JPH0956376A - Promotion of proliferation of microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To promote the proliferation of a microorganism useful for fermenting a wine, a soy sauce, etc., production, etc., of an antibiotic substance by adding β-(6-chloro-3-pyridine)-D,L-alanine to a culture solution or a culture medium and culturing a microorganism. SOLUTION: 6-Chloronicotinic acid chloride is reduced with sodium borohydride in ether to provide 6-chloro-3-pyridylmethanol, which is then oxidized to afford 6-chloro-3-pyridinecarbaldehyde. The resultant 6-chloro-3- pyridinecarbaldehyde is subsequently reacted with N- benzyloxycarbonyl-2-(dimethoxyphosphinyl)-glycine methyl ester in the presence of a base and the resultant product is then reduced with the sodium borohydride. The hydrolysis of the ester and deprotection of amino group are subsequently carried out to provide β-(6-chloro-3-pyridyl)-D,L-alanine, which is further added to a culture solution or a culture medium to culture a microorganism. Thereby, the proliferation of the microorganism useful for fermenting a wine, a soy sauce, etc., production, etc., of an antibiotic substance is efficiently promoted in quantity and time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the production of antibiotics,
The present invention relates to a method for promoting the growth of microorganisms useful for fermentation and the like.

[0002]

BACKGROUND OF THE INVENTION β- (6-Chloro-3-pyridyl)-
D, L-alanine (β- (6-Chloro-3-pyridyl) -D, L-alan
ine; hereinafter referred to as the present compound) is a compound known in the literature. Journal of Medicinal Chemistry [J.
Med. Chem., 14 , 557 (1971)], describes that the compounds inhibit the growth of certain bacteria. Also,
Journal of Medicinal Chemistry [J. Me
d. Chem., 14 , 211 (1971)], a similar β
-(6-Fluoro-3-pyridyl) -D, L-alanine and β- (6-hydroxy-3-pyridyl) -D, L-alanine are tyrosine antagonists against certain bacteria. ), And shows a growth inhibitory activity. As described above, it has been known that the present compound exhibits a growth inhibitory activity against certain bacteria, but the growth promoting effect of microorganisms such as bacteria and yeast has not been known.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a method for promoting the growth of microorganisms which is useful in the production of antibiotics, fermentation and the like.

[0004]

Means for Solving the Problems As a result of intensive studies on the effects of alanine derivatives on microorganisms, the present inventors have found that the compound has a growth promoting effect on microorganisms,
The present invention has been completed. The microorganism obtained by the present invention is
It can be used for the production of useful substances, etc., and contributes to quantitative and time-efficient production of useful microorganisms. Furthermore, the use of this compound has a high utility value because it also has an action of promoting the growth of microorganisms that are placed under growth-deteriorating conditions.

When the present compound is used, it includes a chemically acceptable salt, for example, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid, formic acid. , Acetic acid, haloacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid ,
Examples thereof include acid addition salts with sulfanilic acid or the like, or salts with alkali metals such as sodium and potassium or metals such as aluminum. Further, it includes a metal complex compound, and examples thereof include complex compounds with zinc, nickel, cobalt, copper, iron and the like. This compound has an asymmetric carbon atom and can be used as an optically active substance.

Examples of microorganisms efficiently produced in the present invention include yeasts useful for fermentation of wine, soy sauce and the like, and bacteria useful for production of antibiotics. Examples of yeast include Saccharomyces cerevisiae (Saccharomyces cerevisiae).
ccharomyces), Candida albicans, etc.
The genus dida) is, for example, Bacillus subtil
genus Bacillus, such as is, Pseudmonas aerugino
Pseudmonas genus such as sa, Xanthomonas
The genus Xanthomonas, such as citri, Serrati
Examples include the genus Serratia such as a marcescens. In use, the compound is dissolved in a suitable solvent such as dimethyl sulfoxide (DMSO) and added to the culture medium or medium. The concentration of the present compound used is appropriately selected from the range of 0.1 to 1000 μg / ml, preferably 25 to 100 μg / ml, based on the culture medium or medium.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be specifically described by way of examples as embodiments of the present invention, but the present invention is not limited thereto. Example 1 Growth promoting action against Candida albicans Method: C. albicans IFO 1270 yeast cells were suspended in sterilized physiological saline at a concentration of 1 × 10 ⁷ cells / ml. This compound was dissolved in DMSO at a concentration of 0.1 or 10 mg / ml. Sabouraud's glucose broth (SGB, bacto-pepton) containing 0.1 ml each of bacterial solution and compound solution
1%, glucose 4%) 9.8 ml, and cultured at 37 ° C. for 2 days with shaking. After culturing, the culture turbidity was measured at 650 nm, and the growth promotion rate was calculated according to the following formula. The results are shown in Table 1.

[Equation 1]

Table 1 ────────────────────────────── Compound final concentration Growth promotion rate (μg / ml) (%) ─ ───────────────────────────── This compound 1 2.4 〃 100 110.8 ─────────────── ────────────────

Example 2 Growth-promoting effect on Saccharomyces cerevisiae S. cerevisiae IFO 2376 yeast cells were added to sterilized physiological saline.
The cells were suspended at a density of × 10 ⁶ cells / ml. This compound is D
It was dissolved in MSO at a concentration of 0.04 to 10 mg / ml.
SGB containing 0.1 ml each of bacterial solution and compound solution
In addition to 9.8 ml, the cells were cultured with shaking at 25, 30 or 37 ° C. The culture turbidity was measured with time at 650 nm, and the growth promotion rate was calculated according to the formula shown above. The results are shown in Table 2.

Table 2 ───────────────────────────────── Culture temperature Final concentration Growth promotion rate (%) ─ ────────────── (μg / ml) 24 48 (hours) ────────────────────────── ─────── 25 ℃ 6.3 200.0 100.0 25.0 400.0 3000.0 100.0 9100.0 88100.0 ───────────────────────────────── ─ 30 ℃ 6.3 7.1 14.6 25.0 64.3 221.1 100.0 1221.4 1036.6 ───────────────────────────────── 37 ℃ 6.3 0.0 221.1 25.0 11.1 605.3 100.0 2633.3 7705.3 ──────────────────────────────────

Example 3 Bacterial growth-promoting action Bacterial cells were suspended in sterile physiological saline at 1 × 10 ⁶ cells / ml. The compound was used in sterile water at 0.04 to 10 mg.
It was dissolved so that it became / ml. Sabouraud's glucose broth (S
GB: bacto-pepton 1%, glucose 4%) 9.8 ml,
The culture was performed at 37 ° C with shaking. The culture turbidity was measured with time at 620 nm, and the growth promotion rate was calculated according to the formula shown above. The results are shown in Table 3.

Table 3 ──────────────────────────────────── Compound final concentration Growth promotion rate (%) Bacterial name ─────────────── (μg / ml) 24 48 (hours) ──────────────────────── ───────────── Bacillus subtilis 6.3 107.5 102.3 IFO 3007 25.0 118.4 159.6 100.0 157.1 123.0 ──────────────────────── ──────────── Pseudmonas aeruginosa 6.3 132.2 106.9 IFO 3080 25.0 130.6 113.9 100.0 222.3 126.4 ────────────────────────── ─────────── Xanthomonas citri 6.3 93.1 -69.5 25.0 262.1 772.4 100.0 172.4 260.0 ─────────────────────────── ───────── Serratia marcescens 6.3 108.2 118.2 25.0 176.5 164.1 100.0 643.5 257.3 ── ─────────────────────────────────

Reference Example β- (6-chloro-3-pyridy
1) Synthesis of 1 -D, L-alanine 1) 6-chloronicotinic acid 25.0 g and thionyl chloride 4
5 g was added and the mixture was heated under reflux for 3 hours. Excessive thionyl chloride was distilled off under reduced pressure to obtain 6-chloronicotinic acid chloride. Sodium borohydride (12.0 g) was dissolved in water (50 ml), and 6-chloronicotinic acid chloride in ether was dissolved.
(30 ml) was added dropwise under ice cooling. After completion of the reaction, the mixture was extracted with ethyl acetate, the organic layer was washed with water, dried and concentrated. The precipitated crystals were washed with n-hexane to give 14.1 g of 6-chloro-3-pyridylmethanol. Yield 62%

2) A solution of dimethyl sulfoxide (20.57 g) in methylene chloride (50 ml) was cooled to -60 ° C., and a solution of oxalyl chloride 22.28 g in methylene chloride (30 ml) was added dropwise. A methylene chloride (20 ml) solution of 6-chloro-3-pyridylmethanol was added dropwise and the temperature was raised to -30 ° C.
Triethylamine (100 ml) was added dropwise and the temperature was raised to room temperature. After completion of the reaction, the mixture was extracted with chloroform, the organic layer was washed with water, dried and concentrated. The precipitated crystals were washed with n-hexane to give 6-chloro-3-pyridinecarbaldehyde (10 g). 80% yield

3) 3.90 g of potassium tert-butoxide was suspended in tetrahydrofuran (50 ml), and 10.94 g of N-benzyloxycarbonyl-2- (dimethoxyphosphinyl) -glycine methyl ester (Synthesis, 1984 , 53) was added.
A tetrahydrofuran (50 ml) solution of was added dropwise under ice cooling.
After stirring for 30 minutes, a solution of 6.25 g of 6-chloro-3-pyridinecarbaldehyde in tetrahydrofuran (20 ml) was added, and the mixture was reacted at room temperature for 1 hour. The mixture was extracted with ethyl acetate, the organic layer was washed with water, dried and concentrated. The precipitated crystals were washed with n-hexane to give 8.35 g of 2- (N-benzyloxycarbonylamino-3- (6-chloropyridyl) -acrylic acid methyl ester, yield 80%, melting point (mp) 98-100. ℃

4) 2- (N-benzyloxycarbonylamino-3- (6-chloropyridyl) -acrylic acid
Methyl ester 8.35 g and nickel chloride hexahydrate 5.72
g was dissolved in methanol (300 ml) and cooled to -20 ° C.
To this solution, 9.11 g of sodium borohydride was added little by little, and the mixture was stirred at -20 ° C for 1 hour. After the reaction was completed, the mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. It was extracted with ethyl acetate and treated according to a conventional method. Purification by silica gel column chromatography (ethyl acetate: n-hexane = 1: 1) gave N-benzyloxycarbonyl-β- (6-chloro-3-pyridyl) -D, L-alanine methyl ester (2.2 g). . Yield 26%, paste

5) N-benzyloxycarbonyl-β
-(6-Chloro-3-pyridyl) -D, L-alanine
2.2 g of methyl ester was dissolved in hydrous methanol (50 ml), 1.83 g of potassium hydroxide was added, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, acidified with 1N hydrochloric acid to hydrochloric acid and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure.
The precipitated crystals were washed with ether and N-benzyloxycarbonyl-β- (6-chloro-3-pyridyl) -D, L-
I got alanine. Yield 79%, mp 134-136 ℃

6) N-benzyloxycarbonyl-β
-(6-Chloro-3-pyridyl) -D, L-alanine 5
00 mg and 5% Pd-C 50 mg were added to methanol (16 ml), acetic acid (2
ml) and water (2 ml), and hydrogenated (H ₂ 33 ml) at room temperature under normal pressure. After 1 hour, the catalyst was removed by filtration and the mixture was extracted with ethyl acetate. The aqueous layer was concentrated and the precipitated crystals were collected by filtration and washed with ether to give the desired product β
-(6-Chloro-3-pyridyl) -D, L-alanine 2
80 mg was obtained. Yield 94%, mp 247-251 ° C

[0019]

INDUSTRIAL APPLICABILITY The present compound can be used for the production of microorganisms, useful substances using microorganisms, etc., and contributes to quantitative and temporal efficiency in the production of useful microorganisms. Furthermore, the use of this compound has a high utility value because it also has an action of promoting the growth of microorganisms that are placed under growth-deteriorating conditions.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 1/38 C12R 1: 125) (C12N 1/38 C12R 1: 385) (C12N 1/38 C12R 1:64) (C12N 1/38 C12R 1:43)

Claims (4)

[Claims] 1. β- (6-Chloro-3-pyridyl)-
A method for promoting the growth of a microorganism, which comprises using D, L-alanine. 2. β- (6-chloro-3-pyridyl)-
The method for promoting the growth of microorganisms according to claim 1, wherein D, L-alanine is an optically active substance. 3. β- (6-chloro-3-pyridyl)-
The method for promoting the growth of microorganisms according to claim 1 or 2, wherein D, L-alanine is a chemically acceptable salt. 4. β- (6-chloro-3-pyridyl)-
The method for promoting the growth of microorganisms according to claim 1 or 2, wherein D, L-alanine forms a complex compound.