JP3035675B2 - Modified superoxide dismutase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stable superoxide dismutase modified with a high molecular weight copolymer, and more particularly to a modified superoxide dismutase capable of maintaining its activity in an aqueous system for a long time and having a wide range of applications. It relates to superoxide dismutase.

[0002]

2. Description of the Related Art Conventionally, suppression of the formation of lipid peroxides from sebum
Superoxide dismutase has been studied as a means for preventing the denaturation and deterioration of the keratin protein structure of skin and hair, and for preventing the aging phenomenon such as spots and wrinkles. Superoxide dismutase is an enzyme widely distributed in living organisms such as animals, plants, and microorganisms, and is known to cause disproportionation of superoxide anion by the following reaction.

[0003]

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[0004] Superoxide dismutase contains copper, zinc, iron or manganese in its active center, and has recently been produced in large quantities by genetic engineering techniques.

[0005]

However, when superoxide dismutase is blended directly into an aqueous system,
There are problems such as a decrease in enzyme activity and a tendency to cause irritation and allergy to the skin. In the application of superoxide dismutase, various attempts have been made such as keeping it in a dry state until immediately before use, mixing it with an aqueous solution at the time of use, making it into a liposome, microencapsulating it, and immobilizing it with a polymer compound. However, the method by microencapsulation is a stabilization by cutting off the contact of superoxide dismutase with a base such as an aqueous solution until immediately before use, and does not solve the essential problem. Further, the method of adding a polyhydric alcohol which is effective as a stabilizing agent for other enzymes such as protease had almost no effect on superoxide dismutase. Other methods have not been able to achieve a sufficiently satisfactory stabilizing effect.

An object of the present invention is to provide a modified superoxide dismutase which can maintain its activity in an aqueous system for a long time and has a wide range of application.

[0007]

Means for Solving the Problems The present inventors have developed superoxide dismutase, in which a polymer having an acid anhydride group and a polyoxyalkylene group combined in the same molecule, is chemically active for a long time even in an aqueous system. The present invention was found to last, and the present invention was reached. That is, the present invention provides a modified chemical bond of a copolymer having a molar ratio of the alkenyl ether represented by the formula (1), maleic anhydride and another monomer of 5 to 60:20 to 90: 0 to 30. Superoxide dismutase.

[0008]

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(Z is a residue of a compound having 2 to 8 hydroxyl groups, AO is one or a mixture of two or more oxyalkylene groups having 2 to 18 carbon atoms. May be added at random or may be added at random.
¹ represents an alkenyl group having 2 to 5 carbon atoms, and R ² represents 1 to 24 carbon atoms.
A, b and c are each an average number of added moles of an oxyalkylene group of 0 to 600, and m is 1
An integer of 0 to 7, n is an integer of 0 to 6, m + n = 1 to 7, n /
(1 + m + n) ≦ １ ／ and a + bm + cn = 1 to 10
00. )

In the formula (1), 2 to 8 wherein Z is a residue
Compounds having one hydroxyl group include ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, styrene glycol, and carbon atoms having 8 to 18 carbon atoms.
Glycols such as neopentyl glycol, glycerin, diglycerin, polyglycerin, trimethylolethane, trimethylolpropane,
Polyhydric alcohols such as 1,3,5-pentanetriol, erythritol, pentaerythritol, dipentaerythritol, sorbitol, sorbitan, sorbide, sorbitol and glycerin, adonitol, arabitol, xylitol, mannitol, and partially etherified products thereof Or an esterified product; a sugar such as xylose, arabinose, ribose, rhamnose, glucose, fructose, galactose, mannose, sorbose, cellobiose, maltose, isomaltose, trehalose, sucrose, raffinose, gentianose, melezitose, or a partially etherified product thereof, or Esters; phenols such as catechol, resorcinol, hydroquinone, and phloroglucin.

The oxyalkylene group represented by AO includes oxyethylene, oxypropylene, oxybutylene, oxytetramethylene, oxystyrene, oxydecylene, oxytetradecylene, oxyhexadecylene. And an oxyoctadecylene group. These may be added alone, or two or more may be added simultaneously. When two or more types are added at the same time, block-shaped addition or random addition may be performed.

The oxyalkylene group is necessary to increase the affinity between the copolymer and superoxide dismutase and to improve the stability of superoxide dismutase. It is necessary that a + bm + cn does not exceed 1,000 because the weight ratio of the acid unit becomes low and the reaction with the amino group in the superoxide dismutase becomes difficult to occur.

The alkenyl group having 2 to 5 carbon atoms represented by R ¹ includes vinyl, allyl, methallyl, 1,1-
Examples include a dimethyl-2-propenyl group and a 3-methyl-3-butenyl group. 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. , 2-ethylhexyl, octyl, isononyl, decyl, dodecyl, isotridecyl, tetradecyl, hexadecyl, isocetyl, octadecyl, isostearyl, oleyl, octyldodecyl, docosyl, decyltetradecyl Group, benzyl group, cresyl group, butylphenyl group, dibutylphenyl group, octylphenyl group, nonylphenyl group, dodecylphenyl group, dioctylphenyl group, dinonylphenyl group, styrenated phenyl group, and the like.As the acyl group, 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, arachinic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid, There are acyl groups derived from linolenic acid, erucic acid, benzoic acid, hydroxybenzoic acid, cinnamic acid, gallic acid and the like.

In order for the copolymer of the alkenyl ether of the formula (1) and maleic anhydride to sufficiently bind to superoxide dismutase, an acid anhydride structure is required, and an alkenyl ether having a large amount of free hydroxyl groups is used. To form an ester bond with the acid anhydride unit during polymerization,
As the reactivity with superoxide dismutase decreases or the solubility of the copolymer in the solvent decreases,
It is necessary that n / (1 + m + n) ≦ 1/2.

The copolymer used in the present invention can be easily obtained by copolymerizing the alkenyl ether represented by the formula (1) with maleic anhydride using a radical polymerization catalyst. At that time, another monomer may be added for copolymerization. Other monomers include unsaturated carboxylic acids such as acrylic acid, methacrylic acid, itaconic acid and crotonic acid, aromatic vinyl compounds such as styrene and methylstyrene, vinyl halide compounds such as vinyl chloride and vinylidene chloride, and isobutylene. And olefins such as diisobutylene, and vinyl acetate, acrylonitrile, and acrylamide. These can be added when improving the physical properties such as making the copolymer sticky, but if the ratio is too large, the content of oxyalkylene groups or acid anhydride groups necessary for modification of superoxide dismutase will be increased. Since the amount decreases and the modification cannot be performed well, the proportion of other monomers must be 30 mol% or less of all monomers.

By changing the type of polymerization initiator or the structure of the alkenyl ether of the formula (1), copolymers having various degrees of polymerization can be obtained. The molecular weight is from 10 to 2,000,000, preferably from 3000 to 100,000. In addition, a copolymer having a high hydrophilicity can be obtained by using an oxyalkylene group to which a large number of oxyethylene groups are added.

The method for obtaining the modified superoxide dismutase of the present invention is a method utilizing an amino group in the superoxide dismutase, and the target superoxide dismutase is a copper / zinc type superoxide obtained from bovine erythrocytes. Various types can be used, such as oxidized dismutase, manganese superoxide dismutase derived from microorganisms, copper / zinc superoxide dismutase derived from human erythrocytes, and human copper / zinc superoxide dismutase obtained by genetic recombination. It is not something to be done.

The modified superoxide dismutase of the present invention can be obtained by reacting a copolymer with superoxide dismutase. The ratio at which the copolymer is reacted with superoxide dismutase cannot be specified unconditionally because it varies depending on the content of amino groups in the superoxide dismutase or acid anhydride groups in the copolymer. If the amount of the copolymer is too small compared to the amount of the copolymer, the intended temporal stability will be insufficient because the modification rate will decrease, and if the amount of the copolymer is too large, the initial activity of the modified superoxide dismutase will be remarkable. A preferred range is 50 to 400 parts by weight of the copolymer based on 100 parts by weight of superoxide dismutase.

When the copolymer is water-soluble, a method of directly adding the copolymer to an aqueous solution of superoxide dismutase is preferred. When the copolymer is hardly soluble in water or insoluble in water, In the case of (1), it is easy and preferable to dissolve the copolymer in a solvent such as acetone which is compatible with water, and then add the resulting solution to the aqueous solution of superoxide dismutase.

The pH at the time of the reaction is 6-10, preferably 8-9. This is because when the pH is on the acidic side, the amount of free amino groups in the superoxide dismutase decreases, and the modification rate decreases. The reaction temperature is
If it is too high, the activity of superoxide dismutase in the modification process will decrease, and the hydrolysis of the acid anhydride group will occur more easily than the reaction between the amino group and acid anhydride group of superoxide dismutase, and the modification rate will decrease. Therefore, 0 to 10 ° C. is a preferable range.

[0021]

Industrial Applicability The present invention is a novel modified superoxide dismutase which is a reaction product of a copolymer of an alkenyl ether having a polyoxyalkylene group with maleic anhydride and superoxide dismutase, and has a long activity in aqueous systems. A superoxide dismutase that can be sustained and has low skin irritation and a wide application range can be provided.

[0022]

EXAMPLES The present invention will be described with reference to Production Examples and Examples. Production Example 1 The following compound was dissolved in 1 liter of toluene, and a polymerization reaction was carried out at 80 ± 2 ° C. for 7 hours under a nitrogen atmosphere.

CH ₂ = CHCH ₂₀ (C ₂ H ₄ O) ₃₃ CH ₃ 1524 g (1 mol) Maleic anhydride 103 g (1.05 mol) Benzoyl peroxide 4.8 g (0.02 mol) Then toluene and unreacted maleic anhydride 10 to 30
Distillation at 100 ± 10 ° C under reduced pressure of mmHg, 1450g of copolymer
No.1 was obtained. The obtained copolymer No. 1 was a pale yellow waxy solid, having a melting point of 45 ° C. and a saponification value of 68.5. Production Example 2 19.9 g (0.05 mol) of lauroyl peroxide was dissolved in 1 liter of benzene, heated to 70 ° C. while stirring under a nitrogen atmosphere, and then a mixed solution having the following composition was added dropwise at 70 ± 2 ° C. A polymerization reaction was performed.

CH ₂ = CHCH ₂₀ (C ₂ H ₄ O) ₂₀ C ₄ H ₉ 497 g (0.5 mol)

[0025]

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3 g of maleic anhydride 103 g (1.05 mol) 3 liters of benzene was added dropwise, and after maintaining at the same temperature for 3 hours, benzene and unreacted maleic anhydride were removed under reduced pressure of 10 to 30 mmHg.
The solvent was distilled off at 120 ± 10 ° C. to obtain 1293 g of copolymer No. 2. Copolymer No. 2 was a viscous liquid and had a saponification value of 81.5.

Thereafter, copolymers No. 3 to No. 8 were prepared in the same manner.
Were prepared at the reaction molar ratios and reaction conditions shown in Tables 1-2. The analytical values and solubility are shown in Table 3.

[0028]

[Table 1]

[0029]

[Table 2]

[0030]

[Table 3]

Example Using copper / zinc type superoxide dismutase (3600 units / mg, manufactured by Sigma) obtained from bovine erythrocytes as superoxide dismutase, modification with the copolymer shown in Table 1 was attempted. The activity of the modified superoxide dismutase was measured by the cytochrome C method.

Cytochrome C method 300 mM potassium phosphate buffer (pH 7.8) was placed in a test tube (Note 1)
0.5 ml, 0.3 mM hypoxanthine 0.5 ml, 60 μM cytochrome C (Sigma bovine heart type V) 0.5 ml, superoxide dismutase sample solution 0.3 ml and water 1 ml
And mixed well. Xanthine oxidase
(Note 2) Add 0.2 ml and stir, quickly, optical cell (1 ml)
Moved to

The reduction rate of cytochrome C was determined from the rate of increase in absorbance at 550 nm using a Hitachi spectrophotometer U-3210. Under these conditions, the amount of superoxide dismutase enzyme that inhibits the reduction of cytochrome C by 50% when superoxide dismutase is not added is defined as 1 enzyme unit.
The number of units per mg protein was determined. (Note 1) 300 mM potassium phosphate buffer containing 0.6 mM EDTA-2Na. (Note 2) The reduction rate of cytochrome C (change rate per minute at 550 nm) is about 1% when xanthine oxidase superoxide dismutase is not added.
Determine the concentration to be added to 0.025. Example 1 1 g of superoxide dismutase (3600 units / mg)
Was dissolved in 20 g of a borate buffer (Note 3) having a pH of 8.6, and the temperature of the system was maintained at 3 ° C. 1 g of Copolymer No. 1 was added to the system in the form of a fine powder, and the mixture was stirred at 3 ± 1 ° C. for 30 minutes. Then, another 1 g was added and stirring was continued at the same temperature for 30 minutes. After the completion of the reaction, the pH was adjusted to 7.0 with a 0.1N aqueous solution of sodium hydroxide, and then the inorganic salts were removed using a reverse osmosis membrane.
Dry at 35 ° C for modified superoxide dismutase 1.
8 g were obtained.

The activity of the resulting modified superoxide dismutase measured by the cytochrome C method was 840 units.
s / mg, and the residual activity ratio due to the modification of superoxide dismutase was 70% in terms of pure content. (Note 3) Boric acid buffer (pH 8.6) 0.2M-aqueous sodium hydroxide solution 12.00ml 0.2M-boric acid / potassium chloride aqueous solution 50.00ml The above aqueous solutions were mixed, and purified water was added to make a total volume of 200ml.

The aqueous solution of 0.2 M boric acid / potassium chloride is composed of 12.4 g of boric acid and 14.9 g of potassium chloride per liter of solution.
It is an aqueous solution containing g. FIGS. 1 and 2 show infrared absorption spectra of the modified superoxide dismutase and the raw material superoxide dismutase, respectively.

FIG. 3 shows these gel permeation chromatograms together with copolymer No. 1.
It can be seen that the modified superoxide dismutase has a higher molecular weight. The conditions for gel permeation chromatography are as follows. The above two are connected.

Sample concentration: Superoxide dismutase 0.5 mg / ml Modified superoxide dismutase 1.0 mg / ml Sample injection volume: 100 μl Solvent: 0.1 M phosphate buffer (pH 7.0) Flow rate: 0.5 ml / min Detector: Showa Electric Works, Ltd. RI Differentiometer SE-61 Example 2 Superoxide Dismutase (3600 units / mg) 1g
Was dissolved in 20 g of a pH 8.0 phosphate buffer (Note 4), and the temperature was maintained at 3 ° C. To this was added 0.5 g of copolymer No. 3 (20% acetone solution), and the mixture was stirred at 3 ± 1 ° C. for 30 minutes.
0.5 g (20% acetone solution) was added, and the mixture was stirred at the same temperature for 30 minutes.

Next, water was distilled off at 35 ° C. under reduced pressure to obtain 2.3 g of modified superoxide dismutase. The activity of the resulting modified superoxide dismutase is 972 units
s / mg, and the residual activity rate by modification was 64% in pure content conversion. (Note 4) Phosphate buffer (pH 8.0) 0.2M-aqueous sodium hydroxide solution 46.85ml 0.2M-potassium dihydrogen phosphate aqueous solution 50.00ml The above aqueous solutions were mixed, and purified water was added to make a total volume of 200ml. Examples 3 to 7 and Comparative Examples 1 and 2 Hereinafter, modified superoxide dismutases shown in Table 4 were obtained in the same manner. In Example 5, inorganic salts were removed using a reverse osmosis membrane as in Example 1. Table 4 shows the results of the activity and the stability over time of these modified superoxide dismutases.

Stability test with time 0.5 g of the modified superoxide dismutase was dissolved in 100 ml of a phosphate buffer solution (pH 5) (pH 5) (containing 0.1% of methyl 4-hydroxybenzoate as a preservative) at 40 ° C. After holding for a while, the activity of the modified superoxide dismutase was measured by the cytochrome C method to examine the stability over time.

As a comparative solution, a solution prepared by dissolving 0.5 g of superoxide dismutase in 100 ml of a phosphate buffer at pH 7.0 (containing 0.1% of methyl 4-hydroxybenzoate as a preservative) (Comparative Example 1) The stability over time was also examined for a sample to which 20% of 1,3-butylene glycol was added (Comparative Example 2). Table 4 shows that the modified superoxide dismutase of the present invention has excellent temporal stability in an aqueous system. (Note 5) Phosphate buffer solution (pH 7.0) pH adjusted to 7.0 by mixing 10 mM disodium hydrogen phosphate aqueous solution and 10 mM monosodium hydrogen phosphate aqueous solution.

[0041]

[Table 4]

In the table, SOD: superoxide dismutase Note 1) Units: units / mg 2) Modified SOD for SOD activity (3600 units / mg)
Activity rate (%) 3) Activity rate of modified SOD to initial activity with time after completion of stability test (%) 4) SOD dissolved in phosphate buffer (containing methyl 4-hydroxybenzoate) 5) SOD 20% 1,3-butylene glycol in aqueous solution
Added.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of superoxide dismutase modified with copolymer No. 1.

FIG. 2 is an infrared absorption spectrum of superoxide dismutase.

FIG. 3 Superoxide dismutase, copolymer N
1 is a gel permeation chromatogram of o.1 and modified superoxide dismutase.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61K 7/48 A61K 7/48 (72) Inventor Toru Yasugouchi 2-3-10- 404 Fujisaki, Kawasaki-ku, Kawasaki-shi, Kawasaki, Kanagawa Prefecture (72) Inventor Akinori Suginaka 2-4-10 Murota, Chigasaki City, Kanagawa Prefecture (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 9/00-9/64 A61K ^7/ 00-7/50 BIOSIS (DIALOG) ) CA (STN) WPI (DIALOG)

Claims (1)

(57) [Claims] The molar ratio of the alkenyl ether represented by the formula (1), maleic anhydride and another monomer is 5 to 60:
Superoxide dismutase modified with a copolymer of 20-90: 0-30. Embedded image (Z is a residue of a compound having 2 to 8 hydroxyl groups, AO is one or a mixture of two or more oxyalkylene groups having 2 to 18 carbon atoms, and if two or more, it is added in a block form. R ¹ may have 2 carbon atoms.
An alkenyl group of 5 to 5, R ² is a hydrocarbon group or an acyl group having 1 to 24 carbon atoms, a, b and c are each an average addition mole number of an oxyalkylene group of 0 to 600, m is an integer of 1 to 7,
n is an integer of 0 to 6, m + n = 1 to 7, n / (1 + m +
n) ≦ １ ／ and a + bm + cn = 1 to 1000. )