JPH0728724B2 - New microorganism - Google Patents

Description

The present invention relates to a novel microorganism belonging to the genus Bacillus.
More specifically, the present invention relates to general formula (I) (In the formula, R represents a lower alkyl group and Bzl represents a benzyl group.) The cis-imidazolidinedicarboxylic acid diesters (hereinafter abbreviated as diesters) represented by the general formula (II) (In the formula, R represents a lower alkyl group. Bzl represents a benzyl group. The 4- and 5-positions are 4S and 5R coordinates.) An optically active cis-imidazolidinedicarboxylic acid A novel Pacillus sp. VA-47 having the ability to be converted into a derivative (hereinafter abbreviated as half ester).
Strains (Ministry of Microbiology Research Institute No. 8730) are provided.

In the general formulas (I) and (II), the lower alkyl group includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, n-hexyl groups and the like.
Means C ₁ -C ₆ alkyl group.

The optically active half-esters (I
I) is a very important compound as a synthetic intermediate for biotin and other pharmaceuticals. Heretofore, as a method for producing optically active half-esters (II) by microorganisms, only the method by the genus Chromobacterium (Japanese Patent Laid-Open No. 190395/1983) has been known.

Therefore, the present inventors extensively searched for microorganisms having the ability to convert diesters (I) into optically active half esters (II) in nature. As a result Bacillus
It has been found that some microorganisms belonging to the genus have such ability, and have completed the present invention.

Hereinafter, the microorganism of the present invention will be described in detail.

The mycological properties of the VA-47 strain are as follows.

(A) Morphological properties (broth agar medium and broth medium) (B) Growth condition in various media (C) Physiological properties As shown in the above bacteriological properties, the VA-47 strain is a spore-forming bacillus that grows under aerobic conditions, and therefore, the Barjay's Manual of Determinative Bacteriology (Bergey's Manual of Determinative Bac
teriology) Obviously, it belongs to the genus Bacillus according to the search formula of the 8th edition. Furthermore, when comparing the strains of the same genus with the VA-47 strain, similar strains include Bacillus fastidiosus (Bacillus fastidiosus) and Bacillus alkalophilus (Bacillus alkalophilus). Has a diameter of 1.5 to 2.5 μm and a length of 3 to 6 μm, which is larger than the VA-47 strain, and is different from the VA-47 strain in that it has no nitrate reducing ability.
Bacillus alcalophilus is an alkalophilic strain
It differs from the VA-47 strain in that it does not grow at pH 7 and has no nitrate reducing ability.

Based on the above, the present inventors recognized the VA-47 strain as a new strain belonging to the genus Bacillus and named it Bacillus sp. VA-47. This strain has been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, under the deposit number Microindustrial Research Institute No. 8730.

The microorganism according to the present invention is characterized in that, in addition to the above-mentioned characteristics in mycological properties, it is accumulated in the medium in a large amount by culturing in the presence of diesters (I). Be attached. In addition, mutations such as irradiation of the microorganism with ultraviolet rays, X-rays, γ-rays, chemical mutation treatment agents, phage contact, transformation, transduction, gene recombination by conjugation, gene recombination by cell fusion, and gene introduction by plasmids. The artificial mutants or natural mutants obtained by the treatment are included.

The culture medium for culturing the microorganism used in the present invention includes a carbon source, a nitrogen source, and a carbon source that are commonly used for culturing microorganisms that are usually used.
Various media containing inorganic substances and the like can be used. Examples of the carbon source of the medium include glucose, fructose, sucrose, starch, glycerin, molasses and other carbohydrates, gluconic acid, pyruvic acid, acetic acid, citric acid,
Organic acids such as, glycine, glutamic acid, alanine,
Examples include amino acids such as asparagine and hydrocarbons such as n-paraffin. Examples of the nitrogen source include meat extract, peptone, yeast extract, dry yeast, taste liquid, soybean powder,
Or its hydrolysates, casein or its hydrolysates, various amino acids, various ammonium salts, nitrates and the like. Examples of inorganic substances are phosphates or salts of magnesium, potassium, calcium, sodium, iron, manganese and the like.

Culturing is usually carried out aerobically, and shaking culture or aeration-agitation culture are preferred. The appropriate number of days for culture is usually from half a day to 7 days. The culture temperature is not particularly limited as long as it is a temperature at which the microorganism can grow and convert the diesters (I) into the optically active half esters (II), but usually 25 to 37 ° C. is preferable, and the pH of the culture solution is 6.5. About 8 is preferable.

The best way to obtain optically active half-esters (II) by culturing this strain is to add diesters (I) to the culture solution from the beginning or after the growth, continue the culture, and carry out semi-hydrolysis. It's simple. Substrate concentration is 0.1-10%
The degree is appropriate. In order to isolate and purify the optically active half-esters (I) accumulated in the culture, the isolation and purification means usually used for the isolation and purification of organic compounds may be applied.

The optically active half-esters (II) can also be subjected to semi-hydrolysis using microbial cells collected from the culture broth of this strain by centrifugation or filtration, or by sonication or lysozyme treatment of microbial cells. It is also possible to perform a method in which the esterase is released by freezing, centrifugation is performed under cooling, ammonium sulfate fractionation, precipitation dialysis, and the like, and half-hydrolysis is performed using the esterase. In these cases, the diesters (I) are suspended or dissolved in an aqueous solution or a buffer solution preferably adjusted to pH 6 to 9, and microbial cells or esterases are added to the diesters (I) preferably in the range of 0.001 to The reaction proceeds by adding 0.1 weight ratio and stirring at preferably 10 to 40 ° C., and the reaction is usually completed in 1 hour to several days.

As mentioned above, the aqueous solution is sulfuric acid, hydrochloric acid, mineral acid such as phosphoric acid, acetic acid
Organic acids such as citric acid, inorganic bases such as sodium hydroxide, potassium hydroxide and sodium carbonate, triethylamine,
It means an aqueous solution to which an organic base such as pyridine and a salt such as sodium acetate and sodium chloride are added.

The buffer solution is potassium dihydrogen phosphate / sodium hydroxide, potassium dihydrogen phosphate / sodium monohydrogen phosphate,
It means a general buffer solution such as potassium hydrogen phthalate / hydrochloric acid, glycine / sodium chloride / sodium hydroxide, etc., and there is no particular limitation as long as it does not interfere with the reaction.

If necessary, an alcohol solvent such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, an ether solvent such as ether, tetrahydrofuran, dioxane, benzene,
Aromatic hydrocarbon solvents such as toluene, acetone, methyl ethyl ketone, ketone solvents such as diethyl ketone, amine solvents such as triethylamine and pyridine, polar solvents such as dimethylformamide, dimethyl sulfoxide, etc.,
Surfactants such as sorbitan monopalmitate and sorbitan monolaurate can also be added.

Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited thereto.

Example 1 (Separation of Bacillus sp. VA-47 strain) Small spatula 1 of soil sample collected in Ono-gun, Gifu prefecture
Suspend 1 cup (about 0.5 g) in 10 ml of sterilized physiological saline, stir it thoroughly, and let it stand. About 0.5m of this soil suspension supernatant
l is added to 10 ml of separating medium (Table 1) containing cis-1,3-dibenzyl-2-oxoimidazoline-4,5-dicarboxylic acid dimethyl ester. After culturing with shaking at 28 ° C. for 4 days, 0.5 ml of the same is inoculated into 10 ml of the same medium for separation, and cultivated in the same manner. This operation is repeated 8 times. The final culture sample is diluted with sterile physiological saline to an appropriate number of bacteria, and 1 drop of this is spread on a separation agar medium prepared by adding 2% agar to the separation medium. Colonies formed after culturing at 28 ° C. for 4 days were further separated using the same agar medium for separation, and completely purified to obtain VA-47 strain. The mycological properties of this strain are as described above.

Next, an example in which (4S, 5R) -1,3-dibenzyl-5-methoxycarbonyl-2-oxoimidazolidine-4-carboxylic acid was collected using this strain will be given as a reference example.

Reference Example 1) Soluble starch 1 g, yeast extract 0.5 g, peptone 0.5 g, K _{2
HPO 4 0.1g, MgSO 4 · 7H} 2 O0.02g, CuSO 4 · 5H 2 O0.0005g, Mn
_{Cl 4 · 4H 2 O0.0005g, ZnSO} 4 · 7H 2 O0.0001g, FeSO 4 · 7H 2 O
A solution consisting of 0.0002 g and 100 ml of distilled water was adjusted to pH 7.6 with diluted sodium hydroxide.

This liquid medium was sterilized at 120 ° C for 20 minutes, then inoculated with Bacillus sp. VA-47 strain, and at 28 ° C.
It was shake-cultured for 48 hours. After adding 400 mg of cis-1,3-dibenzyl-2-oxoimidazolidine-4,5-carboxylic acid dimethyl ester thereto, the mixture was further shake-reacted at 28 ° C. for 72 hours.

100 ml of ethyl acetate was added to this reaction solution, which was acidified with diluted hydrochloric acid, and then filtered through Celite. The filtrate was separated, the organic solvent layer was washed with water and dried over anhydrous magnesium sulfate,
It was concentrated under reduced pressure. To the residue was added 20% of 5% heavy sodium hydroxide water, and after dissolution, the mixture was washed with 20 ml of ether and separated.

The aqueous layer was adjusted to pH 2 with diluted sulfuric acid, and the precipitated white crystals were filtered, washed with water and dried. 300 mg of (4S, 5R) -1,3-dibenzyl-5-methoxycarbonyl-2-oxoimidazolidine-4-carboxylic acid was obtained.

The melting point was 149 to 151 ° C ▲ [α] ²⁰ ₃₆₅ ▼ -28.0 ° (C = 1, DMF).

Claims (1)

[Claims] 1. A Bacillus Espi-Paper having the ability to convert cis-imidazolidinedicarboxylic acid diesters into optically active cis-imidazolidinedicarboxylic acid derivatives.
VA-47 strain (Ministry of Microbiology Research Institute No. 8730)