WO2007052780A1

WO2007052780A1 - Composition, protein modifier, and modified protein

Info

Publication number: WO2007052780A1
Application number: PCT/JP2006/322058
Authority: WO
Inventors: Takafumi Kubo; Yasutaka Sumida
Original assignee: Nippon Shokubai Co., Ltd.
Priority date: 2005-11-04
Filing date: 2006-10-30
Publication date: 2007-05-10

Abstract

The present invention is to provide a composition comprising a protein and a surfactant which is useful for various uses such as a detergent, sufficiently suppressing the decrease of activity of a protein, and exhibiting high stability; a protein modifier capable of modifying a protein simply; a modified protein modified with the protein modifier, and a composition comprising the modified protein. The above-mentioned composition is a composition comprising protein and an anionic surfactant, wherein the protein is chemically modified with a polyvalent carboxylic acid having a benzene ring as a protein modifier.

Description

DESCRIPTION

COMPOSITION, PROTEIN MODIFIER, AND MODIFIED PROTEIN

TECHNICAL FIELD

The present invention relates to a composition, a protein modifier, and a modified protein. More specifically, it relates to a useful composition for use when stability to surfactants of a protein is required and so forth, a protein modifier, a modified protein, and a composition comprising the modified protein.

BACKGROUND ART Various kinds of proteins, comprising enzymes which are catalysts derived from nature, are broadly used in industry. Examples of the uses include as a cleansing component in detergents, various catalysts for synthesizing chemicals, a modifier for foods, and pharmaceuticals. However, generally speaking a protein such as an enzyme is denatured, loses its function, or cannot exhibit full performance due to heat, a pH condition, presence of organic compounds, and so forth. In order to overcome these drawbacks, various methods such as modification by amino acid substitutions and chemical modifications have been studied. In the modification by amino acid substitutions, individual investigation for each protein is necessary and requires long hours of effort. In that respect, a chemical modification method is superior in the respect that one method is applicable to a large number of proteins . However, there have been drawbacks of which the modifiers to be obtained are poor in diversity, and a complicated modification process is required.

Further, for example, a composition comprising an enzyme and a surfactant is broadly used in the use of the detergent, and this is because it has superior cleansing effect due to the action of an enzyme to degrade proteins, lipids and so forth, lower their molecular weight and solubilize them into water, together with the action of a surfactant. However, an enzyme such as protease is gradually denatured while stored in a detergent due to the alkaline condition and the presence of surfactants, and loses cleansing performance. Especially, anionic surfactants such as LAS (linear alkyl benzene sulfonate) are known to be a strong denaturants of enzymes (Described in "Enzymes in Detergency" . Edited by Jam H, van Ee. Marcel Dekker, Inc. 1997: page 306) , however, they are often formulated in a detergent with enzymes because they are surfactants having both superior economical efficiency and superior cleansing property. Therefore, improving stability of the enzyme and stable supply of a high cleansing performance are strongly desired for the detergent composition to include such an enzyme. Especially, it is desired to improve the stability of the enzyme toward surfactants such as anionic surfactants .

A chemical modification method of modifying a protein with a polymeric compound such as organic synthetic polymers and natural polysaccharides is generally known as the conventional method to improve the stability of a protein. For example, a method of immobilizing an enzyme such as protease in a water-soluble organic polymer consisting of methyl vinyl ether and maleic anhydride is disclosed in Japanese Patent Laid-Open No. 06-240297. Further, a method of immobilizing polyethylene glycol in an enzyme such as protease is also disclosed in Japanese Patent Laid-Open No. 11-502255. However, in these methods, the effect of improving stability toward surfactants is not sufficient, and the enzyme activity is lost largely after modification. Further, there are problems that a large amount of modifier is necessary and that a complicated modification process is necessary.

On the other hand, a method of improving the stability at high temperature by attaching a monomeric compound having aromatic rings such as pyromellitic acid and aniline to an amino group of cellulase is disclosed in the International Publication No. 02/098824 pamphlet. Further, a method of improving the stability at high temperature in a buffer and the stability in a mixed solvent of water and an organic solvent by modifying trypsin with a carboxylic anhydride comprising benzene rings such as pyromellitic acid is disclosed by

Vinogradov et. al. (The chemical modification of α-chymotrypsin with both hydrophobic and hydrophilic compounds stabilized the enzyme against denaturation in water-organic media, "Protein Engineering", vol. 14, no. 9, p. 683-689, 2001) and by Mozaef et. al . (Protein stabilization via hydrophilization, "Eur. J. Biochem", vol. 173, p. 147-154, 1988). However, there is no disclosure about the superiority in the stability to surfactants of modified enzymes, the cleansing performance of modified enzymes, and the use of modified enzymes for a composition comprising surfactants.

Further, a method of chemically-modifying protease with aliphatic dicarboxylic anhydrides is disclosed in Japanese Patent Laid-Open No. 08-9974, and a method of modifying an enzyme with aromatic compounds such as pyromellitic acid is disclosed in the International Publication No. 03/056001 pamphlet, and a method of modification of alkaline phosphatase with pyromellitic acid and modification of amylase with phthalic acid are described in the examples in the International Publication No. 03/056001 pamphlet, however, there is no disclosure about the superiority in the stability to surfactants. Furthermore, a detergent composition comprising these modified enzymes and surfactants is also described in the pamphlet, however, a combination of the modified enzymes and surfactants, in which modification benefit is obtained, is not disclosed. Moreover, the effect of improving stability toward surfactants is also not sufficient in these methods, and the decrease of activity of the protein could not be suppressed sufficiently. Therefore, in any of these methods, there is still a room for improvement in the respect of reconciling the improvement of stability to surfactants and the protein activity, that is in the respect of sufficiently using both effects of a surfactant and a protein and stably exhibiting a high cleansing performance by the protein having high stability toward the surfactant and maintaining high protein activity.

Further, an antithrombogenic material in which heparin or antithrombin is immobilized in a polymer of butane tetracarboxylic acid and diamine (a ring-open polyimide resin) using a condensing agent such as a carbodiimide compound is disclosed in Japanese Patent Laid-Open No. 58-22065. However, it is complicated and costly because it uses a condensing agent, and there is room for an invention to be able to stabilize a protein simply.

SUMMARY OF THE INVENTION

The present invention considers the above-described present situation, and the object of the present invention is to provide a composition comprising a protein and a surfactant which is useful for various uses such as a detergent, sufficiently suppressing the decrease of activity of a protein, and exhibiting high stability; a protein modifier capable of modifying a protein simply; a modified protein modified by the protein modifier, and a composition comprising the modified protein.

Investigating a composition comprising a protein and a surfactant variously, the inventors of the present invention noticed that a chemically modified protein is useful, for example, as a protein to be included in a composition such as for a detergent because the protein can exist stably against alkaline condition and heat. Then, they found that there is a case that a protein in which a polyvalent carboxylic acid compound is attached to amino groups existing in the protein and mainly derived from lysine has superior stability to surfactants. Especially, they found that when a polyvalent carboxylic acid compound having a benzene ring is used as a modifier, the modified protein exhibits superior stability to surfactants, particularly against anionic surfactants, while it is capable of significantly suppressing the decrease of activity of a protein in a composition comprising a surfactant. Further, they found that a composition comprising a protein modified in such a manner and an anionic surfactant can exhibit desired effects such as high cleansing performance, decomposing performance, and bleaching performance in the various uses such as use for a detergent, use for decomposing insoluble substrates of cellulose and so forth, and use for bleaching pulp, due to the superior characteristics of the composition such as high surface-activating ability and high stability, and considered the composition to be able to solve the above-described problems successfully. -

Further, investigating a protein modifier variously, the inventors of the present invention found that a polymer of a polyvalent carboxylic acid and a connecting agent to connect between the carboxyl groups become a modifier capable of significantly suppressing the decrease of activity of a protein, and that when such polymer has a carboxylic anhydride group in the molecule, the polymer as a modifier can be simply attached to the protein using the carboxylic anhydride group, and the high stabilization of the protein can be realized. Furthermore, using this protein modifier, it becomes possible to modify a protein simply without using an additional reagent such as a condensing agent . Then, they found that a protein modified with such a protein modifier becomes useful in various uses such as use for a detergent, use for decomposing insoluble substrates of cellulose and so forth, and use for bleaching pulp, use for medical products, and these findings have now led to completion of the present invention.

Therefore, the present invention is a composition comprising a protein and an anionic surfactant, wherein the protein is chemically modified with a polyvalent carboxylic acid having a benzene ring as a protein modifier .

The present invention is also a protein modifier, wherein the protein modifier is a polymer of a polyvalent carboxylic acid having three or more carboxyl groups and a connecting agent connecting the carboxyl groups, the protein modifier having at least one carboxylic anhydride group in the molecule.

The present invention is also a protein modifier, the protein modifier being a polyvalent carboxylic acid having three or more carboxyl groups, and having at least one carboxylic anhydride group in the molecule, wherein at least one carboxyl group in the polyvalent carboxylic acid is connected to a monovalent amine by an amide bond or to monohydric alcohol by an ester bond.

The present invention is also a modified protein chemically modified with the above-described protein modifier .

The present invention is also a composition comprising the above-described modified protein.

The present invention is described in detail below. The composition of the present invention comprises a protein and an anionic surfactant. The form of such a composition may be liquid or solid (for example, powder) . However, because the stability of the protein generally causes a problem in liquid, a liquid form is preferable in order to sufficiently obtain the effect of the present invention, which makes the protein sufficiently stabilized.

In the above-described composition, although the protein is not especially limited as long as it shows an effect desired in each use, it is preferably an enzyme.

Examples of the above-described enzyme include hydrolase, oxidase, reductase and lyase. More specifically, examples include protease, lipase, amylase, glucoamylase, cellulase, hemicellulase, glucanase, cutinase, pectinase, ligninase, mannanase, pullulanase, xylanase, chitinase, peroxidase, reductase, oxidase, oxygenase, and laccase. Preferably it is hydrolase and oxidase, and specifically, protease, lipase, amylase, cellulase, peroxidase, mannanase, and xylanase are especially preferable. Among them, protease is the most preferable. Further, commercially available enzymes are preferably used, and they are available from the companies such as Novozymes, Genencor- International, DSM, Nagase ChemteX Corporation, Amano Enzyme Inc. , Takara Bio Inc. , Sigma-Aldrich, Inc., and Henkel KGaA.

Although an protease such as one derived from a micro-organism such as subtilisin and one derived from plants such as papain, can be used as the above-described protease and it is not especially limited, it is preferable to use an protease derived from micro-organisms. Although examples of the protease derived from micro-organisms include proteases derived from Bacillus species and proteases derived from fungi,. it is preferable to use proteases derived from Bacillus species and further preferably proteases , similar to subtilisin. Specifically, Alkalase®, Esperase®, Savinase®, Kannase®, and Everlase® of Novozymes, Purafect®, Purafect OX®, Purafect Prime®, and Properase® of Genencor International, Maxatase®, Maxazyme®, and Ronozyme® of DSM, Bioprase® of Nagase ChemteX Corporation, Opticlean®, Optimase®, Subtilisin A, Subtilisin Carlsberg, Nagase, and so forth are preferable. Among these, Esperase®, Savinase®, Kannase®, Purafect®, Purafect Prime®, Properase®, and Bioprase® are especially preferable. Lipolase®, Lipex®, and Novozyme 435® of Novozymes,

Bakezyme®, Piccantase®, and Lipomax® of DSM, various type of lipase of Amano Enzyme Inc., Lilipase® of Nagase ChemteX Corporation, Lumafirst® of Genencor International, and Lipomax® of Gistprokaze are preferable as the above-described lipase. Among these, Lipolase® and Lipex® are especially preferable.

Termamyl®, Duramyl®, and Stainzyme® of Novozymes, Purastar ST® and Purastar HP® of Genencor International, Bakezyme®, Extrazyme®, Maxamyl®, Hazyme®, andMycolase® of DSM, Craistase® of Amano Enzyme Inc. and so forth are preferable as the above-descrived amelase, and among those, Termamyl®, Duramyl®, Stainzyme®, Purastar ST®, and Puraster HP® are especially preferable.

Calluzyme®, Celluclast® and Carezyme® of Novozymes, Puradax® of Genencor International, Bakezyme®, Rapidase®, and Sitelase® of DSM, and so forth are preferable as the above-described cellulase, and among those, Celluzyme®, Carezyme®, and Puradax® are especially preferable.

The above-described enzymes may be enzymes derived from an appropriate enzyme sources such as plants, animals, bacteria, fungi, and yeasts, and the variants of those enzymes modified by amino acid substitution can also be used. Especially, the effect of chemical modification can be increased by placing amino acids having high reactive side-chains, such as lysine and cysteine, in appropriate positions on the enzyme surface. Further, because many detergents are adjusted to alkaline, an enzyme which exhibits its function in the alkaline range is preferable especially in the case of using the above-described composition for use in a detergent. Although either of the form of powder and solution may be used as the form of the above-described protein, it is preferable to use a protein in the form of solution. Furthermore, one kind or two or more kinds of proteins can be used as the protein included in the composition in the present invention.

The above-described protein is chemically modified with a polyvalent carboxylic acid having a benzene ring as a protein modifier. In the composition of present invention, protein stability to anionic surfactants is improved remarkably by using the protein modified chemically in such a manner. Hereby, because physical properties of the protein and the surfactant can be exhibited more stably, for example, high cleansing performance in the case of using the composition as detergents, high decomposing performance in the case of using the composition for the use of decomposing an insoluble substrate such as cellulose, and high bleaching performance in the case of using the composition for the use of bleaching pulp, can be respectively supplied stably. The anionic surfactants are economically excellent surfactants. Therefore, the composition can be excellent in cost competitiveness.

The above-described polyvalent carboxylic acid is a compound having two or more carboxyl groups. The polyvalent carboxylic acid as a protein modifier is not especially limited as long as it is a compound having a benzene ring and two or more carboxyl groups in the molecule. However, it is preferably a polyvalent carboxylic acid having three or more carboxyl groups .

Further, the above-described polyvalent carboxyl group may form a carboxylic anhydride structure (a structure in which two carboxyl groups are dehydrated, condensed, and bonded, and referred to as an "acid anhydride group" or a "carboxylic anhydride group" below) . Here, the acid anhydride group also includes a carboxylic imide compound (having a structure in which the shared oxygen atom in the acid anhydride group is substituted with a nitrogen atom) . In the case that the acid anhydride group exists, one acid anhydride group is counted as two carboxyl groups. A carboxyl group which is used for attachment with the protein by forming an amide bond or an ester bond is included in one of the above-mentioned "carboxyl group". The definition of "carboxyl group" mentioned above is applied hereafter in the present invention.

Specific examples of the above-described polyvalent carboxylic acid include pyromellitic acid, trimellitic acid, hemimellitic acid, trimesic acid, benzenepentacarboxylic acid, mellitic acid, biphenyltetracarboxylic acid, oxydiphthalic acid, naphthalene tetracarboxylic acid, benzophenone tetracarboxylic acid, benzophenone tricarboxylic acid, perylene tetracarboxylic acid, and diphenylsulfone tetracarboxylic acid. Further, an acid anhydride and an acid halide can be used in addition to the above-described acid-type compounds of the polyvalent carboxylic acid, and an acid anhydride is preferably used in the respects of reactivity and safety.

Among the above-described polyvalent carboxylic acid, pyrorαellitic acid, trirnellitic acid, diphenylsulfone tetracarboxylic acid, and mellitic acid are preferable, and pyromellitic dianhydride, trimellitic anhydride, diphenylsulfonetetracarboxylic dianhydride, and mellitic trianhydride are further preferable from the view point of economy and water-solubility. Further, a derivative of the above-mentioned polyvalent carboxylic acid may be used. The chemical modification of the protein with the above-mentioned polyvalent carboxylic acid means a connection between the polyvalent carboxylic acid and the protein by covalent bond.

Although the method to connect between the above-described polyvalent carboxylic acid and the protein is not limited, it is preferable to activate the carboxyl group in the polyvalent carboxylic acid and to attach the carboxyl group to mainly an amino group (derived from the N-terminal and lysine residues) existing in the protein. Alternatively, the carboxyl group may be attached to the thiol group (derived from cysteine residue) .

Specifically, examples include (1) a method using an acid anhydride of the polyvalent carboxylic acid, (2) a method using an acid halide of the polyvalent carboxylic acid, and (3) a method using the polyvalent carboxylic acid (acid-type) and a condensing agent. Among these, the method (1) using an anhydride of the polyvalent carboxylic acid is preferably used in the respects of easiness and safety of the reaction. In the above-described method (1), the anhydride of the polyvalent carboxylic acid is considered to react mainly with an amino group of the protein, forming an amide bond and a carboxyl group at the same time. The extra acid anhydride group which is not used for the bonding with the protein is considered to produce two carboxyl groups by gradually hydrolyzing when water exists in the system.

In the above-described method (2), the halide of the polyvalent carboxylic acid is considered to react with mainly an amino group in the protein and form an amide bond with the protein while producing hydrogen halide.

In the above-described method (3) , the carboxyl group of the polyvalent carboxylic acid is activated with the condensing agent such as carbodiimide, condensed with an amino group of the protein, and connected by forming an amide bond. N- ( 3-dimethylaminopropyl ) -N' -ethylcarbodiimide hydrochloride and dicyclohexylcarbodiimide is preferable as the carbodiimide compound as the condensing agent.

Furthermore, the above-described protein can be chemically modified together with the above-described one or two or more kinds of other modifiers and methods of modifying.

The modification reaction of the above-described protein with the modifier is preferably performed in a solvent. The solvent is not limited and both water and an organic solvent can be used. However, a solvent having small denaturating effect to protein is preferable and specifically examples include water and glycols such as ethylene glycol, propylene glycol, and glycerin.

Further, the modification reaction is preferably performed in a neutral to alkaline liquid. This is mainly because the amino group in the protein to be mainly modified is made to be in a state of reactive free amine. The pH of the liquid is preferably pH 5 to 11 and more preferably pH 6 to 9. Furthermore, a pH buffer agent may be added to adjust pH. Specifically, boric acid-based, phosphoric acid-based, carbonic acid-based, and amine-based pH buffer agents, and various good buffers can be used. The boric acid-based and amine-based pH buffers are preferable and specifically sodium borate, borax, triethanolamine, and sodium 4- (2-hydroxyethyl) piperazine-1-ethanesulfonate are preferable .

Although the temperature of the above-described modification reaction is not especially limited, the reaction is preferably performed at 0 to 70°C and further preferably 20 to 50°C. Compounds to protect the active center of the protein such as a substrate analogue and stabilizers such as glycols, polysaccharides, and metal ions maybe added in order to improve stability of the protein during modification reaction. After the modification reaction, the modifier which was not attached to the protein may be removed by a method such as dialysis.

In the above-described protein, the amino group (derived from lysine residue and the N-terminal) is mainly modified by the modifier. The modification degree of amino group is preferably 40 to 98%. When it is 40% or more, the effect of improving stability to surfactants of the protein is large, and when it is 98% or less, the extreme decrease of activity of the protein is difficult to occur. More preferably, it is 60 to 90%.

Furthermore, the modification degree of amino group is measured as follows. <Modification degree>

The amino group existing in the protein solution is measured by a method similar to a method using 2, 4, 6, -trinitrobenzene sulfonic acid (TNBS) (Snyder S., Analytical Biochemistry, 64, 284-288, 1975) . That is, 30 mM of TNBS solution 0.025 ml is added to the protein solution (pH9, 50 mM of boric acid buffer solution) 1.2 ml. After it is kept still at room temperature for about an hour, a residual amount of amino group in the protein solution is obtained by measuring absorbance at 420 run. The amount of amino group decreased by the modification treatment (percentage against the total amount of amino group in a unmodified protein solution) is calculated and treated as the modification degree.

The content of the above-described protein to that of the above-described composition is preferably 0.0001% by weight or more and 10% by weight or less as the effective protein in the composition of 100% by weight for example. When it is 0.0001% by weight or more, the desired effect performed by the protein, for example the effect such as cleansing effect, can be obtained sufficiently, and when it is 10% by weight or less, the composition can be made to be superior economically as well.

More preferably, it is 0.005% by weight or more and 2% by weight or less.

Although the form of the above-described composition can be both liquid and powder, liquid is preferable because the effect to be obtained of improving the stability to surfactants is large.

The above-described stability to surfactants of the protein is a scale of how much the protein can keep the activity in the case of contacting with various surfactants, and the larger the value, the more stably the desired effect performed

I by the protein, for example cleansing performance, can be supplied.

Furthermore, the test of the stability to surfactants of the protein in a solution is performed as follows. The

"activity of the protein" used herein means enzyme activity if the protein is an enzyme.

The protein solution is dissolved into a solution comprising various surfactants, and a heat treatment is performed at a fixed temperature in the solution for a fixed time. Protein activities before and after the heat treatment are measured and the ratio of the residual activity (= activity after heating / activity before heating x 100, "%") is treated as the stability to surfactants. Further, the half-life of the activity (the time takes for the activity to be half of the initial activity) is calculated and may be treated as the scale of the stability to surfactants.

Further, an activity of a modified protein as a scale of the effect given to the protein activity by the modification treatment is indicated as relative activity (= activity of a modified protein / activity of a unmodified protein x 100, "%") . Although protein modification often causes a large decrease in the activity in general, for the modified protein in the above-described manner, high relative activity can be kept, and improvement of the activity can be observed as the case may be for the modified protein in the above-described manner.

Further, the enzyme activity of protease is measured as follows.

At 25°C, oligopeptide solution (0.3 mM,

N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroani lide, in a buffer solution) 1 ml as a substrate is added to a solution 0.5 ml made by diluting an enzyme solution with a buffer solution. The amount of the generated p-nitroaniline per unit time is measured from absorbance at 405 nm and is treated as the enzyme activity. The enzyme activity is measured in the range in which the absorbance at 405 nm does not exceed 1.0. Further, the enzyme activity of lipase is measured as follows . <Enzyme Activity of Lipase >

At 25°C, p-nitrophenyl laurate (0.3 mM, in a buffer solution) 1 ml as a substrate is added to a solution 0.5 ml made by diluting an enzyme solution with a buffer solution. The amount of the generated p-nitrophenol per unit time is measured from absorbance at 400 nm and is treated as the enzyme activity. The enzyme activity is measured in the range in which the absorbance at 400 nm does not exceed 1.0. Further, the enzyme activity of cellulase is measured as follows .

The cellulase activity is determined by quantitating p-nitrophenol generated in the reaction from absorbance, using p-nitrophenyl β-D-Cellobioside as a substrate. That is, an enzyme diluted solution 0.1 iαL and a buffer solution (5OmM, pH 7, HEPES) 0.4 ml are mixed and thereto a substrate solution dissolved in a buffer solution (7.5 mM) 1.0 ml is added. The mixed solution is maintained at 40°C ^"for 30 minutes, and then measured for absorbance at 405 nm. , The absorbance is defined as the cellulase activity.

Further, the enzyme activity of amylase is measured as follows . <Enzyme Activity of Amylase> The amylase activity is determined by quantitating the amount of the reducing sugar generated by the reaction, using DNS (dinitrosalicylic acid) method. Soluble starch is used as a substrate. That is, an enzyme diluted solution 0.005 ml and a substrate solution 0.095 ml (1% by weight of starch, Tris-HCl of 5OmM, pH 8) are mixed, and hydrolysis reaction is performed at 50⁰C for 5 minutes . Then, DNS reagent O.lmLis added thereto, and heated to develop color at 100⁰C for 10 minutes. From the absorbance at 510 nm, the activity is determined.

In the above-described modified protein, the stability against surfactants is improved compared to a unmodified protein. Especially, the stability against anionic surfactants such as linear alkylbenzene sulfonate (LAS) and alkylether sulfate (AES) is improved remarkably. At the same time, the improvement of protein activity and the improvement of cleansing performance are observed. These effects are caused by the attachment of the modifier having a benzene ring and a carboxyl group to the protein and are considered to be due to the protecting effect of the protein by the benzene ring, the protecting effect of the protein by the carboxyl group (including carboxylate anion), the effect of the change of charge balance, and the effect of improving penetration into a fiber.

Furthermore, a screening test for detergency is performed as follows. <Screening Test for Detergency >

At 25°C, ion-exchanged water of 70 ml (including calcium chloride of 55 ppm) and a detergent composition comprising a surfactant and a protein is placed in a long glass bottle of 100 ml. Then, a washing step is performed by placing a piece of an artificial stained cloth (EMPAl16, 5 cm x 5 cm, the reflectance has already been measured, made into a cylindrical form and stapled together to improve reproducability) in the glass bottle and stirring at 500 rpm for 20 minutes using a magnetic stirrer. Moreover, a rinsing step is performed by discarding the washing solution, newly adding ion-exchanged water of 70 ml (including calcium chloride of 55 ppm) , and stirring at 500 rpm for 10 minutes. The stained cloth is taken out, dried after water is absorbed, and the reflectance is measured. From the reflectance change, the detergency (%) is calculated using the Kubelka-Munk equation.

A test of lL-scale detergency using Terg-O-tometer is performed based on JIS standard.

Examples of the anionic surfactant comprised in the above-described composition include alkylbenzene sulfonate, polyoxyalkylenealkyl or alkenyl ether sulfate, alkyl or alkenyl sulfate, α-olefinsulfonate, α-sulfofatty acid salt or ester salt, alkane sulfonate, saturated or unsaturated fatty acid salt, alkyl or alkenylether carbonate, amino acid surfactant, N-acylamino acid surfactant, and alkyl or alkenyl phosphoric acid ester or its salt. Moreover, an alkyl group such as a methyl group may be branched from the alkyl group and the alkenyl group of these anionic surfactants. Preferable examples are alkylbenzene sulfonate, polyoxyalkylenealkyl or alkenyl ether sulfate, alkyl or alkenyl sulfate, and saturated or unsaturated fatty acid salt. The above-described alkyl or alkenyl group preferably has 10 to 20 carbon atoms and is a straight chain or includes only one branched chain, and the above-described polyoxyalkylene group is preferably polyoxyethylene or polyoxypropylene containing 1 to 30 oxyalkylene units. Further, an anionic surfactant containing an alkyl group having 10 to 20 carbon atoms is a preferred embodiment. Here, the anionic surfactant containing an alkyl group having 10 to 20 carbon atoms is commomly used for a detergent and economically advantageous. Therefore, in the case that such a surfactant is used in the present invention, the present invention can be advantageous in the respect of cost performance because an extremely high cleansing performance compared to a regular detergent can be supplied stably.

Moreover, one kind of the above-described anionic surfactants may be used, and two kinds or more of those may be used together.

In the above-described composition, the content of the anionic surfactant is, for example, preferably 1 to 70% by weight in composition 100% by weight. It is more preferably 5 to 50% by weight.

Furthermore, the above-described composition may include a surfactant other than the anionic surfactant, such as a nonionic surfactant, a cationic surfactant, and an amphoteric surfactant. One kind or two kinds or more of these surfactants can be used.

Examples of the above-described nonionic surfactant include polyoxyalkylenealkyl or alkenyl ether, polyoxyethylenealkylphenyl ether, higher fatty acid alkanolamide or its alkylene oxide addition, sucrose fatty acid ester, alkylglycoside, fatty acid glycerol monoester, and alkylamine oxide. Moreover, an alkyl group such as a methyl group may be branched from an alkyl group and an alkenyl group of these nonionic surfactants. Preferable examples are polyoxyalkylenealkyl or alkenylether . The above-described alkyl or alkenyl group preferably has 10 to 20 carbon atoms and is a straight chain or includes only one branched chain, and the above-described polyoxyalkylene group is preferably polyoxyethylene or polyoxypropylene containing 1 to 30 oxyalkylene units. An example of the above-described cationic surfactant includes quaternary ammonium salt.

Examples of the above-described amphoteric surfactant include carboxyl or sulfobetaine amphoteric surfactants.

The above-described composition may include one kind or two kinds or more of additives, solvents and so forth which is used normally depending on the use and so forth, and the content is set appropriately depending on the desired performance and so forth. Examples of the additives include a detergent builder, a fluorescent whitening agent, a foaming agent, a bubble inhibitor, a corrosion inhibitor, a rust-preventing agent, a stain suspending agent, a stain discharging agent, a pH control agent, a germicide, a chelating agent, a viscosity control agent, a fragrance, a fiber softener, peroxide, a peroxide stabilizer, a fluorescent agent, a colorant, a foam stabilizer, a polishing agent, a bleaching agent, an enzyme, and a dye.

The above-described composition is also preferably a detergent composition. Such a detergent composition can exhibit superior stability to surfactants, especially the stability to an anionic surfactant, and is capable of stably supplying a high cleansing performance. In this case, the protein included in the above-described detergent composition is preferably an enzyme.

The above-described detergent composition preferably comprises the detergent builder. The content of the detergent builder is, for example, preferably 0.1 to 60% by weight in the detergent composition of 100% by weight. More preferably, it is 1 to 10% by weight in the case that the detergent composition in the present invention is supplied in a liquid form and 1 to 50% by weight in the case that it is supplied in a powder form. The above-described detergent builder is not especially limited, and one kind or two kinds or more of examples can be used comprising organic builders such as various alkaline metal salts, ammonium salts, substituted ammonium polyacetates, carboxylic acid salts, polycarboxylic acid salts, and polyhydroxy sulfonates; and inorganic builders such as silicates, aminosilicates, borates, and carbonates.

Examples of the polyacetates or the polycarboxylic acid salts as the above-described organic builders include ethylenedimanie tertaacetic acid, diethylenetriamine pentaacetic acid, nitrilotriacetic acid, oxydisuccic acid, mellitic acid, glycol acid, benzene polycarboxylic acid, sodium salt of citric acid, potassium salt, ammonium salt, and substituted ammonium salt, polyacrylate, polymaleate and co-polymer of acrylate and maleate The above-described inorganic builders are most preferably sodium salts and potassium salt of carbonic acid, heavy carbonic acid, and silicic acid, alminosilicates such as zeolite, and phosphates such as pyrophosphates and tripolyphosphates . The application of the above-mentioned detergent composition is not especially determined. Examples of the application include fabric detergent, body shampoo, facial cleanser, hair shampoo, oral cavity cleaning agent, dish cleaning agent, auto dish washer detergent contact lens cleaning agent, hard surface washing agent, and cloth cleaning agent. The formulation disclosed in International Publication No. 99/06071 pamphlet, International Publication No . 00/04138 pamphlet, International Publication No . 96/34935 pamphlet and International Publication No. 04/58961 pamphlet and the like may be used as the specific formulation, for example.

The above-described composition is also preferably a composition for degrading an insoluble substrate such as cellulose and protein, or a composition for bleaching pulp. With such a composition, it becomes possible to exhibit superior stability to surfactants, especially to anionic surfactants and to stably supply a high degrading performance and bleaching performance by the synergistic effect of a protein and a surfactant .

The present invention is also a protein modifier, wherein the protein modifier is a polymer of a polyvalent carboxylic acid having three or more carboxyl groups and a connecting agent connecting the carboxyl groups, the protein modifier having at least one carboxylic anhydride group in the molecule. It is preferably a protein modifier, wherein the protein modifier is a polymer of a polyvalent carboxylic acid having three or more carboxyl groups and polyvalent amine and/or polyhydric alcohol, the protein modifier having at least one carboxylic anhydride group in the molecule. More preferably, it is a protein modifier, wherein the protein modifier is a polymer of a carboxylic polyanhydride and a polyvalent amine and/or polyhydlic alcohol, the protein modifier having at least one carboxylic anhydride group in the molecule.

With such a protein modifier, the decrease of activity of protein can be suppressed sufficiently and it becomes possible to realize a high stabilization of protein, and by having at least one carboxylic anhydride group in the molecule, the present invention is advantageous because the modifier can be simply attached to protein using the carboxylic anhydride without using an additional reagent such as a condensing agent. The above-described protein modifier is a modifier with large diversity and capability in design because it can be made by combining freely a component of the polyvalent carboxylic acid and a component of the connecting agent connecting the carboxyl groups. Further, molecular weight can be controlled easily from an oligomer of low molecular weight to a polymer of high molecular weight by changing the ratio of both components.

The polyvalent carboxylic acid in the protein modifier of the above-described polymer is not specially limited as long as it is a polyvalent carboxylic acid having three or more carboxyl groups. Further, a part or all of the carboxyl group may have a carboxylic anhydride structure (and a carboxylic imide structure) or a halogenated structure in the molecule. Specifically, examples include pyromellitic acid, trimellitic acid, hemimellitic acid, trimesic acid, benzenepentacarboxylic acid, mellitic acid, biphenyl tetracarboxylic acid, oxydiphthalic acid, naphthalene tetracarboxylic acid, benzophenone tetracarboxylic acid, benzophenone tricarboxylic acid, perylene tetracarboxylic acid, diphenylsulfone tetracarboxylic acid, bicyclooctene tetracarboxylic acid, butane tetracarboxylic acid, cyclopentane tetracarboxylic acid, cyclohexane tetracarboxylic acid, tetrahydrofuran tetracarboxylic acid, ethylenediamine tetraacetic acid, and diethylenetriamine pentaacetic acid, and so on. Further, in addition to the acid -type compounds of the polyvalent carboxylic acid, derivatives in which a part or all of the carboxyl groups from one or more carboxylic anhydride structure and so forth can be used. In respect of reactivity and safety, it is preferable to use anhydrides. An embodiment in which the above-described polyvalent carboxylic acid is polyvalent anhydride is one of the preferred embodiments in the present invention. Here, the polyvalent anhydride means a carboxylic anhydride which has two or more carboxylic anhydride groups in the molecule, and referred as polyanhydride or carboxylic polyanhydride hereafter .

Among the above-described polyvalent carboxylic acids, polyvalent carboxylic acids comprising 4 or more and 6 or less carboxyl groups, comprising a benzene ring, and having high water-solubility are preferable. Specifically, pyromellitic acid, diphenylsulfontetracarboxylic acid, mellitic acid, butane tetracarboxylic acid, ethylenediamine tetraacetic acid, and diethylenetriamine pentaacetic acid are preferable, and pyromellitic dianhydride, diphenylsulfonetetracarboxylic dianhydride, mellitic trianhydride, butanetetracarboxylic dianhydride, ethylenediamine tetraacetic dianhydride, and diethylenetriaminepentaacetic dianhydride are more preferable .

Among these, an embodiment in which the above-described polyvalent anhydride (polyanhydride) is at least one compound selected from the group consisting of pyromellitic dianhydride, butanetetracarboxylic dianhydride, ethylenediamine tetraacetic dianhydride, and diethylenetriamine pentaacetic dianhydride, is one of the preferred embodiments in the present invention.

Moreover, one kind or two kinds or more of the above-described polyvalent carboxylic acid can be used.

The above-described connecting agent connecting carboxyl groups is a compound having two or more functional groups which can react with -the carboxyl group (or an acid anhydride, an acid halide group, an amide group, an ester group) and connecting the above-described polyvalent carboxylic acid. Moreover, the above-described connecting means a connection between molecules of the polyvalent carboxylic acid and not a connection within a molecule. Specifically, it is a compound having two or more reactive functional groups such as primary and secondary amino groups, a hydroxyl group, an epoxy group, a thiol group, and an isocyanate group. Preferably, it is a compound having two or more amino and/or hydroxyl groups, and examples include polyvalent amine, polyhydric alcohol, amino alcohol and polyhydric phenol. More preferably, it is a polyvalent amine or amino alcohol having at least one amino group of high reactivity.

Moreover, one kind or two kinds or more of the above-described connecting agents connecting carboxyl groups can be used.

Examples of the above-described polyvalent amine include aliphatic polyvalent amine such as methylene dimaine, ethylene diamine, propane diamine, butane diamine, hexamethylene diamine, diamino dodecane, diethyletriamine, bis-hexamethylene triamine, tris (2-aminoethyl) amine, lysine, piperazine, and 1, 4-bis (3-aminopropyl) piperazine; aromatic polyvalent amine such as phenylene diamine, benzidine, diaminostilbene, tolidine, triamino benzene, diaminodiphenyl ether, diamino benzoic acid, diaminobenzene sulfonic acid, diaminobenzene disulfonic acid, and chlorophenylenediamine; and heterocyclic compounds such as pyrazine, pyrimidine, imidazole, diaminopyrimidine, diamino hydroxypyrimidine, triazine, melamine, and diamino triazine. Among the above-described polyvalent amines, ethylene diamine, butane diamine, lysine, 1, 4-bis (3-aminopropyl) piperazine¹, diamino benzoic acid, and diaminobenzene sulfonic acid are preferable. Examples of the above-described polyhydric alcohol and amino alcohol include polyhydric alcohol such as methylene glycol, ethylene glycol, propylene glycol, propanediol, tetramethylene glycol, hexamethylene glycol, diethylene glycol, glycerol, and cyclohexanediol; polymers comprising an alcohol group such as polyethylene glycol and polypropylene glycol; monosaccharides and dissacharides such as glucose, xylose, galactose, fructose, sucrose, trehalose, and sorbitol; alcohol comprising a carboxyl group such as tartaric acid; alcohols comprising an amino group such as triethanolamine, diethanolamine, monoethanolamine, hydroxyethylethylene diamine, threonine, serine, tyrosine and 1, 4-bis (2-hydroxyethyl) piperazine; and so-called good buffers such as N, N-bis (2-hydroxyethyl) -2-aminoethane sulfonic acid. Among them, (poly) ethylene glycol and alcohol comprising an amino group are preferable, and specifically ethylene glycol, diethylene glycol, polyethylene glycol having average molecular weight of 200 to 10000, triethanolamine, diethanolamine, monoethanolamine, hydroxyethylethylene diamine, and 1, 4-bis (2-hydroxyethyl) piperazine are preferable .

Example of the above-described polyvalent phenol include dihydroxybenzene such as hydroquinone and catechol; trihydroxybenzene such as pyrogallol and phloroglucinol; and hydroxyl compounds comprising heterocyclic ring structure such as cyanuric acid.

The above-described polyvalent carboxylic acid and a connecting agent connecting carboxylic groups may be modified with various functional groups such as an alkyl group and an alkylether group and compounds. By the modification, various functional groups can be added to the protein modifier, and furthermore, it becomes possible to add a new function to the modified protein. In the case of adding the compound, the compound is not limited as long as it reacts with at least one compound selected from the group consisting of the above-described polyvalent carboxylic acid and the connecting agents connecting carboxyl groups . However, examples are compounds having a functional group with high reactivity such as epoxide, aldehyde, isocyanate, and halogen. Moreover, at the same time, low molecular weight compounds and polymers having a functional group such as an anionic group, a cationic group, a hydrophilic group, a hydrophobic group, and an affinity group are preferable. Specifically, examples are glycidyl compounds such as (poly) ethyleneglycol (alkyl) glycidyl ether, sulfonated (poly) ethyleneglycol (alkyl) glycidyl ether, (poly) glycerolglycidyl ether, (poly) sorbitolglycidyl ether, glycidylalkyl ether, glycidylphenyl ether, and glycidyl trimethylammonium chloride; and halogen compounds such as

2-haloethyltrimethyl ammonium halide, 4-halobuthyltrimethyl ammonium halide, and 3-halopropane sulfonic acid. Furthermore, a compound comprising a polyethyleneglycol chain is preferable among these and can be introduced in the protein modifier by adding the above-described glycidyl ether and so forth to a composition of the connecting agent such as polyvalent amine and polyhydric alcohol.

The above-described protein modifier, which is the above-described polymer, is a polymer in which the above-described polyvalent carboxylic acid and the above-described connecting agent are polymerized consecutively. The carboxyl group in the above-described polyvalent carboxylic acid reacts with the functional group in the connecting agent and forms a main chain. The main chain structure of the polymer to be produced is the same without depending on the form (including acid type, acid anhydride type, and acid halide type) of the carboxyl group in the polyvalent carboxylic acid compound. That is, at least two carboxyl groups of the three or more carboxyl groups existing in the above-described polyvalent carboxylic acid form an amide bond, an imide bond, or an ester bond with the connecting agent and constitute a main chain structure of the polymer. The carboxyl group which is not used in the formation of the main chain structure may be any form such as an acid anhydride group and an acid halide group.

The existence of this carboxyl group which is not used in the formation of the main chain structure is important to improve various characteristics such as improvement of stability, improvement of solubility and improvement of affinity. Specific examples of the polymer include polyester and polyamide. However, a polymer with high water-solubility is preferable and a polymer which uniformly dissolves into a aqueous solution is further preferable. Specifically, a polymer in which the main chain has amide bonds and the side chain has carboxyl groups (polyamic acid) , and a polymer having ester bonds are preferable. More preferably, it is a polymer having amide bonds of high hydrolysis stability in the main chain of the polymer. The average molecular weight of the polymer is preferably 300 to 500000, more preferably 500 to 50000, most preferably 750 to 10000. The average molecular weight of a polymer can be determined using the GPC (gel permeation chromatography) .

The above-described protein modifier has the merit that the affinity with protein is high compared to a conventional modifier consisting of a polymer of vinyl monomers and a modifier such as polyethylene glycol because the above-described protein modifier is the polymer having an amide bond, an ester bond, and so forth as the main chain. Further, it also has merits that there are plenty of monomers which can be used, that the molecular weight is controlled easily, and that it can be set from an oligomer to a polymer of high molecular weight depending on the purpose.

The synthesizing method of the protein modifier, which is the above-described polymer, is not especially limited. However, depending on the form of the-polyvalent carboxylic acid to be used as a raw material, it is categorized as follows: (1) a method using anhydrides of the polyvalent carboxylic acid, (2) a method using acid halides of the polyvalent carboxylic acid, and (3) a method using the polyvalent carboxylic acid of an acid type. In the respects of easiness and safety of the reaction, the above-described method (1) using acid anhydrides is preferable.

In the above-described method (1), the polyvalent carboxylic acid preferably has two or more acid anhydride groups (such a compound is called "polyanhydride (or polyvalent anhydride)") . When two or more acid anhydride groups exists in the polyvalent carboxylic acid, the addition polymerization of the acid anhydrides with the above-described connecting agent is carried out rapidly and consecutively and produces a polymer forming amide bonds and ester bonds (at the same time, a carboxyl group is formed) . In the case that there are not two or more acid anhydride groups, the condensation polymerization of a carboxyl group and the connecting agent is carried out by a reaction such as dehydration condensation at high temperature. Also, in the above-described method (2), the polyvalent carboxylic acid preferably has two or more acid halide groups. In the above-described method (3), polymerization of a carboxyl group and the connecting agent is performed by a reaction such as dehydration condensation at high temperature. Examples of the polyanhydrides of the polyvalent carboxylic acid used in the above-described method (1) include polymerizable carboxylic anhydrides such as pyromellitic dianhydride, mellitic trianhydride, biphenyltetracarboxylic dianhydride, oxydiphthalic dianhydride, naphthalene tetracarboxylic dianhydride, benzophenone tetracarboxylic dianhydride, perylenetetracarboxylic dianhydride, diphenylsulfone tetracarboxylic dianhydride, bicyclooctene tetracarboxylic dianhydride, butanetetracarboxylic dianhydride, cyclopentane tetracarboxylic dianhydride, cyclohexane tetracarboxylic dianhydride, tetrahydrofuran tetracarboxylic dianhydride, ethylenediamine tetraacetic dianhydride, diethylenetriamine pentaacetic dianhydride. Among these, as described above, pyromellitic dianhydride, butanetetracarboxylic dianhydride, ethylenediamine tetraacetic dianhydride, and diethylenetriamine pentaacetic dianhydride are especially preferable.

In the above-described method ( 1 ) , the molar ratio of the total amount of the acid anhydride group / total amount of the functional group of the connecting agent connecting carboxyl groups is preferably more than 1.0 and more preferably, 1.05 to 5.0, most preferably 1.1 to 3.0. As the above-described molar ratio becomes closer to 1.0, the molecular weight of the polymer increases, and as the molar ratio becomes higher, the molecular weight decreases . Further, by making the molar ratio higher than 1.0, an acid anhydride group can remain at the end of the polymer and is used preferably for attachment with protein. Moreover, in such a manner, the protein modifier of the present invention having at least one carboxylic anhydride group in the molecule can be preferably obtained.

If the protein modifier, which is the above-described polymer, contains no carboxylic anhydride group, an acid anhydride group can be generated between some carboxyl groups by a method using acetic anhydride and the like, or by thermal dehydration. Furthermore, the part of the acid anhydride group remaining at the end of the polymer may be denatured with a compound having one reactive functional group. Examples of such a compound include monovalent amine and monohydric alcohol. By denaturing the end of the polymer with the above-described compound, various functional groups can be introduced at the end of the modifier, and by the synergistic effect with the protein, a further higher modification effect can be obtained.

Here, a reaction equation of a modifier synthesized from pyromellitic dianhydride and ethylenediamine is shown as follows as one example of the modifier with the above-described method (1) .

In the above-described method (1) and the case of using the polyanhydride of the polyvalent carboxylic acid, the condition of the reaction with the connecting agent connecting carboxyl groups is not especially limited. However, it is preferable to add a solution of the connecting agent such as polyvalent amine to a solution of polyanhydrides while stirring. The reaction temperature is preferably in the range of 0°C to 120°C and more preferably, 20°Cto70°C. A solvent and a catalyst may be used in the reaction. The solvent is not especially limited as long as it has low reactivity and amide, sulfoxide, ketone, ether, and so forth can be used as solvent preferably.

Specifically, N,N-dimethylformamide, N-methylpyrrolidone, N, N-dimethylacetaitiide, dimethylsulfoxide, tetrahydrofuran, acetone, methylethylketone, cyclohexanon, ethyleneglycol dimethylether, dioxane, and diethylether are preferable. Among them, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, and ethyleneglycol dimethylether are further preferable. After the reaction, by distilling the solvent, the concentration of the polymer may be increased and collected as a solid. Further, the modifier having a narrow distribution of molecular weight may be made with a method such as crystallization, column chromatography, and dialysis. An example of the catalyst is tertiary amine, and specifically triethylamine, pyridine, and 4-dimethylaminopyridine are preferable. It is preferable that the amount of water existing in the raw material, the solvent, and the reaction product is low, specifically 1% by weight or less is preferable, and 0.1% by weight or less is further preferable. After the reaction, in order to prevent hydrolysis of the remaining carboxylic anhydride group, it is preferable that the reaction product is sealed after nitrogen purge, and then stored away from moisture at low temperature. Moreover, in the synthesis of the above-described modifier, the above-described one kind or two kinds or more of other synthesis methods can be used together.

The attachment of the protein modifier consisting of the above-mentioned polymer to the protein is easily performed with the acid anhydride group existing in the modifier. That is, the amino group existing in the protein (derived from the N terminal and lysine residue) is mainly reacted with the acid anhydride group to form an amide bond. As a result, the modifier is attached to the protein. Alternatively, the acid anhydride group may react with the thiol group (derived from cysteine residue) .

Although the above-mentioned acid anhydride group may be produced after the synthesis of the above-described polymer, it is preferable to use the remaining acid anhydride group. As described above, by controlling the molar ratio of the total amount of the acid anhydride groups / the total amount of the functional groups of the connecting agent, the acid anhydrides can remain at the end. The above-mentioned acid anhydride group existing in the modifier can be quantitated using various methods. Examples of the methods include a method utilizing NMR, and a method of titration using amine. The prompt reduction in the amino group of the protein (increase in modification degree) by mixing the protein with the modifier proves that the acid anhydride group exists in the modifier. The present invention is also a protein modifier, the protein modifier being a polyvalent carboxylic acid having three or more carboxyl groups, and having at least one carboxylic anhydride group in the molecule, wherein at least one carboxyl group in the polyvalent carboxylic acid is connected to a monovalent amine by an amide bond or to monohydric alcohol by an ester bond.

In such the protein modifier, because the carboxyl group in the polyvalent carboxylic acid is connected to monovalent amine or monohydric alcohol, it is possible to give the protein various capabilities such as high stability, activity, and affinity resulting from the functional group in the amine or alcohol. In addition to this, it is possible to modify the protein easily because the modifier has the carboxylic anhydride group in the molecule. Hereby, compared to a modifier in which the carboxyl group is not connected to amine or alcohol

(that is, the carboxyl group remains as it is) , a greater effect can be obtained. If the acid anhydride group is partly denatured, cross-linking between the proteins can be reduced. The cross-linking between the proteins often causes a problem in modification of proteins in use for pharmaceuticals. The attachment of the carboxyl group and the monovalent amine is performed by an amide bond, and further, the attachment of the carboxyl group and the monohydride alcohol is preformed by an ester bond. The specific embodiment and so forth of the above-described polyvalent carboxylic acid having three or more carboxyl groups and the like is described above.

The above-mentioned monovalent amine compound and the above-mentioned monovalent alcohol compound are not especially limited. Preferred examples thereof include compounds containing a functional group such as an alkyl group, a sulfonic acid group, a sulfuric acid group, a carboxyl group, a tertiary amino group, a quaternary ammonium group, a (poly) oxyethylene group, and a (poly) oxypropylene group. Specific examples thereof include alkylamines and alkyl alcohols containing 10 to 18 carbon atoms; nonionic surfactants such as (poly) oxyethylene alkyl ether and (poly) oxypropylene alkyl ether; choline chloride, 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid (and salts thereof) , isethionic acid (and salts thereof) , N- (2-hydroxyethyl) ethylenediamine triacetic acid, taurine and amino acids; tryptophane, phenylalanine, asparagines, histidine and so forth The synthesis method of the above-described protein modifier is not especially limited. However, depending on the form of the polyvalent carboxylic acid to be used as a raw material, examples of the method are as follows: (1) a method using polyanhydrides of the polyvalent carboxylic acid and carrying out an addition reaction with the monovalent amine or the monohydride alcohol and (2) a method using the polyvalent carboxylic acid of an acid type (acid polyvalent carboxylic acid) and carrying out dehydration attachment with the monovalent amine or the monohydride alcohol. Among these, in the respects of easiness and safety of the reaction, method (1) is preferable.

Examples of the method of introducing the carboxylic anhydride group existing in the above-described protein modifier are (1) a method using polyanhydrides of the polyvalent carboxylic acid as a raw material and leaving a part of the anhydride groups and (2) a method of introducing an anhydride group by dehydration of the carboxyl group or by reaction with anhydrides such as acetic anhydride. Among these, in the respect of simplicity, method (1) is preferable. A reaction equation of pyromellitic dianhydride and primary amine is shown as follows as an example of the synthesis of the above-described protein modifier with method (1) . [Formula 2]

Here, in the case of synthesizing the above-described protein modifier by a reaction of the polyanhydride of the polyvalent carboxylic acid and the monovalent amine or the monohydric alcohol, the yield of the modifier is controlled by changing the molar ratio of "the total amount of the anhydrides of a raw material / the total amount of the monovalent amine or the monohydride alcohol". That is, the higher the molar ratio is, the lower the ratio of the reacted monovalent amine or the monohydride alcohol is, and the more the unreacted polyanhydride is. The lower the molar ratio is, higher the ratio of the reacted monovalent amine or the monohydride alcohol, however, the remaining amount of the acid anhydrides which is necessary for attachment with protein decreases. The molar ratio (the total amount of the anhydride group of the raw material / the total amount of the monovalent amine or the monohydride alcohol) is preferably 1.1 to 10 and further preferably, 1.3 to 5. After the reaction, the above-described modifier can be purified and used by removing the unreacted raw material and the compound which is reacted excessively.

The present invention is also a modified protein chemically modified with the above-described protein modifier . The reaction of the above-described protein modifier and the protein can be performed in various conditions described above. The modified protein obtained in such a manner preferably has a half-life of activity of 50 minutes or more at 4O⁰C in a 1% by weight LAS solution. Because the composition comprising the modified protein having such a half-life of activity has high stability and can sufficiently exhibit its functions without the activity decrease. As a result, for example in the case of using it for a detergent, a high cleansing performance can be kept for a long period of time and it can have high stability in storage. The half-life of activity is more preferably 100 minutes or more and further preferably 200 minutes or more.

The above-described modified protein is not especially limited as long as it is the protein chemically modified with the above-described protein modifier, and examples are a modified protein made by modifying the protein such as the above-described protein for detergent, the protein for organic synthesis, the protein for the food industry, the protein for processing fiber and cloth, protein for processing pulp, protein for decomposing starch, protein for producing bio-fuel, the protein for cosmetics, the protein for pharmaceuticals, the protein for diagnostic medicines, and the protein for a sensor. Moreover, among these, it is preferable to use an enzyme and an antibody. Specifically, the above-described enzymes can be used as the protein to be modified with the above-described modifiers. Preferable enzymes are also same as those described above .

The above-described modified protein has the possibility of improving all characteristics such as stability (stability against a surfactant, an oxidizing agent, a chelating agent, an alkaline, an acid, a protease, an organic solvent, and so forth) , substrate selectivity, product selectivity, substrate affinity, cleansing performance, half-life in the blood, allergenicity, irritating effect and trapping performance of metal. However, the stability, especially stability to surfactants and among these stability to anionic surfactants, is improved preferably. Examples of anionic surfactants include the above-described compounds such as alkylbenzene sulfonate and alkylether sulfate. Further, improvement of the activity of protein and improvement of cleansing performance are observed. These effects are considered to be due to the protecting effect resulting from a polymer structure of the above-described modifier, the protecting effect of protein by many carboxyl groups (including carboxylate anion) , the effect of the change of charge balance, and the effect of improving penetration into a fiber . Therefore, the protein to be modified is preferably an enzyme for detergent. Moreover, for the modification of the above-described modified protein, the above-described one kind or two kinds or more of other modifiers and methods of modification can be used together.

The above-described enzyme is preferably used as the above-described enzyme for a detergent. The modified protease modified by the above-described modifier exhibits especially superior stability to LAS (linear alkylbenzene sulfonate) . In the case of measuring the half-life of activity at 4O⁰C in the 1% by weight LAS solution as an evaluation standard of the stability to LAS, protease having the above-described half-life of 50 minutes or more can be obtained. The half-life of protease is preferably 100 minutes or more and more preferably 200 minutes or more. Purafect Prime is known to be protease having high LAS stability, however, the half-life of Purafect Prime is generally 50 minutes or less and therefore protease having a half-life of 50 minutes or more has been demanded. Protease having such superior LAS stability can stably exhibit high cleansing performance in a detergent comprising a surfactant such as LAS .

Moreover, the measurement method of the above-described half-life of activity is performed as follows, unless otherwise specified. The "protein activity" used herein means an enzyme activity if the protein is an enzyme. Measurement Method of Activity Half-life>

Protein (50 to 1000 ppm as solid) , sodium linear benzenesulfonate of 1% by weight as a surfactant, and a solution of pH 8.5 comprising boric acid of 0.1 M are prepared. The protein activity in the solution is measured and is treated as the initial activity (Ai) (t = 0) . Then, the above-described solution is incubated in a water bath at 40°C and the activity measurement is performed by sampling at certain intervals (Activity "At" at time "t") . Using At at which the remaining activity (= At/Ai x 100) is 10 to 80% and t, the half-life of activity T (minutes) is calculated from Equation (1) below. T = t/log₀.s(At/Ai) Equation (1)

Moreover, in the evaluation in which the surfactant and temperature of heating are varied, the half-life of activity is obtained in the same manner as the above-described method except changing the amount and types of the surfactant and temperature for heating.

Then, the present invention is a composition comprising the above-described modified protein. Such a composition is not especially limited as long as the above-described modified protein is included. However, it is preferable to include the above-described surfactant. Further, the above-described detergent builder, additives, solvent and so forth can be included.

The above-described composition is preferably a detergent composition. Such a detergent composition has superior stability to surfactants and includes protein having high cleansing performance (preferably, an enzyme), and hereby, high cleansing performance can be stably exhibited in the detergent comprising surfactants.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is mentioned in more detail below. However, the present invention is not limited to these Examples . The term "%" represents "% by weight" unless otherwise specified.

Descriptions of compounds in the following Tables and the like are as follows.

PMDA: pyromelletic dianhydride

DPSDA: 3, 3' , 4, 4' -diphenyl sulfone tetracarboxylic dianhydride EDTADA: ethylenediaminetetraacetic dianhydride

BTCDA: 1, 2, 3, 4-butanetetracarboxylic dianhydride DTPADA: diethylenetriaminepentaacetic dianhydride BPTCDA: 3, 3' , 4, 4' -biphenyltetracarboxylic dianhydride DPSDA: 3, 3' , 4, 4' -diphenylsulfone tetracarboxylic dianhydride BCODA: bicyclo [2, 2, 2] -octo-7-ene-2, 3, 5, 6-tetracarboxylic dianhydride ODPAN: 4, 4' -oxydiphthalic anhydride

<Connecting agent> EDA: ethylenediamine

HEEDA: N- (2-hydroxyethyl) ethylenediamine

PEG200: polyethylene glycol, average molecular weight of 200

TEA: triethanolamine

BHEP: 1, 4-bis (2-hydroxyethyl) piperazine DETA: diethylenetriamine

BES: N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid

Tween20®: polyoxyethylene sorbitan monolaurate

(Sigma-Aldrich)

DMF: N,N-dimethyl formamide DMSO: dimethyl sulfoxide NMP: N-methyl pyrrolidone

<Denaturing agent (Monohydric alcohol) > HEPES: 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid Brij35@: Polyoxyethylene (23) lauryl ether Brij76®: Polyoxyethylene (10) stearyl ether HEEDT: N- (2-hydroxyethyl) ethylenediamine triacetic acid PEGME350: Polyethylene glycol monomethyl ether, average molecular weight of 350

PEGME3000: Polyethylene glycol monomethyl ether, average molecular weight of 3000

Example 1

An enzyme solution comprising protease (Savinase® liquid, manufactured by Novozymes, obtained from Sigma-Aldrich, Inc., product number P3111) 0.5 ml was placed in a plastic tube of 1.7 ml and adjusted to pH 8.5 with 5% NaOH solution. Then, a solution of the modifier (solution of 0.5M pyromellitic dianhydride (PMDA) in N, N-dimethylformamide (DMF)) 0.01 ml was added and stirred well, and the final volume was adjusted to 1 ml by adding water. This enzyme solution was kept still at room temperature for three hours and a modified enzyme solution was obtained.

The modification degree of the modified enzyme was measured by the above-described method and the result was 81%. Then, the modified enzyme solution 0.01 ml was mixed into a surfactant solution (0.1 M boric acid buffer solution (pH 8.5) containing 1% LAS which is one of anionic surfactant; sodium dodecylbenzenesulfonate as the LAS was used here) 0.49 ml, and a detergent composition was prepared. Using this composition 0.005 ml, the activity of protease was measured with the above-described method (the 50 mM boric acid solution of pH 9 was used as a buffer solution of the activity measurement) , and the half-life of activity at 40°C in the 1% LAS was measured. For comparison, the same experiment was performed on a unmodified enzyme solution (the preparation is in Comparative Example 1), and the activity of protease and the half-life of activity were measured. As a result, the relative activity of the modified enzyme (the activity of the modified enzyme solution to the activity of the unmodified enzyme solution) was 121%, and it was found that the activity was improved by the modification treatment. Further, the half-life of the modified enzyme was 71 minutes and improved largely compared to the unmodified enzyme (Comparative Example 1) of 19 minutes. It was found that the stability to anionic surfactants (LAS) was improved by the modification treatment. Further, the modified enzyme solution was analyzed by SDS-PAGE (polyacrylamide gel electrophoresis) and the difference in molecular weight was compared with the unmodified enzyme. As a result, it was found that the molecular weight of the enzyme increased by about 1000, and it was considered that PMDA of the modifier was chemically attached to the enzyme.

Example 2

PMDA 480 mg (2.2 mmol) was dissolved in DMF 4 ml and treated as a component (1) (a component of the polyvalent carboxylic acid). Then, ethylenediamine (EDA) 88 mg (1.47 mmol) was dissolved into DMF 1 ml and treated as a component (2) (a component of the connecting agent) . While stirring, the component (2) was added to the component (1) at room temperature over about 5 minutes, matured at room temperature for 3 hours, and a modifier 1 (PMDA / EDA polymer, the molar ratio of the functional group (= the total amount of the acid anhydride group/the total amount of the reactive functional group of the connecting agent) was 1.5) was synthesized. Then, the modification of Savinase was performed in the same manner as in Example 1 except using 0.05 ml (5 vol% to the final volume 1 ml) of the synthesized modifier 1 as the modifier solution. Moreover, the modifier was separately added while adjusting with the 5% NaOH solution so that the pH of the enzyme solution did not become lower than 6 when the modifier was added. The modification degree was 79% . Further, when the modified enzyme was analyzed by SDS-PAGE, the molecular weight increased about 3000 on average (there is a distribution of the molecular weight) , and it was considered that PMDA / EDA polymer of the modifier was attached to the enzyme.

Then, when the relative activity was measured in the same manner as in Example 1, the relative activity was 102% . Further, the half-life of activity at 40°C in the 1% LAS was 94 minutes, and it was found that the stabilization effects of the modification by the polymer is higher than that of monomeric PMDA modification. From the result in Comparative Example 2, it was found that the enzyme was attached to the modifier by the residual acid anhydride groups. It was also found that the modifier can exhibit the stabilization effects by binding to the protein.

Examples 3 to 9

Using various carboxylic polyanhydrides (component (1) ) and connecting agents (component (2) )with various solvents and reaction conditions as shown in Table 1, modifiers of polymers 2 to 8 were synthesized in the same manner as in Example 2. Then, using the amount of the modifiers 2 to 8 shown in Table 2, the modification treatment of Saviηase was performed and the modified enzyme solutions were obtained. In the same manner as in Example 1, the modification degree, the relative activity, and the half-life of activity at 40°C in the 1% LAS were measured. The result is shown in Table 2. It was found that all of the modified enzymes have a high stability to LAS.

[Table 1] Synthesis of Modifier (Polymer of Polyvalent carboxylic acid and Connecting agent) J-.

O

* "Molar ratio of functional group = "the total mole of the acid anhydride group of Component (1)" / "the total mole of the primary and secondary amino group and alcohol group of Component (2)"

H-3

(D σ I—¹

(ϋ to

O α H-

Hi

H-

O

0⁾

(Ϊ

H-

O

Hi

CO O⁾ < J-. H- cυ en

♦volume % to the final volume 1 ml

Example 10

The modified Savinase prepared in Examples 1 to 9 was measured for the half-life of activity at 55°C in the same manner as in Example 1, except that AES (alkyl ether sulfate as one of anionic surfactants; polyoxyethylene dodecyl ether sodium sulfate (the number of EO chain=2) was used here) was used instead of the LAS. Similarly, the modified Savinase was measured for the half life of activity at 60⁰C using AE (polyoxyethylene alkyl ether as one of nonionic surfactants; Brij 35® produced by ICI America was used here) instead of the LAS. The results in Table 2 show that the modified Savinase has high stabilization effects particularly on the anionic surfactant.

Example 11

The modified Savinase prepared in Example 1 to 9 was measured for the half-life of activity in commercially available liquid detergents in the same manner as in Example 1 except using undiluted detergents instead of the 1% LAS solution. In this Example, Tide Cold Water® (P & G Corp., hereinafter referred to as "detergent A") and Wisk® (Unilever Corp., hereinafter referred to as "detergent B") were used as commercially available liquid detergents containing anionic surfactants, and Attack® (Kao Corp., hereinafter referred to as "detergent C") which was a nonionic liquid not containing an anionic surfactant was used. The enzymes contained in the detergents were previously inactivated by thermal treatment and then the detergents were subjected to the experiment. The existence of the anionic surfactant in the detergents was determined by the following test method using a cation polymer (Both of the detergent A and the detergent B contained anionic surfactants, no anionic surfactant was detected in the detergent C) . The half life of activity was determined based on the residual activity after heating for 10 days at 40°C. The results in Table 2 show that the modified Savinase has stabilization effects also on the commercially available detergents, and that particularly in the detergents containing anionic surfactants, the modified Savinase has high effects. "Detection method of anionic surfactant" DADMAC (Diallyl dimethyl ammonium chloride) polymer (molecular weight of 200000, Aldrich) was added to a commercially available detergent and sufficiently mixed with each other. Generation of deposits (adducts of the anionic surfactant with the cation polymer) means that the detergent contains an anionic surfactant (no deposits were generated in nonionic surfactants) . The deposits were recovered and the recovered deposits were dried and then measured for weight. Thereby, the content of the anionic surfactant can be estimated. The compound can be indentified based on NMR or IR spectrum of the deposits.'

Examples 12 to 21

In the same manner as in Example 1, the modification of Savinase was performed using various anhydrides of polyvalent carboxylic acid having a benzene ring (various concentrations and amounts) as shown in Table 3. Moreover, the enzyme modification reaction was performed at 0°C in Example 14 and 40°C in Example 15. The modified Savinase was measured for the modification degree, the relative activity, and the half life of activity in the 1% LAS in the same manner as in Example 1. Similarly, the modified Savinase was measured for the half life of activity at 53°C in the detergent A (thermally treated) which was 2-fold diluted with water. The results in Table 3 show that the activity and the stability were improved as compared with those in the unmodified enzyme in Comparative Example 1. Also, it was shown that stabilization effects higher than those in the modification with the polyvalent carboxylic acids not containing a benzene ring (Comparative Examples 3 to 8) could be obtained. ►-3 σ I— ^■ (D

U)

O

Q. H- Hi H- n

0⁾ rt

H- O

O

Hi

CΛ 0⁾

< 4-»

H- (D cn (D

K)

* The modification reaction was performed at 0°C in Example 14 and 40°C in Example 15.

* Maleic anhydride homopolymer: Average molecular weight 5000 (Wako) , Concentration of the modifier solution is shown by monomer concentration.

* Maleic anhydride copolymer: a polymer of maleic anhydride and methylvinylether of 1 : 1, Average molecular weight 20000

(Scientific Polymer Products, inc.).

Example 22

In the same manner as in Example 2, modifiers 9 to 38 of polymers were synthesized in various conditions using various carboxylic polyanhydrides (component (1) ) and connecting agents (component (2)) shown in Table 1.

Examples 23 to 41

Then, using modifiers 9 to 22 and 29 to 32 under condition as shown in Table 4, the modification treatment of Savinase® was performed in the same manner as in Example 2. The modified enzymes were measured for modification degree, relative activity, and half-life of activity in the same manner as in Example 12. The results in Table 4 show that the stability in the enzymes modified with polymers 9 to 22 and 29 to 32 was improved. It was found that the stabilization effects were higher than that in the modification with the monomeric carboxylic anhydrides (Examples 12 to 21) .

[Table 4] Modification of Savinase (3)

Example 42

Apart of the terminal acid anhydride group was denatured with monovalent amine or monohydric alcohol (referred to as

"denaturing agent") to synthesize modifiers . That is, various carboxylic anhydrides and connecting agents (some modifiers contained no connecting agent) and denaturing agents (component

(3) ) , each shown in Table 5, were mixed to synthesize modifiers in the same manner as in Example 2. As a result, partially modified modifiers 39 to 51 were obtained. The denaturing agent reacts with the part of the acid anhydride group and forms a bond such as an ester bond and an amide bond, and is located at the end of the modifier.

_ι rt tr CD σ

O ω

O (Jl

< ^1—1

CD ω

M Φ 3 rt rt tr α>

(D ω

B H-

H- CΛ

<τ> O

Hi

O

O Hi H-

Φ α . — _^

H- O

n

H-

CD Hi

I — ' n Φ

O h

!=r O

I — ' PJ

H rt

H-

(D

M

M •< α

Φ

(D rt

C φ α

^y o^h * "Molar ratio of functional group = "the total mole of the acid anhydride group of Component (1)" / "the total mole of the primary and secondary amino group and alcohol group of Component (2) and (3)"

Examples 43 to 52

The modification treatment of Savinase® was performed in the same manner as in Example 2 except that the partially denatured modifiers 39 to 48 were used in the amounts shown in Table 4 instead of using the modifier 1. Then, the modification degree was measured, and the modified enzyme was evaluated as performed in Example 12. The results in Table 4 shows that the partially denatured modifiers also have high stabilization effects and, in some Examples, show effects higher than those in the undenatured modifiers (for example, comparison of

Example 43 with Example 13, and comparison of Example 47 with Example 35) .

Examples 53 to 57 The modification reaction was performed using Purafect L® (Genencor-Kyowa, liquid form) as protease. That is, an original enzyme solution of Purafect L 0.5 ml was placed in a plastic tube and mixed with 0.25 ml of 2M triethanolamine solution (pH 9) . Then, various modifiers were added at each amount shown in Table 6, mixed, and the final volume was made to 1 ml by adding water. This enzyme solution was kept still at room temperature for one hour and the modified enzyme solution was obtained. t-3 (D σ

(D

O α H- Hi H- O 0⁾ rt H- O

<

(D ^ H- cπ O

C ω

O r+ (D

(D

CΛ (D

Example 58

Then, after the modification degree was measured, the prepared modified enzyme solution 0.005 ml was mixed with the 1% LAS solution 0.495 ml used in Example 1 to prepare a composition. This composition was measured for the relative activity and the half-life of activity at 40⁰C in the 1% LAS in the same manner as in Example 1. Furthermore, the relative activity and the half-life at 53°C were measured in the detergent A in the same manner as in Example 12. However, a 10 itiM boric acid (pH 9) solution comprising calcium chloride of 110 ppm was used here as the buffer solution for measuring the activity (this condition was closer to the actual cleansing condition) . Further, for comparison, an enzyme solution was made in which 0.01 ml of DMSO was added instead of the modifier in the same manner as in Example 1 / Comparative Example 1, and the evaluation was performed on this enzyme solution as a unmodified enzyme solution (Comparative Example 9) . It was found that the stability in the LAS and the detergent A was improved also in the case of using Purafect L as the result shown in Table 6. Furthermore, it was found that the activity was improved drastically in the detergent A.

Examples 59 and 60

The modified Bioprase was obtained by performing an experiment in the same manner as in Example 2 except using Bioprase 30L® (Nagase ChemteX Corporation) liquid as protease and a modifier shown in Table 6 as a modifier. After the modification degree was measured, the relative activity and the half-life at 40°C in the 1% LAS, and the relative activity and the half-life at 53°C in the detergent A, were measured in the same manner as in Example 58 (a unmodified enzyme was evaluated at the same time for comparison, Comparative Example 10) . It was found that the stability in the LAS and the detergent A was improved also in the case of using Bioprase 3OL as the result shown in Table 6. Furthermore, it was found that the activity was improved drastically in the detergent A.

Examples 61 to 71

The modified enzyme was obtained by performing an experiment in the same manner as in Example 53 except using Purafect Prime® (Genencor-Kyowa) liquid as protease and the modifier shown in Table 6 as a modifier . After the modification degree was measured, the relative activity and the half-life at 40°C in the 1% LAS, and the relative activity and the half-life at 53°C in the detergent A, were measured in the same manner as in Example 58 (a unmodified enzyme was evaluated at the same time for comparison, Comparative Example 11) . The stability in the LAS and the detergent A was improved also in the case of using Purafect Prime as the result shown in Table 6, and modified enzymes showing very high LAS stability were obtained.

Example 72

Various proteases modified with PMDA were measured for the half-life of activity in the 1% AE at 60°C as performed in Example 10. Then, the half-life of activity was compared with that in the 1% LAS . The results in Table 6 show that the modified protease has high stabilization effects on the LAS, but has no stabilization effects on the AE as a nonionic surfactant. It appears that the modification with PMDA has stabilization effects specifically on the anionic surfactants such as LAS.

Examples 73 to 77

This time, the modification of lipase was performed. The modified enzyme was obtained by performing an experiment in the same manner as in Example 53 except using Lipex® (Novozymes) liquid as lipase and the modifier shown in Table 7 as a modifier. After the modification degree was measured, the relative activity and the half-life at 40°C in the 1% LAS, and the relative activity and the half-life at 53°C in the detergent A, were measured in the same manner as in Example 58 (a unmodified enzyme was evaluated at the same time for comparison, Comparative Example 12) . It was found that the stability in the LAS and the detergent A was improved also in the case of using Lipex as the result shown in Table 7. Moreover, the buffer solution used here was a 10 mM boric acid (pH 9) solution comprising calcium chloride of 110 ppm and DMSO of 5%.

Example 78

Amano Lipase AK® (Amano Enzyme Inc., obtained from Sigma-Aldrich, Inc., powder form) was dissolved at a concentration of 20 mg/ml into the 10 mM boric acid buffer solution of pH 8. A 0.5 M boric acid buffer solution (pH 8.5) of 0.3 ml was added to this lipase solution 0.5 ml, 0.04 ml of the modifier 2 was added, and the solution was kept still at room temperature for one hour. After the modification degree was measured, the relative activity and the half-life at 55°C were measured in the 0.1 M boric acid buffer solution (pH 9, not including surfactants) . The method of the experiment is the same as in Example 73, the buffer solution was used instead of the surfactant solution, and the heating was performed at

55°C (a unmodified enzyme was evaluated at the same time for comparison, Comparative Example 13) . It was found that the activity and the stability of Lipase AK were improved by the modification as the result shown in Table 8. [Table 8] Modification of Amano Lipase AK®

Example 79

The modification is performed in the same manner as in Example 2 except using Esperase® (Novozymes) liquid as protease and modifier 2 as a modifier. After the modification degree was measured, the relative activity and the half-life of activity at 55°C in the 1% AES were measured in the same manner as in Example 10 (a unmodified enzyme was evaluated at the same time for comparison, Comparative Example 14) . The half-life of activity in the solution containing 0.5% LAS and 0.5% AE (used in Example 10) was also measured.

It was found that the stability to surfactants was improved also in the case of using Esperase as the result shown in Table 9.

[Table 9] Modification of Esperase®

Example 80

Subtilisin Carlsberg (Sigma-Aldrich, Inc., P5380) powder 20 mg as protease was dissolved in 8 ml of boric buffer solution (0.25 M, pH 8.5) . Then, 0.8 ml of this solution was taken and 0.03 ml of the modifier 2 was added at room temperature. Furthermore, the total volume was adjusted to 1 ml by adding water and the modified enzyme solution was obtained. After the modification degree was measured, the relative activity and the half-life of activity at 40⁰C in the 1% LAS solution (same as in Example 1), and the relative activity and the half-life of activity at 55°C in the 0.1 M boric acid buffer solution of pH 8.5 (not including surfactants), were measured (a unmodified enzyme was evaluated at the same time for comparison, Comparative Example 15). The result is shown in Table 10.

[Table 10] Modification of Subtilisin Carlsberg

Example 81

The modified or unmodified Savinase solutions prepared in Examples 7 and 8 and Comparative Example 1 were evaluated for stability and activity in various solutions, as performed in Example 58. The solutions used in this Example were 1%LAS (used in the Example 1) , 1%AS (sodium lauryl sulfate) , a mixture of 0.5% LAS and 0.5% AES (used in Example 10) , a mixture of 0.5% LAS and 0.5% AE (used in Example 10), and 0.5% sodium laurate. Here, these solutions were prepared in a 50 mM Tris-HCL buffer solution (pH 9) . The modified or unmodified Savinase solutions were also evaluated for stability in a 50 mM boric acid buffer solution not containing a surfactant (pH 10) . As shown in results in Table 11, stabilization effects attributed to the modification were observed in the various surfactant solutions and the alkaline buffer solutions . Improvement in the activity in the presence of the fatty acid salt was also observed. (D σ I — ' π>

CD

I—¹

C

CD r+

H- O

O

Hi

¹X

O

H-

Hi

Cn ω

CD

<

H-

Q⁾

CΛ

(D

H-

<

CD h( p-

O

C

CΛ

O h--

C rt p-

O

CΛ

e^{d i} Example 82

Calcium chloride was added to the modified Savinase solution prepared in Example 35 so that the final concentration became 0.5%. Then, the half-life of activity at 53°C was measured in the detergent A which was 2-fold diluted in the same manner as in Example 12. The half-life was 110 minutes and it was found that the stability increased more with the addition of calcium ions (56 minutes without the addition, refer to Example 35 in Table 4) .

Example 83

Using the modified Savinase prepared in Example 3 and the unmodified Savinase in Comparative Example 1, the influence of calcium ions to the activity of protease under the existence of a detergent component was examined. Here, using a 5 mM boric acid (pH 9) as a buffer solution for measuring the activity, the activity was measured under the existence of the detergent A of 1500 ppm in the cases where calcium chloride was added/not added. The activity of the modified enzyme in the presence of the calcium ions was about two times that of the ^' unmodified enzyme as the result shown in Table 12. Further, when calcium was not added, high activity of the modified enzyme was maintained while the activity of the unmodified enzyme largely decreased. It was found that the activity at low concentration of calcium was improved by the modification treatment.

[Table 12] Influence of calcium ions to the activity

* Comparative Example 1: The activity of unmodified and with calcium is shown as 100%. Example 84

Using the modified Savinase shown in Table 13, the influence of pH to the activity under the presence of calcium and a detergent component was examined. Here, using a 10 mM boric acid (including calcium chloride of 110 ppm, pH 9) and a 10 mM HEPES (including calcium chloride of 110 ppm, pH 7) as a buffer solution for measuring the activity, the measurement was performed under the existence of the detergent A of 1500 ppm. The result is shown in Table 13. The effect of improving the activity by the modification, with an especially large improvement at pH 7, was observed.

[Table 13] Influence of pH to the activity

* Comparative Example 1: The activity of unmodified and pH 9 is shown as 100%.

Comparative Example 1

An enzyme solution of Savinase was prepared in the same manner as in Example 1 except DMF 0.01 ml was added instead of the modifier solution in Example 1. This was used as the unmodified enzyme solution and treated as a comparison of various evaluation items such as the relative activity and the half-life of activity.

Comparative Example 2

The modifier 0.1 ml was mixed with water 0.1 ml. This mixture was heated at 50⁰C for 1 hour, and residual acid anhydride groups were denatured with water. Then, Savinase was modified with this denatured modifier 0.1 ml in the same manner as in Example 2 and then the modified Savinase was evaluated. The modification degree was 0%; the half-life of activity at 40⁰C in the 1% LAS was 19 minutes . These results were completely the same as in unmodified Savinase.

Comparative Examples 3 to 8 Savinase was modified in the same manner as in Example 1, except that various anhydrides of polycarboxylic acid not containing a benzene ring at various concentrations and various amounts as shown in Table 3. The obtained modified enzymes were used as objects to be compared in activity evaluation and various stabilization evaluations. Comparative Examples 9 to 15

As mentioned above, unmodified enzyme solutions were prepared in completely the same manner as in the preparation of the modified enzyme using various enzymes, except that 0.01 ml of DMF was added instead of the modifier. These unmodified enzyme solutions were used in each Example as an object to be compared.

Example 85 The following composition (detergent D) was prepared using the modified savinase enzyme.

Detergent D

1.5% LAS (linear dodecyl benzene sodium sulfonate)

1.5% Brij35 (Sigma-Aldrich, Inc.) 0.2% Citric acid

0.2% boric acid the pH was adjusted to 9 with NaOH solution

A detergent composition (a composition (I)) inwhichO.l volume% of trimellitic acid modified enzyme prepared in Example 17 was added to the above-described detergent D and a detergent composition (a composition (2) ) in which 1 volume% of the modified enzyme was added to the above-described detergent D were prepared. Furthermore, a composition (a composition (3)) in which 1 volume% was added and was kept at 30°C for 1 week was prepared. A test of cleansing performance was performed using three kinds of detergent compositions as above.

Moreover, the test of cleansing performance was performed as follows. First, ion-exchanged water 70 ml (including 55 pprti calcium chloride) was placed in a 100 ml glass bottle and kept at 25°C. Then, 0.7 ml of the above-described detergent composition ((1) to (3), in separate bottles) were added. Then, a washing step was performed by placing one piece of artificially stained cloth (EMPA116; cotton stained with blood/milk/carbon black, 5 cm x 5 cm, the reflectance has already been measured, fixed into a cylindrical form and used) in the glass bottle and stirring for 20 minutes at 500 rpm using a stirrer. Furthermore, a rinsing step was performed by discarding the cleansing solution, newly adding ion-exchanged water 70 ml (including 55 ppm calcium chloride) , and stirring at 500 rpm for 10 minutes. The _;stained cloth was taken out, and after it dried, the reflectance is measured. From the reflectances, the detergency (%) was calculated using the Kubelka-Munk equation. Both detergent compositions (1) and (2) showed a high initial detergency compared to the case of the unmodified enzyme

(Comparative Example 16) as the result shown in Table 14.

Further, detergent composition (3) stored for one week kept a high detergency, and an improvement in storage stability was observed. [Table 14] Evaluation of Initial detergency and storage Stability of Modified Savinase

* The value in parentheses in COMPOSITION (3) is the maintained detergency (= COMPOSITION (3) / COMPOSITION (2) x 100) .

Example 86

A test of detergency was performed in the same manner as in Example 85, except using the modified Savinase (PMDA / butadiamine modified) prepared in Example 3 as the modified enzyme. The result is shown in Table 14, and the improvement in the initial detergency and the improvement in stability (storage stability) were observed also in the present modified enzyme. Comparative Example 16

A test of detergency was performed in the same manner as in Example 85, except using the unmodified Savinase prepared in Comparative Example 1 instead of the modified enzyme. The result is shown in Table 14.

Example 87

2500 ppm of the modified Savinase (PMDA / HEPES modified) , which was prepared in Example 43, was added to an undiluted solution of the detergent A (the original contained enzyme was inactivated by the heat treatment) , and a detergent composition E was prepared. Then, a test of detergency was performed in the same manner as in Example 85. However, the experiment was performed using 75 ml of ion-exchanged water (including 110 ppm of calcium chloride) for washing liquor, adding 0.125 ml of the detergent composition E, and adding 0.0375 ml of 2 M monoethalamine solution (pH 8) (final concentration 1 mM) for adjusting pH. The detergency was 57%. Even though it was combined with the commercial detergent, it showed a high detergency compared with the unmodified Savinase (Comparative Example 17, 54%) .

Comparative Example 17 A test of detergency was performed using the unmodified Savinase prepared in Comparative Example 1 instead of the modified Savinase in Example 87. The detergency was 54%.

Examples 88 to 99 Savinase and Esperase were modified with various modifiers shown in Table 15 in the same manner as in Example 1. The modified Savinase and Esperase were measured for modification degree. Amano Lipase AK® was modified with various modifiers shown in Table 15 in the same manner as in Example 78. The modified Amano Lipase AK® was measured for modification degree.

[Table 15] Modification of various enzymes

Example 100

The detergency was evaluated with Terg-O-tometer . The washing step was performed under the conditions of 25°C, 10 minutes, and 100 ppm. The rinsing step was performed under the conditions of 100 rpm, 25°C, and 2 minutes. Used were 7 pieces of EMPA 116 as stain clothes and water (containing 110 ppm of calcium chloride) I L. A detergent F and detergent G (the formulations are shown below) additionally prepared and the commercially available detergents A and B (the enzymes originally contained were inactivated by heating) were used as shown in Table 16. Then, the enzymes at amounts shown in Table 16 were added to the detergents. The detergent B into which the enzyme was added was incubated at 40⁰C for 30 days and then used (heated detergent B) . As the enzymes, the modified/unmodified Savinases prepared in Examples 1, 4, 43, and 88 and Comparative Example 1 were used. The stain clothes before and after the washing were measured for reflectance . The detergency calculated from Kubelka-Munk formula was shown in Table 16. Each of the modified enzymes showed detergency higher than that in the unmodified enzymes. It was also shown that reduction in the detergency after long-term storage was hardly observed and the modified Savinase had excellent storage stability.

Formulation of the detergent F

3.8% straight chain dodecylbenzenesulfonic acid salt

3.8% sodium dodecyl sulfate

3.8% sodium laurate

3.8% Brij30®

1% trisodium citrate

1% ethanolamine

2.5% ethanol

Formulation of the detergent G

7.5% Brij30®

7.5% Brij35®

1% trisodium citrate

1% ethanolamine

2.5% ethanol

The pH was adjusted to 9.5 with hydrochloric acid.

The rest of the component was water.

[Table 16] Detergency Evaluation of Modified Savinase using Terg-O-tometer

Example 101

A solvent (NMP) 50 ml was charged into a 100 ml four-necked flask equipped with a stirring rod and a condenser and then heated to 40°C. Thereto, PMDA 10 g was added and then monoethanolamine 1 g was also added. And about 10 minutes later, the mixture was heated to 50°C. Thereto, PMDA 10 g was further added and then monoethanolamine 3.O g was added. The mixture was matured for 3 hours while the temperature was kept at 50⁰C to obtain a modifier A (a reaction product of PMDA with monoethanolamine) . The reaction was performed under nitrogen bubbling. The average molecular weight (Mw) of modifier A was measured using GPC and determined 870.

Example 102 A liquid Savinase® 50 ml was charged into a beaker.

Thereto, a 15% aqueous solution of potassium hydroxide 4.5 ml was added under stirring. Thereby, the mixture was adjusted to pH 7.6. Then, 1.25 ml of the modifier A prepared in Example 101 was slightly charged into the Savinase solution under stirring. The stirring was continued for one hour at room temperatures to obtain a modified Savinase solution. The modified Savinase solution was subjected to the evaluations as performed in Example 1. The modification degree was 78%; the relative activity was 90%; and the half-life of activity in the 1% LAS was 150 minutes.

Example 103

Cellulase was modified. That is, a 2M aqueous solution of triethanolamine (pH 9) 0.05 ml was added to Celluclast® liquid (Novozymes Corp.) 0.5 ml and mixed. Thereto, 0.05 ml of the modifier A prepared in Example 101 was added and immediately mixed. Water is further added to adjust the final volume to 1 ml. Then, the mixture was kept standing at room temperatures for 1 hour. The solution was measured for modification degree. The modification degree was 67%. Then, the modified enzyme was 50-fold diluted with a 50 mM HEPES buffer (pH 7) . This diluted solution was measured for cellulase activity and half-life of activity at 60°C. The relative activity to the unmodified enzyme was 126% and the half-life of activity was 63 minutes. It was observed that the activity and the stability were improved as compared with those in the unmodified enzyme in Comparative Example 17.

Comparative Example 17 A unmodified enzyme solution was prepared in the same manner as in Example 103, except that NMP 0.05 ml was used instead of the modifier A. Then, the solution was measured for activity . Further, the solution was measured for half-life of activity at 60°C. The half-life of activity was 16 minutes.

Example 104

Amylase was modified. Amylase was modified in completely the same manner as in Example 103, except that Purastar HPAm® liquid (Genencor International) was used instead of the Celluclast. The modification degree was 73%. Then, this modified enzyme solution was 200-fold diluted with a 1% LAS solution (50 mMTris-HCl, pH 8 ) . This diluted solution was measured for activity and half-life of activity at 75°C. As a result, the relative activity to the unmodified enzyme was 86% and the half-life of activity was 37 minutes. It was observed that the stability in the LAS solution was improved as compared with that in the unmodified enzyme in Comparative Example 18.

Comparative Example 18

A unmodified enzyme solution was prepared in the same manner as in Example 104, except that NMP 0.05 ml was added instead of the modifier A. The solution was measured for activity. The solution was also measured for half-life of activity at 75°C. The half-life of activity was 13 minutes. Examples 105 to 109

Glucose oxidase was modified. That is, glucose oxidase (GOD) powders (Sigma) 50 rng were dissolved in a 0.2M boric acid buffer solution (pH 9) 1 ml . As shown in Table 17, the modification was performed by various modifiers . The solution was measured for modification degree.

[Table 17] Modification of various proteins

Examples 110 to 115

The modification was performed using γ-globulin from human serum (Sigma) as a protein which is not an enzyme. The experiment was performed in completely the same manner as in Example 105, except that γ-globulin was used instead of the GOD. The modification was performed by various modifiers shown in Table 17. The solution was measured for modification degree. Examples 116 to 119

The modification was performed using bovine serum albumin

(Sigma) as a protein which is not an enzyme. The experiment was performed in completely the same manner as in Example 105, except that bovine serum albumin was used instead of the GOD.

The modification was performed by various modifies shown in

Table 17. The solution was measured for modification degree.

Example 120 Insoluble cellulose was decomposed using the modified cellulase. That is, Avicel as the insoluble cellulose 50 mg was mixed with a buffer solution containing surfactants 5 ml

(50 mM acetic acid, pH 5, containing each 250 ppm of LAS and

Brij30®) . Thereto, 0.02 ml of the modified Celluclast prepared in Example 103 was added. The mixture was subjected to decomposition reaction at 60⁰C for 1 hour under stirring. After the reaction, the amount of generated reducing sugars was measured using DNS reagent (the concentration was quantified by reference to glucose) . As a result, it was shown that 14 mM reducing sugars was generated and this amount was about 1.4 times that of the unmodified enzyme (Comparative Example 19) .

Comparative Example 19

The same experiment as in Example 120 was performed using the unmodified Cellulclast prepared in Comparative Example 17. The amount of generated reducing sugars was 10 mM.

Example 121

Puradax® (cellulase produced by Genencor International, liquid form) was modified in the completely same manner as in Example 103. The modification degree was 92%. Then, the modified cellulase was evaluated for stability against an anionic surfactant, That is, the modified Puradax solution was 50-fold diluted with 1% LAS solution (5OmM HEPES buffer, pH8 containing 1% sodium linear dodecylbenzene sulfonate) , and measured for -O cellulase activity and half-life at 40 C. The relative activity to the unmodified enzyme was 150%, and the half-life was 100 minutes. The activity and surfactant stability of a cellulase were improved by modification compared with unmodified enzyme (Comparative Example 19)

Example 122

Puradax® was modified with PMDA in the same manner as in Example 103, except that 0.5M PMDA solution (DMF) 0.04 ml was used as the modifier instead of the modifier A. The modified enzyme was evaluated in the same manner as in Example 121. The modification degree was 82%, relative activity was 130%, and the half-life in LAS solution was 60 minutes.

Comparative Example 19

Puradax® solution 0.5ml was mixed with DMF 0.01 ml and water 0.49 ml, and this was treated as unmodified enzyme solution. Same evaluations were performed in the same manner as in Example 121, and the half-life in LAS solution was 15 minutes.

Claims

1. A composition comprising a protein and an anionic surfactant, wherein the protein is chemically modified with a polyvalent carboxylic acid having a benzene ring as a protein modifier.

2. The composition according to Claim 1, wherein the polyvalent carboxylic acid has three or more carboxyl groups.

3. The composition according to Claim 1 or 2, wherein the composition is a detergent composition.

4. A protein modifier, wherein the protein modifier is a polymer of a polyvalent carboxylic acid having three or more carboxyl groups and a connecting agent connecting the carboxyl groups, the protein modifier having at least one carboxylic anhydride group in the molecule.

5. The protein modifier according to Claim 4, wherein the polyvalent carboxylic acid is a carboxylic polyanhydride.

6. The protein modifier according to Claim 5, wherein the carboxylic polyanhydride is at least one compound selected from the group consisting of pyromellitic dianhydride, butanetetracarboxylic dianhydride, ethylenediamine tetraacetic dianhydride, and diethylenetriamine pentaacetic dianhydride.

7. A protein modifier, the protein modifier being a polyvalent carboxylic acid having three or more carboxyl groups, and having at least one carboxylic anhydride group in the molecule, wherein at least one carboxyl group in the polyvalent carboxylic acid is connected to a monovalent amine by an amide bond or to monohydric alcohol by an ester bond.

8. A modified protein chemically modified with the protein modifier of any of Claims 4 to 7.

9. The modified protein according to Claim 8, wherein the modified protein has a half-life of activity of 50 minutes or more at 40°C in a 1% by weight LAS solution.

10. A composition comprising the modified protein of Claim 8 or 9.