JP3090761B2 - Production method of optically active lactic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biological method for producing optically active lactic acid. BACKGROUND ART Optically active lactic acid is used as a chiral center for optically active medical and agricultural chemicals. In particular, demand for D-lactic acid is expected as a raw material for L-2-chloropropionic acid, which is an intermediate of an optically active herbicide. In addition, there are many studies on the development of applications of optically active lactic acid polymers, and their future use as medical materials is expected.

[0002]

2. Description of the Related Art Conventionally, DL by a biological method
-For the method of producing lactic acid from lactonitrile,
A method using a bacterium belonging to the genus Bacillus, Bacterium, Micrococcus or Brevibacterium (Japanese Patent Publication No.
-15120), a method using a bacterium belonging to the genus Corynebacterium (see JP-A-61-56086), a genus Corynebacterium, a genus Nocardia, a bacillus, a bacterium, a genus Micrococcus or Brevibacterium. (See JP-A-61-162191) and a method using microorganisms belonging to the genus Pseudomonas, Arthrobacter, Aspergillus, Penicillium, Cochliobolus or Fusarium (Japanese Patent Application Laid-Open No. 63-222696). Publications) are known. However, these methods relate to the production of racemic lactic acid, not to the production of optically active lactic acid.

On the other hand, as for 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 (Japanese Patent Application Laid-Open No. 84198), a method using a yeast belonging to the genus Tropsis and a microorganism belonging to the genus Alcaligenes, Pseudomonas, Rhodopseudomonas, Corynebacterium, Acinetobacter, Bacillus, Mycobacterium, Rhodococcus or Candida, respectively. Have been. However, these methods do not directly produce a dominant amount of optically active lactic acid from a racemic raw material, and there is no specific example relating to the production of optically active lactic acid from DL-lactonitrile. 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 intensively studied to develop an industrially advantageous method for producing optically active lactic acid from DL-lactonitrile. As a result, they have found that optically active lactic acid can be efficiently obtained by allowing a specific microorganism having a nitrile asymmetric hydrolysis activity to act on DL-lactonitrile in a polar solvent. Lactonitrile has the property of dissociation equilibrium between acetaldehyde and hydrocyanic acid in polar solvents, resulting in a racemization reaction, and conjugation of this racemization mode with the nitrile asymmetric hydrolase of microorganisms Thus, a dominant amount of the desired optically active lactic acid can be directly produced from DL-lactonitrile, and all the raw materials can be stoichiometrically converted to the desired optically active lactic acid. The present invention is based on these findings.

[0005] That is, the present invention relates to an alkaline gene (Alc
aligenes), Corynebacterium
A microorganism belonging to the genus, Microbacterium or Obsumbacterium and having a nitrile hydrolysis activity stereoselective for DL-lactonitrile, or a treated product thereof, in a polar solvent,
-By acting on lactonitrile, the raw material DL
-A significant amount of D-lactic acid or L- directly from lactonitrile
A method for producing optically active lactic acid, characterized by producing lactic acid.

[0006] The microorganism used in the present invention is, specifically,
Alcaligenes (Alcaligenes) sp.BC20
No. 1264), Corynebacterium flavesense (Coryne
ba-cterium flavescens) IAM 1642, Microbac-terium lacticum IAM 164
0 and Obsambacterium proteus (Obsumb-ac
terim proteus) ATCC 12841, and these mutants can also be used.

[0007] Among these microorganisms, Corynebacterium flavescens IAM 1642, Microbacterium lacticum IAM 1640 and Osbacterium proteus ATCC 12841 are known, and are each of the Institute of Applied Microbiology (IAM) of the University of Tokyo and American Type Culture Collection. (ATCC).

[0008] Alkaligenes sp. BC20
No. 11264] was newly isolated from the natural world by the present applicant, and has been deposited with the Institute of Industrial Science and Technology, Microbiological Research Institute (Microtechnical Laboratory) under the above deposit number, and its bacteriological properties are as follows: It is on the street.

Strain BC20 morphology Bacillus Gram stain-spore-motility + flagellar peroxidase + catalase + OF Production of alkalized 3-keto lactose-quinone Q-8

The above 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 cultivation of the microorganism used in the present invention, a medium containing a carbon source, a nitrogen source, and inorganic nutrients necessary for the growth of the microorganism is used. For example, glucose, glycerol, sucrose and the like as carbon sources, yeast extract, peptone, ammonium sulfate and the like as nitrogen sources, dipotassium hydrogen phosphate, magnesium chloride, calcium chloride and secondary chloride as inorganic nutrient sources. Iron, manganese sulfate, zinc sulfate and the like are used. In addition, the nitriles (2-
Chloropropionitrile, acetonitrile, propionitrile, normal butyronitrile, isobutyronitrile, benzonitrile, benzyl cyanide, 3-cyanopyridine, etc.), amides and lactams corresponding to these nitriles (ε-caprolactam, (γ-butyrolactam, etc.) is preferred because higher enzyme activity can be obtained.

The culture is carried out under aerobic conditions at a pH of 4 to 10 and at a temperature of 20
What is necessary is just to carry out at -90 degreeC and culture time for 24-96 hours, controlling in the range suitable for each microorganism.

The hydrolysis reaction of DL-lactonitrile is as follows:
Microbial cells obtained under the above culture conditions or processed products thereof (crushed cells, crude / purified enzymes, immobilized cells / enzymes, etc.)
May be brought into contact with DL-lactonitrile in a polar solvent, whereby a predominant amount (50 to 100%) of the desired D-lactic acid or L-lactic acid is obtained from DL-lactonitrile in high yield. be able to. As the polar solvent, typically, an aqueous medium such as water, physiological saline, and phosphate buffer,
In addition, organic solvents such as lower alcohols such as methanol and ethanol, dimethylformamide and dioxane can be appropriately mixed and used in the above aqueous medium. In addition, this hydrolysis reaction can also be performed using acetaldehyde and hydrocyanic acid instead of DL-lactonitrile.

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

For isolating the desired optically active lactic acid from the reaction solution, known methods such as concentration, electrodialysis, ion exchange, extraction, and crystallization are used after removing the cells by centrifugation or the like. it can.

[0016]

According to the present invention, the desired optically active lactic acid can be directly produced from DL-lactonitrile in a predominant amount (50 to 100%). It is also possible to convert to optically active lactic acid, and to provide a very efficient method for producing optically active lactic acid.

[0017]

The present invention will be described below in detail with reference to examples.

Example 1 (1) Cultivation The strains shown in Table 1 were inoculated into 10 ml of the following medium (in a φ22 mm test tube) and cultured with shaking 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・ 7-hydrate 0.7 mg zinc sulfate ・ 7-hydrate 0.1 mg distilled water 1000 ml

(2) Hydrolysis reaction The cells were collected from the obtained culture by centrifugation, washed with 30 mM phosphate buffer (pH 7.0), and the precipitated cells were reconstituted in 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 cells, and the supernatant was subjected to a column containing MCI-GEL-CRS10W manufactured by Mitsubishi Kasei Corporation as a packing material, or D-lactic acid and L-lactic acid dehydrogenase and nicotine. Lactic acid was quantified and its optical purity was measured by an enzymatic method using amide adenine 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 the composition is all D-form, and -100% ee indicates that the composition is all L-form.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI (C12P 41/00 C12R 1:01)

Claims (1)

(57) [Claims] 1. The genus Alcaligenes, the genus Cory-nebacterium, the genus Microbacterium or the bacterium Osam
bsumbacterium) belonging to the genus, and a microorganism having a stereoselective nitrile hydrolysis activity for DL-lactonitrile or the treated product is allowed to act on DL-lactonitrile in a polar solvent to obtain DL-lactonitrile as a raw material. A method for producing an optically active lactic acid, characterized in that a superior amount of D-lactic acid or L-lactic acid is produced directly from the product.