JPS63173596A - Production of d-lactic acid - Google Patents

Abstract

PURPOSE:To easily obtain D-lactic acid having high optical purify, by culturing a microbial strain assimilating L-lactic acid in a medium containing racemic lactic acid or a mixture of the recemic compound and an optically active lactic acid or by contacting the racemic lactic acid, etc., with cultured microorganism. CONSTITUTION:A culture medium is prepared by using racemic lactic acid or a mixture of racemic lactic acid and an optically active lactic acid as a carbon source and compounding the source with a nitrogen source such as urea, inorganic salt such as MgSO4 and, if necessary, a proliferation promoting factor such as yeast extract. The obtained medium is inoculated with a microbial strain (e.g. Bacillus subtilis IFO 3134) belonging to Bacillus genus and having an assimilation rate of L-lactic acid high than that of D-lactic acid. The strain is cultured in the medium at 3-10pH and 20-60 deg.C for several hours several days under supply of air. The microbial cells are separated and removed from the cultured product and the supernatant liquid is concentrated and refined by ion exchange treatment, solvent extraction, etc., to obtain the objective D-lactic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing D-lactic acid with high optical purity by utilizing the ability of microorganisms to assimilate lactic acid.

(Prior Art) Conventionally, D-lactic acid has been produced by a fermentation method using carbohydrates as a raw material, or by a microbial method using 2-halogenpropionic acid as a raw material.

(Japanese Patent Publication No. 48-1517, Japanese Patent Publication No. 58-36394.

(Japanese Patent Application Laid-open Nos. 58-16688 and 59-31690) [Problems to be solved by the invention] However, there is little knowledge regarding the optical purity of the obtained D-lactic acid, and the largest factor determining this optical purity is D. −
It is based on the characteristics of lactic acid producing bacteria.

When carbohydrates are used as raw materials, D-lactic acid production and accumulation is thought to depend on cell membrane permeability, and L
-Lactic acid and D-lactic acid are mixed.

Therefore, if bacteriolysis occurs, it is expected that I7-lactic acid within the bacterial cells will be released, resulting in a decrease in the optical purity of the lactobacillus. Conventionally, there has been no known method for improving the optical purity of D-lactic acid, which has decreased in optical purity. As a result of intensive studies on the lactic acid assimilation ability of microorganisms, the present inventors found that when a microorganism capable of L-lactic acid assimilation is brought into contact with racemic lactic acid or a mixture of racemic lactic acid and optically active lactic acid, D- It was discovered that lactic acid can be obtained, leading to the present invention.

(Means for Solving the Problems) The present invention involves culturing a racemic form of lactic acid or a mixture of a racemic form and an optically active form with a microorganism capable of assimilating L-lactic acid, or bringing it into contact with a cultured microorganism. This is a characteristic method for producing D-lactic acid.

The starting material used in the present invention is a racemic form of lactic acid or a mixture of a racemic form and an optically active form (D-integrated, L-integrated), and has low optical purity, and monolactic acid, D-lactic acid, etc. can also be used.

Any microorganism can be used in the present invention as long as it has the ability to assimilate L-lactic acid and the rate of L-lactic acid assimilation is faster than the D-lactic acid assimilation rate. Bacillus (Bacillus) is a microorganism with such properties.
acillus), Pseudomonas (Pseudomonas)
Monas ) genus, Escherichia
a) can be selected from among microorganisms belonging to the genus;
Specifically, Bacillus subtilis (Bacillus 5
Pseudomonas ubtilis) IFO3134s Pseudomonaa deha
logenaus) NCIB9061. Pseudomonas alkanoriti force
kanolytica) IFO12319, Escherichia coli (Escherichia coli)
IFO3366 can be mentioned.

To carry out the present invention, the above-mentioned microorganisms are cultured by standing or shaking in an appropriate medium containing a starting material, and the starting material, a racemic form of lactic acid or a mixture of a racemic form and an optically active form, is used as a carbon source. Then, nitrogen sources such as ammonium sulfate, urea, and various amino acids, and inorganic salts such as potassium phosphate and magnesium sulfate may be added, and if necessary, growth promoting factors such as yeast extract and corn staple liquor may be added.

Although optimal conditions for culturing differ depending on the microorganism used, the general conditions are to carry out culturing at a pH of 3 to 10 and a temperature of 20 to 60° C. for several hours to several days. Air, considering the influence of microorganisms on oxygen.
It is also necessary to supply oxygen, inert gas, etc.

Furthermore, in addition to the above-mentioned culture method, the microorganisms used can be cultured using a carbon source that can be used, and the microorganisms collected by means such as centrifugation can be used as a racemic form or a racemic form of lactic acid as a starting material. It can also be carried out by contacting with a mixture with an optically active substance. Although the contact conditions depend on the type of microorganism, contact can be carried out at a pH of 3 to 10 and a temperature of 20 to 60° C. for several hours to several days. D-lactic acid having high optical purity thus obtained can be purified using known methods.

In other words, after removing microbial cells from the reaction solution and adding a concentration operation, D-lactic acid can be recovered and purified by an ion exchange method, a solvent extraction method, or a method in which lactic acid ester is purified by distillation, and then hydrolyzed. .

(Effects of the Invention) According to the present invention, D-lactic acid with high optical purity is brought into contact with L-lactic acid-assimilating bacteria, such as racemic lactic acid or a mixture of racemic lactic acid and optically active lactic acid. (Example) This will be explained below with reference to an example.

Example 1 The liquid culture medium shown in Table 1 was prepared, and 10 ml of the culture medium was placed in a test tube with a diameter of 22 mm, and then sterilized in an autoclave.

Bacillus subtilis (Bacillus subtilis) was added to this medium.
btilis) IFO-3134f - was inoculated with a platinum loop and cultured at 30°C for 4 days.

The cultured solution was centrifuged at 15.00 rpm for 10 minutes, and the lactic acid content of the supernatant was measured. the result,
Although 2.169/l of D-lactic acid remained in the culture solution, no L-lactic acid was detected.

For quantitative determination of L-lactic acid, use "F-kira L-lactic acid" manufactured by Peringer Mannheim Yamanouchi Co., Ltd., and for D-lactic acid, replace the monolactate dehydrogenase in the same kit with D-lactate dehydrogenase. It was quantified.

Table-1 Liquid medium component composition Racemic lactic acid 5g (NH4)2SO42N KH2PO40,8f! Na2HPO4” 12H201,2FMgS04-7
H200,1,9 Yeast extract 0. ! M Water 11 P) 1 7.0 Example-2, 3, 4 Strains used] Monas delrodanas (Pse
udomonas dehalogenaus ) N
CIB-9061y Shoot 9 Pseudomonasalkanolytica
) IFO-12319+ Escherichia coli (
Escherichia coli) IFO-336
6 and cultured in the same manner as in Example-1. Culture 4
The measured values of lactic acid and D-lactic acid after 1 day are shown in Table 2.

Table-2

Claims (1)

[Claims] A method for producing D-lactic acid, which comprises culturing a racemic form of lactic acid or a mixture of a racemic form and an optically active form of lactic acid together with a microorganism capable of assimilating L-lactic acid, or bringing it into contact with a cultured microorganism.