JPS61181391A - Production of d-alpha-amino acid - Google Patents

Abstract

PURPOSE:To obtain the titled substance advantageously, by treating a 5- substituted hydantoin with candida vinaria, a yeast capable of converting the substituted hydantoin into an D-alpha-amino acid and racemizing the 5-substituted hydantoin. CONSTITUTION:Candida vinaria, a yeast capable of converting a 5-substituted hydantoin into D-alpha-amino acid and racemizing the 5-substituted hydantoin is cultivated in a medium containing the 5-substituted hydantoin. The mold of the yeast or a treated material of it is treated with the 5-substituted hydantoin in an aqueous solution. pH in the enzymatic reaction solution is about 6-9, temperature is about 20-60 deg.C, concentration of reaction substrate is about 0.1-10wt% and isolation of D-alpha-amino acid (formula II) formed from the 5- substituted hydantoin (formula I) is carried out by concentration, neutralization, ion exchange resin treatment, etc.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention has the ability to racemize 5-substituted hydantoins, and the ability to convert D-5-substituted hydantoins into D-α-amino acids. The present invention relates to a highly advantageous method for producing D-α-amino acids by using microorganisms of the genus Candida having the following.

(Prior art and its problems) One method for producing D-α-amino acids is to chemically hydrolyze the corresponding 5-substituted hydantoin to produce DL-α-amino acids, which are optically resolved to produce D-α-amino acids. -α-, a method of converting it into an amino acid is known. However, this method requires particularly complicated steps of optical resolution, and the yield is not high. Furthermore, 5-
Optically active N-
After producing the D-alpha-amino acid.

A method of converting D-α-amino acids by chemical treatment is known. However, this method also requires complicated reaction steps and purification steps.

In addition, a culture solution of microorganisms is added to 5-substituted hydantoin.

Methods are also known in which D-α-amino acids are directly produced by reacting with processed cells or bacterial cells, but the yield is not high.

(Means for Solving the Problems) The present inventors have conducted research to find a more efficient method for such conventional manufacturing methods.

A microorganism belonging to the genus Candida was racemized with 5-substituted hydantoin, and D-5-substituted hydantoin was converted into D-α-
It was discovered that it has the ability to convert into amino acids.

However, when microorganisms are applied to 5-substituted hydantoin, N
Methods for converting into -carbamoyl-D-α-amino acids or D-α-amino acids are already known. (Unexamined Japanese Patent Publication No. 51-
139687.53-91189. However, due to the action of microorganisms, L-5=substituted hydantoin is converted to D-α-amino acid, that is, after L-5-substituted hydantoin is racemized and converted to DL-5-substituted hydantoin, D-5
- A method for converting a substituted hydantoin into a D-α-amino acid.

unknown. However, since 5-substituted hydantoins chemically racemize slightly in aqueous solution, L-5
When -substituted hydantoin is used as a raw material, it is possible to observe the production of trace amounts of D-α-amino acids due to the action of microorganisms. However, the production speed is very slow and it cannot be called a practical method.

Thus, it has not been known that the microorganism Candida binaria, which belongs to the genus Candida, has the ability to racemize 5-substituted hydantoins and to convert 5-substituted hydantoins into D-α-amino acids.

This invention was completed as a result of further research based on this knowledge.

That is, the present invention has the general formula O R-CH-C (1) (in the formula,
R is an alkyl group (excluding inpropyl group), substituted alkyl group, aralkyl group, substituted aralkyl group, phenyl group,
Substituted phenyl group, furyl group, pyridyl group, thiacyl group,
The yeast Candida binaria has the ability to convert a 5-substituted hydantoin into a D-α-amino acid and the ability to racemize a 5-substituted hydantoin to a 5-substituted hydantoin represented by (representing an imidazolyl group or an indolylmethyl group) (Candidavinaria) to convert it into D-α-amino acid (wherein R is the same as in formula (1))
This invention relates to a method for producing D-α-amino acid represented by H2.

The microorganisms used in the method of the present invention are collected from soil.

The isolated yeast was identified as Candida binaria based on the mycological characteristics shown below.

1. Form 2) Spore formation: - 26 Physiological properties 1) Growth range: Grows up to a temperature of 37°C.

Does not grow at 39°C.

2) Inorganic nitrogen source: nitrate -, ammonium salt +3)
Vitamin requirement 2-4) Sugar fermentability: D-glucose, lactose.
D-galactose-, lahinose-maltose
-, sucrose - 5) Sugar assimilation = D-glucose + D-galactose + D-ribose - D-xylose - Sucrose - Maltose - Melibiose - Lactose - Ethanol + Glycerol + 5-substituted hydantoin with the candy of the present invention There is a method for making B. da vinaria act on a microorganism by contacting it with cells of a microorganism or a treated product of the cells in an aqueous solution. What is the medium used to culture this microorganism?

In addition to containing 5-substituted hydantoin, it is a conventional medium that can contain assimilable carbon sources, nitrogen sources, and nutrients necessary for the growth of microorganisms.

The culture conditions were aerobic and pH = 4 to 8. Temperature 25~
It may be carried out while controlling the temperature within an appropriate range of 40°C.

The microorganisms used in the present invention were isolated and selected from naturally occurring wild species by examining their ability to convert 5-substituted hydantoins into D-α-amino acids. As a method for testing the ability to convert a 5-substituted hydantoin into a D-α-amino acid, for example, the following method is used.

After collecting the culture solution 5- of the test microorganisms and collecting the bacteria by centrifugation, the collected bacteria were washed with the same volume of sterilized physiological saline, and then treated with L-isopropylhydantoin at a concentration of 0.5% by weight as in 2-. Potassium phosphate buffer (0,1M concentration, p
H=7.5) Disperse in substrate solution and react at 35°C for 24 hours.

The reaction solution was then centrifuged at 10.00 Orpm for 10 minutes to obtain a supernatant, which was separated using a paper chromatograph (developing solution BU-OH: acetic acid: water = 4; 1:1). After that, develop ninhydrin color, cut out 1 colored part and add 7 more.
After extracting the colored part with 5% ethanol solution 5-, wavelength 57
Colorimetrically determined at 0%.

The amino acids produced by the strains found to have the ability to convert hydantoin rings into amino acids as described above were further isolated and purified by conventional methods.

It was verified by measuring the optical rotation.

The microorganism used in the present invention, Cantida binaria, has passed the above-mentioned test.

The enzyme reaction substrate used in the present invention includes various 5-substituted hydantoins, such as 5-methylhydantoin and 5-isobutylhydantoin.

5-5ec-butylhydantoin, 5-methylthioethylhydantoin, 5-7enylhydantoin.

Examples include 5-benzylhydantoin and 5-indolylmethylhydantoin. The concentration of the reaction substrate in the enzyme reaction can range from 0.1 to 10% by weight, and the reaction temperature ranges from 20 to 60°C.

In addition, regarding the pH in the enzyme reaction solution, a practically preferred pH
The range is 6-9. pH = 6 to 11 1 The reaction rate is extremely slow, and if the pH exceeds 9, undesirable side reactions occur. Since the optimum pH of the enzyme for converting into is around 7 to 8, the preferred pH range is 6 to 9.

The D-α-amino acid produced from the 5-substituted hydantoins as described above can be isolated by using known methods such as concentration, neutralization, and ion exchange resin treatment. can be obtained.

Further, when carrying out the present invention, a surfactant may be appropriately used in combination according to common general technical knowledge.

(Operations and Effects of the Invention) The present invention is capable of converting 5-substituted hydantoins into D-α-amino acids by using the yeast Candida vinaria, which has the ability to convert 5-substituted hydantoins into D-α-amino acids and to racemize 5-substituted hydantoins. D-α- easily in high yield
This is an extremely advantageous method for producing D-α-amino acids since amino acids can be obtained.

(Example) The present invention will be specifically explained by the following examples.

The invention is not limited only to these examples.

Example-1 20 d of the culture medium shown in Table-1 was placed in a 250-Erlenmeyer flask, sterilized at 120°C for 15 minutes, and then added with yeast YM medium for 20 minutes.
Candida binaria (Ca.
ndida vinaria) CMT-1012
(FERM P-7845) was inoculated into one platinum loop and cultured at 28°C for 24 hours. This culture solution was centrifuged to collect bacterial cells, and the cells were washed once with the same amount of physiological saline as the culture solution to collect the bacteria. This bacterial cell shows 5-substituted hydantoids 710 shown in Table 2.
0.1M potassium phosphate buffer (pH
= 7.5) (final 5-) at a concentration of 309/l and reacted at 36°C for 20 hours. The various amino acids produced were measured by the method described above, these amino acids were separated and purified, and their optical rotations were measured. As a result, it was confirmed that they were all D-integrated. The results are shown in Table-2.

Table 1 Table 2 Example 2 The same operations as in Example 1 were carried out except that a 5-substituted hydantoin containing L was used as the enzyme reaction substrate. On the other hand, Table 3.4 shows the racemization rate of L-5-substituted hydantoys obtained under the same conditions without the addition of bacterial suspension by measuring the optical rotation.

Table-3 Table-4 *Reaction solution (7) PH=7.5. Temperature: 36°C, 20-hour reaction procedure Amendment Written on March 12, 1985 by the Commissioner of the Japan Patent Office 1. Indication of the case 1985 Patent Application No. 21869 2. Title of the invention: 1. Process for producing α-amino acids 3 , Relationship with the case of the person making the amendment Patent applicant address: 3-2-5-4 Kasumigaseki, Chiyoda-ku, Tokyo;
Column 5 of detailed description of the invention in the specification subject to amendment, content of amendment

Claims (1)

[Claims] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R is an alkyl group (excluding isopropyl group), substituted alkyl group, aralkyl group, substituted aralkyl group, phenyl group, substituted phenyl group , a furyl group, a pyridyl group, a thiazole group, an imidazolyl group, or an indolylmethyl group). The yeast Candidavinaria has the ability to racemize (
FERM P-7845) is converted into D-α-amino acid by the action of the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (R in the formula is the same as in formula (1)) -A method for producing an α-amino acid.