JPS61177991A - Production of d-alpha-amino acid - Google Patents

Abstract

PURPOSE:To produce a D-alpha-amino acid easily in high yield, by reacting a D-5- substituted hydantoin with a microorganism of the genus Hansenula having a special ability. CONSTITUTION:About 30g/l microbial cells of Hansenula polymorpha washed with physiological saline solution is added to a D-5-substituted hydantoin expressed by formula I [R is (substituted) alkyl, (substituted) aralkyl, (substituted) phenyl, furyl, pyridyl, thiazolyl, imidazolyl or indolyl], e.g. D-5- methylhydantoin, as an enzymic reaction substrate adjusted to 0.1-10wt% concentration, and reacted and converted therewith at 20-60 deg.C and 5-9pH for about 20hr. The resultant D-alpha-amino acid is then passed through steps of concentration, neutralization and ion exchange resin treatment to give the aimed D-alpha-amino acid expressed by formula II (R is same as formula I).

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to the use of Hanse
D-α by using microorganisms belonging to the genus
- Conflict regarding a highly advantageous method for producing amino acids.

(Prior art and its problems) One of the methods 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. A method of converting it into an α-amino acid is known. However, this method requires particularly complicated steps of optical resolution, and the yield is not high.

Furthermore, the 5-substituted hydantoin is a culture solution of microorganisms,
A method is known in which optically active N-carbamoyl-D-α-amino acids are produced by the action of processed bacterial cells or enzymes extracted from the bacterial cells, and then converted into D-α-amino acids by chemical treatment. However, this method also requires complicated reaction steps and purification steps.

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

A method of directly producing D-α-amino acids by reacting bacterial cells or a processed product of bacterial cells is also known, but the yield is not high.

(Means for Solving the Problems) The present inventors aim to find a more efficient method for such conventional manufacturing methods (as a result of research).

It has been found that microorganisms belonging to the genus Hansenilla have the ability to convert D-5-substituted hydantoins into D-α-amino acids.

However, when microorganisms are applied to 5-substituted hydantoin, N
-Carbamoyl-D-α-amino acids or methods for converting them into D-α-amino acids are already known. (Unexamined Japanese Patent Publication No. 51-
139687.53-91189゜54-2398)L
However, it has not been previously known that microorganisms belonging to the genus Hansenilla have the ability 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 (wherein R is an alkyl group, a substituted alkyl group, an aralkyl group, a substituted aralkyl group, a phenyl group, a substituted phenyl group,
Hansenilla polymorpha having the ability to convert a 5-substituted hydantoin into a D-α-amino acid into a D-5-substituted hydantoin represented by a furyl group, a pyridyl group, a thiazolyl group, an imidazolyl group, or an indolyl group. (Hansenula polymorpha) (
NH with the general formula R-CH-COOH (wherein R is the same as in formula (1)), which is converted into a D-α-amino acid by the action of FBRMP-8013).

This invention relates to a method for producing D-α-amino acid represented by:

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

The isolated yeast was identified as Hansenilla polymorpha based on the mycological characteristics shown below.

1. Shape 1) Cell shape and size: 1.5-3 x 2-4μ.

oval, white or cream Mu color.

2) Formation of spores... Hat shape 2, physiological properties 1) Growth range: Growth up to a temperature of 42°C 2) Nitrogen source
: Nitrate + ammonium salt + 3) and damine requirement 4) Sugar fermentability: D-glucose + D-galactose + maltose - lactose - lahinose - Shakrose - 5) Sugar assimilation = D-glucose 10-galactose - D-ribose + D-xylose + shefrose + maltose + melichiose - lactose - ethanol + methanol + glycerol + D-5-substituted hydantoin with the 71 nseniera of the present invention.
The method of making polymorpha act is a method in which one microorganism cell or a treated product of the microorganism is brought into contact with the cell in an aqueous solution. The medium used for culturing the present microorganism is a conventional medium containing an assimilable carbon source, a nitrogen source, and inorganic salt nutrients necessary for the growth of the microorganism. Culture conditions were aerobic.

pH=4-9. It is preferable to control the temperature within an appropriate range of 25 to 45°C.

The microorganisms used in the present invention are isolated and selected from naturally occurring wild strains by examining the ability to convert D-5-substituted hydantoins into D-α-amino acids. This D-5-substituted hydantoin was converted into D-α-
For example, the following method can be used to test the ability to convert into amino acids. Collect the culture solution 5- of the test microorganism.

After collecting bacteria by centrifugation, the collected bacteria were washed with the same volume of sterilized physiological saline, and then mixed with D-isopropylhydantoin in potassium phosphate buffer (0, IM concentration) at a concentration of 0.5% by weight. pH=7.5) Disperse in substrate solution and react at 35°C for 24 hours. Then, add the reaction solution to 10. O
A supernatant was obtained by centrifugation at OOrpm for 10 minutes, and the supernatant was subjected to paper chromatography (developing solution Bu-OH:
After separation with acetic acid:water = 4:1:1), color was developed with ninhydrin, one colored part was cut out, and the colored part was extracted with 75 cm of chetanol solution, followed by colorimetric determination at a wavelength of 570 m/m.

Regarding strains recognized as having the ability to convert hydantoin rings into amino acids as described above.

Furthermore, the produced amino acids are isolated and purified using conventional methods. It was verified by measuring the koji luminosity.

The microorganism used in the present invention, Hansenilla polymorpha, passed the above-mentioned test.

The enzyme reaction substrates used in the present invention include various D-5-substituted hydantoins, such as D-5-methylhydantoin, D-5-isopropylhydantoin, and D-5-substituted hydantoin.
Examples include -5-isobutylhydantoin, D-5-secbutylhydantoin, D-5-methylthioethylhydantoin, D-5-phenylhydantoin, D-5-benzylhydantoin, and D-5-indolylmethylhydantoin.

The concentration of reaction substrate in enzyme reaction is 0.1 to 10 times tS
It can be used up to a concentration of . The reaction temperature is the optimum temperature of the microorganism used for the enzyme capable of converting it into D-α-amino acids, and is usually in the range of 20 to 60°C. The pH during the reaction is the optimum pH of the enzyme of the microorganism used that has the ability to convert it into D-α-amino acids, and is usually in the range of pH=5 to 9. Particularly preferably at a temperature of 2
The temperature is 0 to 50°C, and the pH is 6 to 8.5. As described above, D-α-amino acids produced by asymmetrically converting -5-substituted hydantoins are isolated by concentration and neutralization.

The target D-α-amino acid can be obtained by using known methods such as ion exchange resin treatment.

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

(Operations and Effects of the Invention) The present invention can easily convert D-5-substituted hydantoins into D-α-amino acids in high yield by using Hansenilla polymorpha. Since D-α-amino acid can be obtained, D-
This is an extremely advantageous method for producing α-amino acids.

(Example) The present invention will be specifically explained using the following examples, but the present invention is not limited only to these examples.

Example 1 The culture medium shown in Table 1 was cultured in a 250-erlenmeyer flask for 24 hours using a 2° yeast YM medium cultured at 28°C for 40 hours.゛Bacterial cells were collected by centrifugation of this culture solution, washed once with sterilized physiological saline in the same amount as the culture solution, and collected.

0.1 MIJ potassium acid buffer (pH = 7.5) containing 5 l/l of any one of the D-5-substituted hypotoins shown in Table 2...Terminal 5-... 30 to
As a result of adding the amino acids at a concentration of g/l and measuring the intensities at 36° C. for 20 hours, it was confirmed that all of the amino acids produced were in the form of polymers.

The results are shown in Table-2.

Table-1 Table-2 Patent Applicant Mitsui Toatsu Chemical Co., Ltd. Proceedings Amendment Written March 1z, 1985 Director General of the Patent Office 1. Indication of Case Patent Application No. 18660 1985 2. Title of Invention Process for producing α-amino acids 3, Person making the amendment Column 5 of the detailed description of the invention in the specification, Contents of the amendment (1) The statement on page 5, line 12 of the specification [3) Vitamin requirement: ＋" and correct it.

(2) rm/mJ in the 5th line from the bottom of page 7 of the specification is “Tr
Correct it to mJ.

Claims (1)

[Claims] General formula▲ Numerical formula, chemical formula, table, etc.▼...(1) (In the formula, R is an alkyl group, a substituted alkyl group, an aralkyl group, a substituted aralkyl group, a phenyl group, a substituted phenyl group, Hansenula polymorpha has the ability to convert 5-substituted hydantoins into D-α-amino acids (furyl group, pyridyl group, thiazolyl group, imidazolyl group, or indolyl group). polymorpha) (FE
RMP-8013) is converted into D-α-amino acid by the action of the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R is the formula (1)
) A method for producing a D-α-amino acid represented by