JPH06141857A - Modified gamma-gultamyltranspeptidase and its use - Google Patents

Abstract

PURPOSE:To obtain the subject new enzyme freed from dislocation activity, capable of producing 7-aminocephalosporanic acid by acting it on cephalosporin C. CONSTITUTION:The objective gamma-glutamyltranspeptidase modified with a copolymer of the formula [Z is residue of a compound bearing 2 to 8 hydroxyl groups; AO is 2-18C oxyalkylene; R<1> is 2-5C alkenyl; R<2> is 1-24C hydrocarbon group or acyl; (a), (b) and (c) are each average of bearing mole number of oxyalkylene groups, being 0-600; (m) is an integer of 1-7; (n) is an integer of 0-6; (m+n) is 1-7; n/(m+n+1)<=0.5; a+bm+cn=1-1000] with the molar ratio for the constituents: alkenyl ether, maleic anhydride and another monomer being [(5-60):(20-90):(0-30)]. This enzyme can be obtained by reaction between this copolymer and gamma-glutamyltranspeptidase.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a γ-glutamyl transpeptidase modified with a high molecular weight copolymer and a novel method for producing γ-aminocephalosporanic acid using the same.

[0002]

2. Description of the Related Art γ-Glutamyl transpeptidase is known as an enzyme that plays a major role in the γ-glutamyl cycle in which an amino acid is taken into the cell membrane from the outside of the cell membrane in vivo, and it is particularly active in cancer cells. It is an enzyme that is used in the field of diagnostic agents for functional tests of the kidney and liver because of its existence.

This γ-glutamyl transpeptidase reacts with intracellular γ-glutarcysteinyl glycinta tathione and amino acids outside the cell membrane to generate cysteinyl glycine by hydrolysis and rearrangement to produce γ. -A reaction for producing a glutamyl amino acid is carried out at the same time, but this industrial rearrangement reaction has hardly been used so far.

Further, 7-aminocephalosporanic acid is known as a starting material for various β-lactam antibiotics. Currently, it is industrially isolated by fermentative production of cephalosporin C, followed by isolation of amino group and carboxyl group. Protecting the group and via the imino chloride and imino ether intermediates (FMHuber, et al; "Cephalo-sp
orins and Penicillins, Chemistry and Biology ", ed.
by EHFlynn, Academic Press Inc., New York, 197
It is synthesized by a chemical reaction called 2, p.27).

However, this chemical synthesis method is a multi-step reaction, has many by-products, and has a low yield. In addition, 7β- (4-carboxybutanamide) cephalosporanic acid obtained by decarboxylation and deacylation from cephalosporin C by a chemical method was hydrolyzed with an amide bond using penicillin acylase to obtain 7-aminocephalosporan. Method of synthesizing acid (Journal of Japan Agricultural Chemistry Vol. 63 No. 12 1847-18
Page 58 (1989)) has been proposed by Tsuzuki et al. This method uses raw materials obtained by a chemical method, and the enzyme used can only be obtained from Pseudomonas strain SY-77-1, and reverse amidation reaction occurs depending on pH besides hydrolysis reaction. Therefore, there is a problem that the process becomes complicated.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a novel decorative γ-glutamyl transpeptidase which has no transposition activity while retaining the high hydrolysis-degrading property of γ-glutamyl transpeptidase, and the use thereof. It is an object of the present invention to provide a method for efficiently and efficiently producing 7-aminocephalosporanic acid.

[0007]

According to the present invention, the molar ratio of the alkenyl ether represented by the formula (1) to maleic anhydride and the other monomer is 5 to 60:20 to 90: 0 to 30. Γ-glutamyl transpeptidase chemically modified with a copolymer,
And a method for producing 7-aminocephalosporanic acid, which comprises reacting cephalosporin C with the same.

[0008]

[Chemical 2] (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 when two or more are added in a block form. Or it may be added randomly, and R ¹ has 2 carbon atoms.
~ 5 alkenyl group, R ² is a hydrocarbon group or acyl group having 1 to 24 carbon atoms, a and b and c are 0 to 600, respectively, in the average number of moles of added oxyalkylene group, m is an integer of 1 to 7,
n is an integer of 0 to 6, m + n = 1 to 7, n / (m + n +
1) ≦ 1/2, and a + bm + cn = 1 to 10000. In the formula (1), as the compound having 2 to 8 hydroxyl groups with Z as a residue, ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, styrene glycol, alkylene glycol having 8 to 18 carbon atoms, Glycol such as neopentyl glycol, glycerin, diglycerin, polyglycerin, trimethylolethane, trimethylolpropane, 1,3,5-pentanetriol, erythritol, pentaerythritol, dipentaerythritol, sorbitol, sorbitan, sorbide, sorbitol and glycerin. Condensation products, polyhydric alcohols such as adonitol, arabitol, xylitol and mannitol, or their partially etherified products or esterified products; xylose, arabinose, ribose, rhamno. , Glucose, fructose, galactose, mannose, sorbose, cellobiose, maltose, isomaltose, trehalose, sucrose, raffinose, gentianose, merezitose and other sugars, or their partially etherified or esterified compounds; catechol, resorcinol, hydroquinone, phloroglucin, etc. Phenols of.

The oxyalkylene group represented by AO includes oxyethylene group, oxypropylene group, oxybutylene group, oxytetramethylene group, oxystyrene group, oxidedecylene group, oxytetradecylene group, oxyhexadecylene group. , Oxyoctadecylene group and the like. These may be added alone or in combination of two or more. When two or more kinds are added at the same time, block-shaped addition or random-shaped addition may be performed.

The oxyalkylene group enhances the affinity between the copolymer and γ-glutamyl transpeptidase,
And it is necessary to eliminate the rearrangement activity of γ-glutamyl transpeptidase, but if it is too much, the weight ratio of maleic anhydride units in the copolymer becomes low, and γ-
Since it is difficult for the reaction with the amino group in glutamyl transpeptidase to occur, it is necessary that a + bm + cn does not exceed 1000. As the alkenyl group having 2 to 5 carbon atoms represented by R ¹ , a vinyl group, an allyl group, a methallyl group,
1,1-dimethyl-2-propenyl group, 3-methyl-3-
There is a butenyl group.

Examples of the alkyl group having 1 to 24 carbon atoms represented by R ² include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, pentyl group, isopentyl group and hexyl group. , Isoheptyl 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, docosyl group, Decyl tetradecyl group, benzyl group, cresyl group, butylphenyl group, dibutylphenyl group, octylphenyl group, nonylphenyl group, dodecylphenyl group, dioctylphenyl group, dinonylphenyl group, styrenated phenyl group, etc., acyl group As acetic acid, propionic acid, butyric acid Acid, isobutyric acid, caprylic acid, 2-ethylhexanoic acid, isononanoic acid, capric acid, lauric acid, myristic acid, palmitic acid, isopalmitic acid, stearic acid, isostearic acid, arachidic acid, behenic acid, palmitoleic acid, oleic acid , Linoleic acid, linolenic acid, erucic acid, benzoic acid, hydroxybenzoic acid, cinnamic acid, gallic acid and the like.

In order for the copolymer of the alkenyl ether represented by the formula (1) and maleic anhydride to sufficiently bond with γ-glutamyl transpeptidase, an acid anhydride structure is necessary and a free hydroxyl group is required. When a large amount of alkenyl ether is used, an acid anhydride unit and an ester bond are formed during the polymerization, so that the reactivity with γ-glutamyl transpeptidase is decreased and the solubility of the copolymer in a solvent is decreased. Therefore, it is necessary that n / (m + n + 1) ≦ 1/2.

The copolymer used in the present invention can be easily obtained by copolymerizing the alkenyl ether represented by the formula (1) and maleic anhydride using a radical polymerization catalyst. At that time, another monomer may be further 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, isobutylene. , Olefins such as diisobutylene, vinyl acetate, acrylonitrile and acrylamide.

These can be added when improving the physical properties such as making the copolymer tacky, but if the proportion is too large, the polyoxyalkylene group necessary for modification of γ-glutamyl transpeptidase is added. Alternatively, the content of the acid anhydride group decreases, and the modification cannot be performed well. Therefore, the proportion of other monomers needs to be 30 mol% or less of the total monomers.

The copolymer used in the present invention can be obtained by changing the kind of the polymerization initiator or the structure of the alkenyl ether of the formula (1) to obtain copolymers having various degrees of polymerization. The molecular weight is 10 to 2,000,000, preferably 3,000 to 100,000. Further, when an oxyalkylene group to which many oxyethylene groups are added is used, a highly hydrophilic copolymer can be obtained. The method for obtaining the modified γ-glutamyl transpeptidase used in the present invention is a method utilizing the amino group of γ-glutamyl transpeptidase, and the target γ-glutamyl transpeptidase is rat or pig or Derived from kidney and liver of bovine animals, derived from cancer cells of animal liver, Agaricus bisporu
s ) and phaseolus bulgari ( Phaseolus vulgari ), etc.

The modified γ-glutamyl transpeptidase used in the present invention can be obtained by reacting a copolymer with γ-glutamyl transpeptidase. The ratio at the time of reacting the copolymer with γ-glutamyl transpeptidase should be specified unconditionally because it depends on the content of the amino group in γ-glutamyl transpeptidase or the acid anhydride group in the copolymer. However, if the amount of the copolymer is too small compared to the amount of the amino group, the modification rate decreases, so that the target γ-glutamyl transpeptidase without rearrangement activity cannot be obtained, and the copolymerization Since the hydrolysis activity of the modified γ-glutamyl transpeptidase is remarkably reduced when the amount of the combined product is too large, the preferred range is γ-transpeptidase 10
The amount of the copolymer is 50 to 1000 parts by weight based on 0 parts by weight.

When the copolymer is water-soluble, the reaction between the two is
A method of directly adding the copolymer to an aqueous solution of γ-glutamyl transpeptidase is preferable, and when the copolymer is insoluble in water, the copolymer is previously dissolved in a solvent such as acetone which is compatible with water, The method of adding to the γ-glutamyl transpeptidase aqueous solution is easy and is a preferable method.

The pH during 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 γ-glutamyl transpeptidase is small and the modification rate is low. In addition, the reaction temperature is too high, the activity of γ-glutamyl transpeptidase in the modification step is reduced, and the hydrolysis reaction of the acid anhydride group causes the reaction between the amino group and the acid anhydride group of γ-glutamyl transpeptidase. Since it is more likely to occur than the reaction and the modification rate is lowered, 0 to 10 ° C is a preferable range. The cephalosporin C used in the present invention can be produced by a fermentation method using a microorganism such as Candida acremonium ( C. acremonium ).

[0019]

The present invention provides a copolymer of an alkenyl ether having a polyoxyalkylene group and maleic anhydride, and γ
-A novel modified γ-glutamyl transpeptidase, which is a reaction product with glutamyl transpeptidase, and a method for producing 7-aminocephalosporanic acid which causes it to act on cephalosporin C. Since there is no rearrangement activity, 7-aminocephalosporanic acid can be efficiently produced from cephalosporin C.

[0020]

EXAMPLES The present invention will be described with reference to examples. 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 in a nitrogen atmosphere. CH ₂ = CHCH ₂ O (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 10 30
After distilling off at 100 ± 10 ℃ under reduced pressure of mmHg, 1450g of copolymer N
I got o.1. The copolymer No. 1 thus obtained was a pale yellow waxy solid and had a melting point of 45 ° C. and a saponification value of 68.5.

100 mg of γ-glutamyl transpeptidase (Note 1) was dissolved in 0.5 g of borate buffer (Note 2) having a pH of 8.6, and the temperature of the system was kept at 3 ° C. Copolymer No. 1
(200 mg) in the form of fine powder was added to the system and stirred at 3 ± 1 ° C. for 30 minutes, then 200 mg of copolymer No. 1 was added again and the mixture was stirred at the same temperature for 30 minutes. Furthermore, 200 mg of copolymer No. 1
Was added and stirring was continued at the same temperature for 30 minutes. 0.1N after reaction
-The pH was adjusted to 7.0 with aqueous sodium hydroxide solution. Then, after removing the salt derived from the buffer solution using a reverse osmosis membrane,
It was dried at 35 ° C. under reduced pressure to obtain 550 mg of modified γ-glutamyl transpeptidase.

The activity of the obtained γ-glutamyl transpeptidase was determined by the glutathione method (Note 3) for hydrolysis activity and γ-glutamyl-p for translocation activity.
- nitroanilide method (4) result of measurement, the hydrolytic activity (K _m) is 21.2U / mg, transposition activity (V _max) is
It was 0.0001 U / mg. The residual activity rate due to the modification of the raw material γ-glutamyl transpeptidase in terms of pure content was 82% in terms of hydrolysis activity and 0 in terms of rearrangement activity.
%Met. (Note 1) γ-glutamyl transpeptidase

1600 g of azo dye hepatocarcinoma tissue (previously removed necrosis and cryopreserved) was homogenized with 4 liters of 0.08M-MgCl ₂ and adjusted to pH 10.0 with 1N-NaOH.
The mixture was adjusted to 37 ° C and stirred for 2 hours. After centrifugation at 40,000 G for 30 minutes, 3 liters of 1.5% deoxycholic acid was added to the precipitate.
(Dissolved in 0.1 M Tris-hydrochloric acid buffer solution (Note 5) (pH 8.0)) was added, and the mixture was stirred overnight at 0 ° C. After centrifuging at 40,000 G for 30 minutes, collect the supernatant and use 20 liters of distilled water.
Dialysis was performed for 40 hours.

8 liters of the dialyzed solution was cooled to 0 ° C., and acetone previously cooled to −20 ° C. was added with stirring to a final concentration of 15% (V / V), and the whole amount was added and stirred for 30 minutes. After centrifugation at 13,000 G for 30 minutes, the final concentration of 40% in the supernatant
Acetone is added again with stirring so that
Centrifuged at 3,000 G for 30 minutes. 0.1M-Tris-for precipitation
Hydrochloric acid buffer (pH 8.0) was added until dissolved.

Then, reduced glutathione was added at a final concentration of 0.04 M /
Add to make it 1 liter, hold at 56 ℃ for 10 minutes,
It was immediately cooled and centrifuged at 40,000 G for 30 minutes.
The supernatant was removed, and the precipitate was again added with 1 L of 0.1 M Tris-HCl buffer (pH 9.0, containing 1% deoxycholic acid).
Was added and the mixture was stirred at 0 ° C. overnight. After centrifugation at 40,000 G for 30 minutes, ammonium sulfate was added to the supernatant so that the final concentration was 80%, and the mixture was centrifuged at 13,000 G for 30 minutes to collect a brownish brown floating layer. To this, 100 ml of 0.02M-Tris-hydrochloric acid buffer solution (pH 8.0) was added and dissolved uniformly, and then 0.002M-
Dialysis was performed using 10 liters of Tris-HCl buffer (pH 8.0). A 20% streptomycin sulfate solution (0.002M-Tris-hydrochloric acid buffer solution) was added to the obtained crude enzyme solution while stirring.
(dissolved in pH 8.0) was added with a Pasteur pipette until no more white turbidity occurred. After centrifugation at 40,000 G for 30 minutes, ammonium sulfate was added to a final concentration of 40%, the mixture was stirred at 4 ° C. for 30 minutes, and again centrifuged at 18,000 G for 30 minutes to collect the supernatant.

Ammonium sulfate was further added to the supernatant to a final concentration of 80%, the resulting precipitate was centrifuged at 18,000 G for 30 minutes, and then 0.01 M Tris-hydrochloric acid buffer solution (pH 8.0) was dissolved in the precipitate. Added. Then, an equal amount of 1-butanol was added, and the mixture was stirred at 0 ° C for 10 minutes and then kept at 37 ° C for 15 minutes. Centrifuge at 40,000 G for 30 minutes, collect the lower aqueous layer, dialyz with 20 liters of 0.01 M Tris-HCl buffer (pH 8.0), and keep the whole dialysis tube on ammonium sulfate powder at 4 ° C. While shaking slowly for 10 hours. The resulting precipitate was collected by centrifugation at 12,000 G for 30 minutes, and again collected in 0.01 M Tris-HCl buffer (pH 8.0).
Dissolved in.

Next, Sephadex G- which had been previously equilibrated with 0.01 M Tris-hydrochloric acid buffer (pH 8.0, containing 0.15 M NaCl) was used.
The active portion was separated and collected on a 200 column (2.5 × 100 cm) (Pharmacia), and 0.01 M-Tris-hydrochloric acid buffer solution (pH 8.0,
After sufficient dialysis with 0.08M-NaCl), a DEAE-Sephadex-A-50 column (1.7 × 20 cm) equilibrated with 0.01M-Tris-hydrochloric acid buffer (pH 8.0, containing 0.08M-NaCl). ) (Pharmacia) to increase the salt concentration to 0.1-0.3M
The active portion eluting at the salt concentration of M was collected.

The recovered active portion was concentrated to about 2 ml with a collodion pack (made by Sartorius), which is an analytical kit utilizing a collodion membrane, and then a 65% ammonium sulfate solution.
(Dissolved in 0.1 M Tris-HCl buffer (pH 8.0))
After adding 2 ml and stirring, the mixture was centrifuged at 12,000 G for 10 minutes to precipitate the enzyme. Then add 60% ammonium sulfate solution,
The centrifugation was repeated again. Ammonium sulfate concentration of 58
%, 57%, 56%, 55%, and the same operation was repeated.

The supernatant obtained by centrifugation at an ammonium sulfate concentration of 55% was freeze-dried to obtain 20 mg of γ-transpeptidase having a molecular weight of 113,000. The obtained γ-transpeptidase had a hydrolysis activity of 180 U / mg and a rearrangement activity of 22 U / mg. (Note 2) 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 solution was mixed, and the total volume was made up to 200ml. The 0.2 M boric acid / potassium chloride aqueous solution is an aqueous solution containing 12.4 g of boric acid and 14.9 g of potassium chloride in 1 liter of the solution.

[0030] (Note 3) glutathione Method L- ^[glycine-2-3 H] glutathione solution (Note 6) 3.0 ml
Place in a test tube and keep it at 37 ℃ in advance. After adding 0.6 ml of enzyme sample solution I (Note 7) and heating at 37 ℃ for 20 minutes, add protein precipitation reagent (Note 8) to carry out enzyme reaction. Stopped and filtered. Then, cysteinyl- [ ³ H] glycine in the filtrate was fractionated and quantified using a Dowex-1 (acetic acid type) ion exchange column (manufactured by Dow Chemical Co.). The enzyme activity was 1 μm per minute for cysteinyl- [ ³ H] glycine under the above measurement conditions.
The amount of enzyme produced in M / liter was expressed as 1 U / mg.

(Note 4) γ-glutamyl-p-nitroanilide method γ-glutamyl-p-nitroanilide and glycylglycine mixed solution (Note 9) (3.0 ml) was placed in a test tube and preliminarily stored at 37 ° C.
Keep it warm and add 0.6 ml of enzyme sample solution II (Note 10) to 3
After heating at 7 ° C. for 20 minutes, a protein precipitation reagent was added to stop the enzymatic reaction and filtration was performed. Then, the concentration of p-nitroaniline in the filtrate was measured by liquid chromatography.
The enzyme activity is expressed as 1 U / mg of the amount of enzyme which raises the concentration of p-nitroaniline by 1 μM / liter for 1 minute under the above measurement conditions.

(Note 5) Tris-hydrochloric acid buffer solution Tris (hydroxymethyl) aminomethane having a predetermined concentration and a hydrochloric acid aqueous solution having a half concentration thereof are used to adjust the pH to a predetermined value. (Note 6) L- ^[glycine-2-3 H] glutathione solution L- ^[glycine-2-3 H] a 50mM phosphate disodium hydrogen solution 160ml was added to glutathione 1.2g, was completely dissolved by heating, with diluted hydrochloric acid After adjusting the pH to 7.5, purified water was added to bring the total volume to 200 ml. (Note 7) Enzyme sample solution I γ-glutamyl transpeptidase 0.2 to 1.0 U
Diluted with a diluting solution to become / ml. The diluted solution was prepared by dissolving 0.57 g of sodium dihydrogen phosphate in 80 ml of purified water,
After adjusting the pH to 7.5 with dilute hydrochloric acid, add purified water to 10
It's 0 ml.

(Note 8) 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 9) γ-glutamyl-p-nitroanilide and glycylglycine mixed solution To γ-glutamyl-p-nitroanilide 2.6 g and glycylglycine 1.3 g, add 50 ml of 50 mM disodium hydrogen phosphate aqueous solution and heat to complete. It was dissolved and adjusted to pH 8.5 with diluted hydrochloric acid, then purified water was added to bring the total volume to 200 ml.

(Note 10) Enzyme sample solution II 0.2 to 1.0 U of γ-glutamyl transpeptidase to be tested
Diluted with a diluting solution to become / ml. The diluted solution should be dissolved in 50 mM disodium hydrogen phosphate purified water and diluted with dilute hydrochloric acid.
After adjusting the pH to 8.5, purified water was added to make 100 ml. Infrared absorption spectra of the modified γ-glutamyl transpeptidase and the starting γ-glutamyl transpeptidase are shown in FIGS. 1 and 2, respectively.

Example 2 6.5 g of t-butylperoxy-2-ethylhexanoate
(0.05 mol) was dissolved in 1 liter of benzene, the temperature was raised to 70 ° C. under stirring in a nitrogen atmosphere, and then a mixed solution having the following composition was added dropwise to carry out a polymerization reaction at 70 ± 2 ° C. CH ₂ = CHCH ₂ O (C ₂ H ₄ O) ₂ OC ₄ H ₉ 497 g (0.5
Mol)

[0036]

[Chemical 3] 103g of maleic anhydride (1.05
Mol) Benzene 3 liters After dropping the whole amount, the mixture was kept at the same temperature for 3 hours, and then benzene and unreacted maleic anhydride were placed under a reduced pressure of 10 to 30 mmHg.
It was distilled off at 120 ± 10 ° C. to obtain 1028 g of copolymer No. 2. Copolymer No. 2 was a viscous liquid and had a saponification value of 101.5.

Γ-Glutamyl transpeptidase 100 m
g was dissolved in 10 ml of a pH 8.0 phosphate buffer (Note 11), and the temperature was kept at 3 ° C. Add 250 mg of Copolymer No. 2 (20% acetone solution) to this and stir at 3 ± 1 ℃.
The mixture was stirred for 30 minutes, 250 mg was further added, and the mixture was stirred at the same temperature for 30 minutes. Next, the water was distilled off under reduced pressure at 35 ° C., and the modified γ-
520 mg of glutamyl transpeptidase was obtained. The activity of the resulting modified γ-glutamyl transpeptidase is
The hydrolysis activity (K _m ) is 19.3 U / mg, and the rearrangement activity (V
_max ) was 0.0000 U / mg. Γ of raw material in terms of pure content
-The residual activity rate due to modification to glutamyl transpeptidase was 64% in terms of hydrolysis activity and 0% in terms of transposition activity. (Note 11) Phosphate buffer 0.2M-sodium hydroxide aqueous solution 46.85ml 0.2M-potassium dihydrogen phosphate aqueous solution 50.00ml Purified water was added to the above mixture to make a total volume of 200ml.

Examples 3 to 7 and Comparative Example 1 Copolymers No. 3 to No. 7 shown in Tables 1 to 3 in the same manner as described below.
And the modified γ shown in Table 4 using these copolymers.
-Glutamyl transpeptidase was obtained. These residual activities are also shown in Table 4. In addition, Table 4 shows the residual activity using unmodified γ-glutamyl transpeptidase as Comparative Example 1. From Table 4, it can be seen that the modified γ-glutamyl transpeptidase of the present invention has hydrolysis activity but no transposition activity.

[0039]

[Table 1] Note) Items in {} indicate random additions.

[0040]

[Table 2] Note 1) BPO: benzoyl peroxide LPO: lauroyl peroxide, tBEH: t-butylperoxy-2-ethylhexanoate, AIBN: azobisisobutyronitrile. Table 3 below shows the analysis values of the copolymer.

[0041]

[Table 3] Table 4 below shows the composition and activity of modified γ-glutamyl transpeptidases.

[0042]

[Table 4] * 1) Unit: U / mg * 2) Percentage of enzyme pure value based on the activity of the drug substance: Indicates the inactivation rate of the enzyme in the modification reaction.

Example 8 3.5 mg of the modified γ-glutamyl transpeptidase of Example 1 was dissolved in 50 ml of 50 mM cephalosporin C solution (Note 12), and the temperature was kept at 30 ° C. with stirring with a stirrer, The enzyme reaction was carried out for 1 hour while adjusting the pH to 7.6 with N-NaOH. After completion of the reaction, the produced 7-aminocephalosporanic acid was quantified by high performance liquid chromatography.

The existing weight ratio of cephalosporin C to 7-aminocephalosporanic acid in the reaction solution was 25.1: 74.9, and the conversion rate of cephalosporin C to 7-aminocephalosporanic acid was 82%. It was The conditions of high performance liquid chromatography are shown below. Column: Unisil C18 (4 x 150 mm) (manufactured by GL Sciences Inc.) Solvent: 5% ammonium acetate aqueous solution / acetonitrile (100: 2) Flow rate: 0.5 ml / min Detector: Ultraviolet spectrophotometer (260 nm) Sample injection amount: 100 μ L (Note 12) Cephalosporin C solution (50 mM) Purified cephalosporin C 4.15 g 0.2 M-potassium dihydrogen phosphate aqueous solution 50 ml 0.2 M-sodium hydroxide aqueous solution 42.74 ml Copper sulfate (pentahydrate) 1.2 g To the above mixture Purified water was added to make the total volume up to 200 ml and dissolved uniformly.

Examples 9 to 14 In the same manner as in Example 8, 7-aminocephalosporanic acid was produced from cephalosporin C using the modified γ-glutamyl transpeptidase of Examples 2 to 7. The results are shown in Table 5.

Comparative Example 2 In the same manner as in Example 8, except that the modified γ-glutamyl transpeptidase of Example 8 was replaced by a conventional γ-glutamyl transpeptidase, cephalosporins C to 7 were obtained.
-Attempted production of aminocephalosporanic acid. The results are also shown in Table 5.

Comparative Example 3 Instead of the modified γ-glutamyl transpeptidase of Example 8, penicillin amide hydrolase (Penicillin amid) was used.
ohydrolase, EC3.5.1.11) was used to try to produce 7-aminocephalosporanic acid from cephalosporin C in the same manner as in Example 8. The results are also shown in Table 5.

[0048]

[Table 5] Note 1) Conversion: (number of moles of 7-aminocephalosporanic acid after reaction / number of moles of cephalosporin C in solution before reaction) × 100 From Table 5, the modified γ-transpeptidase of the present invention is superior. It can be seen that it has the hydrolytic activity of cephalosporin C.

[Brief description of drawings]

FIG. 1 is an infrared absorption spectrum diagram of γ-glutamyl transpeptidase modified with copolymer No. 1.

FIG. 2 is an infrared absorption spectrum diagram of γ-glutamyl transpeptidase.

Claims (2)

[Claims] 1. The molar ratio of the alkenyl ether represented by formula (1) to maleic anhydride and the other monomer is 5 to 60:
A γ-glutamyl transpeptidase modified with a copolymer of 20 to 90: 0 to 30. [Chemical 1] (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 when two or more are added in a block form. Or it may be added randomly, and R ¹ has 2 carbon atoms.
~ 5 alkenyl group, R ² is a hydrocarbon group or acyl group having 1 to 24 carbon atoms, a and b and c are 0 to 600, respectively, in the average number of moles of added oxyalkylene group, m is an integer of 1 to 7,
n is an integer of 0 to 6, m + n = 1 to 7, n / (m + n +
1) ≦ 1/2, and a + bm + cn = 1 to 1000. 2. A method for producing 7-aminocephalosporanic acid, which comprises reacting cephalosporin C with the modified γ-glutamyl transpeptidase of claim 1.