JP3359868B2 - Wash solution containing protease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cleaning solution containing a protease, which has high stability of the protease and maintains the enzyme activity.

[0002]

2. Description of the Related Art Proteolytic enzymes have been used in medicine, foods, cosmetics, detergents and the like together with lipolytic enzymes, and have produced excellent effects. However, proteolytic enzymes have the problem that they are denatured by heating or in the presence of a surfactant, or are easily deactivated due to self-digestion, and the stability is particularly remarkably reduced in an aqueous solution. In recent years, proteolytic enzymes, which are also incorporated in laundry detergents and household dishwashing detergents, are stable in granular detergents but unstable in liquid detergents.

[0003] In order to overcome these problems,
Several methods have been reported to increase stability by chemically modifying proteolytic enzymes. For example, JP-A-7-155182 describes that stability is enhanced when calcium is contained in a modified protease in which a polysaccharide and a protease are bonded via a triazine ring. JP-A-6-240297 proposes a method for improving the stability of lipase or amylase by modifying lipase or amylase with methyl vinyl ether and maleic anhydride copolymer in the presence of a protease. However, it is described that even if a single enzyme is modified with methyl vinyl ether and maleic anhydride copolymer, the effect is low. In addition, the publication does not describe the stability of the modified protease in the presence of various enzymes, and the stability of the protease must be further improved. In addition, in a phosphate buffer, the dissolution form of the methylvinyl ether-maleic anhydride copolymer is linear, so that the methylvinyl ether-maleic anhydride copolymer is likely to react with the protease at random. Is high. Therefore, it reacts at or near the active site of the protease, losing the effect of the active site, or reacts with the substrate due to steric hindrance of the methyl vinyl ether-maleic anhydride copolymer reacted near the active site. Tends to decrease. Furthermore, since the dissolved modifying enzyme is a linear polymer, the intermolecular distance becomes closer than that of the polymer dissolved in a string form, so that aggregation of the polymer due to entanglement between molecules is likely to occur. . When the modified enzyme is produced, such aggregation of the modified enzyme has serious problems such as precipitation and turbidity, and leads to a decrease in the stability and activity of the enzyme. For this reason, the conventional methods are not sufficient for modifying the protease to maintain the stability and the enzyme activity of the liquid enzyme for a long period of time.

Furthermore, Japanese Patent Application Laid-Open No. 1-153088 discloses that an enzyme modified with a copolymer of maleic anhydride and polyethylene glycol monoallyl ether has high stability and can be used in bioreactors and the like. In addition, JP-A-8-146363 discloses that
Although the enzyme is modified with α-allylpolyoxyalkylene and maleic anhydride copolymer, the stability of such an enzyme modified with maleic anhydride copolymer is not sufficient, and practicality cannot be said. Low. In addition, JP-A-63-15982
No. 2 and JP-A-1-180515 also propose a method of stabilizing the protease by containing a water-miscible polyhydric alcohol. However, there is a drawback in that although the stability is lowered, the stability is lowered, though it is increased by dilution with water. or,
JP-A-6-102474 describes a detergent in which a proteolytic enzyme, a lipolytic enzyme, and a polysaccharide-degrading enzyme are formed on a tablet. However, since this detergent is a tablet, a container for dissolving the tablet, There is a problem that it requires time and effort and is inferior in terms of easy use.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems in the prior art, and to provide a protease-containing cleaning solution in which the stability of the protease is high and the enzyme activity is maintained. And

[0006]

According to the present invention, there is provided a detergent solution containing a protease, wherein the protease comprises polyethylene glycol,
A modifying enzyme chemically modified by a water-soluble polymer selected from the group consisting of polysaccharides, proteins, and copolymers of a compound having a CH ₂ ＣC <group and maleic anhydride; a polyglycerol fatty acid ester and a polyglycerol fatty acid ester; At least one nonionic surfactant selected from oxyethylene alkyl phenyl ethers; anionic surfactants such as sodium myristoyl sarcosine and ammonium lauryl sulfate; and amphoteric surfactants such as alkyldimethylamine oxide and alkyldimethylaminoacetic acid At least one surfactant selected from the group consisting of betaines and a boric acid compound selected from the group consisting of boric acid, metaboric acid, tetraboric acid, borax, metaborate and tetraborate It is characterized by containing.

[0007] The cleaning solution containing the protease of the present invention comprises:
In the above-mentioned feature, the modified enzyme is contained at a ratio of 0.1 to 10% by weight as a modified enzyme solution having an enzyme activity value per unit volume of 10,000 to 200,000 U / ml, and further comprises the modified enzyme The weight ratio of the solution: each of the surfactants and the boric acid compound is 1: 0.05 to 10: 0.5 to 1.
It is characterized by being 0.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Each component contained in the cleaning solution containing a protease according to the present invention will be described. (1) Modification enzyme The protease is chemically modified with a water-soluble polymer, and the protease is derived from a microorganism,
Any of plant-derived and animal-derived ones can be used, and neutral or alkaline ones derived from microorganisms are particularly preferable. Representative examples include the genus Bacillus (B. subtili
s, amyloloquefaciens, cereus, licheniformis, pumil
is, natto, mesentericus, sphaericus), Aspergillus
Genus (A. sojae, oryzae, flavus, sulphureus, candidus,
terricola, melleus, nidulans, sydowi), Streptmyces
Genus (S.fradiae, griseus, moderatus, rectus), Cepha
losporium sp., C. acremonium, Fusarium, Gliocladiu
m, Malbranchea pulchella, penicillium cyano-fulvu
m, P. notatum, Scopulariopsis, Tritirachium album ,,
Achromobacter, Arthrobacter, E.coli, Pseudomonas a
eruginosa, P. maltophilia, Candida lipolytica, Toru
Examples of commercially available proteolytic enzymes include bioseparase manufactured by Nagase Biochemical Co., Ltd., Savinase and Esperase manufactured by Novo Industry Co., Ltd.

In the present invention, examples of the water-soluble polymer that modifies the protease include polyethylene glycol, polysaccharides, proteins, and organic synthetic high-molecular polymers. Are preferably dextran, starch, pullulan, agarose, hydroxypropylcellulose, methylcellulose, ethylcellulose, carboxymethylcellulose and the like, and as the protein, gelatin, casein, milk casein and the like are preferable. As the organic synthetic high molecular polymer, C
A copolymer of a compound having an H ₂ ＣC <group and maleic anhydride is preferred. Specific examples include an alkyl vinyl ether such as methyl vinyl ether-maleic anhydride copolymer, a styrene-maleic anhydride copolymer, and isobutylene. -Maleic anhydride copolymers and the like. The enzyme activity value per unit volume in the detergent solution containing the protease of the present invention is 100.
1010,000 U / ml is preferred, and 500-5,000 U / ml is particularly preferred. Further, the modified enzyme solution having an enzyme activity value of 10,000 to 200,000 U / ml is preferably contained at a ratio of 0.1 to 10% by weight, particularly preferably at a ratio of 0.5 to 5% by weight. .

(2) Surfactant The proteolytic enzyme-containing washing solution of the present invention contains at least one nonionic surfactant selected from polyglycerin fatty acid ester and polyoxyethylene alkylphenyl ether, and an anionic surfactant. Activators myristoyl sarcosine sodium, ammonium lauryl sulfate and
It contains at least one surfactant selected from the group consisting of alkyl dimethyl amine oxide, which is an amphoteric surfactant, and betaine alkyl dimethyl amino acetate, and a plurality of surfactants are respectively selected from the above group. May be contained. Regarding the content (concentration) of these surfactants, the nonionic surfactant, anionic surfactant, and amphoteric surfactant are 0.01 to 10% by weight, particularly 0% by weight, as the active ingredient in the enzyme washing solution. The proportion is preferably about 0.05 to 5% by weight.

(3) Boric acid compound The boric acid compound contained in the proteolytic enzyme-containing washing solution of the present invention includes boric acid such as boric acid, metaboric acid and tetraboric acid, borax, metaborate, and the like. Borates such as tetraborate and the like, the concentration of which is 0.5 to 10%,
Particularly, 1.0 to 5% is preferable.

The ratio of the modified enzyme solution (10,000 to 200,000 U / ml): surfactant: borate compound in the washing solution of the present invention is from 1: 0.01 to 1: 1 from the viewpoint of the stability of the modified enzyme.
20: 0.1-50 (weight ratio) is preferred, and 1: 0.
05 to 10: 0.5 to 10 are particularly preferred.

The above-mentioned modified enzyme of the present invention does not show sufficient stability for practical use when used alone in an aqueous solution. However, the modified enzyme has a specific nonionic surfactant and a specific anionic surfactant. By using at least one surfactant selected from the group consisting of a surfactant and / or an amphoteric surfactant and a boric acid compound, sufficient enzymatic stability sufficient for practical use is exhibited. (Protein removing effect) can be stably used for a long period of time without using protein-containing enzyme-containing cleaning solution without decreasing over time. Although the cleaning solution of the present invention can be used for cleaning various products in which protein adhesion is a problem, it is particularly suitable for cleaning contact lenses, is highly safe, and has a simple operation (for a certain period of time, immersion). This has the advantage that the cleaning can be carried out during storage.

Next, a method for modifying a protease using a water-soluble polymer for obtaining the modification enzyme contained in the protease-containing washing solution of the present invention will be described. (A) Chemical modification with polyethylene glycol (cyanuric chloride method) After reacting polyethylene glycol with cyanuric chloride, adjusting the pH of the resulting reaction product and the protease, and stirring while cooling, the protease A modified enzyme in which the amino group is modified with polyethylene glycol is obtained. (B) Chemical modification with polysaccharide To modify a protease using a polysaccharide, a conventionally known periodate oxidation method, cyanogen bromide method, carbodiimide method, cyanuric chloride method, epichlorohydrin method,
SPDP (N-Succinimidyl 3- [2-pyridyldithio] propio
nate) A reagent method, an active ester method and the like are used. (C) Chemical modification by protein Protein (casein, milk casein, gelatin, etc.)
Is mixed with a protease and glutaraldehyde, and p
After the H adjustment, the mixture is stirred and cooled while being crosslinked to obtain a modified enzyme in which a protease is chemically modified with a protein. (D) Chemical modification with an organic synthetic high molecular polymer Distilled water is added to a copolymer of a compound having a CH ₂ ＣC <group (for example, methyl vinyl ether) and maleic anhydride, a protease, and a boric acid compound. If the pH is adjusted and then stirred while cooling, a modified enzyme in which the proteolytic enzyme is chemically modified with the organic synthetic polymer is obtained.

The cleaning solution of the present invention may contain a preservative in addition to the above-mentioned components. Such a preservative is used for preventing infection of pathogenic microorganisms in medical facilities. Any of them can be used. Such preservatives include quaternary ammonium salt-based disinfectants such as benzalkonium chloride, benzethonium chloride, tisserpyridium chloride, and dialkyldimethylammonium chloride, and glycine-based amphoteric surfactants such as alkylpolyaminoglycine and dialkylamino. It can be selected from the group consisting of ethyl glycine, polyhexamethylene biguanidine hydrochloride which is a biguanide disinfectant, chlorhexidine gluconate or a salt thereof.
More than one species may be used in combination. Among the above compounds, particularly useful disinfectants are benzalkonium chloride, benzethonium chloride, dialkylaminoethylglycine, chlorhexidine gluconate or salts thereof. The amount of the preservative used varies depending on the concentration of the enzyme and the surfactant, the type of the buffer, and the like.
It is preferred to use it in a proportion by weight. In addition, if necessary, various components such as a metal chelating agent, an isotonicity adjusting agent, a polyhydric alcohol, a pH adjusting agent, a hydrophilic polymer, and other enzyme agents such as lipolytic enzymes can be contained.

Next, a method for stabilizing the protease in the enzyme washing solution by using the protease-containing washing solution of the present invention will be described. In this method, a modified enzyme obtained by chemically modifying the above-mentioned protease with the above-mentioned water-soluble polymer is prepared, and the modified enzyme is combined with the above-mentioned nonionic surfactant and the above-mentioned nonionic surfactant. Along with at least one surfactant selected from the group consisting of an anionic surfactant and an amphoteric surfactant, the surfactant is added to the aqueous solution containing the boric acid compound and mixed. At this time, the modifying enzyme may be added in an isolated state or in a dispersed state in a solution, and the pH of the aqueous solution after the boric acid compound is added may be added.
The value is preferably in the range of pH 5 to 12 from the viewpoint of stabilizing the modifying enzyme, and particularly preferably in the range of pH 6 to 10. In such a method, the stability of the protease is significantly improved and the enzyme activity is maintained by the combined use of the surfactant and the boric acid compound, so that the enzyme washing solution can be used stably for a long time. It is.

Hereinafter, the present invention will be described in more detail with reference to Examples of the detergent solution containing a protease, but the present invention is not limited thereto. In addition, regarding the evaluation of the stabilization of the protease in the enzyme washing solution obtained according to the following preparation examples, the protease activity was determined by a known method using casein digestion (for example, Journal of General Phyalology, 30 (1947)).
291), and the residual rate of protease activity was calculated using the following equation. Protease activity residual rate (%) = (protease activity after heat treatment at 60 ° C. for 10 hours / protease activity immediately after solution preparation) × 100

[0018]

EXAMPLES (Example 1) 5-O-methoxy-polyethylene glycol having a molecular weight of 750 to 2,000 was reacted with 5 g of cyanuric chloride to obtain 2-O-.
methoxypolyethylene glycol-4,6-dichloro-s-triazine
(activated PEG 1) as an aqueous solution, 50 g of this 5% solution
And alkaline protease of Bacillus genus (trade name:
50 g of biopulase, manufactured by Nagase Seikagaku Co., Ltd.), adjusted to pH 9.2 with borax, reacted at 4 ° C. for 1 hour, and contained a modified enzyme in which the amino group in the protease was modified with polyethylene glycol. Solution (enzyme activity value 47,300
U / ml). To 100 ml of a 2% boric acid solution, 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglycel monolaurate, and 0.5 g of sodium myristoyl sarcosine (93%) were added, and polyethylene glycol was obtained as a modified carrier. The above modified enzyme solution containing a modified protease (enzyme activity value 47,300
U / ml), and then add borax to adjust the pH of the mixed solution to 8.0 and stir for about 1 hour to wash the enzyme of the present invention (enzyme activity value 946 U / ml). I got After measuring the proteolytic enzyme activity of this enzyme washing solution, the washing solution was put in a sealed container and heated in a thermostat at 60 ° C. for 10 hours. Then, the proteolytic enzyme activity and the cleaning effect of the enzyme cleaning solution after the heat treatment were measured.

Example 2 1 g of dextran T40 was dissolved in 90 ml of a 0.2M sodium hydrogen carbonate solution, and pH was adjusted with borax at 4 ° C. while adding 1.5 g of cyanogen bromide dissolved in 2.5 g of acetonitrile. Was maintained at 9.5. After the completion of the reaction, the pH was adjusted to 8.5.
And dialyzed against buffer. To this, 50 g of the same protease as in Example 1 was added, and after standing overnight, ethanolamine was added to remove the active groups. After dialysis, the modified protease solution (enzyme activity value 27,7
00 U / ml). To 100 ml of a 2% boric acid solution were added 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglycel monolaurate, and 0.5 g of sodium myristoyl sarcosine (93%), and dextran T40 was obtained as a modified carrier. 3.0 g of the modified enzyme solution (enzyme activity value 27,700 U / ml) was added, and then borax was added to adjust the pH of the mixed solution to 8.0,
After stirring for about 1 hour, the enzyme washing solution of the present invention (enzyme activity value 83
0 U / ml). As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

Example 3 1 g of gelatin was mixed with 50 g of the same protease as in Example 1 and 0.2 g of a 25% glutaraldehyde solution, adjusted to pH 8.2 with borax, and reacted at 4 ° C. for 12 hours. And then cross-link the modified enzyme solution (enzyme activity value 34,700 U
/ ml). To 100 ml of a 2% boric acid solution were added 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglyceryl monolaurate, and 0.5 g of sodium myristoyl sarcosine (93%), and the obtained gelatin was used as a modified carrier. 2.5 g of the modified enzyme solution (enzyme activity value 34,700 U / ml) was added, and then the pH of the mixed solution was adjusted to 8.0 by adding borax, and the mixture was stirred for about 1 hour, and the enzyme cleaning solution of the present invention ( An enzyme activity value of 867 U / ml) was obtained. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

Example 4 A copolymer of methyl vinyl ether and maleic anhydride (G
1 g of ANTREZ AN (manufactured by GAF), 50 g of the same protease as in Example 1, 0.1 g of borax and 50 ml of distilled water were mixed, and the mixture was stirred at 4 ° C. for 24 hours to obtain a modified enzyme solution (enzyme activity value 58,900). U / ml). 2% boric acid solution 1
In 00 ml, lauryl dimethylamine oxide (35
%) 0.5 g, decaglycerol monolaurate 0.5 g,
0.5g of myristoyl sarcosine sodium (93%)
, And 1.5 g of the above-mentioned modified enzyme solution (enzyme activity value 58,900 U / ml) obtained using a methyl vinyl ether-maleic anhydride copolymer as a modifying carrier, and then borax is added to adjust the pH of the mixed solution. Was adjusted to 8.0, and the mixture was stirred for about 1 hour, and the enzyme washing solution of the present invention (enzyme activity value: 883 U / ml)
I got As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

Example 5 In 100 ml of a 2% boric acid solution, 0.3 g of betaine lauryldimethylaminoacetate (26%), 0.3 g of polyoxyethylene nonylphenyl ether (100%), and the same as in Example 1 A solution containing the modified enzyme obtained using polyethylene glycol as a modified carrier (enzyme activity value 47,300
U / ml) was added, and borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution of the present invention. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

Example 6 In 100 ml of a 2% boric acid solution, 0.4 g of coconut dimethylamine oxide (30%), 0.4 g of ammonium lauryl sulfate (25%), and the same polyethylene glycol as in Example 1 were used as modified carriers. 2.0 g of a solution containing the above modified enzyme (enzyme activity value 47,300 U / ml) obtained as
Thereafter, borax was added to adjust the pH of the mixed solution to 8.0, and the mixture was stirred for about 1 hour to obtain an enzyme cleaning solution of the present invention. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

Comparative Example 1 An appropriate amount of sodium hydroxide was added to a 0.9% sodium chloride, 0.5% disodium hydrogen phosphate solution to adjust the pH to 8.8.
It was adjusted to 0 and used as a buffer. 0.5 g of lauryl dimethylamine oxide (35%) was added to 100 ml of this buffer,
0.5 g of decaglycerol monolaurate and 0.5 g of myristoyl sarcosine sodium (93%) were added, and a solution containing a modified protease obtained using polyethylene glycol as the modification carrier as in Example 1 (enzyme activity value 4
7,300 U / ml) was added, and then sodium hydroxide was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

(Comparative Example 2) In 100 ml of the same buffer solution (pH 8.0) as in Comparative Example 1,
Lauryl dimethylamine oxide (35%) 0.5
g, 0.5 g of decaglycel monolaurate, and 0.5 g of myristoyl sarcosine sodium (93%), and a solution containing the modified protease obtained using dextran T40 as a modification carrier as in Example 2 (enzyme activity value 2).
7,700 U / ml) was added, and then the pH of the mixed solution was adjusted to 8.0 by adding sodium hydroxide, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

(Comparative Example 3) In 100 ml of the same buffer solution (pH 8.0) as in Comparative Example 1,
Lauryl dimethylamine oxide (35%) 0.5
g, 0.5 g of decaglycel monolaurate and 0.5 g of myristoyl sarcosine sodium (93%), and a solution containing a modified proteolytic enzyme obtained using gelatin as a modification carrier (enzyme activity value 34,700 U / ml
) Was added, and then sodium hydroxide was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

(Comparative Example 4) In 100 ml of the same buffer solution (pH 8.0) as in Comparative Example 1,
Lauryl dimethylamine oxide (35%) 0.5
g, 0.5 g of decaglycel monolaurate and 0.5 g of myristoyl sarcosine sodium (93%), and the modified proteolytic enzyme obtained using the same methyl vinyl ether-maleic anhydride copolymer as in Example 4 as a modification carrier was used. 1.5 g of a solution (enzyme activity value 58,900 U / ml) containing sodium hydroxide was added, and then the pH of the mixed solution was added.
Was adjusted to 8.0 and stirred for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

(Comparative Example 5) 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglyceryl monolaurate, 0.5 g of myristoyl sarcosine sodium (93%) were added to 100 ml of the buffer prepared in the same manner as in Example 1. , And 0.35 g of an unmodified proteolytic enzyme (trade name: Bioprase, manufactured by Nagase Biochemical Co., Ltd.) is added as a proteolytic enzyme.
The pH of the mixed solution was adjusted to 8.0 by adding borax, and
The mixture was stirred for about an hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

Comparative Example 6 A solution containing a modified proteolytic enzyme obtained using polyethylene glycol as a modified carrier (enzyme activity value: 47, 100 ml) in 100 ml of buffer prepared in the same manner as in Example 1 Three
(00 U / ml) was added, and borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

(Comparative Example 7) A solution containing a modified proteolytic enzyme obtained using dextran T40 as a modified carrier (enzyme activity value: 27,700 U) in 100 ml of buffer prepared in the same manner as in Example 1
/ ml), and then borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

Comparative Example 8 A solution containing a modified proteolytic enzyme obtained using gelatin as a modified carrier (enzyme activity value 34,700 U / 100 ml) in 100 ml of buffer prepared in the same manner as in Example 1 2.5 g), and then add borax to adjust the pH of the mixed solution.
Was adjusted to 8.0 and stirred for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

(Comparative Example 9) A modified protease obtained by using a methylvinyl ether-maleic anhydride copolymer as a modified carrier in the same manner as in Example 4 was contained in 100 ml of a buffer solution prepared in the same manner as in Example 1. 1.5 g of a solution (enzyme activity value: 58,900 U / ml) was added, and then borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.

The components contained in the enzyme washing liquids of Examples 1 to 6 and Comparative Examples 1 to 9 are shown in Tables 1 and 2 below.
Summarized in

[0034]

[Table 1]

[0035]

[Table 2]

Using the enzyme washing solutions of Examples 1 to 6 and Comparative Examples 1 to 9, the activity of the protease immediately after solution preparation and the activity of the protease after heat treatment at 60 ° C. for 10 hours were measured. The results obtained are shown in Table 3 below. Table 3 also shows the residual rate (%) of the protease activity calculated using the above formula.

[0037]

[Table 3]

From the experimental results shown in Table 3, it can be seen that the protease-containing cleaning solution of the present invention has excellent enzyme activity even after heating at 60 ° C. for 10 hours.

Next, the above Examples 1 to 6 and Comparative Examples 1 to 9
Using the enzyme cleaning solution of No. 5, the change over time of the cleaning power (dirt removal rate) was measured. (Experimental method) An artificial protein solution having the following composition substantially similar to that of tears was prepared. Albumin (bovine serum) 0.39% γ-globulin (bovine serum) 0.16% Lysozyme (derived from egg white) 0.12% Remaining physiological saline The protein solution contains a dimethylsiloxane group and a fluorine atom The oxygen-permeable hard contact lens was immersed, and the protein was denatured by heating at 60 ° C. while the lens was immersed, so that artificial protein stain was attached to the lens surface. Next, the lens to which the artificial stain was attached in this manner was stained with erythrosine, which specifically stains a protein, and the spectrophotometer (UV-160A manufactured by Shimadzu Corporation) was used.
The absorbance at 545 nm was measured. The absorbance at this time is defined as A. Separately, the lens to which the artificial protein stain was adhered was immersed in an enzyme washing solution for 6 hours, then the lens was washed with water, stained again with erythrosine, and the absorbance at 545 nm was measured with a spectrophotometer. The absorbance at this time is B. The protein stain removal rate is calculated by the following equation. Protein stain removal rate (%) = (B / A) × 100 The protein stain removal rates were measured immediately after preparing the enzyme washing solution and after heating the washing solution at 60 ° C. for 10 hours, respectively, by the above-described experimental method. Table 4 shows the obtained experimental results.

[0040]

[Table 4]

From the experimental results shown in Table 4, it can be seen that the protease-containing cleaning solution of the present invention has an excellent protein stain removal rate even after heating at 60 ° C. for 10 hours.

[0042]

The proteolytic enzyme-containing washing solution of the present invention has high stability of proteolytic enzymes, maintains the enzymatic activity for a long period of time, and has a sufficient effect even when used after being stored for a certain period of time. It has a cleaning effect.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yasushi Nagase 475 Sasaya-machi, Furuya-cho, Nakagyo-ku, Kyoto-shi, Kyoto Sun Contact Lens Co., Ltd. (72) Inventor Kenji Kamiyama Nakagyo-ku, Kyoto-shi, Kyoto 475 Sasaya-machi, Fuya-cho, Toyogawa-kami Sun Contact Lens Co., Ltd. (72) Inventor Akito Tsuchiya 5-116-1, Kosai-cho, Koga-gun, Kiga-gun, Shiga Prefecture (56) References JP-A-9-51793 (JP) JP-A-6-240297 (JP, A) JP-A-8-146363 (JP, A) JP-A-9-241696 (JP, A) JP-A-8-327956 (JP, A) 9-87682 (JP, A) JP-A-7-265075 (JP, A) JP-A-7-155182 (JP, A) JP-A-6-189759 (JP, A) JP-A-6-62846 (JP, A) A) Special table Hei 9-507929 (JP, ) JP-T 2000-500513 (JP, A) WO 97/18288 (WO, A1) (58 ) investigated the field (Int.Cl. ^7, DB name) C11D 17/08 C11D 3/04 C11D 3/37 C11D 3/386 G02C 13/00 Patent file (PATOLIS) JICST file (JOIS)

Claims (2)

(57) [Claims] 1. A method in which a protease is chemically modified with a water-soluble polymer selected from the group consisting of polyethylene glycol, polysaccharides, proteins and a copolymer of maleic anhydride and a compound having a CH ₂ ＣC <group. Modifying enzyme, at least one nonionic surfactant selected from polyglycerin fatty acid ester and polyoxyethylene alkylphenyl ether, and anionic surfactants such as sodium myristoylsarcosine, ammonium lauryl sulfate, and amphoteric surfactant At least one surfactant selected from the group consisting of alkyldimethylamine oxide and betaine alkyldimethylaminoacetate, boric acid, metaboric acid, tetraboric acid, borax, metaborate and tetraborate And a boric acid compound selected from the group consisting of A cleaning solution containing a proteolytic enzyme. 2. The modified enzyme solution containing 0.1 to 10% by weight of the modified enzyme as a modified enzyme solution having an enzyme activity value per unit volume of 10,000 to 200,000 U / ml. The protease-containing cleaning solution according to claim 1, wherein the weight ratio of each surfactant: boric acid compound is 1: 0.05 to 10: 0.5 to 10.