JPH02119796A - Production of l-homophenylalanine - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a raw material for producing medicines, such as beta-lactam antibiotic substances, in high yield by reacting benzylmethylhydantoin with a specific enzymic system or a microorganism having the above-mentioned enzymic system. CONSTITUTION:5-Benzylmethylhydantoin is reacted with an L-specific hydantoinase system consisting of hydantoin hydrolase and N-carbamylamino acid hydrolase or a microorganism having the above-mentioned enzymic system, preferably at 25-40 deg.C and pH8-9 to produce the objective compound. Furthermore, e.g., a bacterial strain HP-27 (FERM P-10346) of the genus Flavobacterium, is cited as the microorganism having the afore-mentioned enzymic system.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to an industrially advantageous method for producing UL-homophenylalanine (L-4-phenyl-2-aminobutyric acid). L-homophenylalanine is useful, for example, as a raw material for pharmaceutical preparations such as so-called ACE inhibitors (enalapril, ramipril, etc.) and β-lactam antibiotics.

(b) Conventional technology Regarding the production method of L-homophenylalanine, asymmetric hydrolysis using an enzyme (K, IJobuo et al., Mem.
Fac, Sci, Kynshu Univ, Se
G, C, 13, 89 (1981)), and a method for splitting homophenylalanine to form diastereomeric salts with brucine after formylation (Vclu Vigneaud et al., J. Biol.

Ohem, 122, 349 (1957-3
8) I know.

- &da, recently, a method of reacting u-acetyl DL-homophenylalanine with optically active phenylethylamine to resolve diastereomer salts (patent application published in 1983-
65646), a method of separating DL homophenylalanine and optically active mandelic acid as diastereomeric salts (Patent Application Publication No. 145256/1983), and an amination method by transamination of 2-ochine-4-phenylbutylene (1986-145256). Patent Application Publication No. 156394/1985 and US Pat. No. 4.525.454 (1985')') are known.

(c) Problems to be Solved by the Invention and Means for Solving the Problems In the conventional techniques, in the method of preparing and resolving diastereomeric salts, the resolving agent is expensive and the operation is complicated. In addition, including the method of asymmetric hydrolysis using enzymes, half of the raw material is left as 9 bodies due to division, which reduces the yield by half, so it is uneconomical unless a method for using 9 bodies is added. be. The transamination method of 2-okino-4-phenylbutyric acid requires an optically active L-amino acid, and ? f. It has drawbacks such as complicated synthesis of raw materials.

Therefore, the present inventors focused on a biochemical method using hydantoin, which is applied to the production of some amino acids, as a method to efficiently obtain fl, L-homophenylalanine. Until now, there was no knowledge of enzymes or microorganisms that act on this. Therefore, we need to explore and discover microorganisms that have a hydantoinase system (consisting of hydantoin hydrolase and N-carbamyl amino acid hydrolase system) that produces L-homophenylalanine from 5-benzylmethylhydantoin, which is a DL-hydantoin corresponding to homophenylalanine. Successfully proceeded with further research, 5
The entire process of the present invention has been established which allows L-homophenylalanine to be produced from -benzylmethylhydantoin in good yield.

B) Effect The microorganism used in the present invention may be a microorganism having an L-specific hydantoinase system that acts on 5-benzylmethylhydantoin.

Such microorganisms include Flavobacterium. More specifically, the Flavobacterium genus bacteria box strain HP
-27, Arthrobacter ureafaciens (Ar
throbacter ureafaciens')
ATC! O7562 is mentioned. Flavobacterium strain HP-27 was isolated from soil by the present inventors. Its mycological properties are as follows.

0.4-0.6 x 0.8-1.0 micron dog rods, no pleomorphism was observed, and they were not motile. Does not produce spores,
Durham negative, no acid fasting. Cultured on broth agar plates, it produces circular, raised colonies with golden edges, and the colonies are pale yellow in color. When cultured on a broth agar slant, it grows pale yellow and translucent. When cultured in broth liquid, it grows uniformly and turbidly. Liquefy gelatin in broth gelatin puncture culture. l) Lidomas is not reduced in the culture of Tomanu milk, and the reaction remains neutral.

It reduces nitrates aerobically, the denitrification reaction is negative, the MR test, ■P test is negative, the test is negative, it does not produce hydrogen sulfide, and it does not decompose starch. KOber's medium is positive for citric acid utilization, but Ohristensen's
It is negative in the culture medium.

Does not utilize nitrates and ammonium salts. It does not produce bath dye, is negative for urease, and positive for both oxidase and catalase. It grows at pH 6-9.15-35°C, hardly grows at 5°C, and does not grow at all at 40°C.

The O-F test is oxidative. D-glucose, D-fructose, maltoine, D-quinlose, D-arabinose, D-mannose, D-galax, sucrose, lactose, trehalone, D-lubitol,
D-mannitol, inositol, glycerin, and starch do not generate acid or gas. Decomposes casein.

Assimilates glucose and quinlose, but does not utilize lactose.

The above various instructions f Bergey'e Manual o
f Systematic Bacteriology
Volume 1 (1984), this strain is determined to belong to the genus Flavobacterium, but there is no match to the bacterial species described in the book. Strain HP-27 is a microorganism. Deposited at the Institute of Industrial Technology.

The entrusted item is Microtechnical Research Institute No. 1o:r4-1s.

Nao, 77 Since the existence of a specific hydantoinase system is known for the production of other amino acids, the genus Flavobacterium [fiti FERM-P6901, )
-y Bobacterium amino acid production (Flavobac
teriumaminogenes') FERM
-P 31ro 6, Arthrobacter taste FERM-p
8191, same (FERM-P 8190).

Arnulobacter genus bacteria FERM-P 7472, Arthrobacter genus bacteria method DSM 3747, etc. are also considered to be effective in the present invention.

In order to culture these microorganisms and obtain specimens with the necessary L-specific hydantoinase fermentation, conventional culture methods may be used, and no special explanation is required for engineers in this field. , a culture with high L-specific hydantoinase activity when microorganisms are grown in a medium containing 5-benzylmethylhydantoin or other 5-hydantoin compounds used as a substrate? You can get it.

In addition, solid media and liquid media can also be used.

The microorganism containing the L-specific hydantoinase activity obtained as described above or the enzyme preparation derived therefrom is
To make benzylmethylhydantoin react, add an enzyme preparation to a solution containing the substrate and incubate until the reaction progresses. Enzyme preparations, bacterial cells, washed bacterial cells, isolated from microbial culture solutions,
It can also be applied to the substrate in the form of physicochemically or biochemically treated microbial cells such as freeze-dried microbial cells, extracts, purified products, immobilized preparations, and the like.

The substrate concentration differs depending on whether it is a batch method or a continuous method, but in a batch method it is generally 01 to 30% in the medium, preferably about 1 to 20%, and in a continuous method the concentration is lower than this. It's better.

The reaction is usually carried out at 5 to 60°C, preferably around 25 to 40°C.
The pH is around 7-10, preferably around 8-9.

The reaction time varies depending on the method of standing, stirring, flowing down, etc., the shape of the enzyme preparation, the concentration of the matrix substrate, etc., and is therefore not uniform, but in the patch method, it is usually about 1 to 1 Do hour.

The progress of the reaction is investigated by analyzing the production of reverse L-homophenylalanine using analytical means such as thin layer chromatography and high performance liquid chromatography. Generally, 5-benzylmethylhydantoin produced by organic synthesis is a racemate, but according to the method of the present invention, this racemate is converted into L-homophenylalanine in high yield and killed.

(E) Examples Example 1 Glucose 5? , ammonium sulfate 5F, potassium phosphate 12, dipotassium phosphate 6? , magnesium sulfate heptahydrate 0.1f, carunium chloride dihydrate 0.01'? ,
Flavobacterium strain HP-27 was inoculated into a 3DOmA (7) Erlenmeyer flask and sterilized with 30 m of a culture medium (pH 7, 2) made by dissolving 1 t of Corn Steed Liquor 50 in tap water. The cells were cultured with shaking at 26°C for 48 hours. Bacterial cells were separated from this culture solution by centrifugation Ia+c, washed twice with phosphorus;
- Suspended in 11 nl of 0.1 molar pI 48.0 phosphate buffer containing -5-benzylmethylhydantoin,
After further shaking culture at 26°C for 72 hours, the bacterial cells were removed by centrifugation, and the supernatant was filled with 66% o.
~/Tn1. L-homophenylalanine was produced at a concentration of .

The ratio of L-isomer in this L-homophenylalanine is 96
%Met.

Example 2 In this example, the bacterial strain is Arthrobacter ureafaciens ATCC! The same procedure as in Example 1 was carried out except that 7562 was used. The concentration of L-homophenylalanine produced in the supernatant of the reaction solution was 24 μr/ml.

Example 6 In Example 1, Arthrobacter globiformis was used as the bacterial strain.
s) The same procedure as in Example 1 was carried out except that ATOO8010 was used. L-homophenylalanine was produced in the reaction supernatant.

In the above examples, the separation and analysis of optical isomers of homophenylalanine was carried out using cation exchange resin (Dowex) from the reaction solution.
50 W-X 8, 200-400 mesh, H type)
After adsorption to 0.1 N 111a2HPO4-H,P
After elution with O, buffer 1 (pH 7, (II)) to obtain a homophenylalanine fraction, a column TSK gel for high performance liquid chromatography manufactured by Tosoh Corporation was used.
o L 1 (46X 250 mm') using f,
The 9-isomer and the L-isomer were separated using 0.5 mM CuSO4 solution overnight as the mobile phase at a flow rate of 1.0 ml per minute, a temperature of 40° C., and ultraviolet absorption for detection.

(F) Effects of the Invention According to the present invention, L-homophenylalanine, which is useful as a raw material for producing pharmaceuticals, can be produced from 5-benzylhydantoin in high yield.

Patent applicant: Piall Co., Ltd. Representative Kiyoshi Koyama Procedural amendment (voluntary) February 2z, 1999

Claims (1)

[Claims] 1,5-benzylmethylhydantoin and hydantoin.
A method for producing L-homophenylalanine, which comprises producing L-homophenylalanine through the action of an L-specific hydantoinase system consisting of hydrolase and N-carbamyl amino acid hydrolase, or a microorganism having this enzyme system. L-homophenylalanine is produced by reacting 2,5-benzylmethylhydantoin with bacteria of the genus Flavobacterium or Arthrobacter, or with hydantoin-degrading enzymes derived from these bacteria. Method for producing phenylalanine. 3. The origin of the microorganism or hydantoin-degrading enzyme to be used is a bacterium with taxonomic properties such as Flavobacterium strain HP-27 (Feikoken Bibliography No. 10346) or Arthrobacter ureafaci. 2. The manufacturing method according to claim 2, which is