JP3271265B2 - Modified protease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an aqueous solution of a protease.
The stabilization method in

[0002]

2. Description of the Related Art Conventionally, proteases have good storage stability in a dry state, but have a short activity in an aqueous solution due to self-digestion. Therefore, the protease is kept in a dry state until immediately before use by microencapsulation, or Methods such as immobilization with a polymer compound have been proposed, and as a method of increasing the stability in an aqueous solution, a method of adding a polyhydric alcohol or the like is disclosed in JP-B-41-152 and JP-B-53-28515. No. 6,086,045.

[0003]

However, methods such as microencapsulation and immobilization with a polymer compound are stabilizations by cutting off the contact between the protease and water until immediately before use, which is an essential problem. It was not a solution, but the aqueous solution could not have stability over time. Further, the method of adding a polyhydric alcohol is limited in its use because the physical properties of the aqueous solution change, and its stability is not sufficient.

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

[0005]

Means for Solving the Problems The present inventors have found that a protease in which a polymer having both an acid anhydride group and a polyoxyalkylene group in the same molecule is chemically bonded has a long-lasting activity even in an aqueous solution. The inventors have found that the present invention has been achieved. That is, the present invention makes protease stable in aqueous solution
In the reaction , the molar ratio of the alkenyl ether represented by the formula (1), maleic anhydride and other monomers is 5 to 60:20 to 90: 0 to 30 parts by weight of the protease. Characterized by modification with 50 to 400 parts by weight of copolymer
This is a method for stabilizing a protease in an aqueous solution .

[0006]

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. 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 having Z as 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 the protease and to prevent the protease from autolyzing. If the amount is too large, the weight ratio of the maleic anhydride unit in the copolymer is reduced. Lower,
It is necessary that a + bm + cn does not exceed 1000, since the reaction with the amino group in the protease hardly occurs.

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, an isoheptyl group, 2-ethylhexyl, octyl, isononyl, decyl, dodecyl, isotridecyl, tetradecyl, hexadecyl, isocetyl, octadecyl, isostearyl, oleyl, octyldodecyl, docosyl, decyltetradecyl Benzyl, cresyl, butylphenyl, dibutylphenyl, octylphenyl, nonylphenyl, dodecylphenyl, dioctylphenyl, dinonylphenyl, styrenated phenyl, etc. , 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, linolenic There are acyl groups derived from acids, 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 the protease, an acid anhydride structure is required, and if an alkenyl ether having a large number of free hydroxyl groups is used, the polymerization will occur. In this case, since an ester bond is formed with the acid anhydride unit, the reactivity with the protease is reduced, and the solubility of the copolymer in the solvent is reduced. Therefore, n / (1 + m + n) ≦ 1/2 It is necessary to be.

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 at the time of improving physical properties such as imparting tackiness to the copolymer, but if the ratio is too large, the content of oxyalkylene groups or acid anhydride groups required for protease modification is reduced. The ratio of other monomers must be 30 mol% or less of the total monomers, since the concentration is lowered and modification cannot be performed well.

The copolymer used in the present invention can obtain copolymers having various degrees of polymerization by changing the type of polymerization initiator or the structure of the alkenyl ether of the formula (1). 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 protease of the present invention is a method utilizing an amino group in the protease, and various proteases such as subtilisin, papain, pepsin, trypsin, chymotrypsin, and thermolysin can be used as the target protease. . The modified protease of the present invention can be obtained by reacting a copolymer with a protease.

The ratio of the reaction between the copolymer and the protease cannot be specified without fail because it varies depending on the content of amino groups in the protease or acid anhydride groups in the copolymer. If the amount of the copolymer is too small compared to the amount, the intended temporal stability becomes insufficient because the modification rate is reduced, and if the amount of the copolymer is too large, the initial activity of the modified protease is significantly reduced. The preferred range is 50 to 400 parts by weight of the copolymer based on 100 parts by weight of the protease.

The reaction between the two is carried out when the copolymer is water-soluble.
A good method is to add the copolymer directly to the protease aqueous solution.If the copolymer is difficult to dissolve in water or is insoluble in water, dissolve the copolymer in advance in a solvent such as acetone that is compatible with water. After that, the method of adding to the aqueous protease solution is easy and a preferable method. Also,
The pH during the reaction is from 6 to 10, preferably from 8 to 9. This is because when the pH is on the acidic side, the amount of free amino groups in the protease decreases, and the modification rate decreases.

On the other hand, if the reaction temperature is too high, the activity of the protease in the modification step is reduced, and the hydrolysis of the acid anhydride group is more likely to occur than the reaction between the amino group and the acid anhydride group of the protease. Because the modification rate decreases,
0 to 10C is a preferred range.

[0018]

The protease of the present invention is stable in an aqueous solution.
According to the method, the activity can be maintained for a long time even in an aqueous solution because there is no autolysis of protease.
The present invention can be used not only in fields where proteases have been used until now, but also in fields where proteases cannot be used, for example, for industrial degradation of proteins.

[0019]

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, the temperature was raised to 70 ° C. while stirring under a nitrogen atmosphere, and 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)

[0022]

Embedded image

103 g (1.05 mol) of maleic anhydride 3 liters of benzene After dropping the whole amount, the mixture was kept at the same temperature for 3 hours, and then 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.

[0025]

[Table 1]

Note) {}: Random additions are shown.

[0027]

[Table 2]

Note 1) BPO: benzoyl peroxide, LPO: lauroyl peroxide tBEH: t-butylperoxy-2-ethylhexanoate, AIBN: azobisisobutyronitrile,

[0029]

[Table 3]

EXAMPLE Subtilisin (Nagase Seikagaku Co., Ltd., trade name: bioprase bulk powder) was used as a protease, and modification with the copolymer shown in Table 1 was attempted. The activity of the modified protease was measured by a casein hydrolytic activity assay. Method for measuring casein hydrolytic activity Transfer 3.0 ml of casein solution (Note 1) to a test tube, and heat at 37 ° C in advance.
Keep warm and add 0.6 ml of enzyme sample solution (Note 2)
After heating at 37 ° C for 20 minutes, protein precipitation reagent (Note 3) 3.0m
The enzyme reaction was stopped by adding l and filtered. 0.8 ml of filtrate
Into a test tube, add 2.0 ml of 0.55 M sodium carbonate aqueous solution and 0.4 ml of Folin reagent (Note 4), and add 30 ml at 37 ° C.
The color was developed by heating for a minute, and the absorbance at 660 nm was measured. Enzyme activity was indicated amount of enzyme to 1 increase the absorbance at 660nm in the above measurement conditions in one minute as 1U _660. (Note 1) Casein solution To 1.2 g of casein (manufactured by Hammarsten), add 160 ml of 50 mM disodium hydrogen phosphate aqueous solution, dissolve completely by heating, adjust the pH to 7.5 with dilute hydrochloric acid, and add purified water. 20
0ml. (Note 2) The enzyme sample solution tested proteases to become 0.2 ~1.0 U ₆₆₀ / ml,
Diluted with diluent. The diluent was prepared by dissolving 0.57 g of sodium dihydrogen phosphate in 80 ml of purified water and diluting to pH 7.
After adjusting to 5, purified water was added to make 100 ml. (Note 3) Protein precipitation reagent Trichloroacetic acid 18.0 g Sodium acetate 18.0 g Acetic acid 19.0 ml The above compound was dissolved in purified water to make 1 liter. (Note 4) Folin reagent A commercially available folin reagent (Merck) diluted three times with purified water. Example 1 5 g of subtilisin (94 U ₆₆₀ / mg) was dissolved in 100 g of a boric acid buffer (pH 5) (Note 5), and the temperature of the system was kept at 3 ° C. To this, 5 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, 5 g was further 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 0.1 N aqueous solution of sodium hydroxide, then the inorganic salts were removed using a reverse osmosis membrane, and dried at 35 ° C under reduced pressure to obtain 9.8 g of modified protease, modified subtilisin. Was.

The activity of the modified protease thus obtained was measured by a casein hydrolysis activity measuring method to be 22 U ₆₆₀ / mg.
The activity remaining ratio by modification of the raw material subtilisin in terms of pure content was 72%. (Note 5) 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 solution was mixed, and purified water was added to make a total volume of 200ml.

Incidentally, an aqueous solution of 0.2M 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. Figure 1 shows the infrared absorption spectra of the modified protease and the starting material subtilisin.
And FIG.

FIG. 3 shows the gel permeation chromatogram together with the copolymer No. 1.
It can be seen that the molecular weight of the modified protease is increased. The conditions for gel permeation chromatography are as follows.

Sample concentration: 0.5 mg / ml subtilisin Modified protease 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 Denko KK RI differential屈計SE-61 example 2 subtilisin (94U ₆₆₀ / mg) 5g phosphate buffer solution of pH 8.0 (Note 6) was dissolved in 100 g, the temperature was maintained at 3 ° C.. To this was added 2.5 g of copolymer No. 3 (20% acetone solution) and 3
The mixture was stirred at ± 1 ° C. for 30 minutes, and 2.5 g (20% acetone solution) was added, followed by stirring at the same temperature for 30 minutes.

Next, water was distilled off at 35 ° C. under reduced pressure to obtain 11.5 g of a modified protease. The activity of the resulting modified protease was 25.4 U ₆₆₀ / mg, and the residual activity ratio by modification of the raw material subtilisin in terms of pure content was 64%. (Note 6) 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 Modified proteases shown in Table 4 were obtained in the same manner as described below. In Example 5, inorganic salts were removed using a reverse osmosis membrane as in Example 1. Table 4 shows the activity of these modified proteases and the results of the stability test over time.

Stability test with time 0.1 g of the modified protease was added to 100 ml of a phosphate buffer (Note 7) at pH 7.0 (methyl 4-hydroxybenzoate was used as a preservative).
0.1%), kept at 40 ° C. for a predetermined time, and measured the activity of the modified protease by a casein hydrolysis activity measurement method to examine the stability over time.

As a comparative example, 0.005 g of subtilisin was added at pH 7.
No. 0 in 100 ml of a phosphate buffer (containing 0.1% of methyl 4-hydroxybenzoate as a preservative) (Comparative Example 1) and a solution in which 5% of glycerin was added thereto (Comparative Example 2). The stability was investigated. Table 4 shows that the modified protease of the present invention has excellent temporal stability in an aqueous solution. (Note 7) Phosphate buffer (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.

[0038]

[Table 4]

In the table, 1) unit: U ₆₆₀ / mg 2) Activity ratio (%) of the modified protease relative to the activity of subtilisin (940 U ₆₆₀ / mg). 3) The activity rate is the activity rate (%) after completion of the stability test over time with respect to the initial activity of the modified protease.

Note) 5 glycerin was added to the subtilisin aqueous solution.
% Added

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of a subtilisin (modified protease) modified with copolymer No. 1.

FIG. 2 is an infrared absorption spectrum of subtilisin.

FIG. 3 is a gel permeation chromatogram of subtilisin, copolymer No. 1, and modified protease.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroko Sonogami 48-18 Sakaemachi, Kita-ku, Tokyo Inside Kose Research Institute, Inc. (72) Inventor Midori 48-18 Sakaemachi, Kita-ku, Tokyo Kobayashi, Inc Kose Research Institute (56) References JP-A-1-153088 (JP, A) JP-A-57-122795 (JP, A) Biotechnology and Bioengineering, (1982) Vol. 24, pp. 4 (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 9/48-9/99 JICST file (JOIS) WPI / L (QUESTEL) BIOSIS (DIALOG)

Claims (1)

(57) [Claims] 1. A method for stabilizing a protease in an aqueous solution.
Per 100 parts by weight of the protease , the molar ratio of the alkenyl ether represented by the formula (1), maleic anhydride and other monomers is 5 to 60:20 to 90: 0 to 30. Modification with 50-400 parts by weight of coalescence
Method of stabilizing ase in aqueous solution . 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. Or may be added randomly, and R ¹ has 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. )