JPH0424992B2 - - Google Patents

Description

[Detailed description of the invention]

(Object of the invention) Industrial application field The present invention relates to a method for producing N-carbomoyl-D-valine or D-valine. For more details,
The present invention relates to a method for producing N-carbamoyl-D-valine or D-valine using L- or DL-isopropylhydantoin as a substrate through the action of microorganisms. The purpose is to produce D-valine, which is an industrially important substance as a raw material for agricultural chemicals, at a low cost. Although N-carbomoyl-D-valine itself has no major industrial uses, it can be easily converted into D-valine chemically or enzymatically, so it can be used as a raw material for producing D-valine. BACKGROUND ART Conventionally, a method for producing N-carbamoyl-D-valine or D-valine from D-isopropylhydantoin by the action of microorganisms is already known. However, there is no known method for producing N-carbamoyl-D-valine and/or D-valine from L-isopropylhydantoin using the action of microorganisms. Isopropylhydantoin is known to slightly racemize in aqueous solution. Therefore, L
- Even if isopropylhydantoin is used as a raw material, it is possible to produce a small amount of N-carbamoyl-D-valine or D-valine through the action of microorganisms. However, the production speed is very slow and it is difficult to call this a practical method. Problems to be Solved by the Invention As a result of intensive research on this point, the present inventors have found that they have the ability to racemize isopropylhydantoin, and that D-isopropylhydantoin can be converted to N-carbamoyl-D-valine or D
- Discovered a microorganism that has the ability to produce valine, and completed the present invention based on this discovery. According to the method of the present invention, D-valine can be produced in high yield using DL-isopropylhydantoin produced by a chemical synthesis method as a raw material, so it is an industrially extremely advantageous method for producing D-valine. Become. Structure of the Invention The microorganisms used in the present invention include microorganisms belonging to the genus Candida, such as Candidaphillyla MT-40361 (Feikoken Bokuyori No. 7763), Candidavinaria MT-40362 ( Microtechnology Research Institute No.
7764), and when these microorganisms are used in the D-valine synthesis reaction, the culture solution of these microorganisms, the cultured cells, or the cell-free extract obtained by crushing the cells, or An immobilized product obtained by immobilizing bacterial cells can be used. The above-mentioned microorganisms are usually cultured under aerobic conditions such as shaking culture or submerged culture with aeration and stirring. The culture temperature is 10-35°C, preferably 25-35°C, and the culture pH is 3-8, preferably 4.5-6.5. As the carbon source and nitrogen source used in the culture medium, any type of carbon source and nitrogen source may be used as long as they are available to the bacteria used. That is, carbon sources include carbohydrates such as glucose, galactose, starch hydrolyzate, and molasses;
Furthermore, glycerol, citric acid, ethanol, etc. can also be used. Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium acetate, or meat extract, yeast extract, malt extract, corn steep liquor,
Natural organic nitrogen sources such as casein hydrolyzate and fruit meal can also be used. Many natural organic nitrogen sources can be both nitrogen and carbon sources. Potassium monohydrogen phosphate as an inorganic substance,
Potassium dihydrogen phosphate, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, and the like may also be advantageously used as required. Furthermore, isopropylhydantoin or N-carbamoylvaline 0.5-10g/, preferably 1-10g/
When added to the culture medium at a concentration of 5 g/ml, the activity of enzymes involved in this reaction in the bacterial cells is increased. There is no particular limit to the concentration of L- or DL-isopropylhydantoin, which is the substrate used in the reaction, but a concentration of 1 to 4% is usually used, and the reaction temperature is 10%.
-50℃, preferably 25-40℃, reaction pH 3-10,
Preferably it is 6-9. Isolation of N-carbamoyl-D-valine or D-valine from the reaction solution can be performed by known methods such as concentration, neutralization, and treatment with an ion exchange resin. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 Candeida phyllilla MT-40361 was inoculated into 100 ml of medium (composition shown in Table 1) in a 500 ml Sakaguchi flask, and cultured with shaking at 30°C for 48 hours. After culturing, collect the bacteria by centrifugation, add 100ml of physiological saline, collect the bacteria again by centrifugation, and reduce the bacterial cells to 0.9%.
The cells were suspended in 50 ml of saline to obtain a cell suspension. After mixing 1 ml of this bacterial cell suspension with 1 ml of 2% L-isopropylhydantoin solution (dissolved in a 0.2 M concentration buffer of the type shown in Table 2), the mixture was heated at 30°C.
The reaction was allowed to proceed with stirring for 17 hours. After the reaction was completed, D-valine was quantified by liquid chromatography. On the other hand, under the above reaction conditions, the racemization rate of L-isopropylhydantoin was determined by measuring the degree of light exposure when no bacterial cell suspension was added. The results obtained are shown in Table 2.

【table】

【table】

[Table] Example 2 DL- instead of L-isopropylhydantoin
The same procedure as in Example 1 was conducted except that isopropylhydantoin was used and the reaction was carried out at 37°C for 21 hours. After the reaction, the produced N-carbamoyl-D
-The amounts of valine and D-valine produced are shown in Table 3. The optical purity of N-carbamoyl-D-valine was 100%.

[Table] Example 3 The same operation as in Example 1 was carried out except that Candeida Vinaria MT-40362 was used instead of Candeida Firilla MT-40361. The results obtained are shown in Table 4.

[Table] Example 4 The same operation as in Example 2 was performed except that Candeida Vinaria MT-40362 was used in place of Candeida Firilla MT-40361. The results obtained are shown in Table 5. The optical purity of N-carbamoyl-D-valine was 100%.

【table】

Claims (1)

[Claims] 1 Belongs to the genus Candeida, has the ability to racemize isopropylhydantoin, and converts D-isopropylhydantoin to N-carbamoyl-
N-carbamoyl-D-, which is characterized by contacting D-valine or a culture solution of a microorganism capable of producing D-valine, cultured cells, or a treated product of a cultured microorganism with L- or DL-isopropylhydantoin. Method for producing valine or D-valine.