JPH05219987A - Production of optically active lactive acid - Google Patents

Abstract

PURPOSE:To extremely efficiently obtain the subject compound useful as a chiral center for optically active medicines and agricultural chemicals by treating DL-lactonitrile with a specific bacterium in a polar solvent. CONSTITUTION:DL-Lactonitrile is treated with a bacterium [e.g. Alcaligenes sp. BC20 (FERM P-11,264)] which belongs to the genus Alcaligenes, Corynebacterium, Microbacterium or Obsumbacterium and has stereoselectively nitrile hydrolyzing activity against DL-lactonitrile, or a treated material of the bacterium in a polar solvent (e.g. water or buffer solution of phosphoric acid) to give an advantageous amount of D-lactic acid or L-lactic acid from DL-lactonitrile. Usually, the amount of DL-lactonitrile in the reaction solution is 0.01-10wt.%, the amount of the bacterium used based on DL-lactonitrile is preferably 0.01-5wt.% calculated as dried amount of cells. The reaction is preferably carried out at pH6-10 at 10-50 deg.C.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a biological process for producing optically active lactic acid. Optically active lactic acid is used as a chiral center for optically active pharmaceuticals and agricultural chemicals, and in particular, D-lactic acid is expected to be in demand as a raw material for L-2-chloropropionic acid, which is an intermediate of optically active herbicides. In addition, there are many studies on application development of polymers of optically active lactic acid, and their future use as medical materials is expected.

[0002]

2. Description of the Related Art Conventionally, DL by biological method
-For a method of producing lactic acid from lactonitrile,
Method using bacteria of the genus Bacillus, bacterium, genus Micrococcus or genus Brevibacterium (Japanese Patent Publication Sho 58)
-15120), a method using a bacterium of the genus Corynebacterium (see JP-A-61-56086), genus Corynebacterium, genus Nocardia, genus Bacillus, genus Bacteridium, genus Micrococcus or Brevibacterium. A method using a bacterium of the genus Urum (see JP-A-61-162191) and a method using a microorganism of the genus Pseudomonas, genus Arthrobacter, Aspergillus, Penicillium, Cochliobolus or Fusarium (JP-A-63-222696). (See the official gazette) and the like are known. However, these methods relate to the production of racemic lactic acid, not the production of optically active lactic acid.

On the other hand, regarding the method for producing an optically active oxyacid, a method for producing an L-α-oxyacid (see Japanese Patent Publication No. 54-14668) and a method for producing an optically active α-substituted organic acid (JP-A-2- 84198), a method using a yeast of the genus Tropsis and a microorganism belonging to the genera Alcaligenes, Pseudomonas, Rhodopseudomonas, Corynebacterium, Acinetobacter, Bacillus, Mycobacterium, Rhodococcus or Candida is known. Has been. However, these methods do not directly produce a predominant amount of an optically active substance from a racemic raw material, and there is no specific example concerning the production of optically active lactic acid from DL-lactonitrile, and the optically active lactic acid is highly efficient. Whether it can be produced with optical purity is completely unknown.

[0004]

DISCLOSURE OF THE INVENTION In view of the above situation, the present inventors have made earnest studies to develop an industrially advantageous production method of optically active lactic acid from DL-lactonitrile. As a result, it was found that optically active lactic acid can be efficiently obtained by allowing DL-lactonitrile to act on a specific microorganism having asymmetric nitrile hydrolysis activity in a polar solvent. Lactonitrile has a property of dissociation equilibrium between acetaldehyde and hydrocyanic acid in a polar solvent, and as a result, a racemization reaction occurs, and this racemization mode is coupled with a nitrile asymmetric hydrolase possessed by a microorganism. As a result, it is possible to directly produce a dominant amount of the target optically active lactic acid from DL-lactonitrile, and it is also possible to convert all the raw materials into the target optically active lactic acid stoichiometrically. The present invention is based on these findings.

That is, according to the present invention,
aligenes), Corynebacterium
A microorganism belonging to the genus, Microbacterium genus or Obsumbacterium genus and having stereoselective nitrile hydrolysis activity for DL-lactonitrile, or a treated product thereof in a polar solvent.
-By acting on lactonitrile, DL as a raw material
-Direct amount of D-lactic acid or L- from lactonitrile
A method for producing optically active lactic acid, which comprises producing lactic acid.

The microorganism used in the present invention is specifically
Alcaligenes sp. BC20
1264], Corynebacterium flavense (Coryne
ba-cterium flavescens) IAM 1642, Microbac-terium lacticum IAM 164
0 and Obsum bacterium Proteus (Obsumb-ac
terim proteus) ATCC 12841, and mutants thereof can also be used.

Among these microorganisms, Corynebacterium flavescens IAM 1642, Microbacterium lacticum IAM 1640 and Obsumobacter proteus ATCC 12841 are known, and the Institute for Applied Microorganisms (IAM) and American Type Culture Collection of the University of Tokyo, respectively. (ATCC).

Alcaligenes sp. BC20 [Microtechnology Research Institute
No. 11264] was newly isolated from the natural world by the present applicant, and has been deposited at the Institute for Microbial Technology (Ministry of Industrial Science), Agency of Industrial Science and Technology with the above deposit number. On the street.

BC20 strain form bacillus Gram stainability-spores-motility + flagella pericardium oxidase + catalase + OF alkalized 3-ketolactose production-quinone Q-8

[0010] The above-mentioned mycological properties are described in Bergey's Manual of S
ystematic Bacteriology 1986, classified according to BC
Twenty strains were identified as bacteria belonging to the genus Alcaligenes.

Next, a general embodiment of the present invention will be described. For culturing the microorganism used in the present invention, a medium containing an assimilable carbon source, a nitrogen source, and inorganic nutrients necessary for the growth of the microorganism is usually used. For example, glucose, glycerol, sucrose, etc. as a carbon source, yeast extract, peptone, ammonium sulfate, etc. as a nitrogen source, dipotassium hydrogen phosphate, magnesium chloride, calcium chloride, second chloride as an inorganic nutrient source. Iron, manganese sulfate, zinc sulfate, etc. are used. In addition, the concentration of nitriles (2-
Chloropropionitrile, acetonitrile, propionitrile, normal butyronitrile, isobutyronitrile, benzonitrile, benzyl cyanide, 3-cyanopyridine, etc.), amides corresponding to these nitriles, lactams (ε-caprolactam, It is preferable to add γ-butyrolactam or the like) because higher enzyme activity can be obtained.

The culture is carried out under aerobic conditions at a pH of 4 to 10 and a temperature of 20.
It may be carried out at a temperature of up to 90 ° C. and a culture time of 24 to 96 hours while controlling within a range suitable for each microorganism.

The hydrolysis reaction of DL-lactonitrile is
Microorganism cells obtained by the above culture conditions or treated products thereof (crushed cells, crude / purified enzyme, immobilized cells / enzyme, etc.)
Can be contacted with DL-lactonitril in a polar solvent, whereby a high yield of the target D-lactic acid or L-lactic acid (50 to 100%) from DL-lactonitrile can be obtained. be able to. The polar solvent is typically water, physiological saline, an aqueous medium such as a phosphate buffer,
In addition, lower alcohols such as methanol and ethanol, organic solvents such as dimethylformamide, dioxane and the like can be appropriately mixed and used in the above aqueous medium. The hydrolysis reaction can also be carried out by using acetaldehyde and hydrocyanic acid instead of DL-lactonitrile.

Normally, DL-lactonitrile in the reaction solution is
0.01 to 10% by weight, the amount of the microorganism used to DL-lactonitrile is 0.01 to 5% by weight as the dry cell amount, and the pH is 3 to
12, preferably 6 to 10, the reaction temperature is freezing point to 70 ° C., preferably 10 to 50 ° C., and the reaction may be performed for 0.5 to 72 hours.

In order to isolate the desired optically active lactic acid from the reaction solution, it is possible to utilize a known method such as concentration, electrodialysis, ion exchange, extraction or crystallization after removing the cells by centrifugation or the like. it can.

[0016]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to directly produce a dominant amount (50 to 100%) of a target optically active lactic acid from DL-lactonitrile, and stoichiometrically target all raw materials. It is also possible to provide a highly efficient method for producing optically active lactic acid.

[0017]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 (1) Culturing 10 ml of the following medium (in a φ22 mm test tube) was inoculated with the strains shown in Table 1 and shake-cultured at 30 ° C. for 2 days.

Medium (pH 7.2) Glycerol 20 g Yeast extract 3 g 2-Chloropropionitrile 1 g Dipotassium hydrogen phosphate 3 g Magnesium chloride 0.2 g Calcium chloride 40 mg Manganese sulfate ・ tetrahydrate 4 mg Ferric chloride・ Heptahydrate 0.7 mg Zinc sulfate ・ Heptahydrate 0.1 mg Distilled water 1000 ml

(2) Hydrolysis reaction The bacterial cells were recovered from the obtained culture broth by centrifugation and washed with 30 mM phosphate buffer (pH 7.0), and the precipitated bacterial cells were reconstituted with 5 ml of the same phosphate buffer. Suspended. DL-lactonitrile having a final concentration of 10 mM was added thereto and reacted at 30 ° C. for 24 hours. After completion of the reaction, the reaction solution was centrifuged to remove the bacterial cells, and the supernatant was used as a column containing MCI-GEL-CRS10W manufactured by Mitsubishi Kasei Co., Ltd. as a packing material, or D-lactic acid and L-lactate dehydrogenase and nicotine. Lactic acid was quantified and its optical purity was measured by an enzymatic method using amidoadenine dinucleotide. The results are shown in Table-1.

[0021]

[Table 1] Note) The optical purity is based on the D form, 100% ee indicates that all D forms, and -100% ee indicates that all L forms.

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Claims (1)

[Claims] 1. A genus of Alcaligenes, a genus of Corynebacterium, a genus of Microbacterium or a genus of Obsum bacterium (O)
A microorganism belonging to the genus bsumbacterium) and having a stereoselective nitrile hydrolysis activity for DL-lactonitrile or a treated product thereof is allowed to act on DL-lactonitrile in a polar solvent to obtain DL-lactonitrile as a raw material. A process for producing an optically active lactic acid, which comprises directly producing a predominant amount of D-lactic acid or L-lactic acid.