JPS6224075B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-arginine by fermentation using microorganisms. The present inventors previously recognized that mutant strains of Brevibacterium flavum and Corynebacterium acetoacidophyllum produce and accumulate L-arginine, and established a method for producing L-arginine.
(French patent 7105791, British patent publication
1278917) The present inventors further conducted various studies to collect mutant strains with high L-arginine production, and as a result, a significant number of mutant strains belonging to the genus Brevibacterium or Corynebacterium and having resistance to argininol were found. The inventors completed the present invention by discovering that L-arginine is produced and accumulated in the culture medium. The parent strain of the mutant strain of the present invention is Brevibacterium
Devaricatum ATCC14020, Brevibacterium flavum ATCC14067, Brevibacterium lactofamentum ATCC13869, Brevibacterium roseum ATCC13825, Corynebacterium acetoacidophyllum
ATCC13870, Corynebacterium rillium
ATCC15990 etc. To collect the mutant strain of the present invention, for example, the above parent strain is treated with 250 μg/ml of N-methyl-N'-nitro-N-nitrosoguanidine at 30°C for 30 minutes, and an argininol-resistant strain is collected and screened. Arginine producing bacteria were collected. A mutant strain with excellent arginine-producing ability was thus collected, and examples of its parent strain and argininol-resistant strains derived from the parent strain are described below. Parent strain Brevibacterium flavum AJ3277
(SD ^r ) (Brevibacterium flavum ATCC14067
) Resistant strain AJ11336 FERM-P 4939 (SD ^r +
Argininol ^r ) Parent strain Corynebacterium acetoacidophyllum AJ3278 (SD ^r ) (Corynebacterium acetoacidophyllum
(derived from ATCC13870) Resistant strain AJ11340 FERM-P 4943 (SD ^r ) +
Argininol ^r ) Parent strain Brevibacterium flavum AJ 11193
(2TA ^r +S.G ^r +His ^- ) (Brevibacterium flavum ATCC14067
) Resistant strain AJ11345 FERM-P 4948 (2TA ^r +
SG ^r +His ^- + argininol ^r ) SD ^r : sulfadiazine resistance, 2TA ^r : 2-
Thiazolealanine resistance, SG ^r : sulfaguanidine resistance, His ^- : histidine requirement. Add argininol to a medium with the following composition,
4 ml of the mixture was dispensed into small test tubes, sterilized, inoculated with each of the above bacterial strains, and cultured with shaking at 31°C for 48 hours. The degree of growth was calculated by measuring the absorbance and the degree of resistance of each strain was investigated. Medium composition: Glucose 2% Urea 0.3〃 Ammonium sulfate 1.0〃 KH ₂ PO ₄ 0.1〃 MgSO ₄・7H ₂ O 0.04〃 Fe and Mn ions 2ppm each Biotin 100μg / Thiamine hydrochloride 200μg / PH7.0

【table】

[Table] Production of L-arginine using these microorganisms is carried out by a conventional method using a conventional nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, growth promoting factors. Examples of carbon sources that can be used include glucose, sucrose, molasses containing the same, sugars such as starch hydrolyzate, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, and hydrocarbons. Ammonium salts, ammonia gas, urea, ammonia, and others can be used as nitrogen sources. The main fermentation conditions are aeration culture, and the fermentation temperature is 24℃.
~37°C, and the number of fermentation days is usually 2 to 7 days. The pH at the start of fermentation and during cultivation is preferably 5.0 to 9.0. To adjust the pH, use inorganic or organic acidic or alkaline substances,
Furthermore, urea, calcium carbonate, ammonia gas, etc. can be used. L-arginine is usually collected from the fermentation broth by a combination of ion exchange resin method and other known methods. The present invention will be specifically explained below using Examples. Example 1 Glucose 10g/dl, ammonium sulfate 6g/dl
dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04g/
dl, FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/
dl, biotin 50μg/, thiamine hydrochloride 20μ
g/, soybean protein acid decomposition solution (total nitrogen 2.4%) 1
ml/dl, calcium carbonate 5g/dl (separate sterilization added)
Prepare a medium containing PH7.0 and add 20ml of it.
was dispensed into a 500ml flask with a shoulder. After sterilization, they were grown on a bouillon slant. Brevibacterium flavum AJ11336 (SD ^r + argininol ^r ) was inoculated and cultured with shaking at 31°C for 72 hours. The amount of arginine produced and accumulated was as follows. Parent strain AJ3277 1.80 g/dl Resistant strain AJ11336 2.50 Furthermore, the supernatant liquid 1 obtained by removing bacterial cells and calcium salts from the cultured liquid by centrifugation was added to a weakly acidic ion exchange resin Amberlite C-50 (NH ₄ ⁺
type) to adsorb L-arginine.
After washing with water, L-arginine was eluted with 2N ammonia water and the eluate was concentrated. The concentrated solution was cooled to obtain 16.2 g of L-arginine crystals. Example 2 Glucose 10g/dl, ammonium sulfate 6g/dl
dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04g/
dl, FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/
dl, biotin 50μg/, thiamine hydrochloride 20μ
g/, soy protein decomposition solution (total nitrogen 2.4%) 1 mg/
dl, prepare a medium containing 5 g/dl of calcium carbonate (separately added for sterilization) and adjust the pH to 7.0, and add 20 ml of it to 500 ml.
Dispense into ml shoulder flasks. After sterilization, Brevibacterium flavum AJ11345 (SG ^r + 2TA ^r + His ^- + Argininol ^r ), which had been grown in advance on a bouillon slant, was inoculated and cultured with shaking at 31°C for 72 hours. The amount of arginine produced and accumulated was as follows. Parent strain AJ11193 3.3 g/dl Resistant strain AJ11345 3.7 The cultured solution was treated in the same manner as in Example 1 to obtain 21.5 g of L-arginine crystals. Example 3 Glucose 10g/dl, ammonium sulfate 6g/dl
dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04g/
dl, FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/
dl, biotin 50μg/, thiamine hydrochloride 20μ
g/, soy protein decomposition solution (total nitrogen 2.4%) 1 mg/
dl, calcium carbonate 5g/dl (separate sterilization added), PH7.0
A culture medium adjusted to the following conditions was prepared, and 20 ml of the medium was dispensed into a 500 ml shoulder flask. After sterilization, Corynebacterium acetoacidophyllum AJ11340 (SD ^r + Argininol ^r ), which had been grown in advance on a bouillon slant, was inoculated and cultured with shaking at 31°C for 72 hours.
The amount of arginine produced and accumulated was as follows. Parent strain AJ3278 1.70 g/dl Resistant strain AJ11340 1.95 The cultured solution was treated in the same manner as in Example 1 to obtain 12.7 g of L-arginine crystals.

Claims (1)

[Claims] 1. Cultivating a microorganism of the genus Brevibacterium or Corynebacterium that is resistant to argininol and has L-arginine production ability,
A method for producing L-arginine using a microorganism, which comprises collecting L-arginine produced and accumulated in a culture medium.