JPH0728725B2 - New microorganism - Google Patents

Description

The present invention relates to a novel microorganism belonging to the alkalophilic Bacillus genus. 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-position and 5-position are 4S and 5R coordinates.) Optically active cis-imidazolidinedicarboxylic acid The present invention provides a novel alkaline basophilus Bacillus sp. Strain VA-62 strain (Ministry of Industrial Science, Microbiology Co. No. 8729) having the ability to convert to a derivative (hereinafter referred to as half ester).

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. Conventionally, as a method for producing optically active half-esters (II) by microorganisms, a method based on the genus Chromobacterium has been disclosed (Japanese Patent Application Laid-Open No. 5-312058).
Only 8-190395) was 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, they have found that some of the microorganisms belonging to the alkalophilic Bacillus genus have such ability, and completed the present invention.

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

The mycological properties of the VA-62 strain are as follows.

(A) Morphological properties (broth agar medium and broth medium) (B) Growth form in various media (C) Physiological properties As shown in the above bacteriological properties, the VA-62 strain is a spore-forming bacillus that grows under aerobic conditions, and therefore, the Barjays 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, comparing the strains of the same genus with the VA-62 strain, Bacillus brevis (Ba
cillus brevis) and Bacillus alcalophilus (Bac
illus alkalophilus), but Bacillus brevis differs from the VA-62 strain in that the optimum temperature for growth is 40 to 60 ° C., it has no starch hydrolyzing power, and its salt tolerance is 5% or less NaCl. Bacillus alcalophilus is the same as the VA-62 strain in that it grows only in an alkaline medium, but it does not form a swelling spore with swelling and has no nitrate reducing ability, so it is different from the VA-62 strain. different.

From the above, the present inventors recognized the VA-62 strain as a new alkalophilic strain belonging to the genus Bacillus and named it Bacillus sp. VA-62. 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. 8729.

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 medium for culturing the microorganism used in the present invention includes a carbon source and a nitrogen 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 hydrolysates thereof, casei or hydrolysates thereof, 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 the microorganism can grow and convert the diesters (I) into the optically active half esters (II), but it is preferably 20 to 30 ° C., and the culture solution pH is 8 -10 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 by using microbial cells collected from the culture solution of this strain by centrifugation or filtration, or by sonication or lysozyme treatment of the cells of the microorganism. It is also possible to carry out a method in which an alkali-resistant esterase is obtained by liberating the product by centrifugation under cooling, ammonium sulfate fractionation, precipitation dialysis and the like, and using this to perform semi-hydrolysis. In these cases, the diesters (I) are preferably suspended or dissolved in an aqueous solution or a buffer solution prepared to have a pH of 8 to 10, and microbial cells or an alkaline esterase resistant to the diesters (I) are preferred. Is added 0.001 to 0.1 weight ratio, preferably 10 to 40
The reaction proceeds by stirring at ℃, usually 1 hour ~
The reaction ends in a few 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-62 strain) Small spatula 1 of soil sample collected in Takarazuka City, Hyogo 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 3 times. The final culture sample is diluted with sterile physiological saline to an appropriate number of bacteria, and 1 drop of this is smeared on a separation agar medium in which agar is added to the above-mentioned separation medium so that the content of agar is 2%. Colonies generated after culturing at 28 ° C. for 4 days were further separated using the same agar medium for separation, and completely purified to obtain VA-62 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 O 0.02g, CuSO 4 · 5H 2 O 0.0005
g, MnCl ₄ / 4H ₂ O 0.0005g, ZnSO ₄ / 7H ₂ O 0.0001g, FeSO ₄
A solution of 0.0002 g of 7H ₂ O and 100 ml of distilled water was adjusted to pH 9.0 with diluted sodium hydroxide.

This liquid medium was sterilized at 120 ° C for 20 minutes, and then inoculated with Bacilus sp. VA-62 strain, and at 4 ° C at 28 ° C.
It was shake-cultured for 8 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, dried over anhydrous magnesium sulfate, and 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, the precipitated white crystals were filtered, washed with water and dried. 320 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. Bacillus sp. Which has an optical activity of converting cis-imidazolidinedicarboxylic acid diesters into cis-imidazolidinedicarboxylic acid derivatives.
VA-62 strain (Microtechnology Research Institute, Microbiology No. 8729)