of the present invention and estimate allergenicity.

Existing several interior animal model of body that the enzyme 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 and enzyme is a Brown Norway strain.Brown Norway rat produces IgE as allergic response.

The BALB/C mice strain is suitable for determining that the IgE that is caused by subcutaneous injection replys.

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 rat, rabbit etc.) also can be used for similar research.

Brief description of the drawings

Fig. 1 has shown PD498-SPEG, the PD498 and the glycine-SPEG15 of unmodified that modifies using, after 000 immunity, and the kinetics that specific anti-PD498 IgE replys in the BALB/C mice.

Fig. 2 has shown the IgG1 level of Dunkin Hartley cavy of the PD498 of the PD498-SPEG that uses modification in the tracheae and unmodified.

Fig. 3 has shown IgG1 level (the ■ 3.0 μ g that 3 μ g, 30 μ g and 300 μ g modify in the Dunkin Hartley cavy IT dose response study PD498-SPEG 5000 causes; ▲ 30 μ g;  300 μ g).Because the dosage of 0.3 μ g does not produce and does not reply, the Therefore, omited 0.3 μ g dose curve.

Fig. 4 has shown IgG1 level (the ■ 0.3 μ g that the parent PD498 of 0.3 μ g, 3.0 μ g and 30 μ g unmodifieds causes in the Dunkin Hartley cavy IT dose response study; ▲ 3.0 μ g;  30 μ g).

Detailed Description Of The Invention

The purpose of this invention is to provide the coupled enzyme that is suitable for skin nursing.

As mentioned above, know, with polymerizable molecular and enzyme coupling, can improve the stability of polypeptide (comprising enzyme) and reduce its sensitizability. When with polymerizable molecular and enzyme coupling, a forfeiture that problem is enzymatic activity of appearance.

According to above-mentioned EP 471,125 (Kanebo), with bacillus protein enzyme Esperase  (can be provided by Novo Nordisk A/S) by triazine ring and 40kDa glucan (embodiment 1) and 50kDa amylopectin (embodiment 2) combination.

Said bacillus protein enzyme (being Esperase ) has 3 can reach amino (NH₂) linking group, polymerizable molecular (being polysaccharide in this case) can coupling thereon. These several linking groups are two amino (i.e. two lysine residues on the 3D body structure surface) and a N terminal amino group. (modification rate will be 68%-71% when 3 polymerizable moleculars are coupled on the said protease when being no more than, by TNBS method (Haynes etc., (1967), biochemistry, 6, P.641) measure), it is said that the residue enzymatic activity that keeps is between 45% (referring to embodiment 4)-67% (referring to embodiment 3).

Inventor of the present invention finds: when use has the coupled enzyme of the activity that is suitable for skin nursing, to enzyme and polymer molecule certain requirement must be arranged, with the stability of acquisition raising and the sensitizability of reduction, and still kept significant residue enzymatic activity simultaneously. Inventor of the present invention finds: with the number of the polymerizable molecular of enzyme surface coupling and weight must with weight and/or the surface area balance of enzyme. And the position of coupling polymerization molecule (on the surface) is also very important.

Polymerizable molecular number and enzyme weight

The present invention is based on following rule: the surface area of enzyme and/or weight are more big, just must have more many polymerizable moleculars to be coupled on the surface of enzyme, with the stability that to improve, remain the sensitizability of enzymatic activity and/or reduction significantly.

Be coupled on the big enzyme with high surface area iff few polymerizable molecular, so said few polymerizable molecular can not cover (namely hiding/cover) lip-deep epi-position of enzyme, and this epi-position can cause the immune response that causes antibody (especially IgE antibody) to form.

Above-mentioned EP 471,125 (Kanebo) has described seldom (namely the most nearly 3) big (namely 40 and 50kDa) polymerizable molecular and has been coupled to the microbial protease Esperase with about 28kDa molecular weight^The situation on surface.

First aspect of the present invention relates to a kind of coupled enzyme that is suitable for skin nursing, and it is that 4-70 the polymerizable molecular covalent coupling of 1-35kDa is the surface of the parent enzyme of 15-100kDa to molecular weight that this enzyme has molecular weight.

According to the present invention, will have the enzyme (as come from bacillus bacterialprotease) and 4-20 polymerizable molecular covalent coupling of 15-35kDa molecular weight (this to many microbial enzymes relatively typical case).

In other words, coupled enzyme can have 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 covalent couplings in the parent enzyme 3D body structure surface polymerizable molecular of (comprising the N terminal amino group).

According to the present invention, the preferred proportion between the number of the polymerizable molecular of the weight of enzyme and/or surface area, coupling and the weight of polymer is as shown in table 1.

Table 1

Enzyme molecular weight (Mw) kDa	Number with the polymerizable molecular of enzyme coupling	The mean molecule quantity kDa of polymerizable molecular
			15-35	4-20	1-35
35-60	7-40	1-35
			60-80	10-50	1-35
80-100	15-70	1-35
			Greater than 100	Greater than 20	1-35

According to the present invention, the molecular weight of polymerizable molecular can be 1-35kDa. Yet, if polymerizable molecular is littler and/or still less, the epi-position on enzyme surface may not fully be covered, thereby cause immune response. According to the present invention, the preferred molecular weight of polymerizable molecular is 4-25kDa, and 6-25kDa especially is such as 8-20kDa.

All polymer molecular weights of touching upon all are mean molecule quantities.

The position of the polymerizable molecular of coupling

In fact, all ionogens (such 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 (namely amino) number is generally equal to the number that lysine residue in the enzyme primary structure adds the N-terminal amino group on the surface of enzyme.

When selecting to want to carry out in conjunction with when being used for the parent enzyme of skin care compositions and methods and product, preferably use has the enzyme of the number of linking group shown in the table 1.

The remaining enzymatic activity of sensitizability and maintenance

Than other oroteins and polypeptide, especially for enzyme, because the interaction between the avtive spot in the activity of enzyme and substrate and the enzymatic structure crack is relevant, therefore minimizing immune system replying and keep some conflict between the significant remaining enzymatic activity enzyme.

According to the present invention, the residual activity that " significantly " keeps has referred to keep more than 20%, 30% or 40% of enzymatic activity, is more preferably more than 50%, 60% or 70%, also will be more preferably 70% or 80%, up to 80%-90%, or even up to 100%.

Be not subjected to the restriction of any theory, the enzymatic activity loss of coupled enzyme may be owing to the big/catalysis crack of reaggregation molecule space obstruction substrate arrival enzyme hinders substrate and reaches the result that avtive spot causes. Might (at least part of) be because disadvantageous structural change has taken place the 3D structure of enzyme also. When with few big/when the reaggregation molecule is coupled to the enzyme surface, might cause in the different piece of enzyme molecule inhomogeneous interaction. This may cause the enzymatic structure part to depart from its normal configuration, and this in most of the cases can cause the enzymatic activity loss.

The modified protein enzyme of describing in the EP 471,125 (Kanebo) have seldom (namely the most nearly 3 polymerizable moleculars) heavy/polymerizable molecular (namely 40 and 50kDa polysaccharide) is coupled on the amino on enzyme surface greatly. The enzymatic activity loss of observing (being the remaining enzymatic activity of 45%-67%) may be because the inhomogeneous interaction on the different piece of enzyme surface causes that enzymatic structure departs from its normal parent's state configuration. And, on the enzyme surface coupling big/the reaggregation molecule also may stop substrate to arrive the avtive spot of enzyme, the enzymatic activity that causes keeping reduces.

People be sure of that when more littler/light polymerizable molecular was coupled on the surface of enzyme, the adverse effect of polymerizable molecular was just less obvious, because to the more balance/equably distribution in the lip-deep bigger zone of enzyme of the influential power of enzymatic structure tool. Like this, polymerizable molecular is just less obvious to the impact of loss of activity.

Thereby the more poly that preferably will have a relative low-molecular-weight (being 1-35kDa) closes molecule (namely greater than 4) and is coupled on the surface of enzyme (having at enzyme in the situation of molecular weight of 15-35kDa).

In the preferred embodiment of the invention, polymerizable molecular is distributed widely on the enzyme surface except the zone of contiguous avtive spot. In the context of the present invention, " be distributed widely in " and refer to that its residing position can be so that be coupled to the different piece that the polymerizable molecular of enzyme linking group covers the enzyme surface (preferably the whole surface except avtive spot or close to whole surface), to guarantee that said discernible relevant epi-position is covered, thus not by immune antibody recognition. It is believed that: the surface area of enzyme and antibody interaction is approximately 500 ²(26 * 19 ) (referring to Sheriff etc., (1987), NAS's journal, 84 volumes, p.8075).

Preferably, the two or more linking groups on the enzyme should be mutually not close, causes possibly only polymerizable molecular of energy coupling because linking group is close to mutually.

Loss minimum in order to ensure enzymatic activity preferably is not coupled to polymerizable molecular in the short range of avtive spot.This distance depends on the size of polymerizable molecular.Because do not wish to be hindered the arrival avtive spot by big polymerizable molecular, like this, polymerizable molecular is huge more, and institute's link coupled polymerizable molecular just should be far away more from the distance of avtive spot.

Usually it seems: with avtive spot 5 of distance enzyme, preferably do not have polymerizable molecular to be coupled on the said enzyme in the scope of 10 to good.

And, according to the present invention, can by the known epi-position of immune system recognition or approach can by the known epi-position of immune system recognition or approach said epi-position coupling the enzyme of polymerizable molecular also be regarded as very beneficial.If the position of epi-position is unknown, then many polymerizable moleculars are coupled to the enzyme surface can and linking group on be rather favourable.Preferably said linking group is distributed widely in the surface of enzyme with the distance that is fit to from avtive spot.According to the present invention, preferably meet the coupled enzyme that above-mentioned relevant coupling polymerization molecule requires in the enzyme surface arrangement.Especially preferably do not have or have only that only a few polymerizable molecular (being 0-2) is coupled in the avtive spot 0-5 scope apart from enzyme, the enzyme in the 0-10 scope preferably.

Polymerizable molecular

The polymerizable molecular that is coupled on the enzyme can be any suitable polymerizable molecular, comprises that natural [as polyvalent alcohol (being poly-OH), polyamines (is poly-NH with the synthetic homopolymer ₂) and poly carboxylic acid (being 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 oxides (PAO), as polyalkylene glycol (FAG), 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, polyvinyl alcohol (PVA), polycarboxylate, poly-(V-Pyrol RC), poly-D, L-amino acid, ethene-copolymer-maleic anhydride, styrene-maleic 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, the synthesising biological polymeric gel, carrageenin (carrageenan), pectin, alginic acid hydrolysate and biological polymer.

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

Generally it seems, polyalkylene oxides (PAO) (as polyethylene oxide, as PEG, mPEG particularly) be preferred polymerizable molecular, because these polymerizable moleculars are compared with polysaccharide (as dextran, amylopectin etc.), the crosslinked active group of the energy that they had seldom.

According to the present invention,, preferably use methoxy poly (ethylene glycol) (mPEG) although can use all above-mentioned polymerizable moleculars.This is because such fact: methoxyl group ethylene glycol only has a reactive terminal that can be connected with enzyme.Thereby crosslinked possibility is more not obvious relatively.And this will make more homogeneous of product, the easier control of the reaction of polymerizable molecular and enzyme.

The activation of polymkeric substance

If will not have activity, just must use appropriate methodology to make its activation with enzyme link coupled polymerizable molecular.Polymerizable molecular can be by joint and enzyme coupling.The joint that is fit to is known those of skill in the art.

Be used for the activated polymerization molecule and be used to connect method of protein and chemistry has 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, biepoxides, epoxy chloropropane, 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 bound 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.).Selected linking group on functional groups amino, hydroxyl, sulfydryl, carboxyl, aldehyde or sulfhedryl in selecting activation and combining chemical process on the necessary consideration polymkeric substance and the protein, activation with combine chemistry and generally include: the i) activation of polymkeric substance, ii) combination, the 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 enzyme 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 bound 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, JacobyW.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, 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 muriate) 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.High in conjunction with the productive rate except having, that reaction conditions also requires usually is relatively gentleer (neutrality or slight alkalinity pH value are to avoid sex change and active few or destruction not), 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 bioconjugation chemistry, 6,150-165).Epoxy compounds 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), bioconjugation 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 imide thrones, can obtain acid amides from PEG acid (United States Patent (USP) no.5,349,001, (1994), Greenwald etc.).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 binding substances 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).

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 (WO95/11924, Greenwald etc.).Lysine residue also can be used as main chain.

Parent enzyme

Above-mentioned binding substances of the present invention can use any suitable technology as known in the art to prepare based on selected parent enzyme.

Term " parent " enzyme is meant any not link coupled enzyme (i.e. the enzyme that will modify).This enzyme preferably can be microbe-derived, as bacterium, filamentous fungus or yeast source.

Parent enzyme can be naturally occurring (or wild-type) enzyme or its variant.

The parent enzyme that evaluation/selection is fit to

Be of some use aspect the suitable parent enzyme that the three-dimensional structure of enzyme will be modified in evaluation/selection.Three-dimensional structure can be the model structure of X-ray structure, nucleus magnetic resonance structure or foundation.The Brookhaven database can be used as the source of X-ray and nucleus magnetic resonance structure.

If have one or more 3D structures of one or more homology enzymes that at least 30% sequence is equal to known with said enzyme, then can set up model structure by the one skilled in the art.Several software packages are arranged,, can be used for making up model structure as " Homo1ogy95.0 " software package of Biosym.

Making up the needed exemplary operation of model structure is: to the homologous sequence that has the 3D structure carry out the sequence contrast, determine structure conservative region (SCR), determine SCR be equal to sequence, in structural database searching structure fragment/ring with the replacement region of variability, determine being equal to sequence, making the structure precision of these zones by energy minimization.The known zone of containing big inset (〉=3 residues) with respect to known 3D structure quite is difficult to simulation, must predict its structure carefully.

Obtain the 3D-structure of said enzyme or based on behind the structural models of setting up with the homology of known structure, this structure promptly is to determine the necessary prerequisite of suitable parent enzyme (when modifying, these enzyme allergenicities reduce, and fully keep remaining enzymatic activity).

The preferred enzyme that is used for skin care products is those enzymes that remarkable enzymatic activity is arranged in the pH value scope that skin care products use.

Enzymic activity

Parent enzyme can have any activity of the skin care of becoming known for.Related enzyme 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, particularly subtilisin, and lipolytic enzyme; Transferring enzyme, (E.C.2), as trans-glutaminases (TG enzyme); Isomerase (E.C.5) is as protein disulfide isomerase (PDI).

Lytic enzyme

Proteolytic ferment

Related proteolytic ferment comprise be selected from following group, to have above-mentioned character (be the number of linking group, the position of linking group etc.) enzyme: acid aspartate protease, L-Cysteine HCL Anhydrous, serine protease, as subtilisin, or metalloprotease.

Specific examples with suitable parent protease of suitable number coupling group is shown in Table 2:

Table 2

Enzyme	The number of linking group	Molecular weight kDa	Reference
				PD498	????13	????29	?Seq.ID?No.2 ?WO?93/24623
Savinase	????6	????27	Von der Osten etc., (1993), the biotechnology magazine, 28, p.55+
				Proteinase K	????9	????29	Gunkel etc., (1989), european journal of biological chemistry, 179, p.185-194
Proteolytic enzyme R	????5	????29	Samal etc., (1990), molecular microbiology, 4, p.1789-1792
				Proteolytic enzyme T	????14	????29	Samal etc., (1989), gene, 85, P.329-333
Subtilisin DY	????13	????27	Betzel etc., (1993), Arch.Biophys, 302, no.2, P.499-502
				Lion?Y	????15	????46	?SEQ?ID?NO.4 ?JP?04197?182-A
Rennilase		????39	Can obtain from Novo NordiskA/S
				Ja16	????5	????28	?WO?92/17576
Thermolysin	????12	????34	The natural neontology 238 of Titani etc. (1972) is P.35-37 with SEQ ID NO 5
				Alcalase  (natural subtilisin Carlberg variant)	????10	????27	Von der Osten etc., (1993), the biotechnology magazine, 28, P.55+

Subtilisin PD498 has the molecular weight of 29kDa, and is shown among the SEQ IDNO.2.PD498 has 12 Methionin groups in its surface and can be used for connecting, and also has a N-terminal amino.As mentioned above, in the preferred enzyme, Methionin is distributed widely in the enzyme surface.Be positioned at scope apart from avtive spot 0～10 without any a lysine residue among the PD498, this makes PD498 be particularly suitable for being processed into modified forms.In addition, all lysine residues extensively distribution (promptly away from avtive spot) on the surface of this enzyme.

Subtilisin DY has the molecular weight of 27kDa, and 12 amino (being lysine residue) are arranged in its surface, also has a N-terminal amino (referring to SEQ ID NO.3).

Parent protease Lion Y has the molecular weight of 46kDa, and 14 amino (being lysine residue) are arranged in its surface, also has a N-terminal amino (referring to SEQ ID NO.4).

Neutral metal proteolytic enzyme thermolysin has the molecular weight of 34kDa, and 11 amino (being lysine residue) are arranged in its surface, also has a N-terminal amino (referring to SEQ IDNO.5).

Lipolytic enzyme

Related lipolytic enzyme comprise pubescence humicola lanuginosa (Humicola lanuginosa) lipase (as in EP 258 068 and EP 305 216, describe that is a kind of), Humicolainsolens, Rhizomucor miehei lipase (as described in EP 238 023), colter enzyme lipolytic enzyme (WO 96/13578), mycocandida lipase is (as C.antarctica lipase, C.antarctica lipase A or the B that in EP 214 761, describes for example), (for example Pseudomonas alcaligenes, pseudomonas pseudoalcaligenes lipase are described in EP 218 272 for Rhodopseudomonas lipase; Onion pseudomonas lipase is described in EP 331 376; Disclosed pseudomonas lipase among the WO95/14783), genus bacillus lipase (subtilis lipase (Dartois etc. for example, (1993) Biochemica et Biophysica acta, 1131,253-260), bacstearothermophilus lipase (JP 64/744992) and bacillus pumilus lipase (WO 91/16422)).The lipolytic enzyme of other type comprises at, the at of for example in WO88/09367, describing that is derived from pseudomonas mendocina, or be derived from the at (as described in the WO 90/09446) of Fusariumsolani pisi.

Oxydo-reductase

Laccase

Disclosed laccase among the WO 96/00290 that related laccase is included in Novo Nordisk and the WO95/33836.

Transferring enzyme

Trans-glutaminases

The transferring enzyme that is fit to is included in disclosed any trans-glutaminases among WO 96/06931 (Novo Nordisk A/S) and the WO96/22366 (Novo Nordisk A/S).

Isomerase

Protein disulfide isomerase

The protein disulfide isomerase that is fit to is included in those PDI that describe among the WO 95/01425 (Novo NordiskA/S), but is not limited to this.

Be suitable for the enzymic activity of skin care

Second aspect of the present invention relates to the skin care compositions that comprises coupled enzyme of the present invention 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).

Trans-glutaminases

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) make up to go up acceptable carrier, 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 effect.

Skin care products of the present invention

The 3rd aspect of the present invention relates to the skin care products that comprise skin care compositions of the present invention.Term " skin care products " as above defines.

Skin care products of the present invention can comprise the coupled enzyme of the present invention of significant quantity.This significant quantity known to the one skilled in the art often accounts for the 0-5% of final skin care products.

Related skin care products of the present invention include but not limited to following products: soap, makeup, cold cream, skin-care gel, skin care milk, clean lotion for skin care, cleansing cream, cleaning lotion, clean skin milk, cold cream, cold soap, the cosmetic base-material, the cleaning breast, facial mask, contain the zinc astringent, T district essence, hand cream, face powder, whitening powder, soap powder, cake soap, transparent soap, lipstick, lipstick, nutrient substance, foundation cream, face powder, eye shadow powder, foundation cream, the nail varnish sanitising agent, hair tonic, hairdressing liquid, hair-cream, hair gel, mayonnaise agent, hair setting goods, hair dye, hair coloring agent, scalp moisturizing agent (scalp treatment), shampoo, balm, hair conditioner, hair jelly, suntan oil (sur oil), sunscreen products, shaving foam and gel, shaving cream, baby oil, the acne care article, antiperspirant, wormer, deodorant etc.

Conventional skin care products prescription

Term " composition that is used for skin care products " is meant and comprises all effective constituents that become 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 prescriptions have been summarized important skin care products prescription involved in the present invention.

About 0.5 dyestuff of perfumed soap composition example % surfactant soap (sodium salt) 83-87 chelating agent edetate 0.1-0.3 consistency modifiers sodium chloride＜0.1 Optical Bleaching Agent＜0.1 antioxidant 2; Two (1,1-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 ether 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 composition example % foam bath % shower 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

The ethoxylized fatty alcohol 0.5-5 0-4 protease enzyme/quaternized hydroxypropyl cellulose of lipase 0-5 0-5 composition example % bubble bath % shower 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 vegetable extract 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

Cold cream (water-in-oil-type and oil-in-water-type) composition example % water-in-oil-type/oil-in-water emulsifiers Arlacel-83 3-5-

Aluminum stearate 1-2-

Trolamine stearic acid-1-2

Cetyl alcohol/Stearyl alcohol polyoxyethylene glycol-1-3

Ether adipose-derived Wickenol 111 1-5 0-3 thing

Cetyl alcohol/Stearyl alcohol-0-2

2-Standamul G 2-10 3-7

Stearic/palmitic acids-0-3

Caprylic/capric triglyceride 5-10-

Vinlub-0-5 wetting agent glycerine 1-5 1-5

Sorbitol Powder 1-5 1-5

Poly-(hydroxycarboxylic acid) 0.5-2-

Propylene glycol-0-3 stablizer sal epsom 0-0.8-sanitas p-Hydroxybenzoate 0.2-0.4 0.2-0.4 protease enzyme/lipase 0-5 0-5 moisture difference difference

Be used for the refreshing body water % skin lotion emulsifying agent cetyl alcohol of the refreshing body water (oil-in-water-type) of moistening skin and skin lotion composition example %/Stearyl alcohol polyoxyethylene glycol 1-3-

Ether

Arlacel-20 0.5-1-

Sodium stearate-1-2

Sodium Lauryl Sulphate BP/USP-0.5-2 adipose-derived 2-Standamul G 1-3 0-5 thing

Paraffin oil (paraflin oils)-20-25

Beeswax 0.5-1-

The different monooctyl ester 3-7 of stearic acid-

Wickenol 111-2-5 wetting agent glycerine 3-5 5-10

Sorbitol Powder-0-5 thickening material polyacrylic ester 0-0.3 0-1

Methylhydroxypropylcellulose 0-0.3 0-0.5 sanitas p-Hydroxybenzoate 0.2-0.4 0.2-0.4 protease enzyme/lipase 0-5 0-5 moisture difference difference

Profit is showed composition example % tensio-active agent lauryl magnesium sulfate 0.2-0.5 fatting agent adipic acid-di-n butyl ester 1-2 solubilizing agent Viscotrol C polyglycol ether 0.1-1 cleaning and salubrious component ethanol 0-15 wetting agent glycerine 0-5

Sorbyl alcohol 0-5 sanitas p-Hydroxybenzoate 0.2-0.4 astringent matter vegetable extract 1-5 counter irritant panthenol 0-1

Wallantoin 0-0.2

Vegetable extract 0.5-3 protease enzyme/lipase 0-5 moisture difference

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

Fatty acid distribution of coconut oil acyl aminopropyl diformazan 2-5

The base trimethyl-glycine

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 composition example % hair conditioner % hair conditioning

Agent surfactivity fatty alcohol polyglycol ether 0.1-0.2 1.5-2.5 agent

Varisoft 300 0.5-1-

Dimethyl benzyl stearyl ammonium chlorination-0.5-1

The list of thing fatting agent glycerine/two spermaceti acid/stearic 0.5-1.5 1.5-2.5

The acid esters denseness is regulated fatty alcohol 1-2.5 2.5-3.5 agent thickener methylhydroxypropylcellulose 0.3-0.6 0.4-0.8 conditioner quaternized hydroxyethylcellulos 0.1-0.3 0.3-0.4 anticorrisive agent p-hydroxybenzoate 0.1-0.3 0.1-0.3 dyestuff＜0.1＜0.1pH conditioning agent acid/alkali 0.1-1 0.1-1 spices 0.2-0.5 0.2-0.5 protease enzyme/lipase 0-5 0-5 moisture difference difference

Hair dye composition example % component 1: alkaline cream rinse tensio-active agent lauryl ether sulfate 1-4

The Viscotrol C 1-2 consistency modifiers Fatty Alcohol(C12-C14 and C12-C18) 8-10 reductive agent S-WAT 0.8-1.2 damping fluid ammonium chloride 0.5-1 sequestrant 1-hydroxyl ethane-1 of ethoxylation, 1-di 2 ethylhexyl phosphonic acid 0.1-0.2 alkaline agent ammonia 1.2-2 oxidation dye developer (Developing agent) 1

Coupling agent 1 enzyme laccase 0-5 moisture difference component I I: hydrogen peroxide dispersion surfactant lauryl sulfate 0.5-1 oxidants hydrogen peroxide 6-9 stabilizing agent 1-hydroxyl ethane-1,1-di 2 ethylhexyl phosphonic acid 1-1.5 thickener polyacrylate 3-5 enzyme laccase 0-5 moisture difference

Shaving cream composition example % soap palmitinic acid/stearic acid 30-40

Potassium hydroxide 5-7

Sodium hydroxide 1-2 lipid fraction Oleum Cocois 5-10

Polyoxyethylene glycol 0-2 stablizer sodium tetraborate 0-0.5

Water glass 0-0.5

The castor oil 0.5-1 astringent vegetable extract 1-10 counter-stimulus panthenol 0-0.5 of sorbierite 0-3 protease enzyme 0-5 moisture difference shaving liquid composition example % disinfectant ethanol 40-80 fatting agent adipic acid-di-n butyl ester 1-2 solubilizer ethoxylation

Vegetable extract stablizer glycerine 0-5

Sorbyl alcohol 0-5

Propylene glycol 0-3 protease enzyme 0-5 water difference hair oil composition example % consistency modifiers Fatty Alcohol(C12-C14 and C12-C18) 4-5

The Wool wax alcohol 3-6 mineral fat Vaseline 45-52 of ethoxylation

Branched paraffins 10-18 antioxidant 2, two (1, the 1-the dimethyl ethyl)-0.5-1 of 6-

4-methylphenol (BHT) spices 0.2-0.4 dyestuff 0.1 enzyme lipase 0-5 emollient glycerine difference typing water constituent example % solvent isopropyl alcohol 12-20 film-forming components vinyl pyrrolidone/vinyl acetate 2-3.5

Ester copolymer tenderizer vinyl pyrrolidone/dimethylamino 0.2-1

The castor oil 0.1-0.5 spices 0.1-0.2 dyestuff of base ethyl methacrylate conditioner protein hydrolysate 0.2-0.5 antistatic additive hexadecyltrimethylammonium chloride 0.1-0.5 emulsifying agent ethoxylation＜0.1 enzyme lipase 0-5 moisture difference

Last aspect of the present invention relates to the purposes of coupled enzyme of the present invention, when using skin care products, replys the sensitizability that has reduced skin care products by reducing IgE.

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 subtilisin type proteolytic enzyme genus bacillus mutation, that be shown in SEQ ID NO:4 (Detzel etc. (1993), the biophysics archives, 302 volumes, 2, P.499-502).

ELISA reagent:

The Chinese People's Anti-Japanese Military and Political College mouse Ig (Dako, DK, P162, the #031 of horseradish peroxidase-labeled; Extent of dilution 1: 1000).

Mouse anti rat IgE (Serotec MCA193; Extent of dilution 1: 200).

Rat anti-mouse IgE (Serotec MCA419; Extent of dilution 1: 100).

Biotin labeled mouse anti rat IgG1 monoclonal antibody (Zymed03-9140; Extent of dilution 1: 1000)

Biotin labeled rat anti-mouse IgGl monoclonal antibody (Serotec MCA336B; Extent of dilution 1: 1000)

Streptavidin-horseradish peroxidase (Kirkegard and Perry 14-30-00; Extent of dilution 1: 1000).

Solution:

Stop bath (DMG damping fluid)

Sodium Tetraborate, borax (Sigma)

3,3-dimethylated pentanedioic acid (Sigma)

CaCl ₂(Sigma)

Tresyl muriate (2,2,2-trifluoro ethyl sulfonyl chloride) (Fluka)

Polysorbas20: polyoxyethylene sorbitan monolaurate (Merck catalog number (Cat.No.) 822184)

1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) (Fluka)

N-hydroxy-succinamide (Fluka art.56480)

Carbonyl chloride (Fluka art.79380)

Lactose (Merck 7656)

PMSF (phenyl methyl sulfonic acid fluoride) Sigma

Succinyl--Ala-Ala-proline(Pro)-phenylalanine-acyl group p-Nitroaniline (Suc-AAPF-pNP) Sigma NO.S-7388, molecular weight 624.6g/ mole.

Painted substrate:

OPD: O-Phenylene Diamine, (Kementec catalog number (Cat.No.) 4260)

Experimental animal:

Brown Norway rat (derives from Charles River, DE)

Brown Norway rat (BN) claims to such an extent that body weight is greater than 250 grams when the experiment beginning, records to be approximately 450 grams when experiment finishes.

Dunkin Hartley cavy, (derive from Charles River, Wiga GmbhSulzfeld 1, Sandhofer Weg, DE).

Male Dunkin Hartley (being Parainfluenza 3, E.cuniculi, the seronegativity of Kpneumonia and P multocida).Animal claims to such an extent that body weight is the 350-450 gram when the experiment beginning

Female BALB/C mice (approximately 20g) (available from Bomholdtgaard, Ry, DK)

Equipment

XCEL?II(Novex)

ELISA readout instrument (UVmax, Molecular Devices)

HPLC(Waters)

PFLC(Pharmacia)

The Superdex-75 post, Mono-Q, Mono S is (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, SW)

The Amicon sulculus

Method:

The immunity of BALB/C mice

Contain 25 μ l PD498, PD498-SPEG 5,000 and glycine-SPEG-15 respectively by subcutaneous injection, 000 50 μ l 0.9% (weight/volume) NaCl solution carry out immunity to female Balb/C mouse (20 gram).The protein mass of each batch is measured by NanoOrange quantification of protein test (Molecular Probes Europe N-6666).Immunity is carried out once in per two weeks, carries out three months altogether.In when week after immunity, collect blood sample (200 μ l) from eyes.By blood coagulation and centrifugal acquisition serum.

Determine the ELISA method of IgGl antibody relative concentration in the BALB/C mice

1) wrap by the ELIAS plate with 1 μ g protein/ml of being cushioned in the liquid of bag, under 4 ℃, be incubated overnight, or incubation at least 3 hours at room temperature.50 μ l/ holes.Slightly shake.

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

3) with antigen and 1/2 extent of dilution serum incubation in dilution buffer liquid.All solution adds in the hole after preparation immediately.Stay some holes only to handle (blank) with dilution buffer liquid.At room temperature incubation is at least 1 hour.50 μ l/ holes.Slightly shake.Wash plate is 3 times in lavation buffer solution.

4) biotin labeled rat anti-mouse IgGl monoclonal antibody of dilution or biotin labeled mouse anti rat IgGl monoclonal antibody in dilution buffer liquid.At room temperature incubation is at least 1 hour.50 μ l/ holes.Slightly shake.Wash plate is 3 times in lavation buffer solution.

5) in dilution buffer liquid, dilute Streptavidin-horseradish peroxidase.At room temperature incubation is at least 1 hour.50 μ l/ holes.Slightly shake.Wash plate is 3 times in lavation buffer solution.

6) 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.

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

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

Determine the ELISA method of IgE antibody relative concentration in the BALB/C mice

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

1) wraps by elisa plate with 10 μ g rat anti-mouse IgE or mouse anti rat IgE/ml 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 mouse/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.

At room temperature incubation is 30 minutes.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.At room temperature incubation is 30 minutes.Slightly shake.Wash plate is 3 times in lavation buffer solution.

6) with the anti-pIg antibody of dilution buffer liquid dilution horseradish peroxidase bonded.50 μ l/ holes.At room temperature incubation is 30 minutes.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 492nm to dull and stereotyped reading, with the reading of 620nm as reference.Data also show with the Lotus computed in software.

Be used to measure the ELISA method of the positive cavy of IgGl

Wrap by ELISA microtiter plate at 1: 8000 with the anti-PD498 of rabbit in the carbonate buffer solution (pH 9.6), and under 4 ℃, be incubated overnight.Second day, sealed each plate 1 hour with 2%BSA, and wash each plate 3 times with the PBS polysorbas20.

1 μ g/ml PD498 is added in each plate, and incubation 1 hour is used PBS polysorbas20 wash plate 3 times again.

All guinea pig serum samples and contrast are added on the elisa plate with 2 μ l serum and 98 μ l PBS, incubation 1 hour, and with PBS polysorbas20 wash plate 3 times.

Then the anti-cavy IgG1 of goat (1: 4000 extent of dilution in the PBS damping fluid (NordicImmunology 44-682)) is added on the plate, incubation 1 hour, and wash each plate with the PBS polysorbas20.

The anti-goat of rabbit 1: 8000 (Sigma A4187) that adds alkali phosphatase enzyme mark, incubation 1 hour with PBS polysorbas20 wash plate twice, is used diethanol amine damping fluid wash plate 1 time.

Under 37 ℃, use the p-nitrophenyl phosphoric acid ester that the alkaline phosphatase of mark was developed the color 30 minutes, perhaps up to demonstrating suitable color.

Use and stop the medium (K that comprises EDTA ₂HPO ₄/ HaH ₃Damping fluid (pH 10)) termination reaction, and use the ELISA readout instrument to read the numerical value of OD 405/650.

On all elisa plates, include the double blinding sample.

Positive and negative serum value is calculated as the standard deviation that average blind value adds 2 times.The accuracy that reaches like this is 95%.

(IT) stimulates in the tracheae of rat

The disposable syringe that use has 2  inchage metal probes 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.Stimulate animal 4 times, stimulate for the last time between the bloodletting 5 days at interval.

Experimental animal is 10 every group a Brown Norway rat (BN).Weight is tested about 450 grams of weight when finishing greater than 250 grams when the experiment beginning.

(IT) stimulates in the tracheae of cavy

The disposable syringe that use has 2  inchage metal probes carries out using molecule in the tracheae.This probe inserts in the tracheae of cavy into 1cm under epiglottis greatly, places the molecular solution of 0.1ml.One week stimulated animal once, 10 weeks of continued stimulus.

ELISA IgE pilot system (being used for Brown Norway rat)

Use three-layer sandwich ELISA to determine the relative concentration of specific antibody.

Use immune molecule as envelope antigen (10 μ g/ml in the neutral phosphonic phthalate buffer, 50 μ l/ holes), under 4 ℃, be incubated overnight.All binding sites that residue in hole surface sealed 30 minutes in room temperature (RT) with 2% skim-milk (200 μ l/ hole) in the phosphate buffered saline buffer at least.With 8-passage liquid getting device, by 50 μ l/ holes, 3 times of serial dilution degree that 10 times of extent of dilution of all serum that desire is tested with this antigen reach subsequently add in the flat board.Each dilution is to carry out in the phosphate buffered saline buffer that contains 0.5% skim-milk and 0.05% polysorbas20, in stirring on the platform incubation under the room temperature 2 hours.Probe molecule is biotinylated mouse anti rat IgE (50 μ l/ hole), and dilution is 2000 times in the phosphate buffered saline buffer with 0.5% skim-milk and 0.05% polysorbas20, in stirring on the platform incubation under the RT 2 hours.The operation of contrast (blank) is identical, but does not have rat blood serum.Incubation was being stirred on the platform 1 hour in Streptavidin-horseradish peroxidase 50 μ l/ holes of 2000 times of dilutions.The chromogenic substrate in 50 μ l/ holes is OPD (6mg) and the H in every 10ml citrate buffer (pH5.2) ₂O ₂(solution of 4 μ l 30%).Add 2N H by 100 μ l/ holes ₂SO ₄With termination reaction.Utilize SLT to read the value at 486nm place, the value of using the 620nm place is as reference.Data Lotus computed in software and demonstration.

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

Analyze 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

Substrate: 100mg suc-AAPF-pNa is dissolved in the 1ml dimethyl sulfoxide (DMSO) (DMSO).This solution of 100 μ l 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 N-succinimdyl carbonate activation mPEG 15,000

MPEG 15,000 is suspended in (4ml/g mPEG) in the toluene, and distillation removes 20% with the azeotropic drying reactant under standard atmosphere pressure.When solution is cooled to 30 ℃, add methylene dichloride (the dry mPEG of 1ml/g), and add the carbonyl chloride (1.93M 5 moles/mole mPEG) in the toluene, mixture at room temperature stirs and spends the night.Mixture is evaporated to drying, and the purpose product of gained is a wax shape piece.

After evaporation, add methylene dichloride and toluene (1: 2, the dry mPEG of 3ml/g) to dissolve white solid again.Add N-hydroxy-succinamide (2 moles/mole mPEG) with solid form, add triethylamine (1.1 moles/mole mPEG) then.Mixture was stirred 3 hours.Mixture is not limpid at first, and it is limpid to become then, forms little precipitation at last.Mixture is evaporated to drying, recrystallization in ethyl acetate (10ml), heat filtering desalts and insoluble micro substance to remove.Place limpid liquid, it was at room temperature slowly cooled off 16 hours, then in refrigerator overnight.Leach white precipitate,, and carry out drying, obtain the product of 98% (w/w) with some cold ethyl acetate washings.Nucleus magnetic resonance shows has 80-90% to be activated and 5o/oo (w/w) HNEt ₃Cl.MPEG 15,000 (CDCl ₃) ¹H-NMR is: δ 1.42t (I=4.8 CH ₃IHNEt ₃Cl), 2.84s (I=3.7 succinimide), 3.10dq (I=3.4 CH ₂IHNEt ₃Cl), 3.38s (I=2.7 CH ₃IOMe), 3.40*dd (I=4.5o/oo, ¹³C satellite), 3.64bs (I=1364 main peak), 3.89*dd (I=4.8o/oo ¹³C satellite), 4.47dd (I=1.8, the CH among the PEG ₂).After in moisture eliminator, storing 4 months under 22 ℃, do not see variation.

Embodiment 2

With N-succinimdyl carbonate activation mPEG 5,000

MPEG 5,000 carries out as described in embodiment 1 with the activation of N-succinimdyl carbonate.

Embodiment 3

The combination of PD498 proteolytic enzyme and activatory mPEG 5,000

In the final volume of 20ml, in 50mM Sodium Tetraborate (pH10), with 200mg PD498 and 1.8g with N-succinimdyl carbonate activatory mPEG 5,000 (according to embodiment 2 preparations) incubation together.Stir by magnetic, at room temperature react.Reaction times is 1 hour.By adding DMG damping fluid to final concentration is 5mM Methyl glutarate, 1mM CaCl ₂(pH5.0) comes termination reaction with the 50mM borate.

The molecular weight of gained derivative approximately is 100kDa, is equivalent to every mole of PD498 and has connected about 13 moles of mPEG.

With parent enzyme relatively, coupled enzyme to the remaining activity of peptide substrates (succinyl--Ala-Ala-Pro-Phe-acyl group p-Nitroaniline) near 100%.

Embodiment 4

The combination of subtilisin DY proteolytic enzyme and activatory mPEG 5,000

Use and identical method described in the embodiment 3, with subtilisin DY proteolytic enzyme and carry out combination through N-succinimdyl carbonate activatory mPEG 5,000.

Embodiment 5

BALB/C mice subcutaneous (SC) is followed the trail of

Use as above-mentioned embodiment and describe the PD498-SPEG 5,000 of prepared modification, parent PD498 and glycine-SPEG 15,000 subcutaneous stimulation BALB/C mice of unmodified.

The serum of immune mouse detects in specific IgE ELISA (as mentioned above), whether can the activated immune answering system to analyze all molecules, cause that specific IgE replys (referring to Fig. 1).

Biweekly totally four times the immunization IgE that is enough to cause PD498 replys.

Immunization protocol biweekly continues 3 months.Last what study, carried out seven immunity.As shown in Figure 1, in the BALB/C mice of the parent PD498 of unmodified immunity, anti-PD498 IgE level all increased before the 5th immunity, kept the level of quite stable then.Comparatively speaking, not detecting specific IgE in the mouse that crosses with PD498-SPEG 5,000 immunity of modifying replys.

Embodiment 6

Follow the trail of in the allergenicity tracheae of PD498-SPEG 5,000 in the cavy

By dispenser in the tracheae, stimulate Dunkin Hartley cavys with the PD498 of 1.0 μ g purifying and the PD498-SPEG 5,000 of 1.0 μ g modification.

During detect following the trail of with specific IgG1 ELISA (as mentioned above) through the serum of the Dunkin of immunity Hartley cavy, whether can the activated immune answering system to analyze this molecule, cause that the specific IgG 1 that is designated as allergic response replys (referring to Fig. 2).The mensuration level is 1: 50.

Fig. 2 has shown the IgG1 level of Dunkin Hartley cavy during the tracking in 10 weeks.As can be seen from the figure, in No. 7 tapping (Tap-7) that was equivalent to for the 7th week before, detect IgG1 level less than the PD498 that modifies.Behind the PD498 continued stimulus of modifying, the IgG1 level does not significantly improve.

Embodiment 7

Follow the trail of in the tracheae of dose-response in the cavy (IT)

The potential allergic response of the PD498-SPEG 5,000 that in cavy, modifies by trace detection in the tracheae.One week of cavy stimulates once continuous 10 weeks.

Before first time tracheae internal stimulus, use the ELISA that is used for cavy as mentioned above, the blood sample of collecting from every Dunkin Hartley cavy is carried out blood testing.Doing like this is non-specific binding in order to ensure there is not serum in ELISA.

10 one group cavy carries out the tracheae internal stimulus with the following material of 0.3 microgram, 3 micrograms, 30 micrograms, 300 micrograms:

■ parent PD498 and

The PD498-SPEG 5,000 that ■ modifies.

Following solution is used for blind check:

■ 0.9%NaCl (being used for the blind check of parent PD498),

The 300 microgram PEGs 5,000 of ■ in 0.9%NaCl (being equivalent to PD498-SPEG5, the PEG amount in 000) (blind check of the PD498-SPEG that is used to modify).

The serum of all test cavys of check in IgG1 ELISA (as mentioned above).Provided the interior result of tracking of tracheae of the PD498-SPEG 5,000 that modifies among Fig. 3.Provided the tracking result of the parent PD498 of unmodified among Fig. 4.

By comparison diagram 3 and Fig. 4 as can be seen: with compare with the cavy of parent enzyme tracheae internal stimulus, decrease with replying of causing of the enzyme tracheae internal stimulus cavy of modifying.

According to the disclosed content in front, it will be obvious to those skilled in the art that when enforcement is of the present invention under the situation that does not depart from essence of the present invention or scope, many changes and modification to be arranged.Therefore, scope of the present invention should be according to the content understanding of following claim definition.

Sequence table (1) general information: (i) applicant:

(A) title: Novo Nordisk A/S

(B) street: Novo Alle

(C) city: Bagsveard

(E) country: Denmark

(F) zip code (ZIP): DK-2880

(G) phone: 45 4,444 8888

(H) fax: 45 4,449 3256 (ii) denominations of invention: the coupled enzyme that is used for skin care is sequence number (iii): 4 (iv) computer-reader forms:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release #1.0, version #1.30 (EPO) (2) are about the information of SEQ ID NO:1: (i) sequence signature:

(A) length: 840 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA (genome) (vi) primary source:

(B) bacterial strain: genus bacillus PD498, NCIMB No.40484 (ix) feature:

(A) title/keyword: CDS

(B) position: 1..840, (xi) sequence description: SEQ ID NO:1:TGG TCA CCG AAT GAC CCT TAC TAT TCT GCT TAC CAG TAT GGA CCA CAA 48Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln 15 10 15AAC ACC TCA ACC CCT GCT GCC TGG GAT GTA ACC CGT GGA AGC AGC ACT 96Asn Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr

20??????????????????25??????????????????30CAA?ACG?GTG?GCG?GTC?CTT?GAT?TCC?GGA?GTG?GAT?TAT?AAC?CAC?CCT?GAT????144Gln?Thr?Val?Ala?Val?Leu?Asp?Ser?Gly?Val?Asp?Tyr?Asn?His?Pro?Asp

35??????????????????40??????????????????45CTT?GCA?AGA?AAA?GTA?ATA?AAA?GGG?TAC?GAC?TTT?ATC?GAC?AGG?GAC?AAT????192Lau?Ala?Arg?Lys?Val?Ile?Lys?Gly?Tyr?Asp?Phe?Ile?Asp?Arg?Asp?Asn

50??????????????????55??????????????????60AAC?CCA?ATG?GAT?CTT?AAC?GGA?CAT?GGT?ACC?CAT?GTT?GCC?GGT?ACT?GTT????240Asn?Pro?Met?Asp?Lau?Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Thr?Val?65??????????????????70??????????????????75??????????????????80GCT?GCT?GAT?ACG?AAC?AAT?GGA?ATT?GGC?GTA?GCC?GGT?ATG?GCA?CCA?GAT????288Ala?Ala?Asp?Thr?Asn?Asn?Gly?Ile?Gly?Val?Ala?Gly?Met?Ala?Pro?Asp

85??????????????????90??????????????????95ACG?AAG?ATC?CTT?GCC?GTA?CGG?GTC?CTT?GAT?GCC?AAT?GGA?AGT?GGC?TCA????336Thr?Lys?Ile?Leu?Ala?Val?Arg?Val?Leu?Asp?Ala?Asn?Gly?Ser?Gly?Ser

100?????????????????105?????????????????110CTT?GAC?AGC?ATT?GCC?TCA?GGT?ATC?CGC?TAT?GCT?GCT?GAT?CAA?GGG?GCA????384Leu?Asp?Ser?Ile?Ala?Ser?Gly?Ile?Arg?Tyr?Ala?Ala?Asp?Gln?Gly?Ala

115?????????????????120?????????????????125AAG?GTA?CTC?AAC?CTC?TCC?CTT?GGT?TGC?GAA?TGC?AAC?TCC?ACA?ACT?CTT????432Lys?Val?Leu?Asn?Leu?Ser?Leu?Gly?Cys?Glu?cys?Asn?Ser?Thr?Thr?Leu

130?????????????????135?????????????????140AAG?AGT?GCC?GTC?GAC?TAT?GCA?TGG?AAC?AAA?GGA?GCT?GTA?GTC?GTT?GCT????480Lys?Ser?Ala?Val?Asp?Tyr?Ala?Trp?Asn?Lys?Gly?Ala?Val?Val?Val?Ala145?????????????????150?????????????????155?????????????????160GCT?GCA?GGG?AAT?GAC?AAT?GTA?TCC?CGT?ACA?TTC?CAA?CCA?GCT?TCT?TAC????528Ala?Ala?Gly?Asn?Asp?Asn?Val?Ser?Arg?Thr?Phe?Gln?Pro?Ala?Ser?Tyr

165?????????????????170?????????????????175CCT?AAT?GCC?ATT?GCA?GTA?GGT?GCC?ATT?GAC?TCC?AAT?GAT?CGA?AAA?GCA????576Pro?Asn?Ala?Ile?Ala?Val?Gly?Ala?Ile?Asp?Ser?Asn?Asp?Arg?Lys?Ala

180?????????????????185?????????????????190TCA?TTC?TCC?AAT?TAC?GGA?ACG?TGG?GTG?GAT?GTC?ACT?GCT?CCA?GGT?GTG????624Ser?Phe?Ser?Asn?Tyr?Gly?Thr?Trp?Val?Asp?Val?Thr?Ala?Pro?Gly?Val

195?????????????????200?????????????????205AAC?ATA?GCA?TCA?ACC?GTT?CCG?AAT?AAT?GGC?TAC?TCC?TAC?ATG?TCT?GGT????672Asn?Ile?Ala?Ser?Thr?Val?Pro?Asn?Asn?Gly?Tyr?Ser?Tyr?Met?Ser?Gly

210?????????????????215?????????????????220ACG?TCC?ATG?GCA?TCC?CCT?CAC?GTG?GCC?GGT?TTG?GCT?GCT?TTG?TTG?GCA????720Thr?Ser?Met?Ala?Ser?Pro?His?Val?Ala?Gly?Leu?Ala?Ala?Leu?Leu?Ala225?????????????????230?????????????????235?????????????????240AGT?CAA?GGT?AAG?AAT?AAC?GTA?CAA?ATC?CGC?CAG?GCC?ATT?GAG?CAA?ACC????768Ser?Gln?Gly?Lys?Asn?Asn?Val?Gln?Ile?Arg?Gln?Ala?Ile?Glu?Gln?Thr

245?????????????????250?????????????????255GCC?GAT?AAG?ATC?TCT?GGC?ACT?GGA?ACA?AAC?TTC?AAG?TAT?GGT?AAA?ATC????816Ala?Asp?Lys?Ile?Ser?Gly?Thr?Gly?Thr?Asn?Phe?Lys?Tyr?Gly?Lys?Ile

260?????????????????265?????????????????270AAC?TCA?AAC?AAA?GCT?GTA?AGA?TAC????????????????????????????????????840Asn?Ser?Asn?Lys?Ala?Val?Arg?Tyr

275 280 (2) information: (i) sequence signature about SEQ ID NO:2:

(A) length: 280 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): protein (xi) sequence description: SEQ ID NO:2:Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln 15 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 (2) information: (i) sequence signature about SEQ ID NO:3:

(A) length: 274 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): protein (vi) primary source:

(B) bacterial strain: genus bacillus mutation (xi) sequence description: SEQ ID NO:3:Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val1 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?Val65??????????????????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?Serl45?????????????????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?????????????????255Gly?Asp?Ser?Phe?Tyr?Tyr?Gly?Lys?Gly?Leu?Ile?Asn?Val?Glu?Ala?Ala

260 265 270Ala Gln (2) are about the information of SEQ ID NO:4: (i) sequence signature:

(A) length: 433 amino acid

(B) type: amino acid

(C) chain:

(D) topological framework: linearity is molecule type (ii): protein (vi) primary source:

(B) bacterial strain: genus bacillus Y kind (xi) sequence description: SEQ ID NO:4:Asn Asp Val Ala Arg Gly Ile Val Lys Ala Asp Val Ala Gln Asn Asn1 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?Ala65??????????????????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?Ser?Phe

325?????????????????330?????????????????335Gln?Ala?Gln?Ala?Gly?Lys?Pro?Leu?Lys?Ile?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 (2) are about the information of SEQ ID NO:5: (i) sequence signature:

(A) length: 316 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): protein (vi) primary source:

(B) bacterial strain: Bacillus Thermoproteolyticus (xi) sequence description: SEQ ID NO:5:Ile Thr Gly Thr Ser Thr Val Gly Val Gly Arg Gly Val Leu Gly Asp1 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?Thr65??????????????????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 (23)

1. a coupled enzyme is characterized in that, it is that 4-70 the polymerizable molecular covalent coupling of 1-35kDa is the surface of the parent enzyme of 15-100kDa to molecular weight that molecular weight is arranged on it.

2. according to the coupled enzyme of claim 1, it is characterized in that it is on the surface of described enzyme of 15-35kDa to molecular weight that 4-20 polymerizable molecular covalent coupling arranged.

3. according to any one coupled enzyme in the claim 2,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 polymerizable moleculars are wherein arranged, 13-18 polymerizable molecular preferably, covalent coupling is to the surface of parent enzyme 3-D structure.

4. according to the coupled enzyme of claim 1, wherein there be 7-40, preferably 10-30 polymerizable molecular is coupled on the surface of described parent enzyme that molecular weight is 35-60kDa.

5. according to the coupled enzyme of claim 1, wherein there be 10-50, preferably 13-40 polymerizable molecular is coupled on the surface of described parent enzyme that molecular weight is 60-80kDa.

6. according to the coupled enzyme of claim 1, wherein there be 15-70, preferably 18-60 polymerizable molecular is coupled on the surface of described parent enzyme that molecular weight is 80-100kDa.

7. according to any one coupled enzyme among the claim 1-6, the molecular weight of wherein said polymerizable molecular is 1-35kDa, as 4-25kDa, and preferably 6-25kDa, especially 8-20kDa.

8. according to the coupled enzyme of claim 1-7, wherein said polymerizable molecular is selected from the group that comprises natural or synthetic homopolymer and heteropolymer.

9. according to the coupled enzyme of claim 8, wherein said polymer molecule is selected from the group that comprises the synthesized polymer molecule, and described synthesized polymer molecule comprises the PEG of branch, polyvinyl alcohol (PVA), poly carboxylic acid, poly-(V-Pyrol RC) and poly-D, L-amino acid.

10. according to the coupled enzyme of claim 8, wherein said polymerizable molecular is selected from the group that comprises naturally occurring polymerizable molecular, and described naturally occurring polymerizable molecular comprises dextran, comprises Sensor Chip CM 5; Mierocrystalline cellulose is as methylcellulose gum, carboxymethyl cellulose, ethyl cellulose, Natvosol, hydroxypropylcellulose); And the hydrolysate of chitosan; Starch is as hydroxyethylamyle, hydroxypropylated starch; Glycogen, agarose, guar gum, inulin, amylopectin, synthesising biological polymeric gel, carrageenin, pectin and alginic acid.

11. according to any one coupled enzyme among the claim 1-10, wherein said enzyme is coupled on the activatory polymkeric substance on one or more in the following groups: amino, hydroxyl, sulfydryl, carboxyl, aldehyde radical or sulfhedryl.

12. according to any one coupled enzyme among the claim 1-11, as described in wherein said polymerizable molecular is coupled to by joint (as triazine ring) on the enzyme.

13. according to any one coupled enzyme among the claim 1-12, wherein said enzyme is microbe-derived, as bacterium, filamentous fungus or yeast source.

14. according to any one coupled enzyme among the claim 1-13, wherein said enzyme is a kind of lytic enzyme, comprises proteolytic enzyme (as subtilisin) and lipase.

15. according to the coupled enzyme of claim 14, wherein said parent protease is selected from and comprises PD498, Savinase ^, Proteinase K, proteolytic enzyme R, Thermitase, subtilisin DY, Lion Y, Alcalase ^, proteolytic enzyme T and JA16 group.

16. according to the coupled enzyme of claim 16, wherein said enzyme is the PD498 shown in the SEQ ID NO.1, or the proteolytic enzyme subtilisin DY of the subtilisin type shown in the SEQ ID NO.3, or the Lion Y shown in the SEQ ID N0.4.

17. according to any one coupled enzyme among the claim 1-13, wherein said enzyme is a kind of oxydo-reductase, comprises laccase and superoxide-dismutase.

18. according to any one coupled enzyme among the claim 1-17, wherein said polymerizable molecular is by being positioned at the lip-deep amino (NH of enzyme ₂) be coupled on the described enzyme.

19. according to the coupled enzyme of claim 18, wherein said polymerizable molecular is by the N-terminal amino group or be positioned at the lip-deep lysine residue of enzyme and be coupled on the described enzyme.

20. according to the coupled enzyme of claim 1-19, wherein said polymerizable molecular be coupled on the described enzyme apart from enzyme active sites greater than 5 , be preferably more than the position of 10 .

21. a skin care compositions wherein comprises according to coupled enzyme of any one and all multicomponents that become known for skin care products among the claim 1-20.

22. skin care products, wherein comprise the skin care compositions according to claim 21, described care products is selected from down group: soap, makeup, cold cream, skin-care gel, skin care milk, clean lotion for skin care, cleansing cream, cleaning lotion, clean skin milk, cold cream, cold soap, the cosmetic base-material, the cleaning breast, facial mask, contain the zinc astringent, T district essence, hand cream, face powder, whitening powder, soap powder, cake soap, transparent soap, lipstick, lipstick, nutrient substance, foundation cream, cosmetic end powder, eye shadow powder, foundation cream, the nail varnish sanitising agent, hair tonic, hairdressing liquid, hair-cream, hair gel, mayonnaise agent, hair setting goods, hair dye, hair coloring agent, the scalp moisturizing agent, shampoo, balm, hair conditioner, hair jelly, suntan oil, sunscreen products, shaving foam and gel, shaving cream, baby oil, the acne care article, antiperspirant, wormer, deodorant etc.

23. be used to reduce the purposes of skin care products sensitizability according to the coupled enzyme of any one among the claim 1-20.

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