JP2014088534A - Liquid detergent composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid detergent composition capable of efficiently suppressing a protease activity in the liquid detergent composition during storage even in high concentration of the liquid detergent composition, of which reduction of detergency is low even when a long period passes.SOLUTION: A liquid detergent composition contains protease (A), a following protease activity inhibitor (B), a surfactant (C) and a solvent (D), and the content of (A), (B), (C) and (D) is (A) of 0.00001 to 0.1 wt.%, (B) of 0.0000001 to 5 wt.%, (C) of 36 to 94 wt.% and (D) of 0.9 to 63.9999899 wt.% on the basis of the total weight of (A), (B), (C) and (D). The protease activity inhibitor (B): a protease activity inhibitor having an amino acid sequence of SEQ ID NO:1.

Description

  The present invention relates to a liquid detergent composition.

Protease is a general term for a group of enzymes that catalyze the hydrolysis of peptide bonds, and is widely known in microorganisms, animals and plants. Its application fields include clothing cleaners, automatic dishwasher cleaners, contact lens cleaners, bathing agents, exfoliating cosmetics, food modifiers (baking, meat softening, marine processing, etc. ), Beer fining agents, leather tanning agents, photographic film gelatin removers, digestion aids and anti-inflammatory agents, and the like, are actively used in many fields.
As is well known, enzymes such as protease are now indispensable for detergents due to their high degradability. On the other hand, the demand for detergents is changing from conventional solid detergents to liquid detergents in terms of ease of use. However, the protease hydrolyzes the protease itself and other enzymes in the liquid detergent, so that the activity of the protease itself and other enzymes is remarkably lowered with time, and the detergency of the liquid detergent is remarkably lowered. is there. Therefore, in order to suppress hydrolysis of the protease itself and other proteins, research on protease activity inhibitors that inhibit protease activity has been conducted. For example, it is known that boronic acid inhibits the activities of serine protease and subtilisin (for example, Non-Patent Document 1 and Patent Document 1).
However, the liquid detergent whose demand is currently increasing is a liquid detergent containing a high concentration (above 35% by weight) of a surfactant, and a low concentration (below 35% by weight) of a normal surfactant. Compared with liquid detergents, protease autolysis is faster. For this reason, even if boronic acid is used, protease degradation cannot be effectively inhibited, and a method that can sufficiently inhibit protease activity during storage has not been developed for liquid detergents with high surfactant concentrations. Therefore, it is desired to develop a high-concentration liquid detergent with a small deterioration in detergency.

Japanese National Patent Publication No. 11-507680

Molecular & Cellular Biochemistry, 51, 1983, p5-p32

  Therefore, the present invention can sufficiently suppress the protease activity in the liquid detergent composition at the time of storage even in a high concentration liquid detergent composition, and the liquid detergent composition has a small decrease in detergency even after a long period of time from preparation. The issue is to provide goods.

As a result of studies to achieve the above object, the present inventors have completed the present invention. That is, the liquid detergent composition of the present invention is a liquid detergent composition containing protease (A), the following protease activity inhibitor (B), surfactant (C) and solvent (D), , (B), (C), and (D), the content of (A) is 0.00001 to 0.1 based on the total weight of (A), (B), (C), and (D). The gist is that the weight percent is 0.0000001 to 5 wt%, (C) is 36 to 94 wt%, and (D) is 0.9 to 63.9999899 wt%.
Protease activity inhibitor (B): Protease activity inhibitor having the amino acid sequence of SEQ ID NO: 1.

  The liquid detergent composition of the present invention has a small decrease in detergency even after a long period of time since its preparation.

The liquid detergent composition of the present invention is a liquid detergent composition containing protease (A), the following protease activity inhibitor (B), surfactant (C) and solvent (D),
The content of (A), (B), (C) and (D) is 0.00001 to (A) based on the total weight of (A), (B), (C) and (D). The liquid detergent composition is 0.1% by weight, (B) is 0.0000001 to 5% by weight, (C) is 36 to 94% by weight, and (D) is 0.9 to 63.9999899% by weight.
Protease activity inhibitor (B): Protease activity inhibitor having the amino acid sequence of SEQ ID NO: 1.

  In the present invention, as the protease (A), one generally known as a protease can be used, and a low temperature optimal protease having an optimal temperature for protease activity at a low temperature (0 to 50 ° C.) and a high temperature (50 ° C.). A high temperature optimal protease having an optimal temperature. (A) is preferably a serine protease, more preferably subtilisin, from the viewpoint of effectiveness of activity. Examples of commercially available products include Alkalase, Sabinase, Everase, PTN manufactured by Novozymes, and Purefect and Purefect OXP manufactured by Genencor.

In the present invention, the protease activity inhibitor (B) is a protease activity inhibitor having the amino acid sequence of SEQ ID NO: 1.
(B) includes a protease activity inhibitor (B-1) extracted from a natural product and a protease activity inhibitor (B-2) produced by utilizing a gene recombination technique.

The protease activity inhibitor (B-1) extracted from a natural product is a protease activity inhibitor having the amino acid sequence of SEQ ID NO: 1, and is a natural product (for example, plant or animal cell, extracellular tissue, seed, A protease activity inhibitor extracted from the inside and outside secretion of microorganisms).
The extraction method includes steps of separating general proteins from natural products such as disruption of cell walls and cell membranes, centrifugation, ammonium sulfate fractionation, chromatography and dialysis.
The amino acid sequence of (B-1) can be determined by a general method such as a peptide mapping method.
In addition, whether or not a protein extracted from a natural product falls under (B-1) is determined by determining the amino acid sequence of the extracted protein by a general method such as a peptide mapping method, and providing homology provided by National Center for Biotechnology Information. Using the blastp algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the sex analysis program “BLAST”, it is confirmed whether it matches the amino acid sequence of SEQ ID NO: 1.

  As a protease activity inhibitor (B-2) produced by the host, a gene transformed by the genetic recombination method described in Japanese Patent No. 3338441 is used to transform the appropriate host, and the recombinant host is cultured. And those obtained by harvesting from the culture. Specifically, by cloning a gene sequence containing a gene sequence encoding the protein whose amino acid sequence is SEQ ID NO: 1, transforming into a suitable host, culturing the recombinant host, and collecting from the culture can get.

  For gene cloning, a general gene recombination technique may be used, and examples include a cDNA library method and a method using an artificially synthesized gene. The gene mutation means may be a generally used site-specific mutation method, and specifically includes a method using Quick Change Site-Directed Mutagenesis Kit (Agilent Technology).

  The method for producing (B-2) using the above mutant gene uses a recombinant vector in which the above mutant gene is incorporated in any vector suitable for expressing the gene in the target host. To obtain a transformant containing the recombinant vector. What is necessary is just to culture | cultivate the obtained transformant and extract | collect (B-2) from the said culture solution.

In the present invention, the recombinant vector can be obtained by inserting the mutant gene into an appropriate vector.
Various vectors are known and there are many commercially available products. A person skilled in the art can easily select an appropriate vector according to the type of host. Specific examples of vectors include the pET series and the pUC series.
The preparation method of the recombinant vector itself is a well-known conventional method. Specific methods for inserting a mutated gene into an appropriate vector and transforming a host include an electroporation method and a calcium method.

Examples of the host in the present invention include animal cells, microorganisms and plant cells.
Examples of animal cells include, but are not limited to, insect cells, monkey cells COS-7, Vero, mouse L cells, rat GH3, human FL cells, and CHO cells.
Insect cells include, but are not limited to, Sf9 cells and Sf21 cells.
Although it does not specifically limit as microorganisms, Bacteria, yeast, etc. are mentioned.
Bacteria include eubacteria and archaea.
Eubacteria include gram negative bacteria and gram positive bacteria. Examples of gram-negative bacteria include Escherichia, Thermus, Rhizobium, Pseudomonas, Shewanella, Vibromo, Vibromo, Vibromo Examples include Salmonella, Acetobacter genus, Synechocystis genus, and the like. Gram-positive bacteria include the genus Bacillus (genus Bacillus), the genus Streptomyces (genus Streptomyces), the genus Corynebacterium (genus Corynebacterium), the genus Brevibacillus, the genus Bifidobacterium, and the genus Bifidobacterium. Examples include the genus Coccus (genus Lactococcus), the genus Enterococcus (genus Enterococcus), the genus Pediococcus (genus Pediococcus), the genus Leuconostoc (genus Leuconostoc) and the genus Streptomyces (genus Streptomyces).
Although it does not specifically limit as a plant cell, BY-2 cell etc. are mentioned.

  In the present invention, the host is preferably a microorganism from the viewpoint of ease of cloning, and more preferably Escherichia, Thermus, Rhizobium, Pseudomonas. , Shewanella, Vibrio, Salmonella, Acetobacter and Synechocystis, particularly preferably Escherichia escherichia These are the genus Shewanella, the genus Bacillus (genus Bacillus), and the genus Brevibacillus (genus Brevibacillus).

  The culture may be carried out by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients that can assimilate microorganisms, and following a conventional method.

  In the present invention, the method for collecting and purifying (B-2) from the culture solution can be performed according to a conventional method. For example, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be prepared from the obtained culture supernatant by conventional means. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.

  Surfactant (C) is nonionic surfactant (C-1), anionic surfactant (C-2), cationic surfactant (C-3) and amphoteric surfactant (C-4). Is mentioned.

  As nonionic surfactant (C-1), alkylene oxide addition type nonionic surfactant (C-1-1), polyhydric alcohol type nonionic surfactant (C-1-2) and Fatty acid alkanolamide (C-1-3) etc. are mentioned.

  As (C-1-1), higher alcohol (8 to 18 carbon atoms) alkylene (2 to 4 carbon atoms, preferably 2) oxide adduct (1 to 30 added moles per active hydrogen), Alkyl (carbon number 1-12) phenol ethylene oxide (hereinafter abbreviated as EO) adduct (addition mole number 1-30), higher amine (carbon number 8-22) alkylene (carbon number 2-4, preferably 2 ) Oxide adduct (addition moles per active hydrogen 1-40), fatty acid (carbon number 8-18) EO adduct (addition moles 1-60 active hydrogen), polypropylene glycol (molecular weight 200) -4000) EO adduct (addition mole number per active hydrogen 1-50), polyoxyethylene (degree of polymerization = 3-30) alkyl (carbon number 6-20) allyl ether and sorbita Multivalent (2 to 8 valences) such as monolaurate EO adduct (1-30 added moles per active hydrogen) and sorbitan monooleate EO adduct (1-30 added moles per active hydrogen) Or more) Fatty acid (carbon number 8-24) ester EO adduct (number of added moles per active hydrogen 1-30) of alcohol (carbon number 2-30).

  As (C-1-2), fatty acids of polyvalent (2 to 8 or more) alcohols (2 to 30 or more carbon atoms) such as glycerol monostearate, glycerol monooleate, sorbitan monolaurate and sorbitan monooleate (C8-24) ester etc. are mentioned.

  (C-1-3) includes alkanolamides of fatty acids having 12 or more carbon atoms, and specific examples include coconut oil fatty acid diethanolamide, lauric acid diethanolamide, and lauric acid monoethanolamide.

  Examples of the anionic surfactant (C-2) include alkyl (carbon number 8 to 24) ether carboxylic acid or a salt thereof, alkyl (carbon number 8 to 24) (poly) oxyethylene ether carboxylic acid or a salt thereof [(poly ) Oxyethylene (degree of polymerization = 1-100) sodium lauryl ether acetate and (poly) oxyethylene (degree of polymerization = 1-100) disodium lauryl sulfosuccinate], alkyl (carbon number 8-24) sulfate, alkyl (Carbon number 8-24) (poly) oxyethylene sulfate ester salt [sodium lauryl sulfate, lauryl (poly) oxyethylene (degree of polymerization = 1-100) sodium sulfate and lauryl (poly) oxyethylene (degree of polymerization = 1-100) ) Sulfuric acid-triethanolamine salt, etc.], palm oil fatty acid monoethanolamide sulfate sulfone Sodium, alkyl (carbon number 8-24) phenyl sulfonate [sodium dodecylbenzenesulfonate, etc.], alkyl (carbon number 8-24) phosphate ester salt, alkyl (carbon number 8-24) (poly) oxyethylene phosphorus Acid ester salts [sodium lauryl phosphate and (poly) oxyethylene (degree of polymerization = 1-100) sodium lauryl ether phosphate, etc.], fatty acid salts [sodium laurate, triethanolamine laurate, etc.], acylated amino acid salts [ Palm oil fatty acid methyl taurine sodium, palm oil fatty acid sarcosine sodium, coconut oil fatty acid sarcosine triethanolamine, N-coconut oil fatty acid acyl-L-glutamic acid triethanolamine, N-coconut oil fatty acid acyl-L-glutamic acid sodium and lauroyl Methyl-β- Alanine sodium, etc.].

  As the cationic surfactant (C-3), quaternary ammonium salt type [stearyl trimethyl ammonium chloride, behenyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride and ethyl lanolin sulfate fatty acid aminopropylethyl dimethyl ammonium etc.] and amine salts Type [diethylaminoethylamide stearate lactate, dilaurylamine hydrochloride, oleylamine lactate, etc.] and the like.

  As the amphoteric surfactant (C-4), a betaine-type amphoteric surfactant [coconut oil fatty acid amidopropyl dimethylaminoacetic acid betaine, lauryldimethylaminoacetic acid betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazoli Nium betaine, lauryl hydroxysulfo betaine, lauroylamidoethyl hydroxyethyl carboxymethyl betaine hydroxypropyl sodium phosphate and the like] and amino acid type amphoteric surfactants [sodium β-laurylaminopropionate and the like].

  As (C), 1 type (s) or 2 or more types can be used. When two or more kinds are used, the combination includes, for example, a combination of a nonionic surfactant and an anionic surfactant, a nonionic surfactant and a cationic surfactant, and a nonionic surfactant and an amphoteric surfactant. Etc.

As (C), 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.
The nonionic surfactant is preferably an aliphatic alcohol (8 to 24 carbon atoms) EO adduct (degree of polymerization = 1 to 100), more preferably an aliphatic alcohol (12 to 18 carbon atoms) from the viewpoint of detergency. EO adduct (degree of polymerization = 4-20), then more preferably aliphatic alcohol (carbon number 12-15) EO adduct (degree of polymerization = 8-12), particularly preferably oleyl alcohol EO11 mol adduct is there.
As an anionic surfactant, from a viewpoint of detergency, an alkylphenyl sulfonate having 8 to 24 carbon atoms, a fatty acid salt, an alkyl sulfate ester having 8 to 24 carbon atoms, and an alkyl (poly) having 8 to 24 carbon atoms. Preferred are oxyethylene sulfate salts, more preferably alkyl phenyl sulfonates having 12 to 16 carbon atoms and fatty acid salts having 8 to 16 carbon atoms, and more preferably dodecylbenzenesulfonic acid monoethanolamine salts and lauric acid. Sodium.

As the solvent (D), water and hydrophilic solvents {monoalcohols having 1 to 3 carbon atoms (methanol, ethanol, isopropyl alcohol, etc.), polyols having 1 to 10 carbon atoms (ethylene glycol, diethylene glycol, propylene glycol, etc.)} and These mixtures etc. are mentioned.
The solvent (D) is preferably water from the viewpoint of detergency.
Moreover, it does not specifically limit as water, A tap water, ion-exchange water, distilled water, reverse osmosis water, etc. are mentioned. Moreover, the buffer aqueous solution etc. which contain a pH adjuster in water are mentioned.
As the pH adjuster, a conventional pH adjuster can be used, borate buffer, phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, sulfuric acid, hydrochloric acid, citric acid, lactic acid, pyruvic acid, formic acid, sodium chloride, chloride. Examples include potassium, monoethanolamine and diethanolamine.

The content (% by weight) of protease (A) contained in the liquid detergent composition of the present invention is 0.00001 to 0.1, and preferably 0.0001 to 0.01 from the viewpoint of detergency. .
The content (% by weight) of the protease activity inhibitor (B) is 0.0000001 to 5, preferably from 0.00001 to 3, more preferably from 0.001 to 3, from the viewpoint of maintaining the detergency for a long period of time. 3.
The content (% by weight) of the surfactant (C) is 36 to 94, preferably 40 to 90, particularly preferably 61 to 80, from the viewpoint of detergency.
The content (% by weight) of the solvent (D) is 0.9 to 63.9999899, and from the viewpoint of detergency, it is preferably 6.99 to 59.99989, particularly preferably 1699 to 38.9899. is there.
The molar ratio of the protease activity inhibitor (B) to the protease (A) {number of moles of (B) / number of moles of (A)} is preferably from 0.01 to 30 from the viewpoint of maintaining the washability for a long period of time. More preferably, it is 0.01-10, Most preferably, it is 0.3-3.

  If necessary, the liquid detergent composition may contain other known components such as builders, chelating agents, hydrophilic solvents, antifoaming agents, fluorescent whitening agents, bleaching agents and softening agents described in JP-A-2004-27181. Further, it may contain a disinfectant, a fragrance, a colorant and the like.

  Builders include polycarboxylates (such as acrylate and maleate homopolymers), polyvalent carboxylates (such as citric acid and malic acid), and alkali builders (caustic soda, soda ash, ammonia, triethanol, etc.) Amine, sodium tripolyphosphate and sodium silicate). Examples of the chelating agent include EDTA and NTA. Examples of antifoaming agents include silicone-based antifoaming agents, polyoxyalkylene-based antifoaming agents, and mineral oil-based antifoaming agents.

Among the other components, the content of the builder and the chelating agent is preferably 10% by weight or less, more preferably 5% by weight or less, based on the total weight of the liquid detergent composition, the builder and the chelating agent.
The content of optical brightener, bleach, softener, enzyme, disinfectant, fragrance, colorant and antifoaming agent is liquid detergent composition, optical brightener, bleach, softener, disinfectant, Preferably it is 5 weight% or less based on the total weight of a fragrance | flavor, a coloring agent, and an antifoamer, More preferably, it is 2 weight% or less.

  In the liquid detergent composition, in addition to the above, if necessary, salt (G), sugar (H), amino acid (I), fatty acid (K), oil (L), other low molecular organic compounds (J) and protease Protein (M) other than can be contained.

  Examples of the salt (G) include inorganic salts {sodium chloride, sodium borate, calcium chloride, magnesium chloride, magnesium sulfate, ammonium sulfate, etc.} and organic salts (sodium formate, etc.).

  Examples of the sugar (H) include trehalose, sucrose, dextrin, cyclodextrin, maltose, fructose, hyaluronic acid and chondroitin sulfate.

  As amino acid (I), glycine, alanine, aspartic acid, asparagine, phenylalanine, tryptophan, tyrosine, leucine, lysine, histidine, cysteine, glutamine, glutamic acid, isoleucine, methionine, proline, serine, threonine, valine and their salts Can be mentioned.

  Examples of the fatty acid (K) include oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid.

  Examples of the oil (L) include monoglycerides, diglycerides, and triglycerides of the above fatty acids (K).

  Examples of other low molecular weight organic compounds (J) include benzyl acetate, methyl salicylic acid, benzyl salicylic acid, hydroxybenzoic acid, cinnamic acid, caffeic acid, catechins, ascorbic acid and carotenoids.

  The protein (M) other than protease is not particularly limited, and examples include enzymes other than protease, recombinant proteins, antibodies and peptides. Specifically, enzymes other than protease {cellulase (endolase etc.), Amylase, lipase, peroxidase and the like}, serum albumin, collagen, casein, gelatin, silk peptide and the like.

The content (% by weight) of the salt (G) contained in the liquid detergent composition of the present invention is preferably from 0 to 10, more preferably from 0 to 5, especially from the weight of the liquid detergent composition from the viewpoint of detergency. Preferably it is 0-3.
The content (% by weight) of sugar (H) contained in the liquid detergent composition of the present invention is preferably 0 to 50%, more preferably 0 to 30%, based on the weight of the liquid detergent composition from the viewpoint of detergency. Especially preferably, it is 0 to 20%.
From the viewpoint of detergency, the content (% by weight) of amino acid (I) contained in the liquid detergent composition of the present invention is preferably 0 to 50%, more preferably 0 to 30%. Especially preferably, it is 0 to 20%.
The content (% by weight) of other low-molecular organic compounds (J) contained in the liquid detergent composition of the present invention is preferably 0 to 50% with respect to the weight of the liquid detergent composition from the viewpoint of detergency, and Preferably it is 0 to 30%, particularly preferably 0 to 20%.
The content (% by weight) of the fatty acid (K) contained in the liquid detergent composition of the present invention is preferably 0 to 50%, more preferably 0 to 30% based on the weight of the liquid detergent composition from the viewpoint of detergency. Especially preferably, it is 0 to 20%.
From the viewpoint of detergency, the content (% by weight) of the fat (L) contained in the liquid detergent composition of the present invention is preferably 0 to 50%, more preferably 0 to 30% with respect to the weight of the liquid detergent composition. %, Particularly preferably 0 to 20%.
The content (% by weight) of protein (M) other than protease contained in the liquid detergent composition of the present invention is preferably from 0 to 50%, more preferably from the weight of the liquid detergent composition, from the viewpoint of detergency. 0 to 30%, particularly preferably 0 to 20%.

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) Into a mixing tank equipped with a stirrer and a heating / cooling device, the surfactant (C), the solvent (D) and other components are charged without any particular limitation in the charging order, until uniform at 20 to 50 ° C. Stir.
(2) Add protease activity inhibitor (B) and stir at 20-50 ° C. for 30 minutes to 2 hours.
(3) Protease (A) is added and further stirred at 20-50 ° C. until uniform.

  The liquid detergent composition of the present invention can be used as a detergent for clothing, a detergent for automatic dishwashers, a detergent for contact lenses, and the like.

The method for using the liquid detergent composition may be the same as the method for using the conventional liquid detergent composition, and is not particularly limited. An example of usage as a cleaning agent for clothing 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.

  The present invention will be further described with reference to the following examples and comparative examples, but the present invention is not limited thereto.

<Production Example 1: Preparation of plasmid expressing protease activity inhibitor (B)>
Gene encoding the amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 5) (restriction enzyme NcoI added to the 5 ′ end and restriction enzyme BamHI recognition sequence added to the 3 ′ end, and artificial synthesis was requested from Hokkaido System Science Co., Ltd. ) Were treated with restriction enzymes NcoI and BamHI to bind to the NcoI restriction enzyme site and the BamHI restriction enzyme site of the pET-22b plasmid (Novagen), thereby preparing a plasmid (P1) that expresses the protein of SEQ ID NO: 2. Using this plasmid and a primer for mutagenesis (SEQ ID NO: 3 and 4) for substituting methionine at position 48 with glycine, mutagenesis was performed on the plasmid (P1) under the following conditions. Specifically, 0.5 μL (10 ng) of plasmid (P1), 0.75 μL (7.5 pg) of each 10 μM primer for mutagenesis, 2.5 μL of 10 times concentration buffer for PCR (manufactured by Takara Bio Inc.), 2 mM deoxynucleotide After mixing 2 μL of 3-phosphate (dNTP) mixture (Takara Bio), DNA polymerase ExTaq 0.25 μL (Takara Bio) and 17 μL of deionized water, PCR was performed using TaKaRa Thermal Cycler Dice (Takara Bio). The reaction conditions were heat denaturation at 94 ° C. for 2 minutes, followed by 30 cycles of 98 ° C. for 10 seconds, 50 ° C. for 10 seconds, and 68 ° C. for 6.5 minutes. After purifying the obtained PCR product with QIAquick Gel Extraction Kit (manufactured by Qiagen) (50 μL), 6 μL of 10 times concentrated DpnI buffer solution and 3 μL of restriction enzyme DpnI (manufactured by Takara Bio) were added for 1 hour at 37 ° C. The template was disassembled. The obtained restriction enzyme-treated PCR product (5 μL) was subjected to transformation into E. coli DH5α. That is, 5 μL of restriction enzyme-treated PCR product was converted to 100 μL of E. coli. It was added to Coli DH5α competent cell (manufactured by TOYOBO), stored on ice for 30 minutes, and then heated at 42 ° C. for 90 seconds. Here, 900 μL of SOC medium (manufactured by TOYOBO) was added, followed by static culture at 37 ° C. for 1 hour. 100 μL of the culture solution was applied to an LB / ampicillin plate and cultured overnight at 37 degrees. The appearing colonies were cultured in 1 mL of LB medium for 12 hours, and then subjected to Quantumprep Miniprep Kit (manufactured by Bio-Rad) to purify the plasmid (P2) expressing the protease activity inhibitor (B) of SEQ ID NO: 1 (100 μL) . The obtained plasmid was analyzed by a DNA sequence analysis service (Macrogen Japan), and it was confirmed that the mutation was introduced.

<Production Example 2: Production of protease activity inhibitor (B)>
The protease activity inhibitor expression plasmid (P2) obtained in Production Example 1 was transformed into E. coli E. coli. Coli BL21 (DE3) was transformed in the same manner. The obtained protease activity inhibitor-expressing strain was inoculated into 1 mL of LB culture solution (containing 100 mg / L of ampicillin) and cultured at 30 ° C. for 12 hours to prepare an overnight culture solution. 0.5 ml of LB culture solution ( 5 ml of ampicillin (containing 100 mg / L) was inoculated and cultured with shaking at 30 ° C. for 3 hours to prepare a preculture solution. The pre-culture solution is 50 mL of culture solution {the content of each component in 50 mL of water is 1.2 g of yeast extract (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.6 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) 47 g, monopotassium phosphate 0.11 g, ammonium sulfate 0.35 g, disodium phosphate 12 hydrate 0.66 g, sodium citrate dihydrate 0.02 g, glycerol 0.2 g, lactalbumin hydrolyzate 1.5 g Defoaming agent (manufactured by Shin-Etsu Silicone, “KM-70”) 0.3 g, 1 mM magnesium sulfate, trace metal solution (calcium chloride 18.9 μg, iron (III) chloride 500 μg, zinc sulfate heptahydrate 9.0 μg, Copper sulfate 5.1 μg, manganese chloride tetrahydrate 6.7 μg, cobalt chloride 4.9 μg, ethylenediaminetetraacetic acid tetrasodium 200 μg), 100 mg / L Was inoculated into Pishirin} microbial culture apparatus and culture was performed while maintaining the pH6.8,30 ℃ by using the (Able Co., Ltd., product name "BioJr.8"). After the start of culture, 0.15 mL of 1M IPTG solution was added. From 14 hours after the start of the culture, glycerol / protein solution (50% glycerol, 50 g / L lactalbumin hydrolyzate, 33 g / L antifoam (manufactured by Shin-Etsu Silicone, “KM-70”), 100 mg / L ampicillin) was added dropwise. Started. The culture solution (Q-1) was collected 48 hours after the start of the culture.
The obtained culture solution (Q-1) was separated with a carrier for purification of His-tag (Ni Sepharose 6 Fast Flow manufactured by GE Healthcare) to obtain a solution (R-1) of a protease activity inhibitor (B). . The amount of the protease activity inhibitor (B) in (R-1) was measured by SDS-PAGE and found to be 1 g / L. Moreover, it was about 8000 when the molecular mass of (B) was calculated | required by SDS-PAGE.

<Examples 1-9>
It mix | blended at 30 degreeC in the ratio of Table 1, and obtained the liquid detergent composition (S-1)-(S-9) of this invention.

<Comparative Examples 1-3>
The liquid detergent composition (S'-1)-(S'-3) for a comparison was obtained by mix | blending at the ratio of Table 1 at 30 degreeC.

In Table 1, the following was used for protease (A) and other proteins (M).
○ Protease (A)
Sabinase aqueous solution: manufactured by Novozymes, trade name “Sabinase 16L”, molecular mass: 26700, estimated by SDS-PAGE to have a sabinase content of 1 g / L ○ Other proteins (M)
Endolase aqueous solution: manufactured by Novozymes, trade name “Endolase”, SDS-PAGE estimated endrase content to be 1 g / L

<Measurement of residual activity of protease after dilution after storage of liquid detergent composition at 37 ° C.>
About the liquid detergent compositions obtained in Examples and Comparative Examples, the liquid detergent compositions (S-1) to (S-9) and (S′-1) to (S′−) after storage at 37 ° C. for 1 week. 3) is diluted with 10 μL, 9990 μL of 0.1 M Tris / HCl + 1 mM CaCl 2 buffer (pH 8, 25 ° C.), and solutions (T-1) to (T-9) and (T′-1) to (T′-3) are diluted. ). To 700 μL of the solutions (T-1) to (T-9) and (T′-1) to (T′-3), 50 mg / mL of azocasein (0.1 M Tris / HCl + 1 mM CaCl ₂ buffer (pH 8, 25 ° C.)) The solution (U-1) to (U-9) and (U′-1) to (U′-3) were prepared by adding 70 μL each of the lysate and Nacalai Tesque), and the enzyme reaction was started. Immediately after preparing solutions (U-1) to (U-9) and (U′-1) to (U′-3) and three times every 15 minutes, 150 μL of the reaction solution was taken out, and a 15% trichloroacetic acid solution Mixed with 200 μL. The mixture was centrifuged at 15,000 × g for 5 minutes, 400 μL of 0.1N NaOH was added to the supernatant, and the absorbance at 405 nm was measured using a spectrophotometer. The absorbance immediately after the preparation of the solutions (U-1) to (U-9) and (U′-1) to (U′-3) is Aλ ₀ and the solutions (U-1) to (U-9) and (U '-1) to (U'-3) are plotted with the absorbance after A hour being Aλ _h , the change in absorbance ΔAλ (ΔAλ = Aλ _h −Aλ ₀ ) on the vertical axis, and the time h plotted on the horizontal axis. Thus, the slope of the straight line (coefficient v ₁ ) was obtained. Incidentally, as a blank, creating liquid detergent composition immediately after Comparative Example 1 (S'-1) the slope of the straight line was determined using the (coefficient v ₁₎ as the slope of the straight line (coefficient v _0), the following equation (1 Table 1 shows the results of the determination of the residual activity of the proteases of the liquid detergent compositions (S-1) to (S-9) and (S′-1) to (S′-3).
Residual activity (%) after storage at 37 ° C. for 1 week = v ₁ / v ₀ × 100 (1)

<Measurement of residual activity of protease after dilution at 37 ° C. for 3 months>
In the above “Measurement of protease activity at dilution after storage at 37 ° C. for 1 week”, “Liquid detergent composition stored at 37 ° C. for 3 months” instead of “Liquid detergent composition after storage at 37 ° C. for 1 week” In the same manner except that the product is used, the liquid detergent compositions (S-1) to (S-9) and (S′-1) to (S′-3) were subjected to residual activity after storage at 37 ° C. for 3 months ( %). The results are shown in Table 1.

<Durability of activity>
From the residual activity after storage at 37 ° C. for 1 week and the residual activity after storage at 37 ° C. for 3 months, the sustainability of the activity was calculated according to the following formula, and the results are shown in Table 1.
Persistence of activity (%) = {remaining activity after storage at 37 ° C. for 3 months} / {remaining activity after storage at 37 ° C. for 1 week} × 100

  From the results of Table 1, the liquid detergent compositions of Comparative Examples 1 to 3 have a sustained activity of 50% or less and the protease activity is halved, whereas the liquid detergent compositions of the present invention of Examples 1 to 9 It can be seen that the activity persistence is as high as 62% or more and the protease activity is difficult to decrease.

<Detergency test>
<Detergency after storage at 37 ° C for 1 week>
About the liquid detergent compositions obtained in Examples and Comparative Examples, the liquid detergent compositions (S-1) to (S-9) and (S′-1) to (S′−) after storage at 37 ° C. for 1 week. Immediately after the preparation of 3), 0.8 g of the liquid detergent composition was dissolved in 999.2 g of water to obtain a solution. Into this solution, 5 wet artificially contaminated cloths (4 cm × 4 cm) were added, washed and rinsed under the following conditions using a targotometer (manufactured by Daiei Chemical Co., Ltd.), then taken out of the cloth, and a gear oven ( A test cloth was obtained by drying at 70 ° C. for 60 minutes using GPS-222 manufactured by Tabai. Next, using a multi-light source spectrocolorimeter (manufactured by Suga Test Instruments Co., Ltd.), the reflectance of 540 nm of this test cloth is a total of four places (total of five test cloths, two on each side). (20 places) was measured, the average value was obtained, and the detergency (%) after storage at 37 ° C. for 1 week was calculated by the following formula. The results are shown in Table 1.
(Cleaning conditions)
Time: 10 minutes, temperature: 25 ° C., rotation speed: 120 rpm
(Rinsing conditions)
Time: 1 minute, temperature: 25 ° C., rotation speed: 120 rpm
(Cleanability)
Detergency after storage at 37 ° C. for 1 week (%) = {(R _W −R _S ) / (R _I −R _S )} × 100
R _I represents the reflectance of the cleaning cloth, R _W represents the reflectance of the cleaning cloth, and R _S represents the reflectance of the contaminated cloth.
Moreover, 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 2.

<Detergency after storage at 37 ° C for 3 months>
For the liquid detergent compositions obtained in Examples and Comparative Examples, in the above <detergency after storage at 37 ° C. for 1 week>, instead of “liquid detergent composition after storage at 37 ° C. for 1 week”, “37 ° C. In the same manner except that the “liquid detergent composition after 3 months storage” was used, the detergency after storage at 37 ° C. for 3 months was calculated. The results are shown in Table 1.

<Durability of detergency>
From the cleaning properties after storage at 37 ° C. for 1 week and the cleaning properties after storage at 37 ° C. for 3 months, the sustainability of the cleaning properties was calculated according to the following formula, and the results are shown in Table 1.
Persistence of detergency (%) = {detergency after storage at 37 ° C. for 3 months} / {detergency after storage at 37 ° C. for 1 week} × 100

From the results shown in Table 1, it can be seen that the liquid detergent composition of Comparative Example 1 containing no protease activity inhibitor has a very low detergency of 53%, and the detergency was lowered after long-term storage. Moreover, it turns out that the liquid detergent composition of the comparative example 2 containing the conventional inhibitor has a low washability of 66%, and the washability is lowered by long-term storage. Further, Comparative Example 3 contains a protease activity inhibitor (B), which is outside the scope of the present invention, but has a low detergency of 63% and does not sufficiently inhibit protease activity. It can be seen that the properties are reduced.
On the other hand, Examples 1 to 9, which are liquid detergent compositions of the present invention, have a high detergency of 73% or more, and it is understood that the detergency is not easily lowered after long-term storage.

  Since the liquid detergent composition of the present invention is less likely to deteriorate after long-term storage, the detergent for clothing, the detergent for automatic dishwashers, and the cleaning for contact lenses that require a long period of time from manufacture to home use. It can be suitably used as an agent, and is particularly useful as a liquid detergent for clothing.

Claims (3)

A liquid detergent composition comprising protease (A), the following protease activity inhibitor (B), surfactant (C) and solvent (D),
The content of (A), (B), (C) and (D) is 0.00001 to (A) based on the total weight of (A), (B), (C) and (D). A liquid detergent composition comprising 0.1% by weight, (B) 0.0000001 to 5% by weight, (C) 36 to 94% by weight, and (D) 0.9 to 63.9999899% by weight.
Protease activity inhibitor (B): Protease activity inhibitor having the amino acid sequence of SEQ ID NO: 1. The liquid detergent composition according to claim 1, wherein the molar ratio of the protease activity inhibitor (B) to the protease (A) {number of moles of (B) / number of moles of (A)} is 0.01-10. Furthermore, the liquid detergent composition of Claim 1 or 2 containing protein (M) other than protease.