JPH11253156A - Modified catalase, modified catalase composition and production of modified catalase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a modified catalase having enhanced long-term stability against a peroxo acid salt, capable of being mass-produced by a simple operation and useful for a catalyst for a bioreactor by modifying the catalase with a specific copolymer. SOLUTION: This modified catalase is modified by a copolymer composed of (i) one or mare kinds of alkenyl ethers of the formula [Z is a residue of a compound having 2-8 hydroxy groups; AO is a 2-8C oxyalkylene; R<1> is a 2-5C alkenyl; R<2> is a 1-24C hydrocarbon or an acyl; (a) and (b) are each 0-1,000; (m) is 1-1; (a)+(b).(m) is 1-1,000], (ii) maleic anhydride and (iii) one or more kinds of monomers selected from an unsaturated carboxylic acid, a vinyl compound and an olefin, regulated so that the ratio of the components (i):(ii):(iii) may be (5-60):(20-90):(0-30), preferably of 20-1,000 pts.wt. (based on 100 pts.wt. catalase) and stable against a peroxo acid salt. The modified catalase is obtained by modifying the catalase under a condition of (-5)-10 deg.C and pH 6-10.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a modified catalase modified with a unique copolymer and having high stability, a modified catalase composition containing the modified catalase, and a method for producing the modified catalase.

[0002]

2. Description of the Related Art Generally, proteins are stable in vivo. However, once the protein is removed from the body and purified, it is unstable, heated, or
Denatures when exposed to acids, alkalis or organic solvents, losing protein-specific functions. This is because the specific higher-order structure of the protein is destroyed. Catalase is well known as an enzyme that catalyzes the decomposition of hydrogen peroxide, but since catalase is also a protein, this is no exception. Therefore, the protein is chemically modified with a synthetic polymer or the like, and the modified enzyme stabilized with heating or acid, alkali or organic solvent, etc., is applied to the respective fields such as medicine, pharmacy, engineering and agriculture. Considerations have been added.

[0003] The expected functions of the modified protein include the loss of antigenicity and prolonged residence time in the blood, which are problems when administering the protein as a drug to blood, and the use of a drug such as an antibiotic or an anticancer drug and an antibody. Examples include a coupled missile therapy, a bioreactor catalyst modified with an amphiphilic synthetic polymer and made soluble in an organic solvent, and the like.

[0004] Modifiers used for stabilizing enzymes include, for example, 2,4-bis (o
-Methoxypolyethylene glycol) -6-chloro-s
-Examples of modification with triazines have been reported (Biochem.
Biophys. Res. Commun. Vol. 125, No. 2, 761-766, 198
Four). However, this is for solubilizing catalase in an organic solvent, and the modification method also requires several steps, so that it is difficult to produce the modified enzyme in large quantities.

[0005] Further, other modified enzymes that have stabilized the enzyme include those obtained by modifying a protease (Japanese Patent Laid-Open No. 6-20 / 1994).
No. 5675) and a modified asparaginase (JP-A-5-336966). However, catalase itself is inactivated in the presence of a peroxoacid salt such as sodium peroxocarbonate and sodium peroxoboronate, so that catalase cannot be expected to catalyze in the presence of a peroxoacid salt.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to a storage stability (hereinafter simply referred to as "catalase") for a long period of time even in the presence of peroxoacid salts such as sodium peroxocarbonate and sodium peroxoborate without impairing the original properties of catalase. It is an object of the present invention to provide a modified catalase, a modified catalase composition, and a method for producing a modified catalase, which have large storage stability) and can be mass-produced by a simple operation.

[0007]

Means for Solving the Problems The present inventors have developed a catalase modified by using a copolymer having at least a polyoxyalkylene group and an acid anhydride group in the same molecule,
The present inventors have obtained a new finding that the activity is stable without loss of activity over a long period of time even in the presence of a peroxoacid salt such as sodium peroxocarbonate and sodium peroxoboronate, and the present invention has been achieved based on this new finding.

The first aspect of the present invention is a compound selected from the group consisting of one or more of the alkenyl ethers represented by the formula (1) (a), maleic anhydride (b) and unsaturated carboxylic acids, vinyl compounds and olefins. (A), (b):
(C) is a modified catalase characterized by being modified with a copolymer (A) having a molar ratio of 5 to 60:20 to 90: 0 to 30 and being stable to peroxoacid salts. .

[0009]

Embedded image

(Z is a residue of a compound having 2 to 8 hydroxyl groups, AO is one or more oxyalkylene groups having 2 to 18 carbon atoms, and two or more AOs are added simultaneously. AO may be either block-like addition or random addition, and R ¹ has 2 to 5 carbon atoms.
R ² is a hydrocarbon group or an acyl group having 1 to 24 carbon atoms, a and b are each 0 to 100
0 and m are 1 to 7, and a + bm is 1 to 1000. )

The second invention comprises the modified catalase of the first invention and an ethylenically unsaturated monomer and a monomer having a carboxyl group or a monomer having an acid anhydride group. A modified catalase composition comprising a copolymer (B) or a salt of the copolymer (B).

The third invention is characterized in that the temperature is -5 to 10 ° C. and the pH is 6
A method for producing a modified catalase according to the first aspect of the present invention, wherein catalase is modified with the copolymer (A) under the conditions of:

[0013]

DETAILED DESCRIPTION OF THE INVENTION In the above formula (1), compounds having 2 to 8 hydroxyl groups having Z as a residue include ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, styrene glycol,
Glycols such as alkylene glycols and neopentyl glycols having 8 to 18 carbon atoms; glycerin, diglycerin, polyglycerin, trimethylolethane,
Polyhydric alcohols such as trimethylolpropane, 1,3,5-pentanetriol, erythritol, pentaerythritol, dipentaerythritol, sorbitol, sorbitan, sorbite, condensates of sorbitol and glycerin, adonitol, arabitol, xylitol and mannitol, and Partially etherified and partially esterified products of these polyhydric alcohols; such as xylose, arabinose, ribose, rhamnose, glucose, fructose, galactose, mannose, sorbose, cellobiose, maltose, isomaltose, trehalose, sucrose, raffinose, gentianose and merezitose. Sugars and etherified and esterified products of these sugars; catechol, resorcinol, hydro Phenols such as non and phloroglucinol and the like. Of these, glycols and polyhydric alcohols are preferred, and ethylene glycol, propylene glycol and glycerin are particularly preferred.

The oxyalkylene group having 2 to 18 carbon atoms represented by AO includes oxyethylene group, oxypropylene group, oxybutylene group, oxytetramethylene group, oxystyrene group, oxydecylene group, oxytetradodecane group. Examples include a silene group, an oxyhexadecylene group, and an oxyoctadecylene group. Further, among the oxyalkylene groups having 2 to 18 carbon atoms represented by AO, oxyalkylene groups having 2 to 4 carbon atoms are preferable, and each of oxyethylene group, oxypropylene group, oxybutylene group and oxytetramethylene group is preferable. Is particularly preferred. One or more of these may be added simultaneously. When two or more types are added at the same time, either block addition or random addition may be used.

In the formula (1), the proportion of the oxyethylene group (—C ₂ H ₄ O—) in the oxyalkylene group having 2 to 18 carbon atoms represented by AO is preferably 20% by weight.

In the formula, a and b represent the average number of moles of the oxyalkylene group having 2 to 18 carbon atoms, respectively. The oxyalkylene group having 2 to 18 carbon atoms is necessary for increasing the affinity between the copolymer (A) and catalase and improving the stability of catalase.
Exceeds 1,000, the weight ratio of the maleic anhydride unit in the copolymer becomes small, the reaction with the amino group or the terminal amino group of the lysine unit in the catalase hardly occurs, and the desired modified catalase cannot be obtained. In order to obtain a desired modified catalase, a + bm is preferably from 1 to 500, particularly preferably from 1 to 200.

Examples of the alkenyl group having 2 to 5 carbon atoms represented by R ¹ include a vinyl group, an allyl group, a methallyl group,
There are a 1,1-dimethyl-2-propenyl group and a 3-methyl-3-butenyl group. Among these, the number of carbon atoms of 3 exemplified for each of the allyl group and the methallyl group is 3
Preferred are -4 alkenyl groups.

Examples of the hydrocarbon group having 1 to 24 carbon atoms represented by R ₂ include an alkyl group having 1 to 24 carbon atoms and an aromatic hydrocarbon group having 1 to 24 carbon atoms. Examples of the alkyl group having 1 to 24 carbon atoms represented by R ₂ include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a hexyl group, and an isoheptyl group. Group,
2-ethylhexyl group, octyl group, isononyl group, decyl group, dodecyl group, isotridecyl group, tetradecyl group, hexadecyl group, isocetyl group, octadecyl group,
Isostearyl group, oleyl group, octyldodecyl group,
There are a docosyl group and a decyltetradecyl group, and examples of the aromatic hydrocarbon group having 1 to 24 carbon atoms include a benzyl group, a tolyl group, a butylphenyl group, a dibutylphenyl group, an octylphenyl group, a nonylphenyl group, and a dodecylphenyl group. And dioctylphenyl and dinonylphenyl groups.

The acyl group having 1 to 24 carbon atoms represented by R ² includes acetic acid, propionic acid, butyric acid, isobutyric acid,
Caprylic acid, 2-ethylhexanoic acid, isononanoic acid, capric acid, lauric acid, myristic acid, palmitic acid,
Isopalmitic acid, stearic acid, isostearic acid,
There are acyl groups from each of arachiic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, erucic acid, benzoic acid, hydroxybenzoic acid, cinnamic acid and gallic acid. Among them, a hydrocarbon group having 1 to 4 carbon atoms or an acyl group is preferable.

One or more of the alkenyl ethers represented by the formula (1) can be used. Alkenyl ether (a) represented by the formula (1): maleic anhydride (b): at least one monomer (c) selected from the group consisting of unsaturated carboxylic acids, vinyl compounds and olefins in a molar ratio. , 5-60: 20-90: 0-3
In order for the copolymer (A) having a ratio of 0, preferably 20 to 60:40 to 70: 0 to 20 to sufficiently bind to catalase, an acid anhydride structure is required.

Representative examples of monomers selected from the group consisting of unsaturated carboxylic acids, vinyl compounds and olefins include unsaturated carboxylic acids such as acrylic acid, methacrylic acid, itaconic acid and crotonic acid, styrene and methylstyrene. And vinyl halides such as vinyl chloride and vinylidene chloride; olefins such as isobutylene and diisobutylene; and vinyl acetate.

When the monomer (c) is contained in the copolymer (A), if the proportion of the monomer (c) is excessive, the polyoxyethylene chain required for the modification of catalase may be used. And the content of each of the acid anhydrides is reduced, so that catalase is not sufficiently modified. Therefore, these monomers (c) have a total amount of the monomers constituting the copolymer (A). Should not exceed 30 mol% in

The copolymer (A) used in the present invention is obtained by copolymerizing an alkenyl ether represented by the formula (1) and maleic anhydride by a conventional method using a radical copolymerization catalyst (polymerization initiator). Can be easily obtained by: At this time, the monomer (c) may be present to copolymerize the three. Alternatively, the monomer (c) can be polymerized after radical copolymerization of the alkenyl ether represented by the formula (1) and maleic anhydride.

The radical copolymerization catalyst may be a commonly used one, such as lauroyl peroxide, t
organic peroxides such as tert-butyl peroxy-2-ethylhexanoate, benzoyl peroxide and tert-butyl perbenzoate, inorganic peroxides such as hydrogen peroxide and potassium persulfate, azobisisobutyronitrile,
Azo and diazo compounds such as diazoaminoazobenzene and aromatic sulfinic acids, alkylmercury and alkyllead may be used.

The copolymer (A) used in the present invention can be made into various copolymers by appropriately selecting the type of radical copolymerization catalyst, polymerization conditions, and the type of alkenyl represented by the formula (1). be able to. Its weight average molecular weight is
0.5 × 10 ⁴ ~200 × 10 ⁴ nm, preferably, ^0.5
It is about × 10 ⁴ to 15 × 10 ⁴ . When an alkenyl ether represented by the formula (1) having a relatively large number of oxyethylene groups is used, usually, a solid copolymer can be obtained at room temperature to room temperature.

The raw material catalase used in the present invention is:
Those obtained from aerobic cells of animals, plants and microorganisms are used without any particular limitation. In addition, commercially available products can be used as they are.

The modified catalase of the first invention is obtained by reacting the above-mentioned copolymer (A) with catalase, and the amino acid or terminal amino group of the lysine unit of catalase and the above-mentioned copolymer (A ) Are combined. The ratio of the copolymer (A) to catalase varies depending on the number of amino groups and terminal amino groups of lysine units in catalase and the content of acid anhydride in the copolymer, and can be specified unconditionally. However, when the amount of the copolymer (A) is too small compared to the number of amino groups, the modification rate decreases and the stability of catalase becomes insufficient. If the amount is too large, the modification rate becomes too high, and the expected activity of catalase is remarkably reduced, and in any case, it is not suitable for practical use. Therefore, in practical use, usually, the amount of the copolymer (A) is preferably about 20 to 1000 parts by weight, particularly preferably about 50 to 1000 parts by weight, based on 100 parts by weight of catalase.

Catalase can be reacted with the copolymer (A) by a method known per se to modify the catalase with the copolymer (A). That is, the aqueous catalase solution and the copolymer (A) are mixed. When the copolymer (A) is hardly soluble or insoluble in water, the copolymer (A)
Can be mixed with an aqueous solution of catalase previously dissolved in an organic solvent compatible with water such as acetone and toluene.

Further, depending on the copolymer (A), if the entire amount thereof is mixed with catalase at once, the modification rate of catalase is reduced, and there is a risk that the desired storage stability of the modified catalase may not be obtained. In order to avoid danger, it is preferable to add the copolymer (A) in portions to the aqueous catalase solution. The number of divisions varies depending on the composition and amount of the copolymer (A), but is usually 2 to 2.
Five times is preferred, but not limited to these times. The amount of the copolymer (A) to be divided may be different each time or may be the same. In addition, it does not prevent mixing the whole amount of the copolymer (A) with catalase at once.

Further, if the reaction is carried out under strong acidity, the reactivity of lysine residues in catalase decreases, and the modification rate decreases,
If the reaction is carried out under strong alkaline conditions, hydrolysis of the acid anhydride group of the copolymer (A) is preferentially performed, and the modification rate is reduced. Therefore, the reaction is carried out slightly acidic to slightly alkaline, but usually, pH
6-10 are preferable, and pH7-9 are particularly preferable.

Further, if the reaction temperature is too high, the activity of catalase decreases in the modification step, and the hydrolysis reaction of the acid anhydride group tends to occur in preference to the reaction between the amino group and the acid anhydride group of catalase. And the modification rate decreases. On the other hand, if the reaction temperature is too low, the copolymer (A) solidifies, which is not suitable for practical use. Therefore, the reaction temperature
It is practically preferable that the temperature be about 5 to 10 ° C.
It is particularly preferred that the temperature be about ° C.

Further, depending on the composition of the copolymer (A) to be used, the modified catalase of the first invention of the present invention may be paste-like and difficult to use when used. In such a case, in order to make it easier to use, prior to use, the paste-form modified catalase should be prepared in advance, for example, without affecting the storage stability of the modified catalase, such as sodium sulfate and sodium chloride. It is preferable that the modified catalase is powdered by adding such an inorganic salt.

In order to further increase the storage stability of the modified catalase of the first invention, the modified catalase of the first invention, which is the second invention, is combined with an ethylenically unsaturated monomer and a carboxyl group. A modified catalase composition comprising a copolymer (B) comprising a monomer having the monomer or a monomer having an acid anhydride group, or a salt of the copolymer (B).

The ethylenically unsaturated monomer constituting the copolymer (B) in the second invention is, for example, ethylene,
It means ethylene series hydrocarbons (olefins) such as propylene, butylene and isobutylene, and aromatic vinyl compounds such as, for example, styrene and α-methylstyrene. Above all, each of isobutylene and styrene is preferred, with isobutylene being particularly preferred. Representative examples of the monomer having a carboxyl group or an acid anhydride that constitutes the copolymer (B) include acrylic acid, methacrylic acid, itaconic acid, crotonic acid, maleic acid, and maleic anhydride. Of these, each of maleic acid and maleic anhydride is particularly preferred.

In the copolymer (B), the ratio of the ethylenically unsaturated monomer to the monomer having a carboxyl group or an acid anhydride is usually from 10 to 80:90 by molar ratio.
20, preferably 30 to 70: 70 to 30. Commercial products can be used as the copolymer (B). Further, as a preferable copolymer (B), a copolymer having a number average molecular weight of 5 × 10 ^{4 to} 17 × 10 ⁴ and composed of isobutylene and maleic anhydride can be exemplified.

Examples of the salt of the copolymer (B) include alkali metal salts such as lithium salt, sodium salt and potassium salt, methylamine salt, dimethylamine salt, ethylamine salt, monoethanolamine salt, diethanolamine salt, triethanolamine salt and triethanolamine salt. Examples thereof include amine salts such as an ethanolamine salt and a methylairanolamine salt, and an ammonium salt. Of these, an alkali metal salt and an ammonium salt are preferable.

The copolymer (B) is preferably used in an amount of 0.1 to 1000 parts by weight, particularly preferably 1 to 1000 parts by weight, based on 100 parts by weight of the modified catalase of the first invention.
Mixing 500 parts by weight, most preferably 5-250 parts by weight, results in a modified catalase composition with very good stability.

[0038] The method for obtaining the modified catalase composition of the second invention is not particularly limited, and can be a method known per se. That is, for example, the aqueous solution of the modified catalase is directly mixed with the copolymer (B), or the aqueous solution in which the copolymer (B) is dissolved in water in advance and the aqueous solution of the modified catalase are mixed. Can also.

When the copolymer (B) is hardly soluble or insoluble in water, the copolymer (B) is converted into an organic solvent having good compatibility with water, such as acetone and toluene. It is also possible to mix a previously dissolved solution with an aqueous solution of the modified catalase. Water and an organic solvent are distilled off from the above mixture under reduced pressure to obtain a rubbery or powdery or granular modified catalase composition. If desired, this powder can be granulated into granules.

[0040]

The present invention will be described in more detail with reference to the following production examples and examples, but the invention is not limited to these production examples and examples. Production Example 1 Production of Copolymer (A) The following compound was dissolved in 1 liter of toluene, and heated at 80 ± 2 ° C. for 7 hours in a nitrogen atmosphere to carry out a polymerization reaction.

CH ₂ ＣＨCHCH ₂ O (C ₂ H ₄ O) ₁₁ CH ₃ 556 g (1.0 mol) Maleic anhydride 103 g (1.05 mol) tert-butylperoxy-2-ethylhexanoate 4 0.3 g (0.02 mol)

Then, toluene and unreacted maleic anhydride were distilled off at 100 ± 10 ° C. under a reduced pressure of 10 to 30 mmHg to obtain 528 g of copolymer (A) No. 1. The obtained copolymer (A) No. 1 was a brown, transparent liquid having a kinematic viscosity of 206.2 cSt at 100 ° C. and a saponification value of 182.0 KOH mg / g.
Met.

Production Example 2 The following compound was dissolved in 2 liters of toluene, and heated at 80 ± 2 ° C. for 9 hours in a nitrogen atmosphere to carry out a polymerization reaction.

CH ₂ ＣC (CH ₃ ) CH ₂ O (C ₂ H ₄ O) ₄₄ CH ₃ 2022 g (1.0 mol) Maleic anhydride 103 g (1.05 mol) tert-butylperoxy-2- Ethyl hexanoate 10.8 g (0.05 mol)

Then, toluene and unreacted maleic anhydride were distilled off at 110 ± 10 ° C. under a reduced pressure of 10 to 30 mmHg to obtain 928 g of copolymer (A) No. 2. The obtained copolymer (A) No. 2 was a white solid at 25 ° C., and had a saponification value of 36.1 KOH mg / g.

Production Example 3 Except that the alkenyl ether shown in Table 1 and the maleic anhydride, monomer (c) and catalyst shown in Table 2 were used in the molar ratios shown in Tables 1 and 2, respectively. Copolymer (A) Nos. 3 to 8 were prepared in the same manner as in Production Example 2. Table 3 shows properties of the copolymer (A) Nos. 3 to 8, such as weight average molecular weight, saponification value, shape, and solubility in organic solvents.

[0047]

[Table 1]

Note) The symbol in the square indicates that it is a random addition.

[0049]

[Table 2]

Note) BPO: benzoyl peroxide LPO: lauroyl peroxide tBEH: t-butylperoxy-2-ethylhexanoate

[0051]

[Table 3]

In the following examples, the modified catalase was a catalase (Ask Super: trade name, a product of Mitsubishi Gas Chemical Co., Ltd.), and the copolymers (A) Nos. 1 to 8 obtained in the above production examples. Is modified with The activity of the resulting modified catalase was determined by converting the modified catalase and hydrogen peroxide contained in an aqueous solution of hydrogen peroxide at a predetermined concentration to 30 ° C.
And the amount of residual hydrogen peroxide was analyzed by potassium iodide-potassium thiosulfate titration, and the catalase activity was determined from the decomposed hydrogen peroxide. One unit of catalase is defined as a catalase activity that degrades 1 micromol (μmol) of hydrogen peroxide per minute.

Preservation test and results of unmodified catalase 100,000 units of unmodified catalase and 100 g of sodium peroxocarbonate were uniformly mixed and stored in a thermostat kept at 30 ° C., 40 ° C. and 50 ° C., respectively. And its catalase activity (hereinafter referred to as residual activity) was measured, and the residual relative catalase activity was calculated from the residual activity. Here, the “residual relative catalase activity” is calculated as “percentage of the residual activity to the initial activity of the modified catalase”. The results are shown in FIG.

Preservation test method of modified catalase of the present invention 100,000 units of modified catalase were added to 100 g of sodium peroxocarbonate and mixed uniformly to obtain a composition for stability evaluation. This mixture is brought to 30 ° C., 40 ° C. and 50 ° C.
The samples were stored in a temperature bath maintained at each of the temperatures.

After 6 hours, 1 day, 4 days, 1 week, 2 weeks, 3 weeks, 5 weeks and 7 weeks from the start of storage, the residual activity was measured, and the residual relative catalase activity was calculated from the residual activity. The storage test method of the modified catalase composition of the present invention was in accordance with the above-described storage test method of the modified catalase of the present invention. In each of FIGS. 1 to 11, a line connected by a white circle (丸), a line connected by a white triangle (△), and a line connected by a cross (×) are respectively “Storage at 30 ° C”, “Storage at 40 ° C”
And “Storage at 50 ° C.”.

Example 1 5 g of catalase was dissolved in 100 g of a borate buffer having a pH of 9.0, and the temperature of the catalase solution was maintained at 3 ° C. To this catalase solution, 2.5 g of the copolymer (A) No. 1 obtained in the above Preparation Example was added in the form of fine powder, and 3 ± 1 ° C.
, And 2.5 g of the copolymer (A) No. 1 obtained in the above reference example was added as a fine powder, followed by stirring at the same temperature for 30 minutes. Subsequently, water was distilled off at 25 ° C. under reduced pressure to obtain 9.2 g of modified catalase. Since the obtained modified catalase was in a paste form, 325 parts by weight of sodium sulfate was added to 100 parts by weight of the modified catalase, and the powdery modified catalase obtained by mixing was subjected to a storage test.

Boric acid-based buffer solution (pH 9.0) 0.05 M-borax aqueous solution 90 ml 0.2 M-boric acid / 0.05 M-sodium chloride aqueous solution 10 ml A liquid obtained by mixing the above two aqueous solutions. The same applies to borate-based buffers in the following examples.

As a result of measuring the catalase activity of the resulting modified catalase, it was found that the modified catalase retained 1/5 of the activity of the starting catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase and 100 g of sodium peroxocarbonate was stored in an oven at 30 ° C., 40 ° C., and 50 ° C., and a storage test was performed. The catalase activity during the period was measured, and the remaining relative catalase activity was determined from this activity. The results obtained are shown in FIG.

Example 2 5 g of catalase was dissolved in 100 g of ion-exchanged water.
The catalase solution was adjusted to pH 7.0 with 0.01N aqueous sodium hydroxide and the temperature was maintained at 3 ° C.
The copolymer (A) No. obtained in the above Production Example was added to this solution.
After adding 10 g of fine powder and stirring at 3 ± 1 ° C. for 30 minutes, 5 g of copolymer (A) No. 2 obtained in the above-mentioned Preparation Example was further added as fine powder and the same temperature was added. For 30 minutes. Then, water is distilled off at 25 ° C. under reduced pressure,
19.5 g of modified catalase were obtained.

As a result of measuring the catalase activity of the resulting modified catalase, it was found that the modified catalase retained one-third the activity of the starting catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase and 100 g of sodium peroxocarbonate was stored in an oven at 30 ° C., 40 ° C., and 50 ° C., and subjected to a storage test to perform a storage period. The catalase activity was measured, and the residual relative catalase activity was determined from this activity. FIG. 3 shows the obtained results.

Example 3 5 g of catalase was dissolved in 100 g of ion-exchanged water.
The catalase solution was adjusted to pH 7.5 with 0.01 N aqueous sodium hydroxide and the temperature was maintained at 3 ° C.
The copolymer (A) No. obtained in the above Production Example was added to this solution.
After adding 5 g of 3 and stirring at 3 ± 1 ° C. for 30 minutes,
Further, 5 g of the copolymer (A) No. 3 obtained in the above Production Example was added, and the mixture was stirred at the same temperature for 30 minutes. Then, water was distilled off at 25 ° C. under reduced pressure, and 13.8 g of modified catalase was removed.
I got

As a result of measuring the catalase activity of the resulting modified catalase, it was found that the modified catalase retained one-fourth the activity of the starting catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase and 100 g of sodium peroxocarbonate was stored in an oven at 30 ° C., 40 ° C., and 50 ° C., and subjected to a storage test to perform a storage period. The catalase activity was measured, and the residual relative catalase activity was determined from this activity. FIG. 4 shows the obtained results.

Example 4 5 g of catalase was dissolved in 100 g of ion-exchanged water.
The catalase solution was adjusted to pH 7.5 with 0.01 N aqueous sodium hydroxide and the temperature was maintained at 3 ° C.
The copolymer (A) No. obtained in the above Production Example was added to this solution.
After stirring for 30 minutes at 3 ± 1 ° C., the copolymer (A) No. 4 obtained in the above Production Example was further added.
4g was added and stirred at the same temperature for 30 minutes. Then
The water is distilled off at 25 ° C. under reduced pressure to give modified catalase 1
8.3 g were obtained.

As a result of measuring the catalase activity of the resulting modified catalase, it was found that the activity was one-third that of the starting catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase and 100 g of sodium peroxocarbonate was stored in an oven at 30 ° C., 40 ° C., and 50 ° C., and subjected to a storage test to perform a storage period. The catalase activity was measured, and the residual relative catalase activity was determined from this activity. FIG. 5 shows the obtained results.

Example 5 5 g of catalase was dissolved in 100 g of a borate buffer having a pH of 9.0, and the temperature of the catalase solution was maintained at 3 ° C. To this catalase solution, 5 g of the copolymer (A) No. 5 obtained in the above-mentioned Production Example was added, and the mixture was stirred at 3 ± 1 ° C. for 30 minutes. No.)
5g was added and stirred at the same temperature for 30 minutes. Then
The water is distilled off at 25 ° C. under reduced pressure to give modified catalase 1
4.3 g were obtained.

As a result of measuring the catalase activity of the obtained modified catalase, it was found that the modified catalase retained one-fifth the activity of the starting catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase and 100 g of sodium peroxocarbonate was stored in an oven at 30 ° C., 40 ° C., and 50 ° C., and subjected to a storage test to perform a storage period. The catalase activity was measured, and the residual relative catalase activity was determined from this activity. The results obtained are shown in FIG.

Example 6 5 g of catalase was dissolved in 100 g of a borate buffer at pH 9.0, and the temperature of the catalase solution was maintained at 3 ° C. To this catalase solution, 10 g of the copolymer (A) No. 6 obtained in the above-mentioned Production Example was added as a fine powder, and the mixture was stirred at 3 ± 1 ° C. for 30 minutes. 5 g of polymer (A) No. 6 was added as a fine powder and stirred at the same temperature for 30 minutes. Subsequently, water was distilled off at 25 ° C. under reduced pressure to obtain 19.2 g of modified catalase.

As a result of measuring the catalase activity of the obtained modified catalase, it was found that the activity was 1/6 that of the raw catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase and 100 g of sodium peroxocarbonate was stored in an oven at 30 ° C., 40 ° C., and 50 ° C., and subjected to a storage test to perform a storage period. The catalase activity was measured, and the residual relative catalase activity was determined from this activity. FIG. 7 shows the obtained result.

Example 7 5 g of catalase was dissolved in 100 g of a borate buffer having a pH of 9.0, and the temperature of the catalase solution was maintained at 3 ° C. To this catalase solution was added 10 g of the copolymer (A) No. 7 obtained in the above-mentioned Production Example, and the mixture was stirred at 3 ± 1 ° C. for 30 minutes. A)
5 g of No. 7 was added, and the mixture was stirred at the same temperature for 30 minutes. Subsequently, water was distilled off at 25 ° C. under reduced pressure to obtain 19.3 g of modified catalase.

As a result of measuring the catalase activity of the obtained modified catalase, it was found that the modified catalase retained one-fourth the activity of the starting catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase and 100 g of sodium peroxocarbonate was stored in an oven at 30 ° C., 40 ° C., and 50 ° C., and subjected to a storage test to perform a storage period. The catalase activity was measured, and the residual relative catalase activity was determined from this activity. FIG. 8 shows the obtained results.

Example 8 5 g of catalase was dissolved in 100 g of a borate buffer at pH 9.0, and the temperature of the catalase solution was maintained at 3 ° C. To this catalase solution, 5 g of the copolymer (A) No. 8 obtained in the above Production Example was added in the form of a fine powder.
After stirring for 0 minutes, 5 g of the copolymer (A) No. 8 obtained in the above Production Example was added as a fine powder, followed by stirring at the same temperature for 30 minutes. Then, water is distilled off at 25 ° C. under reduced pressure,
14.3 g of modified catalase were obtained.

As a result of measuring the catalase activity of the resulting modified catalase, it was found that the modified catalase retained one-third the activity of the starting catalase. 100,000 units of this modified catalase and 100 g of sodium peroxocarbonate are mixed uniformly,
A storage test was performed by storing in a temperature bath maintained at 0 ° C. and 50 ° C., respectively, and the catalase activity during the storage period was measured, and the residual relative catalase activity was determined from this activity. FIG. 9 shows the obtained results.

FIG. 1 shows that unmodified catalase was treated at 30 ° C.
It can be seen that the storage stability at 40 ° C. and 50 ° C. is extremely low for peroxoacid salts. FIG.
9 that the modified catalase of the present invention was treated at 30 ° C.
Each of the storage at 0 ° C. and 50 ° C. has a long-term stability to the peroxoacid salt, and has a large storage stability, particularly a large storage stability at 30 ° C. and 40 ° C., respectively. I understand.

Example 9 5 g of catalase was dissolved in 100 g of ion-exchanged water.
The catalase solution was adjusted to pH 7.0 with 0.01N aqueous sodium hydroxide and the temperature was maintained at 3 ° C.
The copolymer (A) No. obtained in the above Production Example was added to this solution.
After adding 10 g of a fine powder and stirring at 3 ± 1 ° C. for 30 minutes, 5 g of the copolymer (A) No. 2 obtained in the above Preparation Example was further added as a fine powder and the same temperature was added. For 30 minutes to obtain a modified catalase solution.

To this modified catalase solution, 10 g of an isobutylene / maleic anhydride copolymer (trade name of Isovan-104 Kuraray Co., Ltd., number average molecular weight 6.5 to 7.5 × 10 ⁴ ) was used as a copolymer (B). Ion exchange water 5
After adding an aqueous solution of isobutylene / maleic anhydride copolymer dissolved in 0 g and stirring for 10 minutes, water was distilled off at 25 ° C. under reduced pressure to obtain a modified catalase composition 28.
5 g were obtained.

As a result of measuring the catalase activity of the obtained modified catalase composition, it was found that 1/9 the activity of the starting catalase was retained. A mixture obtained by uniformly mixing 100,000 units of the modified catalase composition and 100 g of sodium peroxocarbonate was treated at 30 ° C, 40 ° C and 50 ° C.
A storage test was performed by storing in a temperature bath maintained at each of the temperatures of ℃, the catalase activity during the storage period was measured, and the remaining relative catalase activity was determined from this activity. The obtained result is
As shown in FIG.

Example 10 5 g of catalase was dissolved in 100 g of ion-exchanged water.
The catalase solution was adjusted to pH 7.0 with 0.01N aqueous sodium hydroxide and the temperature was maintained at 3 ° C.
The copolymer (A) No. obtained in the above Production Example was added to this solution.
After adding 10 g of fine powder and stirring at 3 ± 1 ° C. for 30 minutes, 5 g of copolymer (A) No. 2 obtained in the above-mentioned Preparation Example was further added as fine powder and the same temperature was added. For 30 minutes to obtain a modified catalase solution.

To this modified catalase solution, 10 g of a copolymer (B) of isobutylene / maleic anhydride copolymer (Isovan-104, trade name of Kuraray Co., Ltd., number average molecular weight 6.5 to 7.5 × 10 ⁴ ) was added. Ion exchange water 5
After adding an aqueous solution of isobutylene / maleic anhydride copolymer dissolved in 0 g and stirring for 10 minutes, water was distilled off at 25 ° C. under reduced pressure to obtain a modified catalase composition 29.
3 g were obtained.

As a result of measuring the catalase activity of the obtained modified catalase composition, it was found that the activity was 1/8 of that of the starting catalase. A mixture obtained by uniformly mixing 100,000 units of the modified catalase composition and 100 g of sodium peroxocarbonate was treated at 30 ° C, 40 ° C and 50 ° C.
A storage test was performed by storing in a temperature bath maintained at each of the temperatures of ℃, the catalase activity during the storage period was measured, and the remaining relative catalase activity was determined from this activity. The obtained result is
As shown in FIG.

From each of FIGS. 10 and 11, the modified catalase composition of the present invention was obtained at 30 ° C., 40 ° C. and 5 ° C.
Each storage at 0 ° C. has a long-term stability with respect to the peroxoacid salt, and has a large storage stability, especially at 30 ° C. and 40 ° C., and a storage stability. Is much larger than the storage stability of the modified catalase of the present invention.

[0081]

Industrial Applicability The modified catalase of the present invention and the modified catalase composition containing the modified catalase are easy to produce, and are more effective than catalase itself and conventional modified catalase for peroxoacid salts such as sodium peroxocarbonate. Long-term stability has been increased and storage stability has been increased.

[Brief description of the drawings]

Fig. 1 Results of storage test of unmodified catalase

FIG. 2 shows the results of a storage test of the modified catalase of Example 1.

FIG. 3 shows the results of a storage test of the modified catalase of Example 2.

FIG. 4 shows the results of a storage test of the modified catalase of Example 3.

FIG. 5 shows the results of a storage test of the modified catalase of Example 4.

FIG. 6 shows the results of a storage test of the modified catalase of Example 5.

FIG. 7 shows the results of a storage test of the modified catalase of Example 6.

FIG. 8 shows the results of a storage test of the modified catalase of Example 7.

FIG. 9 shows the results of a storage test of the modified catalase of Example 8.

FIG. 10 shows the results of a storage test of the modified catalase composition of Example 9.

FIG. 11 shows the results of a storage test of the modified catalase composition of Example 10.

Continuation of the front page (72) Inventor Shunsuke Ohashi 3-32-6, Shirayuri, Izumi-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Toru Yasukawachi 1-1-1, Chigasaki-higashi, 1-3-1, Chigasaki-Higashi, Tsuzuki-ku, Yokohama-shi, Kanagawa Prefecture, Room 401.

Claims (8)

[Claims] 1. An alkenyl ether represented by the formula (1), one or more of which (a), maleic anhydride (b) and at least one selected from the group consisting of unsaturated carboxylic acids, vinyl compounds and olefins. Modified with a copolymer (A) composed of different kinds of monomers (C), wherein the (A) :( B) :( C) is in a molar ratio of 5-60: 20-90: 0-30. Modified catalase characterized by being stable to peroxoacid salts. Embedded image (Z is a residue of a compound having 2 to 8 hydroxyl groups, AO is 1
A species or two or more kinds of oxyalkylene group having 2 to 18 carbon atoms, when two or more AO is added at the same time the AO may be either block-like addition and added randomly, R ¹ is carbon An alkenyl group having 2 to 5 atoms, R ² is a hydrocarbon group or an acyl group having 1 to 24 carbon atoms, a and b are each 0 to 1000;
7, a + bm is 1 to 1000. ) 2. The alkenyl ether constituting the copolymer (A) is a compound represented by the formula (1) wherein R ¹ has 3 to 4 carbon atoms.
Alkyl group, AO is modified catalase according to claim 1, wherein the oxyalkylene group of 2 to 4 carbon atoms, R ² is a hydrocarbon group or an acyl group having 1 to 4 carbon atoms. 3. The alkenyl ether constituting the copolymer (A) has a ratio of oxyethylene group of 20% by weight or more of oxyalkylene groups having 2 to 18 carbon atoms represented by AO in the formula (1). The modified catalase according to claim 1, which is 4. The modified catalase according to claim 1, wherein the ratio of the copolymer (A) is 20 to 1000 parts by weight based on 100 parts by weight of the catalase. 5. The modified catalase according to claim 1, wherein the modified catalase comprises an ethylenically unsaturated monomer and a monomer having a carboxyl group or a monomer having an acid anhydride group. A modified catalase composition comprising the copolymer (B) or a salt of the copolymer (B). 6. The copolymer (B) is composed of isobutylene and maleic anhydride, and has a number average molecular weight of 5 × 10 ^4.
The modified catalase composition according to claim 5, which has a size of up to 17 x 10 ⁴ . 7. The modified catalase composition according to claim 5, wherein the proportion of the copolymer (B) is 0.1 to 1000 parts by weight based on 100 parts by weight of the modified catalase. 8. The method of claim 1, wherein the catalase is modified with the copolymer (A) at a temperature of -5 to 10 ° C. and a pH of 6 to 10. 4
A method for producing the modified catalase according to the above.