JP3106265B2 - Modified lysozyme - 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 lysozyme modified with a high molecular weight copolymer, and more particularly, to a modified lysozyme which can maintain its activity in an aqueous system for a long time and has a wide range of application. .

[0002]

2. Description of the Related Art Lysozyme is a lytic enzyme that hydrolyzes the β1 → 4 bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), which is present in mucopeptides of bacterial cell walls. This enzyme is widely found in the animal and plant kingdoms, of which hen egg white lysozyme is the most studied. Its molecular weight is 14,300,
It is a relatively small protein consisting of 129 amino acids. It has been subjected to X-ray crystallography for the first time as an enzyme, and its three-dimensional structure has been clarified.

The main pharmacological actions of lysozyme include (1) antiviral action against viral diseases, (2) antiinfective action against bacterial and protozoan infections, (3) hemostatic action, (4) antitumor and antiinflammatory actions (5) Tissue repair action is known. Lysozyme applications include anti-virus, anti-infection,
Attempts have been made to use tissue-repairing effects as cosmetics in facial cleansers, basic cosmetics, etc.In addition, it is used in toothpaste and mouthwashes to control bacteria that cause dental caries, prevent viral stomatitis, and prevent infection from the oral cavity There is an attempt for the purpose.

[0004]

However, when lysozyme is directly incorporated into an aqueous system, the enzyme activity decreases,
There are problems such as easy irritation and allergy to the skin. In the application of lysozyme, various attempts have been made such as leaving 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. The method of microencapsulation is a stabilization by cutting off the contact between lysozyme and a base such as an aqueous solution until immediately before use, and does not solve an essential problem. Also,
The method of adding polysaccharides such as polyhydric alcohol, maltose, and sorbitol is effective for stabilizing other enzymes such as protease, but has little effect on lysozyme. However, other methods have not been able to obtain a sufficiently satisfactory stabilizing effect.

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

[0006]

Means for Solving the Problems The present inventors have found that lysozyme, in which a polymer having an acid anhydride group and a polyoxyalkylene group combined in the same molecule, is chemically bonded, has a long-lasting activity even in an aqueous system. And arrived at the present invention. That is, in the present invention, the molar ratio of the alkenyl ether represented by the formula (1), maleic anhydride and another monomer is 5%.
The modified lysozyme is obtained by chemically bonding a copolymer having a ratio of 6060: 20 to 90: 0-30.

[0007]

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 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 lysozyme and to improve the stability of lysozyme. The weight percentage is lower,
Since the reaction with the amino group in the lysozyme hardly occurs, it is necessary that a + bm + cn does not exceed 1,000.

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 be sufficiently bonded to lysozyme, an acid anhydride structure is required, and if an alkenyl ether having a large amount of free hydroxyl groups is used, the polymerization will proceed. In this case, since an ester bond is formed with the acid anhydride unit, the reactivity with lysozyme is reduced, or 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 proportion is too large, the content of oxyalkylene groups or acid anhydride groups required for lysozyme 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 lysozyme of the present invention is a method utilizing an amino group in the lysozyme. The target lysozyme is lysozyme derived from chicken egg white, as well as egg white such as turkey and duck. And plants derived from plants such as papaya and figs, and Bacillus subtilis (Bacilluss subtilis).
Various substances such as those derived from microorganisms such as ubtilis) can be used, but are not particularly limited thereto.

The modified lysozyme of the present invention can be obtained by reacting a copolymer with lysozyme.
The ratio when reacting the copolymer with lysozyme cannot be specified unconditionally because it varies depending on the content of amino groups in the lysozyme or acid anhydride groups in the copolymer, but it cannot be specified unconditionally. If the amount of the copolymer is too small, the modification time decreases, and the intended temporal stability becomes insufficient.If the amount of the copolymer is too large, the initial activity of the modified lysozyme is significantly reduced. Is 50 to 400 parts by weight of the copolymer based on 100 parts by weight of lysozyme.

The reaction between the two is preferably carried out by directly adding the copolymer to an aqueous solution of lysozyme when the copolymer is water-soluble, and when the copolymer is hardly soluble in water or insoluble in water. Is an easy and preferred method in which a copolymer is dissolved in a solvent such as acetone which is compatible with water in advance and then added to an aqueous lysozyme solution. The pH at the time of the reaction is 6 to 10, preferably 8 to 9.
This is because when the pH is on the acidic side, the amount of free amino groups in lysozyme decreases, and the modification rate decreases.

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

[0020]

Industrial Applicability The present invention is a novel modified lysozyme which is a reaction product of a copolymer of an alkenyl ether having a polyoxyalkylene group with maleic anhydride and lysozyme, and has a long lasting activity even in an aqueous system. It is possible to provide lysozyme having low skin irritation and a wide application range.

[0021]

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 and heated to 70 ° C. while stirring under a nitrogen atmosphere. The polymerization reaction was performed at 2 ° C. CH ₂ = CHCH ₂₀ (C ₂ H ₄ O) ₂₀ C ₄ H ₉ 497 g (0.5 mol)

[0024]

Embedded image

3 g of maleic anhydride 103 g (1.05 mol) After adding 3 liters of benzene in total, 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.

[0027]

[Table 1]

[0028]

[Table 2]

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

[0030]

[Table 3]

Example Using chicken egg white lysozyme as lysozyme, Table 1
The modification with a copolymer was attempted. The activity of the modified lysozyme was determined by the activity of Micrococcus lysodictis (Micrococcus l
ysodeikticus) It was measured by the following measurement method using dried cells. Activity measuring method 3.0m substrate solution (Note 1) pre-incubated at 37 ℃
To this was added 0.1 ml of a sample solution (Note 2), and the mixture was stirred. The mixture was transferred to an optical cell (1 ml), and the decrease in absorbance at a wavelength of 640 nm due to lysis was measured over time using a Hitachi spectrophotometer U-3210.
The same procedure was applied to the standard lysozyme (Note 3), and the amount of lysozyme in the sample was calculated according to the following equation.

[0032] (Note 1) Micrococcus lysodetics (M.lysod)
eikticus) to 75 mM sodium phosphate (pH
6.2) Add (Note 4), shake, suspend, and add dry cells or the same buffer so that the absorbance at 640 nm wavelength becomes 1.00 using the same buffer as a control.

(Note 2) Dissolve in purified water so that the amount of lysozyme is about 5 to 15 μg / g. (Note 3) Standard product of the National Institutes of Health control No.831 (Note 4) 75 mM Na ₂ HPO ₄ and 75 mM NaH ₂ PO ₄ are mixed to obtain a pH of 6.2.
Adjust to Example 1 1 g of lysozyme (titer: 1.64 mg / g) was dissolved in 20 g of a boric acid buffer (pH 5) (note 5), and the temperature of the system was kept 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, then the inorganic salts were removed using a reverse osmosis membrane, and dried at 35 ° C under reduced pressure to obtain 2.0 g of modified lysozyme.

The result of measuring the activity of the obtained modified lysozyme was 0.41 mg / g, and the residual activity ratio by the modification of lysozyme was 75% in terms of a pure content. (Note 5) Boric acid buffer (pH 8.6) 0.2M-sodium hydroxide aqueous 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 the infrared absorption spectra of the modified lysozyme and the raw material lysozyme, respectively.

FIG. 3 shows these gel permeation chromatograms together with copolymer No. 1.
From this figure, it can be seen that the molecular weight of the modified lysozyme has increased. The conditions for gel permeation chromatography are as follows.

Sample concentration: 0.5 mg / ml lysozyme 1.0 mg / ml modified lysozyme Sample injection volume: 100 μl Solvent: 0.1 M phosphate buffer (pH 7.0) Flow rate: 0.5 ml / min Detector: RI, Showa Denko KK Example 2 1 g of lysozyme (titer: 1.64 mg / g) was dissolved in 20 g of a pH 8.0 phosphate buffer (Note 6), and the temperature was kept at 3 ° C. To this, 0.5 g (20% acetone solution) of Copolymer No. 3 was added, and the mixture was stirred at 3 ± 1 ° C. for 30 minutes. Further, 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 2.1 g of modified lysozyme. The activity of the resulting modified lysozyme was 0.435 mg / g, and the residual activity by modification was 63% in pure content. (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 lysozyme shown in Table 4 was obtained in the same manner as described below.
In Examples 3, 4, 5, and 6, 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 lysozymes.

Stability test with time 0.5 g of modified lysozyme was added to a phosphate buffer (pH 7) of pH 7.0 (Note 7).
0ml (Methyl 4-hydroxybenzoate 0.1% as preservative
Was maintained at 40 ° C. for a predetermined time, and the activity of the modified lysozyme was measured to examine the stability over time. As a comparative solution, 0.15 g of lysozyme was added to 100 ml of a phosphate buffer at pH 7.0 (containing 0.1% of methyl 4-hydroxybenzoate as a preservative).
(Comparative Example 1) and sorbitol
The stability with time was also examined for the one to which 30% was added (Comparative Example 2).

Table 4 shows that the modified lysozyme of the present invention has excellent temporal stability in an aqueous system. (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.

[0042]

[Table 4]

Note 1) Unit: mg / g 2) Modified lysozyme activity rate (%) relative to lysozyme activity (titer 1.64 mg / g) 3) Activity rate (%) relative to initial activity of modified lysozyme 4) Phosphoric acid Dissolved in a system buffer (containing methyl 4-hydroxybenzoate) 5) A solution obtained by adding 30% of sorbitol to an aqueous lysozyme solution.

[Brief description of the drawings]

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

FIG. 2 is an infrared absorption spectrum diagram of lysozyme.

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

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FI A61P 31/12 A61P 35/00 35/00 43/00 43/00 A61K 37/54 (72) Inventor Yoshiura Adachi Kita, Tokyo 48-18 Sakaemachi-ku, Kose Research Institute Co., Ltd. (72) Inventor Tohru Yasugouchi 2-3-10-, Fujisaki, Kawasaki-ku, Kawasaki-shi, Kanagawa 404 10 (56) References JP-A 1-153088 (JP, A) JP-A 1-165377 (JP, A) JP-B 55-10235 (JP, B1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9/00-9/99 A61K 31/00-48/00 A61P 1/00-43/00 BIOSIS (DIALOG) CA (STN) MEDLINE (STN) REGISTRY (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:
Lysozyme 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. )