JPS6248379A - Production of cephalosporin c acylase - Google Patents

Abstract

PURPOSE:To produce cephalosporin C acylase, having specific hydrolytic action and useful for the field of utilizing enzymes and medicines, etc., by cultivating a microorganism of the genus Pseudomonas in a culture medium. CONSTITUTION:A microorganism of Pseudomonas sp. SE-83 separated from soil is inoculated into a culture medium containing an organic acid as a carbon source, yeast extract as a nirogen source, calcium salt as an inorganic material, etc., to carry out aerobic liquid cultivation at 25-32 deg.C for 2-4 days. The resultant culture is then subjected to treatment, e.g. centrifugal separation, salting out, dialysis, gel filtration, etc., to give an enzyme having properties of hydrolyzing a compound expressed by formula I (R is -OCOCH3, -H or -OH) into a compound expressed by formula II and D-alpha-aminoadipic acid, about 9 optimum pH, 4-4.5 isoelectric point and an inhibitor of p-chloromercury benzoate.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to 7-amitusephalosborane acid (hereinafter referred to as l'
The present invention relates to a method for producing Kuraki enzyme, which is used in the enzymatic production method of -7-ACAJ) and its derivatives.

(Problems to be Solved by the Prior Art and the Invention) Cephalosporin C obtained by fermentation production is produced by binding to the 7-position amine group and then removing the acyl group.
-ACAKN/'L1 Various cephalosporin antibiotics have been synthesized using this as a starting material and are in practical use as pharmaceuticals.

The methods used industrially to remove the N-acyl group of cephalosporin C are (1) chemical methods;
(2) Enzymatic methods via intermediates. Among these methods, the chemical method is 1, for example,
This method is described in No. 1-13862 or Fi Patent Publication No. 45-40899. In addition, an enzymatic method via an intermediate is described in 2. For example, cicephalosvorin Ci7β-(5-carboxy-5-oxopentanamide)cephalosporanic acid is induced by the method of Japanese Patent Publication No. 55-12910, and b. 7β-(4-carboxybutanamide)cephalosporanic acid (hereinafter referred to as 7β-(4-carboxybutanamide)cephalosporanic acid) by the action of hydrogen oxide.

1-GL-7ACAJ) K induction and further microbial enzymes, e.g.
Biological chemistry) 45
This is a method for producing 7-ACA by using an enzyme described in Vol., p. 1561 (1981). these two 7
- All ACA sentencing methods have in common that they require multiple reaction steps.

Therefore, cephalosporin C can be combined with 7-ACA and D-
Using an enzyme that hydrolyzes α-aminoadipic acid,
7-ACAi from cephalosporin C in a single reaction step
It has been predicted that if FF could be produced, it would be extremely advantageous industrially.

To date, methods for producing 7-ACA from cephalosporin C using microbial cell-1fc using the enzymatic action of the processed product have been reported (1) U.S. Patent No. 5,259,594.
(1966), (2) % opening 52-1432
Four methods are known: (3) the method described in JP-A No. 53-94095, and (4) the method described in JP-A No. 59-44592.

Among these, the method of US Pat.
k or Flavobacterium spp.
This is a method using bacteria of C. cterium), as described by Flynn, “Cephalosporanic Acid and Penicillins J.
(E, HoFlynn; Cephalosporin
s and Penncillins, Academ
icPress, New York, 1972), page 37, it is known that this patent cannot be retested. Mayu, Alternaria sp. (Al
ternaria) is a species of the genus Aspergillus (Asp
JP-A-52-14 using fungi of S. ergillus
The method described in No. 52139, Pseudomonas putida (P
BN (seudomonas putida) closely related bacteria
The method described in JP-A-53-94095 using the -188 strain, and the method described in JP-A-53-94095 using the
) The method described in JP-A No. 59-44392 using fungi uses 7-ACA using the enzymatic action of microorganisms.
However, the substance of the enzyme that catalyzes the enzymatic action is not described.

That is, these patents do not contain any description regarding the production of fractionated silk of the enzyme that catalyzes the enzyme action.

・Is the enzymatic action a one-step reaction catalyzed by a single enzyme?
Or it is not clear whether it is a multi-stage reaction catalyzed by multiple enzymes. Conventionally, it was not predicted that this multi-stage reaction system exists in microbial cells, but the present inventors have previously discovered a microorganism that possesses this multi-stage reaction system.
No. 475). Furthermore, in these patents, D-α-
Because the production of 7-minoadibic acid is clearly under pressure and does not respond,
The enzymatic action converts cephalosporin C into 7-ACA and D-α.
-7 Is it due to an enzyme that hydrolyzes it to minoadipic acid?
It is clear that the problem is caused by an unknown enzyme, and in the above patent, cephalosporin C to 7-A
Utilizing an enzymatic action to produce CA does not necessarily mean discovering and utilizing an enzyme that hydrolyzes the cephalosporin Ci7-ACA and D-α-aminoadipine cIRK. .

The amide bond at the 6-position of the penicillin compound or the 7-position of the cephalosporin compound is hydrolyzed to produce the respective mother nucleus 6-7 minobenicilanic acid compound or 7-AC.
Enzymes that produce compound A are collectively called acylases, and many enzymes with different substrate specificities are known. However, the cephalosporins Ci7-ACA and D-α-
The existence of acylase, which hydrolyzes aminoadipic acid (hereinafter referred to as cephalosporin C acylase + FCpc acylase), has not been clarified despite the efforts of many researchers over many years. It is something that

Therefore, the present inventors have been searching for producing bacteria for the purpose of producing CPC acylase.

(Elaborate means and effects for solving all problems) The present inventors previously discovered that Pseudomonas sp.
NAS sp, ) S'E-83 (FER No. 7649) has two types of acylases (hereinafter referred to as ``acylase I'' and ``acylase ■'' that hydrolyze GL-7ACA into 7-ACA and geltaric acid. We discovered that the acylase type ``CpC acylase'' produces a CpC acylase, and developed an enzymatic production method for 7-ACA using cells of this bacterium or processed cells. Invented the invention (% Application No. 59-141475), and cloned the DNA fragment carrying the CPC acylase production genetic information of this bacterium into Escherichia coli, and revealed its nucleotide sequence. issue).

Furthermore, the present inventors have discovered that the bacterial strain 5E-495, which was isolated from soil by Shintomo, contains two types of 7-silases (hereinafter referred to as
Of these, acylase I (2) type is a novel CPC acylase that is different from the CPC acylase produced by the Bz-ss strain. I discovered something.

The present invention based on these discoveries is based on the general formula (I) (wherein,
R is 100OCR, -H is 10H'fr: Represents. ] is hydrolyzed into D-α-aminoadipic acid with a compound represented by the general 2〇 (in the formula, %R is the same as above, having a k taste).
This method is characterized by culturing bacteria belonging to the genus Pseudomonas that are isolated from soil and producing c-acylase, and collecting the enzyme from the culture.

7 discovered first? -8E-83 strain was identified as Pseudomonas deminuta as a result of isolation research.
Although it is closely related to Pseudomonas diminuta), it differs from Pseudomonas deminuta in its citric acid auxotrophy and auxotrophy, and has been determined to be a new species (Japanese Patent Application No. 141475/1982).

The newly discovered strain 5E-495 is a bacterium isolated from soil in the Yamaguchi region, and its mycological properties are as follows.

Mycological Properties of Strain 5E-495 (I) Morphological Properties Cells cultured on broth agar had 1.1) to 1.2 x 1.
It is a bacillus with a diameter of 9 to 2.1 microns, and moves with polar hairs. It does not produce spores and shows no pleomorphism.

Durham staining is negative.

■Cultural properties (1) Meat juice agar culture: The bacterial cells appear yellowish white and grow.

No production of diffusible pigments is observed. It exhibits neither viscosity nor migration.

(2) Broth liquid culture: The entire medium becomes slightly cloudy. No bacterial membrane formation was observed.

(3) Gelatin liquefaction puncture culture: No liquefaction of gelatin was observed (25C, 14 days).

(4) Lidomus Milk Medium II: No liquefaction of casein was observed.

(LID Physiological properties (11 Nitrate reduction: Positive (2) Denitrification reaction: Negative (3) MR test: Negative +41 VP test: Positive (5) Indole production: Negative (6) Hydrogen sulfide production: Negative (Kadenbun Hydrolysis of: Negative (8) Utilization of citric acid: Negative (9) Utilization of inorganic nitrogen source: Use ammonium salt as an N source.

Formation of αC dye No formation of two dyes was observed.

Uυ oxidase: positive Q3 catatase double positive (13 Growth range: Grows well at 25C to 30C.

It will not grow above 37C.

04 Attitude towards oxygen: Growth under anaerobic conditions is not observed 0 Q40F test (Hureyfunn method): [17 No acid production is observed regardless of the presence or absence of halafine.

Utilization of αQ carbon sources (1) Carbon sources used: glutamic acid, aspartic acid (11) Carbon sources not used: nigerose, arabinose, xylose, mannose, galactose, inodite, maltose, sucrose, glycerin sorbitol, mannitol (17) ) Auxotrophy: Pantothenic acid requirement Arginine decomposition: Negative α'J IJ resin decarboxylation: Negative Ornithine decarboxylation: Negative C++ Aesculin decomposition: Negative or higher Mycological properties・Determinative bacteriology (Bergey
's Manual of Determinati
veBacteriology) 8th edition (1974)
%-r New 7 Le Op Clinical Microbiology (Manual of C11nical M
icrobiology) 5th edition (1980), and Bersey's Manual of Systematic
Batteriology (Bergey's Manual
of Systematic Bacteriolo
gy) (1984), and the following conclusions were obtained.

This strain can be identified as belonging to the genus Pseudomonas because it is a Durham-negative bacillus, does not produce spores, and is motile by motile hairs, is absolutely aerobic, and does not have the ability to ferment glucose. Mayu.

Due to its negative glucose oxidation and oxidase positive properties, Pseudomonas alcaligenes
nas alcaligenes), Pseudomonas pseudomonas
seudoalcaligenes), Pseudomonas testosteronii (Pseudomonas te)
stosteroni).

Pseudomonas deminuta and Pseudomonas
Pseudomonas vesic
ularis). Furthermore, since it has monopolar hairs, is negative for fuctose oxidation, negative for estarin decomposition, negative for nitrate reduction, and exhibits auxotrophy, it can be determined that this strain is closely related to Pseudomonas deminuta species. From the above results, it is concluded that strains 5E-83 and 5E-495 belong to closely related species of the genus Pseudomonas.

The BE-495 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as a Pseudomonas sp.
as sp, ) S E-495 (Fei FE
RM BP-818).

Next, the bacteria of the genus Pseudomonas according to the present invention were introduced into 1
A method for producing CPC acylase of the present invention will be explained. Cultivation can be performed according to known methods. As the culture medium, those known as nutrient sources for general microorganisms are used, such as organic acids of various types as a carbon source and soybean flour as a nitrogen source.

Wheat germ, meat extract, peptone, cornstap liquor, yeast extract, etc. can be used. In addition to other salts such as magnesium salts, phosphates, and calcium salts, additives necessary for the growth and activity of the bacteria may be used in combination as necessary.

As a culture method, an aerobic liquid culture method is suitable.

The culture temperature may be selected within the range of 25 to 32C, and the culture time varies depending on the culture conditions, but usually takes 2 to 4 days.

The CPC acylase of the present invention is produced from the obtained culture by appropriately combining all known purification methods. That is, c
Since most of PC acid 2-ase is normally present inside the cells, the bacterial cells are first collected from the above-mentioned culture by means such as centrifugation, and then subjected to physical treatment such as ultrasonication or enzyme treatment. Add to disrupt the cells. Furthermore, after removing hard particles by means such as centrifugation, salting out, ion exchange chromatography, and affinity chromatography 1
It is prepared by appropriately combining purification means such as hydrofluoric chromatography or gel filtration with means such as dialysis, ultrafiltration, centrifugation, concentration, or freeze-drying.

It can be seen that the acylase type 7 of the 5E-83 strain and the acylase I(2) type of the 5E-495 strain are not the same substance because they behave slightly differently in the above purification process. For example, in DEAE Sephadex column chromatography, the above two CPC acylases are eluted at different salinities. i.e.% 0.1
Equilibrate with M phosphate buffer (pH 8,0) and
When CPC acylase is adsorbed onto a fully packed column of EAE Sephadex A-50 (Pharmacia) and then eluted using the 4-degree salt gradient elution method, acylase type II is eluted at a salt concentration of approximately 0.17M. However, acylase type I(2) is eluted at a salt concentration of about J12M.

The properties of CPC acylase produced by the method of the present invention and the enzyme activity measurement method Fi are as follows.

(I) Properties (1) Action CPC acylase is one of the penicillin amide hydrolases (E, C, 5, 5, 1, 11), and is a compound (
It is characterized in that it hydrolyzes I) into compound (1) and D-α-7mino-7-divic acid.

This enzyme hydrolyzes compound (1) to form compound 0 and D-α
The production of -7minoadibic acid has been demonstrated for the enzyme preparations purified by the method described above. That is, Compound 0 was identified and quantified using high performance liquid chromatography as described below, and -1y'c and D-α-7 minoadivic acids were identified and quantified using an amino acid analyzer. The optical activity of α-1 minoadipic acid was determined based on the following experiment using L- and D-amino acid oxidases.

(2) Substrate specificity CPC acylase #'i of the 5E-85 strain and 5E-495 strain, in addition to the above actions, GL-7ACA.

7β-(5-carboxypentanamide) cephalosporanic acid and 7β-(3-carboxypropanamide)
Hydrolyzes to cephalosporan fi'17-ACA and dicarboxylic acid. On the other hand, the N-acetyl compound of cephalosporin C and 7β-(phenylacetamido)cephalosporanic acid are not hydrolyzed. In addition, in both strains, the GL-7ACA hydrolyzing activity of the enzyme is in the range of 15 to 25 times the cephalosporin C hydrolyzing activity.

(3) Optimum pH The optimum pH of the enzymes is approximately 9. As an example%
The effect of pH on the reaction rate of CPC acylase of strain 5E-85 was measured using GL-7ACA as a substrate, and the results are shown in FIG. The buffers used were citrate buffer for pH 5-6, phosphate buffer for pH 6-8, and phosphate buffer for pH 8-1.
0 is glycine buffer.

(4) Stable pH range The enzyme has a pH of 7 to 10 in the presence of dithiothreitol.
All are stable within the range of . As an example.

Figure 2 shows the residual activity of CPC acylase of the SE-85 strain after treatment at 50C for 2 hours in the presence of 0.5mM dithiothreitol. The buffer used is the same as that used in (3) above.

(5) Inhibitors Both enzymes are inhibited by p-chloromercury-benzoate, but not by phenylmethylsulfonyl fluoride.

(6) Isoelectric Point The isoelectric points of the # element determined by the chromatofocusing method using Pharmacia's Polybuffer are all in the range of I) H4 to 4.5.

From the above results, the cp of the 5E-85 strain and 5E-495 strain
Although c-acylase is a different substance,! #It can be seen that the elementary chemical sex trade is very similar.

■ Enzyme activity measurement method (11 Cephalosporin C hydrolysis activity measurement method) 0.1M phosphate buffer containing 50mM cephalosporin C (p
An enzyme solution (pH 8,0) containing 2 mM of H8,0)1- and dithiothreitol) is mixed and reacted at 37C for 10 minutes. 67 Thiacetic acid aqueous solution (pH2,0) 0
．． After the reaction is stopped by adding 4- and solid matter is removed by centrifugation and membrane filtration, the produced 7-ACA is quantified by high performance liquid chromatography to determine the enzyme activity. The high performance liquid chromatography conditions are as follows.

Column Two Micro Bondapac cμ-Bondapac
k) C, Column (manufactured by Waters) Mobile phase: A mixture of 98 volumes of ammonium 5-thiacetate and 2 volumes of acetonitrile Detection wavelength: 260 nm (21 Method for measuring hydrolysis activity of compounds (I) other than cephalosporin C Compound (I) ) as a substrate, an enzymatic reaction was carried out in the same manner as in method (I) above, and the resulting compound ■ was converted to N by the following method.
- Phenylacetyl compounds are derived and quantified using high performance liquid chromatography.

That is, baking soda is added to a certain amount of aqueous solution containing Compound 0 to keep it alkaline. To this, 1/1 volume of acetone and 7-enyl acetyl chloride, which is about 5 times the mole of the estimated amount of Compound 2, are added and reacted at room temperature. let After 30 minutes, extract with % volume ether to remove excess phenylacetyl chloride. The aqueous layer is adjusted to pH 2, extracted twice with equal volumes of ethyl acetate, and the ethyl acetate layers are combined and dried under reduced pressure. 4 left
is dissolved in a certain amount of methanol, and the resulting N-phenylacetylated product is analyzed by high performance liquid chromatography and quantified by comparison with a standard sample. The column is Micro Bonder Pack C1,
Fia, o5MIJ acid buffer (
A mixture of pH 7.0) and methanol is used as appropriate. Detection is performed at 260 nm, and t of the product is determined from the quantitative value of the 9N-phenylacetylated product produced and the recovery rate obtained in a control experiment using a compound at a known concentration, and the enzyme activity is calculated.

+31GL-7ACA hydrolysis activity measurement method GL-7A
Using CA as a substrate, an enzymatic reaction is carried out in the same manner as in method 11 above, and the produced 7-ACA is fixed by a colorimetric method using p-dimethylaminobenzaldehyde. That is, 67% of the 7-ACA is added to enzyme reaction solution 2-. Acetic acid aqueous solution (pH2,0
) 0.4- is added to stop the reaction. Then p-
Methanol solution of dimethylaminobenzaldehyde (5
? Add 0.41 nt of /l) and stir. After removing the solid matter by centrifugation, the yellow color of the produced compound is detected at a wavelength of 40.
7-A from the calibration curve created in advance with a constant ψ of 0 nm.
The amount of CA produced is determined, and further, the enzyme activity is calculated. Enzyme activity is expressed as an enzyme that produces 1 micromole (μmol) of 7-ACA per minute under the enzyme reaction conditions described above.
The unit is ft1.

Example C) Next, the present invention will be explained using examples.

Example 1 A medium (pH 7,
0) Pour 50 tons of 15 tons into a jar fermentor, kill at 120C for 30 minutes, and inoculate Pseudomonas sp. 5E-83Q, which was precultured in the same medium, to a concentration of 2%. After culturing at 25C for 48 hours, the bacterial cells were collected by centrifugation to obtain wet bacterial cells 521. The GL-ZACA hydrolysis activity catalyzed by two types of acylases (acylase lff1 and acylase type II) contained in this bacterial cell was approximately 0.86 molecules/1 wet bacterial cell. This wet bacterial cell 527 was dissolved in 0.1M phosphate buffer (pH 8,0) at 200-
111! ! ! After turbidity and ultrasonic cell disruption treatment at 5C, solid matter was removed to prepare an enzyme solution. Next, add 5 to this enzyme solution.
c. Under cooling, ammonium sulfate was gradually added to a 30% saturation concentration while stirring, and the precipitate formed was removed.

Ammonium sulfate was added to the supernatant fraction to a saturation concentration of 60%, and after stirring for 1 hour, the precipitate fraction was collected and dissolved in 0.1M phosphate buffer (pH 8,0) 100-. This enzyme solution was dialyzed against the same buffer solution at 5c overnight, and then DEAE-Sephadex A-50, which had been equilibrated with the same buffer solution (
When passing through a column packed with 600- (manufactured by Pharmacia), the activity is adsorbed and fC8 activity is eluted using 5 times the column volume of 0.4 MIJ acid buffer (pH 8,
0) (i- stool was used, and the sodium chloride concentration was increased linearly with a final 6 degrees of 0.5M gradient elution. As a result, acylase type I was dissolved in the bath at a sodium chloride concentration of around 0.05M. It was found that Cpc acylase (acylase E1.) was eluted at around 0.17 M of sodium chloride.
It was found that type I acylase and CPC acylase were present in approximately equal amounts. FIG. 6 shows the elution pattern of DEAE Sephadex column chromatography. Then, when the CpC acylase fraction was collected, 18 units of G
L-7ACA hydrolysis activity was recovered. Both fractions were again subjected to DEAE Sephadex column chromatography under the same conditions to obtain a preparation of the enzyme. The protein content in the sample was 45m9, and the enzyme activity was GL-7AC.
The A hydrolysis activity was about 9 units, and the cephalosporin C hydrolysis activity was about 0.5 units. In addition, the protein ψ is calculated by the Lowry method,
Bovine serum albumin was measured as a standard.

Example 2 Pseudomonas sp. 5E was grown in the same manner as in Example 1.
-495 was cultured and the bacterial cells were collected by centrifugation, and wet bacterial cells 501 were obtained. There are two types of acylases contained in this bacterial cell (acylase type ■ and acylase I (type 21)).
The GL-7ACA hydrolysis activity catalyzed by the GL-7ACA was approximately 0.52 units 72-wet cells. This wet bacterial body 501
was subjected to ultrasonication, ammonium sulfate fractionation, and DEA-Sephadex column chromatography purification in the same manner as in Example 1. As a result, acylase type I has a salt concentration of 0.0.
It was found that CPC acylase (acylase type I(2)) was eluted at a salt concentration of around 0.12M. Furthermore, it was found that acylase type I and CPC acylase were present in y-equal amounts. DE in Figure 4
The elution pattern of AE Sephadex column chromatography is shown.

The cpc acylase fraction was then collected, and 10 units of GL-7ACA hydrolysis activity was recovered. These two fractions were again subjected to DEAE Sephadex column chromatography K under the same conditions to obtain a purified enzyme preparation. The protein content in the obtained sample was 40 μ, and the enzyme content was GL-7A.
It is about 4.8 units in terms of CA hydrolysis activity, and about 0.2 units in terms of se7alosbo+)yC hydrolysis activity.
It was 4 units.

(Effect of the invention) The present inventors have synthesized compound (I) with compound 0 and [)-(χ-
We searched for microorganisms that produce an enzyme (CPC acylase) that hydrolyzes aminoadipic acid, and found Pseudomonas sp. 5E-83 and Pseudomonas sp. 5E-
I found 495. Based on these discoveries, a method for producing CpC acylase, which is used in the enzymatic production of compound (1), was established as shown in the examples.

[Brief explanation of drawings]

Figure 1 is a graph showing the optimum pn of CPC acylase, Figure 2 is a pH stability curve of CpC acylase, and Figure 3 is a graph showing the elution pattern of CpC acylase of Pseudomonas sp. 5E-85 in DEAE Sephadex column chromatography. , Figure 4 shows the DE of CPC acylase of Pseudomonas sp. 5F-495.
It is a graph showing the elution pattern in AE Cef7dex column chromatography. Figure 1 Figure 2 Protein C”&/ml) (Δ---Δ) Salt concentration (M)
(----)

Claims (3)

[Claims] (1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R represents -OCOCH_3, -H or -OH.) General formula (II) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) (In the formula, R represents -OCOCH_3, -H or -OH.) A compound represented by the formula and cephalosporin C acylase which hydrolyzes it to D-α-aminoadipic acid. A method for producing cephalosporin C acylase by a fermentation method, which comprises culturing the producing bacteria of the genus Pseudomonas and collecting the enzyme from the culture. (2) The method according to claim 1, wherein the bacterium of the genus Pseudomonas is Pseudomonas sp. SE-83 (FERM BP-817, FERM BP-817). (3) Bacteria of the genus Pseudomonas is Pseudomonas sp. SE-495 (
FERM BP-818) The method according to claim 1.