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WO2011024862A1 - Stabilisateur de solution aqueuse de protéines, solution aqueuse de protéines et détergent liquide contenant tous deux le stabilisateur, et méthode de stabilisation de protéines utilisant le stabilisateur - Google Patents

Stabilisateur de solution aqueuse de protéines, solution aqueuse de protéines et détergent liquide contenant tous deux le stabilisateur, et méthode de stabilisation de protéines utilisant le stabilisateur Download PDF

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Publication number
WO2011024862A1
WO2011024862A1 PCT/JP2010/064398 JP2010064398W WO2011024862A1 WO 2011024862 A1 WO2011024862 A1 WO 2011024862A1 JP 2010064398 W JP2010064398 W JP 2010064398W WO 2011024862 A1 WO2011024862 A1 WO 2011024862A1
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Prior art keywords
protein
weight
stabilizer
aqueous solution
atom
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English (en)
Japanese (ja)
Inventor
山口俊一郎
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Sanyo Chemical Industries Ltd
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Sanyo Chemical Industries Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38663—Stabilised liquid enzyme compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present invention relates to a protein aqueous solution stabilizer, a protein aqueous solution and a liquid detergent composition containing the stabilizer, and a protein stabilization method using the stabilizer.
  • Proteins such as enzymes, antibodies, and peptides are widely used as detergents, diagnostic / testing agents, and pharmaceuticals. In these products, it is important that the physiological activity (titer) is not impaired during the manufacturing process and storage period.
  • a method of supplying a purified protein preparation has been adopted as a method of supplying a protein without reducing its physiological activity.
  • lyophilization is generally performed. Many proteins have the property of being easily inactivated by heat, but the freeze-drying method can stabilize the protein without applying heat.
  • the lyophilization method cannot be used for a protein that is denatured by dehydration, and there are drawbacks such as deterioration due to moisture absorption or oxidation during the lyophilization process.
  • drawbacks such as deterioration due to moisture absorption or oxidation during the lyophilization process.
  • a freeze-dried preparation is used by dissolving as an aqueous solution at the time of use, there is a problem of complexity that an aqueous solution in which a protein preparation is adjusted to a necessary concentration must be prepared each time. For these reasons, techniques for stabilizing proteins in aqueous solutions have been published.
  • Patent Document 1 a technique for containing a polyhydric alcohol such as glycerin in an aqueous solution containing urease peroxidase (Patent Document 1) and an aqueous solution containing cholesterol oxidase to stabilize cholesterol oxidase
  • examples include a technique of adding a saccharide such as bovine serum albumin or glucose or an amino acid such as lysine (Patent Document 2).
  • a saccharide such as bovine serum albumin or glucose or an amino acid such as lysine
  • Patent Document 2 an amino acid
  • these are all methods for stabilizing a specific protein, and it is difficult to say that they are versatile. They are general-purpose stabilizers and stabilizers that can be applied to all proteins and maintain their activity for a long time. There is no way.
  • enzymes are known as constituents of detergents such as clothing detergents. Dirt varies depending on the type of clothing. Among soils, complex soils such as sebum soils, protein soils and particle soils are considered difficult to clean. Conventionally, it has been known that a detergent containing an enzyme such as a surfactant excellent in sebum dirt removal and particle dirt dispersion and a protease excellent in protein dirt degradation is effective against such dirt. .
  • enzymes are indispensable for detergents because of their high degradability.
  • detergents are changing from conventional solid detergents to liquid detergents in terms of ease of use.
  • the enzyme has a problem that its enzyme activity is poor in an aqueous solution and the enzyme activity is remarkably lowered during storage. Therefore, it has become a big problem to provide a liquid detergent that has a high enzyme activity and can maintain its detergency.
  • Non-patent document 1 describes that boronic acid inhibits serine protease and subtilisin.
  • Patent Document 3 describes that 4-substituted phenylboronic acid inhibits protease and sabinase.
  • Patent Document 4 also describes adding a polyol (eg, 1,2-propanediol, sorbitol, glycerol) to a liquid detergent.
  • a polyol eg, 1,2-propanediol, sorbitol, glycerol
  • liquid detergents containing these compositions have certain effects, they cannot sufficiently suppress the decrease in enzyme activity during storage, and it is impossible to obtain detergency sufficient to meet consumer needs.
  • “enzyme activity has good persistence” means that the difference between the enzyme activity measured after storage for a certain period of time and the enzyme activity measured immediately before storage is small and shows a certain enzyme activity. Means.
  • a protein aqueous solution stabilizer that can suppress a decrease in the physiological activity of the protein due to aggregation of the protein in the aqueous solution and stabilize the aqueous solution of the protein for a long time, and the enzyme activity of the enzyme is good. It is an object to provide a liquid detergent composition that can maintain a cleanability for a long period of time.
  • the present invention is a protein aqueous solution stabilizer comprising the organic compound (A) that satisfies the following conditions (1) to (3): (1) When a proton is added to the molecule of the organic compound (A), at least one atom (Z) in the molecule has an atom (Z) and an atom (Y) bonded to the atom (Z). The ⁇ bond formed by ⁇ electrons has a resonance structure, There are four or more ⁇ electrons involved in the ⁇ bond of this resonance structure, which are electrons possessed by the atom (Z) and atom (Y). (2) The ionic strength of the 0.1 moldm ⁇ 3 solution is 0.
  • the molecular weight is less than 300.
  • the protein aqueous solution of this invention makes it a summary to contain the stabilizer of this protein aqueous solution, protein, and water.
  • the gist of the protein stabilization method of the present invention is that the aqueous protein stabilizer, protein and water are mixed and stirred for 1 minute to 2 hours.
  • the liquid detergent composition of this invention makes it a summary to contain the stabilizer of this protein aqueous solution, enzyme (C), surfactant (D), and water.
  • the protein aqueous solution stabilizer of the present invention stabilizes the protein in the aqueous solution and does not decrease the physiological activity. Therefore, the protein aqueous solution containing the protein aqueous solution stabilizer of the present invention can maintain physiological activity for a long period of time. In addition, the liquid detergent composition of the present invention can maintain cleanability for a long time.
  • the present invention is a protein aqueous solution stabilizer, comprising an organic compound (A) that satisfies the following conditions (1) to (3): (1) When a proton is added to the molecule of the organic compound (A), at least one atom (Z) in the molecule has an atom (Z) and an atom (Y) bonded to the atom (Z). The ⁇ bond formed by ⁇ electrons has a resonance structure, There are four or more ⁇ electrons involved in the ⁇ bond of this resonance structure, which are electrons possessed by the atom (Z) and atom (Y). (2) The ionic strength of the 0.1 moldm ⁇ 3 solution is 0. (1) The molecular weight is less than 300.
  • the proteins are stored as they are as an aqueous solution, they will cause aggregation, hydrolysis and the like, and the titer will be significantly reduced.
  • the above compound (A) having a specific chemical structure is present. Can be used as a stabilizer for protein aqueous solutions.
  • the condition (1) will be described.
  • the organic compound (A) is guanidine represented by the following general formula (1)
  • the carbon atom (I) is bonded to the nitrogen atoms (II), (III) and (IV).
  • the carbon atom (I) becomes a cation and has a vacant orbit
  • the nitrogen atoms (II) to (IV) each have a lone electron pair in the sp 3 hybrid orbital.
  • These three lone electron pairs can form a ⁇ bond with the carbon atom (I) and have a resonance structure.
  • the carbon atom (I) in the guanidine molecule is an atom (Z)
  • the atom (Y) bonded to the atom (Z) is a nitrogen atom (II) to (IV)
  • the number of ⁇ electrons that the atom (Z) ⁇ carbon atom (I) ⁇ and atom (Y) ⁇ nitrogen atoms (II) to (IV) ⁇ have and participate in the ⁇ bond of this resonance structure.
  • protons are added to the guanidine molecule, nitrogen atoms (II) to (IV) are bonded to carbon atom (I) and two hydrogen atoms, respectively, and each lone pair forms a ⁇ bond.
  • Examples of the compound satisfying the condition (1) include guanidinium salts, guanazine, guanamine, formamidine, heterocyclic compounds (pyrrole, imidazole, pyridine, pyrimidine, indole, quinoline, isoquinoline, purine, etc.), organic phosphoric acid ( And phosphate esters of alcohols having 1 to 30 carbon atoms).
  • Examples of the organic compound (A) satisfying the above conditions (1) to (3) of the present invention include a heterocyclic compound (A-1), an organic phosphoric acid (A-2), nitrogen having 1 to 20 carbon atoms, oxygen And a compound (A-3) having two or more sulfur atoms and a salt thereof (A-4).
  • Examples of the heterocyclic compound (A-1) include nitrogen-containing heterocyclic compounds, oxygen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds, and phosphorus-containing heterocyclic compounds.
  • pyrrole imidazole, pyridine, pyrimidine, indole
  • examples include quinoline, isoquinoline, purine, furan, thiophene, oxazole, thiazole, isoxazole, and isothiazole.
  • organic phosphoric acid (A-2) examples include organic phosphoric acids having 1 to 20 carbon atoms, such as methyl phosphoric acid, ethyl phosphoric acid, isopropyl phosphoric acid, butyl phosphoric acid, dimethyl phosphoric acid, diethyl phosphoric acid, dipropyl phosphoric acid, Examples include dibutyl phosphoric acid, methyl pyrophosphoric acid, and ethyl pyrophosphoric acid.
  • Examples of the compound (A-3) having two or more nitrogen, oxygen and sulfur atoms having 1 to 20 carbon atoms include the compound (a) represented by the following general formula (2), an amidino group-containing compound and an amide group-containing compound Examples thereof include guanidine, urea, thiourea, formamidine, methylamidine, biguanide and the like.
  • Examples of these salts (A-4) include salts of the compound (a) represented by the following general formula (2).
  • Examples of the salt include inorganic acid salts such as hydrochloride, phosphate, and sulfate, and organic acid salts such as carbonate, citrate, and acetate.
  • the organic compound (A) is preferably a compound (a) represented by the following general formula (2) and a salt of the compound (a) from the viewpoint of sustained activity. .
  • X represents an imino group, an oxygen atom or a sulfur atom.
  • Specific examples of the compound (a) represented by the general formula (2) include guanidine, urea and thiourea.
  • Examples of the salt of the compound (a) represented by the general formula (2) include guanidine salts.
  • Examples of the salt include hydrochloride, carbonate, borate, sulfate and phosphate.
  • the compound (a) and the salt of the compound (a) are preferably a guanidine salt and urea, more preferably a guanidine salt, and further preferably a guanidine hydrochloride from the viewpoint of protein stabilization.
  • the content (% by weight) of the organic compound (A) contained in the stabilizer of the present invention is preferably 10 to 100% by weight, more preferably It is 20 to 90% by weight, more preferably 30 to 80% by weight, and particularly preferably 30 to 70% by weight.
  • the content (% by weight) of the organic compound (A) contained in the stabilizer of the present invention is 2 to 6000% by weight with respect to the weight of the protein when the stabilizer is used from the viewpoint of protein stabilization. It is preferably contained so as to be 5 to 500% by weight, more preferably 10 to 100% by weight.
  • the stabilizer of the present invention can further contain a compound (B) represented by the following general formula (3).
  • Q represents an alkyl group, and a part of hydrogen atoms in the alkyl group may be substituted with groups other than hydrogen atoms.
  • alkyl group of Q examples include an alkyl group having 1 to 22 carbon atoms, specifically, methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, octyl group, nonyl group, decyl group, A dodecyl group, a cetyl group, a stearyl group, a behenyl group, etc. are mentioned.
  • Some of the hydrogen atoms in these alkyl groups may be substituted with groups other than hydrogen atoms.
  • substituents other than hydrogen atoms include amino groups, carboxyl groups, amide groups, ester groups, imino groups, and hydroxyl groups.
  • the number of substituents is preferably 1 to 3, more preferably 2 to 3.
  • the compound (B) represents arginine.
  • Examples of the compound (B) include arginine or a salt thereof (B-1) and an arginine derivative or a salt thereof (B-2).
  • Arginine or its salt includes arginine, arginine inorganic acid salt (hydrochloride, borate, phosphate, pyrophosphate, sulfate, silicate, etc.) and arginine organic acid salt (formic acid) Salt, acetate, oxalate, lactate, citrate, trimellitic acid salt and pyromellitic acid salt).
  • the arginine derivative is a derivative in which the ⁇ -amino group or ⁇ -carboxyl group of arginine represented by the following general formula (4) or both of these groups are substituted.
  • the substitution of the ⁇ -amino group is performed on the N-alkylcarbonyl-amide group (Y-1) represented by the following general formula (5) or the imino group (Y-2) represented by the following general formula (6).
  • the substitution of the ⁇ -carboxyl group is an ester group (Z-1) represented by the following general formula (7) or an N-alkylamide group (Z-2) represented by the following general formula (8) Is a replacement.
  • arginine derivative or its salt (B-2) at least one of an ⁇ -amino group and an ⁇ -carboxyl group is substituted. That is, when Y is an amino group, Z is an ester group (Z-1) represented by the following general formula (7) or an N-alkylamide group (Z-2) represented by the following general formula (8). And when Z is a carboxyl group, Y represents an N-alkylcarbonyl-amide group (Y-1) represented by the following general formula (5) or an imino group (Y— 2).
  • Y is an amino group, an N-alkylcarbonyl-amide group (Y-1) represented by the following general formula (5) or an imino group (Y—) represented by the following general formula (6). 2).
  • Z represents a carboxyl group, an ester group (Z-1) represented by the following general formula (7), or an N-alkylamide group (Z-2) represented by the following general formula (8).
  • R 1 represents a hydrogen atom or a monovalent hydrocarbon group having 1 to 36 carbon atoms, and this hydrocarbon group has a part of the hydrogen atom as a functional group other than a hydrogen atom. May be substituted.
  • the hydrocarbon group of R 1 in the N-alkylcarbonyl-amide group (Y-1) represented by the general formula (5) is a monovalent hydrocarbon group having 1 to 36 carbon atoms, and is linear or branched Aliphatic hydrocarbon groups, alicyclic hydrocarbon groups and aromatic hydrocarbon groups.
  • Linear aliphatic hydrocarbon groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, lauryl, palmityl, stearyl, oleyl and Examples include a behenyl group.
  • Examples of the branched aliphatic hydrocarbon group include an isopropyl group and a t-butyl group.
  • Examples of the alicyclic hydrocarbon group include a cyclohexyl group, a methylcyclohexyl group, and a cyclohexylmethyl group.
  • Examples of the aromatic hydrocarbon group include a phenyl group, a methylphenyl group, a benzyl group, a phenylethyl group, and a methylbenzyl group.
  • a linear aliphatic hydrocarbon group is preferable, a methyl group and an ethyl group are more preferable, and a methyl group is most preferable.
  • substituents other than hydrogen atoms include amino groups, carboxyl groups, amide groups, ester groups, imino groups, and hydroxyl groups.
  • N-alkylcarbonyl-amide group (Y-1) represented by the general formula (5) include formamide group, acetylamide group, propionic acid amide group, butyric acid amide group, hexylic acid amide group, and cyclohexyl.
  • Examples include an acid amide group, an octylic acid amide group, and a benzoylamide group.
  • R 2 and R 3 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 36 carbon atoms, and these hydrocarbon groups are other than hydrogen atoms.
  • the functional group may be substituted.
  • R 2 and R 3 include the same hydrocarbon group as R 1, and these hydrocarbon groups are the same as R 1 , Some may be substituted with other functional groups.
  • Examples of the imino group (Y-2) represented by the general formula (6) include a methylimino group.
  • R 4 represents a hydrocarbon group having 1 to 36 carbon atoms, a residue obtained by removing one hydroxyl group from a polyhydric alcohol or sugar.
  • this hydrocarbon group part of the hydrogen atoms may be substituted with another functional group such as a hydroxyl group, a methoxyl group, an ethoxyl group, a nitro group, or a hydroxyphenyl group.
  • the hydrocarbon group when R 4 is a hydrocarbon group having 1 to 36 carbon atoms, the hydrocarbon group includes the same hydrocarbon group as the above R 1 It is.
  • R 4 is a hydrocarbon group having 1 to 36 carbon atoms, among these hydrocarbon groups, a linear aliphatic hydrocarbon group is preferable from the viewpoint of stabilization of the protein-containing aqueous solution, and a methyl group and more preferably An ethyl group, most preferably an ethyl group.
  • the polyhydric alcohol includes divalent to trivalent alcohols, and examples thereof include ethylene glycol, propylene glycol, diethylene glycol, and glycerin.
  • examples of the sugar include glucose, sucrose, sorbitol, mannitol and trehalose.
  • R 5 represents a hydrogen atom or a hydrocarbon group having 1 to 36 carbon atoms, and in this hydrocarbon group, part of the hydrogen atom is substituted with another functional group other than a hydrogen atom. May be.
  • R 5 is a hydrocarbon group having 1 to 36 carbon atoms
  • the hydrocarbon group is the same as the carbon atom as R 1.
  • a hydrogen group is included, and these hydrocarbon groups may be partially substituted with other functional groups in the same manner as R 1 .
  • R 5 is a hydrocarbon group having 1 to 36 carbon atoms
  • a linear aliphatic hydrocarbon group is preferable from the viewpoint of stabilization of the protein-containing aqueous solution, more preferably a methyl group and An ethyl group, most preferably a methyl group.
  • the arginine derivative or a salt thereof (B-2) is a salt of an arginine derivative
  • examples of the salt include inorganic acid salts (hydrochloride, borate, phosphate, pyrophosphate, sulfate, silicate, etc.) and Organic acid salts (formate, acetate, oxalate, lactate, citrate, trimellitic acid, pyromellitic acid, etc.) can be mentioned.
  • arginine derivative or a salt thereof (B-2) include N- ⁇ -acetylarginine ethyl ester hydrochloride.
  • the compound (B) contained in the stabilizer of the present invention is preferably an arginine derivative or a salt thereof (B-2), more preferably an arginine ⁇ -amino group and ⁇ -amino group, from the viewpoint of sustained activity.
  • the content (% by weight) of the compound (B) contained in the stabilizer of the present invention is preferably 10 to 90% by weight, more preferably 20%. -80% by weight, and more preferably 30-70% by weight.
  • the content (% by weight) of the compound (B) contained in the stabilizer of the present invention is 2 to 500% by weight based on the weight of the protein when the stabilizer is used, from the viewpoint of protein stabilization. It is preferably contained so as to be 5 to 200% by weight, more preferably 10 to 100% by weight.
  • the stabilizer of the present invention may contain only the organic compound (A), but preferably contains the organic compound (A) and the compound (B) from the viewpoint of protein stabilization.
  • the weight ratio of the organic compound (A) to the compound (B) (weight of the organic compound (A) / compound (B)
  • the weight is preferably from 0.1 to 9, more preferably from 0.2 to 8, particularly preferably from 0.5 to 5.
  • the stabilizer of the present invention includes, in addition to the organic compound (A) and the compound (B), a surfactant (E), an inorganic salt (F), a polyhydric alcohol (G), a sugar (H), and arginine.
  • a surfactant (E) for example, a surfactant (E), an inorganic salt (F), a polyhydric alcohol (G), a sugar (H), and arginine.
  • An amino acid (I) and other organic compounds (J) may be contained.
  • Surfactants (E) include anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants, such as polyethylene glycol, polyoxypropylene / polyoxyethylene copolymers, Examples thereof include sorbitan alkyl ester ethylene oxide adduct, aliphatic alcohol ethylene oxide adduct, fatty acid ethylene oxide adduct, and alkylamine ethylene oxide adduct.
  • Examples of the inorganic salt (F) include sodium chloride, sodium borate, calcium chloride, magnesium chloride, sodium formate, magnesium sulfate, and ammonium sulfate.
  • polyhydric alcohol (G) examples include ethylene glycol, propylene glycol, and glycerin.
  • sugar (H) examples include trehalose, sucrose, dextrin, cyclodextrin, maltose, fructose, hyaluronic acid and chondroitin sulfate.
  • amino acids (I) other than arginine examples include glycine, alanine, aspartic acid, asparagine, phenylalanine, tryptophan, tyrosine, leucine, lysine, histidine, and salts thereof.
  • organic compounds (J) are not particularly limited, and examples include serum albumin, collagen, casein, gelatin, and silk peptide.
  • the content (% by weight) of the surfactant (E) contained in the stabilizer of the present invention is preferably 0 to 50% by weight, more preferably based on the weight of the stabilizer from the viewpoint of protein stabilization. Is 0 to 30% by weight, more preferably 0 to 20% by weight. From the viewpoint of protein stabilization, the content (% by weight) of the inorganic salt (F) contained in the stabilizer of the present invention is preferably 0 to 20% by weight, more preferably It is 0 to 15% by weight, and further preferably 0 to 10% by weight.
  • the content (% by weight) of the polyhydric alcohol (G) contained in the stabilizer of the present invention is preferably 0 to 70% by weight, more preferably Is 0 to 60% by weight, more preferably 0 to 50% by weight.
  • the content (% by weight) of the saccharide (H) contained in the stabilizer of the present invention is preferably 0 to 50% by weight, more preferably 0%, based on the weight of the stabilizer, from the viewpoint of protein stabilization. -30 wt%, then more preferably 0-20 wt%.
  • the content (% by weight) of amino acid (I) other than arginine contained in the stabilizer of the present invention is preferably 0 to 30% by weight with respect to the weight of the stabilizer from the viewpoint of protein stabilization. Preferably it is 0 to 20% by weight, then more preferably 0 to 10% by weight.
  • the content (% by weight) of the other organic compound (J) contained in the stabilizer of the present invention is preferably 0 to 10% by weight with respect to the weight of the stabilizer from the viewpoint of protein stabilization. Preferably it is 0 to 5% by weight, then more preferably 0 to 3% by weight.
  • Another embodiment of the present invention is an aqueous protein solution containing the protein aqueous solution stabilizer, protein and water.
  • the content (% by weight) of the stabilizer is preferably 0.1 to 50% by weight based on the weight of the protein aqueous solution from the viewpoint of protein stabilization.
  • the protein content (% by weight) is preferably 0.01 to 2% by weight based on the weight of the protein aqueous solution from the viewpoint of long-term stability.
  • the water content (% by weight) is preferably 48 to 99.8% by weight based on the weight of the protein aqueous solution from the viewpoint of protein stabilization.
  • the content (% by weight) of the organic compound (A) in the stabilizer is preferably 0.01 to 40% by weight based on the weight of the protein aqueous solution from the viewpoint of protein stabilization.
  • the weight ratio of the protein to the organic compound (A) in the stabilizer is preferably 2 to 6000, more preferably from the viewpoint of protein stabilization. Is from 5 to 500, and more preferably from 10 to 100.
  • the content (% by weight) of the compound (B) in the stabilizer is preferably 0.01 to 20% by weight based on the weight of the aqueous protein solution from the viewpoint of protein stabilization.
  • the weight ratio of the protein to the compound (B) in the stabilizer is preferably 2 to 500, more preferably 5 from the viewpoint of protein stabilization. ⁇ 200, and more preferably 10 ⁇ 100.
  • the protein to which the protein aqueous solution stabilizer of the present invention can be applied is not particularly limited, and examples thereof include enzymes, recombinant proteins, antibodies and peptides.
  • Enzymes include oxidoreductases ⁇ cholesterol oxidase, glucose oxidase, ascorbate oxidase, peroxidase, etc. ⁇ , hydrolases ⁇ lysozyme, protease, serine protease, amylase, lipase, cellulase, glucoamylase, etc. ⁇ , isomerase ⁇ glucose isomerase, etc.
  • Etc. ⁇ transferase ⁇ acyltransferase, sulfotransferase, etc. ⁇ , synthase ⁇ fatty acid synthase, phosphate synthase, citrate synthase, etc. ⁇ and elimination enzyme ⁇ pectin lyase etc. ⁇ .
  • Recombinant proteins include protein preparations ⁇ interferon ⁇ , interferon ⁇ , interleukin 1-12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), sodium And diuretic peptides, blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like ⁇ and vaccines ⁇ hepatitis A vaccine, hepatitis B vaccine and hepatitis C vaccine ⁇ and the like.
  • antibodies include monoclonal antibodies and polyclonal antibodies.
  • the peptide is not particularly limited in amino acid composition, and examples thereof include dipeptides and tripeptides. Among these proteins, enzymes are preferable from the viewpoint of long-term stabilization of activity.
  • the water contained in the protein aqueous solution of the present invention is not particularly limited, and examples thereof include tap water, ion exchange water, distilled water, and reverse osmosis water.
  • a buffering agent may be added to the protein-containing aqueous solution of the present invention as long as the stabilizing effect is not impaired.
  • the buffer include known buffers, and specific examples include Tris buffer (Tris buffer), HEPES buffer, and MOPS buffer. Among these, an alkaline buffer is preferable, and a tris buffer is particularly preferable from the viewpoint of availability.
  • the stabilizer may be added to the aqueous protein solution as it is or dissolved in water. May be added to the aqueous solution of the stabilizer, or the protein, stabilizer and water may be mixed together.
  • a stabilizer is added to water to prepare an aqueous solution.
  • the enzyme aqueous solution after separation and purification is added to the aqueous solution.
  • the protein stabilization method of the present invention is a method of stabilizing a protein by mixing the above-mentioned protein stabilizer, the above-mentioned protein and water and stirring for 1 minute to 2 hours.
  • the amount of stabilizer added the amount of protein, the amount of water, the weight ratio of protein to organic compound (A) in the stabilizer, and the amount of protein in stabilizer
  • the weight ratio with the compound (B) is the same as that described in the protein aqueous solution, and the preferred range is also the same.
  • the stabilizer when the stabilizer, protein and water are mixed, the stabilizer may be added as it is or dissolved in water to the protein aqueous solution, or the protein may be added to the stabilizer aqueous solution. You may add, and you may mix protein, a stabilizer, and water together.
  • the liquid detergent composition of the present invention is a liquid detergent composition containing a protein aqueous solution stabilizer, an enzyme (C), a surfactant (D) and water.
  • the enzyme When an enzyme is stored in a liquid detergent, the enzyme causes aggregation, hydrolysis and the like, and the enzyme activity (titer) is remarkably reduced.
  • the protein aqueous solution stabilizer is used as a liquid detergent composition. It can be solved by adding to.
  • the stabilizer for the aqueous protein solution contained in the liquid detergent composition of the present invention is the stabilizer for the aqueous protein solution of the present invention described above.
  • the stabilizer for the aqueous protein solution contained in the liquid detergent composition of the present invention contains the organic compound (A).
  • the organic compound (A) is preferably a guanidine salt and urea, more preferably a guanidine salt, and still more preferably a guanidine hydrochloride from the viewpoint of sustaining enzyme activity.
  • the content (% by weight) of the stabilizer contained in the liquid detergent composition of the present invention is preferably 1 to 40% by weight with respect to the weight of the liquid detergent composition from the viewpoint of sustaining enzyme activity, Preferably it is 2 to 35% by weight, then more preferably 3 to 30% by weight, particularly preferably 5 to 20% by weight.
  • the content (% by weight) of the organic compound (A) contained in the liquid detergent composition of the present invention is 0.01 to 30% by weight with respect to the weight of the liquid detergent composition from the viewpoint of sustaining enzyme activity. More preferably, it is 0.02 to 10% by weight, next more preferably 0.03 to 5% by weight, and particularly preferably 0.05 to 3% by weight.
  • the content (% by weight) of the organic compound (A) contained in the liquid detergent composition of the present invention is 1 to 1000% by weight with respect to the weight of the enzyme from the viewpoint of sustaining the enzyme activity. It is preferably contained so as to be 5 to 500% by weight, and more preferably 10 to 300% by weight.
  • the stabilizer for the aqueous protein solution contained in the liquid detergent composition of the present invention may contain the compound (B).
  • the compound (B) an arginine derivative or a salt thereof (B-2) is preferable from the viewpoint of sustaining enzyme activity, and more preferably, both the ⁇ -amino group and ⁇ -carboxyl group of arginine are substituted.
  • Particularly preferred is N- ⁇ -acetylarginine ethyl ester hydrochloride.
  • the content (% by weight) of the compound (B) contained in the liquid detergent composition of the present invention is 0.01 to 30% by weight with respect to the weight of the liquid detergent composition from the viewpoint of sustaining enzyme activity. Preferably, it is 0.03 to 10% by weight, and further preferably 0.05 to 5% by weight.
  • the content (% by weight) of the compound (B) contained in the liquid detergent composition of the present invention is 1 to 1000% by weight with respect to the weight of the enzyme from the viewpoint of sustaining the enzyme activity. More preferably, it is contained in an amount of 5 to 500% by weight, and further preferably in an amount of 10 to 300% by weight.
  • the stabilizer contained in the liquid detergent composition of this invention should contain only an organic compound (A), it contains an organic compound (A) and a compound (B) from a viewpoint of the sustainability of the enzyme activity of an enzyme. It is preferable to do.
  • the weight ratio of the organic compound (A) to the compound (B) is 0.1 to 9 is preferable, more preferably 0.2 to 8, and particularly preferably 0.5 to 5.
  • the enzyme (C) that is an essential component in the present invention includes protease (C-1), cellulase (C-2), amylase (C-3), lipase (C-4) and oxidoreductase (C-5). Can be mentioned.
  • Protease (C-1) includes those of animal, plant or microbial origin, and those of microbial origin are preferred from the viewpoint of availability. Proteases also include chemically or genetically modified variants. Among proteases, serine proteases are preferable from the viewpoint of detergency, and alkaline microbial proteases and trypsin-like proteases are more preferable.
  • Alkaline microbial proteases include subtilisins, particularly those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168.
  • Trypsin-like proteases include trypsin (eg, of porcine or bovine origin) and Fusarium protease.
  • Cellulases include those of bacterial or fungal origin. Cellulases also include chemically or genetically modified variants. Cellulases include those disclosed in US Pat. No. 4,435,307 as fungal cellulases produced from Humicola insolens. Also particularly suitable cellulases are cellulases useful for color care, including cellulases described in European Patent Application No. 0 495 257. Examples of commercially available cellulases include Novozymes Celluzyme TM produced by Humicola insolens strain and Kao KAC-500 (B) TM .
  • Amylase (C-3) includes those of bacterial or fungal origin. Amylases also include chemically or genetically modified variants. Examples of the amylase include B.I. described in detail in British Patent No. 1,296,839. And ⁇ -amylase obtained from a special strain of B. licheniformis. Commercially available amylases, Novozymes of Duramyl TM, Termamyl TM, etc. Fungamyl TM and BAN TM and Gist-Brocades Inc., Rapidase TM and Maxamyl P TM, and the like.
  • Lipases include those of bacterial or fungal origin. Lipases also include chemically or genetically modified variants. Examples of lipases include Humicola lanuginosa lipase (European Patent 258 068 and European Patent 305 216), Rhizomucor miehei lipase and Candida (Candida Europe) Patent No. 238 023), C.I. Antactica lipase A and B, Pseudomonas lipase (European Patent No. 214,761), P. P. pseudoalcaligenes and P. p. P. alcaligenes lipase (European Patent No. 218,272), P. a. P. cepacia lipase (European Patent No.
  • Lipases Genencor M1 Lipase TM, Luma fast TM and Lipomax TM, Novozymes of Lipolase TM and Lipolase Ultra TM and Amano Enzyme Inc. of Lipase P "Amano" TM, and the like.
  • Oxidoreductase (C-5) includes peroxidase and oxidase (eg laccase).
  • Peroxidases include those of plant, bacterial or fungal origin. Peroxidases also include chemically or genetically modified variants. Peroxidases include Coprinus (e.g. from C. cinereus or C. macrolithus strains), Bacillus (from B. pumilus strains). And the peroxidase described in WO 91/05858 are preferable, and the peroxidase described in WO 91/05858 is particularly preferable.
  • Laccases include those of bacterial or fungal origin. Laccases include Trametes [eg T. et al. T. vilosa or T.
  • protease (C-1), cellulase (C-2), amylase (C-3), and lipase (C-4) from the viewpoint of detergency against protein stains, fat stains, particle stains and carbohydrate stains. ) Is preferred, and protease (C-1) is more preferred.
  • the enzyme (C) contained in the liquid detergent composition can contain two or more kinds from the viewpoint of detergency.
  • examples of combinations of two or more include protease and cellulase, protease and cellulase and lipase, protease and amylase, and protease, cellulase and amylase.
  • the content of the enzyme (C) contained in the liquid detergent composition of the present invention is preferably 0.01 to 5% by weight, more preferably 0.05, based on the weight of the liquid detergent composition. It is ⁇ 2% by weight, particularly preferably 0.1 to 1% by weight.
  • the surfactant (D), which is an essential component of the present invention, is a nonionic surfactant (D-1), an anionic surfactant (D-2), a cationic surfactant (D-3), and an amphoteric surfactant.
  • Examples of the cationic surfactant (D-3) include quaternary ammonium salt types [stearyl trimethyl ammonium chloride, behenyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, ethyl lanolin sulfate fatty acid aminopropylethyl dimethyl ammonium, etc.] and amine salts Type [diethylaminoethylamide stearate lactate, dilaurylamine hydrochloride, oleylamine lactate, etc.] and the like.
  • amphoteric surfactant (D-4) a betaine-type amphoteric surfactant [coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine, lauryldimethylaminoacetic acid betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazoli Nitrobetaine, laurylhydroxysulfobetaine, lauroylamidoethylhydroxyethylcarboxymethylbetaine hydroxypropyl sodium phosphate, etc.] and amino acid type amphoteric surfactants [sodium ⁇ -laurylaminopropionate, etc.].
  • surfactant (D) 1 type (s) or 2 or more types can be used.
  • combinations thereof include, for example, a nonionic surfactant and an anionic surfactant, a nonionic surfactant and a cationic surfactant, and a nonionic surfactant And a combination of amphoteric surfactants.
  • the surfactant (D) from the viewpoint of detergency, it is preferable to use a nonionic surfactant alone or a combination of a nonionic surfactant and an anionic surfactant.
  • an anionic surfactant from the viewpoint of detergency, an alkylphenyl sulfonate having 8 to 24 carbon atoms, a fatty acid salt, an alkyl sulfate salt having 8 to 24 carbon atoms, and an alkyl (poly) having 8 to 24 carbon atoms.
  • Oxyethylene sulfate ester salts are preferred, more preferably alkyl phenyl sulfonates having 12 to 16 carbon atoms and fatty acid salts having 8 to 16 carbon atoms, and even more preferably dodecylbenzenesulfonic acid monoethanolamine salts and lauric acid Sodium.
  • the content of the surfactant (D) contained in the liquid detergent composition of the present invention is preferably 5 to 80% by weight, more preferably 10 to 50%, based on the weight of the liquid detergent composition. % By weight, particularly preferably 20 to 40% by weight.
  • Water that is an essential component of the present invention is not particularly limited, and examples thereof include tap water, ion exchange water, distilled water, and reverse osmosis water.
  • the content of water contained in the liquid detergent composition of the present invention is preferably 5 to 90% by weight, more preferably 13 to 80% by weight, particularly preferably from the weight of the liquid detergent composition, from the viewpoint of detergency. Is 29 to 70% by weight.
  • the liquid detergent composition of the present invention includes an inorganic salt (F), a polyhydric alcohol (G ), Sugar (H), amino acids other than arginine (I), builder (K), alkali agent (L) and chelating agent (M).
  • Examples of the inorganic salt (F) include sodium chloride, sodium borate, calcium chloride, magnesium chloride, sodium formate, magnesium sulfate, and ammonium sulfate.
  • polyhydric alcohol (G) examples include ethylene glycol, propylene glycol, and glycerin.
  • sugar (H) examples include trehalose, sucrose, dextrin, cyclodextrin, maltose, fructose, hyaluronic acid and chondroitin sulfate.
  • amino acids (I) other than arginine examples include glycine, alanine, aspartic acid, asparagine, phenylalanine, tryptophan, tyrosine, leucine, lysine, histidine, and salts thereof.
  • Examples of the builder (K) include poly (meth) acrylate and polycarboxylic acid ⁇ for example, oxalic acid, citric acid, succinic acid and malic acid ⁇ .
  • alkali agent (L) examples include caustic soda, soda ash, ammonia, triethanolamine, diethanolamine, monoethanolamine, and sodium tripolyphosphate.
  • chelating agent known ones used for liquid detergents can be used.
  • aminopolyacetic acid or salts thereof such as nitrilotriacetic acid, iminodiacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, glycol etherdiaminetetraacetic acid, hydroxyethyliminodiacetic acid, triethylenetetraaminehexaacetic acid and diencoric acid, diglycol Acids, oxydisuccinic acid, carboxymethyloxysuccinic acid, citric acid, lactic acid, tartaric acid, oxalic acid, malic acid, oxydisuccinic acid, gluconic acid, carboxymethyl succinic acid and carboxymethyl tartaric acid and their salts and aminotri (methylene Phosphonic acid), 1-hydroxyethylidene-1,1-diphosphonic acid, ethylenediaminetetra (methylenephosphonic acid), diethylenetriaminepent
  • the content (% by weight) of the inorganic salt (F) contained in the liquid detergent composition of the present invention is preferably 0.01 to 10% by weight with respect to the weight of the liquid detergent composition from the viewpoint of detergency. More preferred is 0.05 to 5% by weight, and still more preferred is 0.1 to 3% by weight.
  • the content (% by weight) of the polyhydric alcohol (G) contained in the liquid detergent composition of the present invention is 0 to 20% by weight with respect to the weight of the liquid detergent composition from the viewpoint of the uniformity of the liquid detergent composition. %, More preferably 0 to 10% by weight, and still more preferably 0 to 5% by weight.
  • the content (% by weight) of the sugar (H) contained in the liquid detergent composition of the present invention is preferably 0 to 5% by weight, more preferably from the weight of the liquid detergent composition, from the viewpoint of detergency. It is 0 to 3% by weight, and more preferably 0 to 1% by weight.
  • the content (% by weight) of the amino acid (I) other than arginine contained in the liquid detergent composition of the present invention is preferably 0 to 10% by weight with respect to the weight of the liquid detergent composition from the viewpoint of detergency. More preferably, it is 0 to 5% by weight, and further preferably 0 to 3% by weight.
  • the content (% by weight) of the builder (K) contained in the liquid detergent composition of the present invention is preferably from 0 to 5% by weight, more preferably from the weight of the liquid detergent composition, from the viewpoint of detergency. It is 0 to 3% by weight, and more preferably 0 to 1% by weight.
  • the content (% by weight) of the alkaline agent (L) contained in the liquid detergent composition of the present invention is preferably from 0 to 5% by weight, more preferably from the weight of the liquid detergent composition, from the viewpoint of detergency. Is 0.1 to 4% by weight, more preferably 0.5 to 3% by weight.
  • the content (% by weight) of the chelating agent (M) contained in the liquid detergent composition of the present invention is preferably from 0 to 5% by weight, more preferably from the weight of the liquid detergent composition, from the viewpoint of detergency. Is 0 to 3% by weight, and more preferably 0 to 2% by weight.
  • the pH of the liquid detergent composition of the present invention is preferably 7 to 11 and more preferably 7 to 10 with a 1% (w / w) aqueous solution from the viewpoint of detergency.
  • the liquid detergent composition of this invention is obtained by mixing each component, and a manufacturing method is not specifically limited. An example is shown below. (1) Add surfactant (D), organic compound (A) and, if necessary, compound (B) to water and stir at 25 ° C. until uniform. (2) A predetermined amount of components other than the enzyme (C) is added and dissolved uniformly. (3) Finally, the enzyme (C) is added and dissolved to produce a liquid detergent composition.
  • the method of using the liquid detergent composition of the present invention may be the same as the method of using the conventional liquid detergent composition, and is not particularly limited. An example is shown below. (1) Tap water in a washing machine containing laundry, add the liquid detergent composition at 25 ° C., and gently stir to dissolve. (2) Wash the laundry with a washing machine. (3) Drain the liquid from the washing machine and rinse with tap water once or twice. (4) Appropriate dehydration.
  • N- ⁇ -acetylarginine ⁇ Arginine acetamide manufactured by MP Bio-Japan Co., Ltd. ⁇ 12.6 parts by weight (0.05 mole part), 1 part of methanesulfonic acid and 92 parts by weight of ethanol (2 mole parts) were uniformly mixed.
  • the mixture was heated and stirred at 80 ° C. for 5 hours, concentrated by an evaporator, and then neutralized by adding 5.2 parts by weight (0.05 mol part) of hydrochloric acid (concentration: 35% by weight). Thereafter, it was recrystallized from water and dried under reduced pressure ⁇ 60 ° C., 20 Pa ⁇ to obtain N- ⁇ -acetylarginine ethyl ester hydrochloride as compound (B).
  • Example 1 100 mg of guanidine hydrochloride ⁇ manufactured by Wako Pure Chemical Industries, Ltd.) which is an organic compound (A) was used as a stabilizer (N-1).
  • Example 2 ⁇ Examples 2 to 15>
  • stabilizers (N-2) to (N-15) were obtained in the same manner as in Example 1 except that the organic compounds (A) and (B) had the compositions and amounts shown in Table 1.
  • the organic compound (A) urea ⁇ manufactured by Wako Pure Chemical Industries, Ltd. ⁇ and pyridine ⁇ manufactured by Wako Pure Chemical Industries, Ltd. ⁇ were used in addition to guanidine hydrochloride.
  • Comparative Examples 2 to 4 ⁇ Comparative Examples 2 to 4>
  • the stabilizers (HN-2) to (HN-4) were used in the same manner as Comparative Example 1 except that propylene glycol was used in the composition and amount shown in Table 2.
  • glycerin ⁇ manufactured by Wako Pure Chemical Industries, Ltd. ⁇ and trehalose ⁇ manufactured by Wako Pure Chemical Industries, Ltd. ⁇ which are conventional stabilizers, were used, respectively.
  • Stabilizers (N-1) to (N-15) of Examples 1 to 15 and stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 4 were each 50 mmol / L Tris buffer.
  • ⁇ Made by Wako Pure Chemical Industries, Ltd. ⁇ (pH 7)
  • each cellulase ⁇ manufactured by Wako Pure Chemical Industries, Ltd. ⁇ -1) to (S-15) and (HS-1) to (HS-4) were obtained, respectively.
  • the obtained cellulase aqueous solution was sealed in a thermostat at 25 ° C.
  • protease aqueous solution was sealed in a thermostatic chamber at 25 ° C. for 2 days, 5 days, 2 weeks, and 1 month, and the enzyme activity was measured by the following method. 1 mL each of protease aqueous solution was measured into a 15 mL test tube, and 0.5 wt% casein solution (0.5 g of casein manufactured by Wako Pure Chemical Industries, Ltd. dissolved in 100 mL of 0.05 mol / L Tris buffer) 5 mL was added and left at 25 ° C. for 10 minutes.
  • casein solution 0.5 wt% casein solution (0.5 g of casein manufactured by Wako Pure Chemical Industries, Ltd. dissolved in 100 mL of 0.05 mol / L Tris buffer) 5 mL was added and left at 25 ° C. for 10 minutes.
  • TCA solution trichloroacetic acid ⁇ manufactured by Wako Pure Chemical Industries, Ltd. ⁇ was dissolved in ion-exchanged water
  • the mixture was treated with a centrifuge (manufactured by KUBOTA, cooling centrifuge 3922) at 2500 rpm for 20 minutes, and 2 mL of the remaining supernatant was measured into another 15 mL test tube.
  • Stabilizers (N-1) to (N-15) of Examples 1 to 15 and stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 4 were each 50 mmol / L Tris buffer.
  • ⁇ Wako Pure Chemical Industries, Ltd. ⁇ (pH 7)
  • each lipase ⁇ manufactured by Wako Pure Chemical Industries, Ltd. ⁇ 5.0 mg is added and mixed uniformly to obtain an aqueous lipase solution (L -1) to (L-15) and (HL-1) to (HL-4) were obtained, respectively.
  • Stabilizers (N-1) to (N-15) of Examples 1 to 15 and stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 4 were each 50 mmol / L Tris buffer.
  • ⁇ Made by Wako Pure Chemical Industries, Ltd. ⁇ (pH 7) In addition to 1.0 mL, uniformly mixed. Further, 5.0 mg of lipase ⁇ made by Wako Pure Chemical Industries, Ltd. ⁇ is added and mixed uniformly, and an amylase aqueous solution (AM -1) to (AM-15) and (HAM-1) to (HAM-4) were obtained, respectively.
  • the obtained amylase aqueous solution was sealed in a thermostat at 25 ° C.
  • Example 16 to 35 Organic compound (A), compound (B), enzyme (C), surfactant (D), alkaline agent (L) and water in the proportions of Table 3 (in Table 3, the unit of each component represents wt%) Were mixed at 25 ° C. to obtain liquid detergent compositions of Examples.
  • RI represents the reflectance of the cleaning cloth
  • RW represents the reflectance of the cleaning cloth
  • RS represents the reflectance of the contaminated cloth.
  • the used wet artificial soiling cloth is a wet artificial soiling cloth (reflectance at 540 nm of 40 ⁇ 5%) manufactured by the Japan Laundry Science Association having the soil composition shown in Table 5. The reflectance of the clean cloth at 540 nm is 82.5 ⁇ 0.2%.
  • the liquid detergent compositions of Examples 16 to 19 containing the organic compound (A) showed a significant reduction in detergency after storage at 25 ° C. for 3 months. It can be seen that can be maintained. Further, in Examples 20 to 31 containing the compound (B), it can be seen that the detergency is hardly lowered and the detergency can be maintained for a long time even after storage at 25 ° C. for 3 months. In Examples 32 to 35 in which a plurality of enzymes are used in combination, the detergency is hardly lowered, and the detergency is further improved as compared with the case where the enzyme is used alone.
  • the protein aqueous solution stabilizer of the present invention stabilizes the protein and does not decrease the activity for a long period of time, it can be used effectively in the fields of pharmaceuticals, foods, detergents and biochemistry.
  • it can be used for protein pharmaceutical liquid preparations, enzyme liquid preparations, industrial enzyme aqueous solutions, liquid detergents, beverages, measuring reagents for diagnostic agents, protein standard solutions, and the like.
  • the liquid detergent composition of the present invention has good enzyme activity sustainability and can maintain cleanability for a long period of time, and can be used particularly for liquid detergent compositions for clothing.

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Abstract

L'invention porte: sur un stabilisateur de solution aqueuse de protéines pouvant empêcher la détérioration des propriétés physiologiques de protéines due à l'agrégation, ou analogue, des protéines présentes dans la solution aqueuse, et pouvant stabiliser la solution aqueuse de protéines pendant une longue période; et sur un détergent liquide dans lequel l'activité enzymatique d'une enzyme peut se maintenir de manière satisfaisante, et dont les performances de lavage peuvent se maintenir pendant une longue période. Le stabilisateur de la solution aqueuse de protéines contient un composé organique (A) qui satisfait aux conditions 1 à 3 suivantes: (1) quand un proton est ajouté à la molécule du composé organique (A), une liaison p formée d'un électron p d'au moins un atome (Z) de la molécule et d'un d'électron p d'au moins un atome (Y) lié à l'atome (Z), présente une structure résonante dans l'atome (Z), et il existe au moins quatre électrons p partagés entre l'atome (Z) et l'atome (Y), et impliqués dans la liaison p présentant la structure résonante; (2) une solution à 0,1 moldm-3 du composé organique (A) a une force ionique de 0,1 ou plus; et (3) le composé organique (A) a un poids moléculaire de moins de 300.
PCT/JP2010/064398 2009-08-31 2010-08-25 Stabilisateur de solution aqueuse de protéines, solution aqueuse de protéines et détergent liquide contenant tous deux le stabilisateur, et méthode de stabilisation de protéines utilisant le stabilisateur Ceased WO2011024862A1 (fr)

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WO2014115804A1 (fr) * 2013-01-25 2014-07-31 ライオン株式会社 Préparation enzymatique pour détergent, détergent liquide et procédé de fabrication correspondant
US11633476B2 (en) 2017-05-02 2023-04-25 Merck Sharp & Dohme Llc Stable formulations of programmed death receptor 1 (PD-1) antibodies and methods of use thereof
US11845798B2 (en) 2017-05-02 2023-12-19 Merck Sharp & Dohme Llc Formulations of anti-LAG3 antibodies and co-formulations of anti-LAG3 antibodies and anti-PD-1 antibodies
US12319735B2 (en) 2018-11-07 2025-06-03 Merck Sharp & Dohme Llc Co-formulations of anti-LAG3 antibodies and anti-PD-1 antibodies
US12509509B2 (en) 2012-12-13 2025-12-30 Merck Sharp & Dohme Llc Solution formulations of engineered anti-IL-23p19 antibodies

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