JPH0488978A - Culture of bacteria - Google Patents

Abstract

PURPOSE:To culture a microbial strain capable of producing a dehalogenase useful as a synthetic raw material for various pharmaceuticals and physiologically active substances in increased enzymatic activity by adding a compound such as cyclohexanol to a culture liquid. CONSTITUTION:The objective culture is carried out by adding at least one kind of compound selected from cyclohexanol, 2,3-dihalo-1-propanol and dichloroethanol to a culture liquid of a dehalogenase-producing microbial strain (bacteria belonging to genus Microbacterium or genus Corynebacterium). The amount of the compound is selected to get a concentration of preferably 0.01-1.0 (W/V)% in the culture liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for culturing highly active dehalogenase-producing bacteria. The dehalogenase is useful as a raw material for the synthesis of various pharmaceuticals and physiologically active substances, for example, a raw material for the synthesis of L-lunitine (R)-(-)-3.
- Used in the production of halo 1,2-propanediol (
Patent Application No. 1-100173 and Patent Application No. 1-10017
(See Specification No. 4).

(Prior art and problems) (R)-(-)-3-halo 1 by biological method,
A method for producing 2-propanediol is to react racemic (R,5)-3-halo 1,2-propanediol with microorganisms to produce (S)-(+)-3-halo 1,2-
Selectively metabolize propanediol, (R)-(-)
-3-halo 1,2-propanediol (refer to Japanese Patent Application Laid-open No. 158494/1983), racemic (R,5)-2,3
-By acting on dichloro-1-propanol (R) -
A method for fractionating (-)-3-halo-1,2-propanediol is known (see Japanese Patent Application Laid-Open No. 62-69993), but these can be obtained because they are made from racemic substances ( The yield of R)-(-)-3-halo 1,2-propanediol based on the raw material is less than 50%, and this cannot be an economically advantageous production method.

The applicant previously obtained optically active (R)-(-)-3-halo-1,2-propanediol from the inexpensive brochiral compound 1,3-dihalo-2-propanol by the action of dehalogenase. A patent application has been filed for a method for producing with a theoretical optical yield of 100% (see Japanese Patent Application No. 1-100173), but in order to make this method more industrially advantageous, It has been desired to develop an effective enzyme inducer that can express enzyme activity even higher.

(Summary of the Invention) Therefore, the present inventors have diligently investigated the culture conditions for culturing the enzyme-producing bacteria with higher dehalogenase activity.
Cyclohexanol, enzyme inducer in the culture medium
It was discovered that dehalogenating enzyme-producing bacteria with high enzymatic activity could be obtained by adding 2,3-dihalo-1-propanol, dichloroethanol, etc., and the present invention was completed.

Furthermore, as a result of further studies, it was found that these compounds are difficult to assimilate by the dehalogenating enzyme-producing bacteria, and not only does it not require complicated operations to control the concentration of these compounds, but the HCI produced as a result of assimilation is It has been found that this method has an advantage in that it is possible to avoid deactivation of the enzyme due to a decrease in pH due to oxidation.

That is, the present invention provides cyclohexanol,
2. A method for culturing dehalogenase-producing bacteria, which comprises adding at least one compound selected from 3-dihalo-1-propanol and dichloroethanol.

(Detailed Description of the Invention) The dehalogenase used in the present invention is 1,3-dihalo2-
It is an enzyme that can ultimately convert one halogen atom of propanol into a hydroxyl group. Specifically, examples include enzymes produced by the Microbacterium genus strain I-4701, and the Corynebacterium strain N-653 and N-1074. These microorganisms are Microbacterium sp.
No. 2642 (Corynebacterium sp, N-653) and Koken No. 26
It has been deposited as No. 43 (Corynebacterium sp, N-1074) at the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology, and its mycological properties are as follows.

L'111 Table Morphology Peripheral cells of the colony Durham staining Spores Motility Color of the flagellar colony Oxidase Catalase F Growth in anaerobic conditions Cell wall diamino acid glycolyl test Starch decomposition Gelatin Liquefaction Nitrate reduction Arginine utilization Pleomorphic bacilli No elongation + Approved + Very yellow-orange side hair + + Lysine + (glycolyl type) + + Urea decomposition Heat resistance in skim milk medium 60°C, 30 minutes Acid production Inulin Glycerol Glucose Sucrose Trehalose Raffinose Morphology Surrounding cells of the colony Durham staining Spore motility oxidase catalase F + No polymorphic rod growth + Not observed + + Hydrogen sulfide production Growth in anaerobic conditions Catalase + Cell wall diamino acid glucoryl test Starch decomposition Gelatin Liquefied cellulose decomposition Urea decomposition In acid-fast skim milk medium Heat resistance of 63°C for 130 minutes and 72°C for 115 minutes [River 1 Morphology Peripheral cells of colony Duram staining Spore motility Oxidase diacetylbutyric acid (acetyl type) + Polymorphic rods not elongated + Not observed + F Growth under anaerobic conditions Cell wall diamino acid glucoryl diaminobutyrate g
Test-(ycechi IL type) Starch decomposition 10 Gelatin Decomposition of liquefied cellulose Urea decomposition Heat resistance in acid-fast skim milk medium 63°C for 130 minutes 72°C for 315 minutes or more Mycological properties・Systematic Tsuku Bacteriology Vo12 (1
986) (Bergy's manual of S
ystematic Bacteriology Vo
1, 2 (1986)), N-
The 4701 strain was identified as a bacterium belonging to the genus Micronacterium, and the N-653 strain and N-1074 strain were identified as bacteria belonging to the genus Corynebacterium.

Generally, any culture solution composition that can be used in the present invention can be used as long as the above-mentioned microorganisms can grow therein. for example,
Carbon sources include sugars such as glucose, fructose, sucrose, and maltose; alcohols such as glycerol and L-ethanol such as acetic acid and citric acid; nitrogen sources include peptone, meat extract, yeast extract, protein hydrolysates, and amino acids. In addition to general natural nitrogen sources such as, various inorganic and organic acid ammonium salts can be used;
Trace metal salts, vitamins, etc. are used as appropriate.

The concentrations of cyclohexanol, 2.3 dihalo-1-propanol (2,3-dichloro-1-propanol, 2,3-dibromo-1-propanol, etc.) and dichloroethanol used in the present invention are particularly limited. Although not a certain amount, 0.01 to 1.0 (II/V)% is preferable,
These can be added all at once or in portions. Moreover, even if these compounds are added alone, 2
A combination of more than one species may be added. Furthermore, these compounds may be added at the beginning of the culture or during the culture.

The dehalogenating enzyme-producing bacteria may be cultured using the above culture solution according to a conventional method, for example, at a temperature of 20 to 45"C1pH4
Incubate aerobically for 10-96 hours at a temperature of ~10.

According to the culture method of the present invention, enzyme-producing bacteria having highly active dehalogenating enzymes can be produced. This can greatly contribute to the industrial production of dehalogenase-producing bacteria useful for the production of dehalogenating enzymes.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited only to these Examples.

Example 1 Glucose 1%, peptone 0.5%, meat extract 0.3%
A medium containing 0.3% yeast extract was adjusted to pH 7.0 and dispensed into 500-Erlenmeyer flasks in 100d portions.
Cyclohexanol, 2,3-dichloro-1-, which was sterilized at 20°C for 15 minutes and then sterilized with a membrane filter.
A solution of propanol and dichloroethanol was added to a predetermined concentration. Further, as a control, the same operation was performed in the case where these compounds were not added and in the case where 3-chloro-1,2-propanediol, which had been sterilized with a membrane filter, was added instead of these compounds.

Inoculating the dehalogenating enzyme producing microbacterium Microbacterium strain N-4701 (Kaikoken Jokyo No. 2644) into the above medium,
Shaking culture was performed at 30°C for 48 hours. The dehalogenase activity of the dehalogenase-producing bacteria thus obtained was measured as follows.

That is, bacterial cells were collected from this culture solution 40d by centrifugation, and 50IIM Tris-H1SO, buffer (pH 8,
0) After washing twice at 40d, 2M) Lis-11cI ll
The cells were suspended in 20 d of liquid pi(8,0) to obtain a bacterial cell suspension. The reaction was started by adding 20i of 2 (edge/V)% 1.3-dichloro-2-propanol solution to this bacterial cell suspension, and after 2 hours, 3-chloro-1,2-propanediol was produced. was determined by gas chromatography.

The results are shown in Table-1.

Claims (1)

[Claims] 1. Cyclohexanol, 2,3-dihalo- in the culture solution
A method for cultivating a dehalogenating enzyme-producing bacterium characterized by adding at least one compound selected from 1-propanol and dichloroethanol; 2, the dehalogenating enzyme-producing bacterium is a microbacterium of the genus Microbacterium or the genus Corynebacterium; 2. The culture method according to claim 1, wherein the bacteria belong to .