JPH0527388B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Fields] L-arginine is an important component of liver function promoters, amino acid infusions, comprehensive amino acid preparations, and the like. The present invention improves the method for producing L-arginine by fermentation. [Prior Art] In order for microorganisms of the genus Brevibacterium and Corynebacterium to have the ability to produce L-arginine, they must have resistance to 2-thiazolealanine (hereinafter abbreviated as 2-TA), arginine hydroxamate, etc. I know that it would be better if I let them be granted. Furthermore, in addition to the above-mentioned drug resistance, it is possible to impart resistance to drugs such as sulfur drugs, arginol resistance, or 8-azaguanine, and to impart resistance to drugs such as L-histidine, L-proline, L-threonine, L-tryptophan, or L-lysine. It is known that the ability to produce L-arginine is improved by imparting requirements for amino acids. [Problems to be Explained by the Invention] It is an important problem to improve the fermentation yield in order to reduce the production cost of L-arginine. [Means for Solving the Problems] The present invention has been made to solve the above-mentioned problems, and is aimed at solving the problems described above.
As a result of research to improve microorganisms capable of producing arginine and find strains with even higher fermentation yields, we found that α-amino-β-hydroxyvaleric acid (hereinafter referred to as α-amino-β-hydroxyvaleric acid)
Abbreviated as AHV. ) among the strains that have conferred resistance to
L-arginine production with higher yield than conventional L-arginine producing bacteria
They discovered that there are strains of bacteria that produce arginine. That is, the present invention cultivates a microorganism belonging to the Brevibacterium genus or Corynebacterium genus and having AHV resistance and L-arginine production ability in a liquid medium, and L-arginine produced and accumulated in the medium. The present invention relates to a method for producing L-arginine, which comprises collecting L-arginine. The microorganism used in the present invention belongs to the genus Brevibacterium or Corynebacterium,
This is a mutant strain that is resistant to AHV and has the ability to produce L-arginine. To obtain the mutant strain of the present invention, the following wild strain may be first endowed with L-arginine production ability, and then AHV resistance may be imparted,
Alternatively, AHV resistance may be imparted first, and then L-arginine production ability may be imparted. The wild strain that serves as the parent strain of this mutant strain is a bacterium of the genus Brevibacterium or Corynebacterium that is particularly known as a coryneform L-glutamic acid-producing bacterium, and includes, for example, the following.

[Mutation induction method]

Brevibacterium flavum grown on broth agar slants at 30°C for 24 hours.
AJ11169FERM-P4161 (2-TA resistant strain derived from ATCC14067) and Corynebacterium grumemicum AJ12092FERM-P7273 (ATCC13032
2-TA resistant strain) was suspended in M/30 phosphate buffer, and 500 μg/ml of N-methyl-N' was added to the cell suspension with a cell concentration of 10 ⁸ to 10 ⁹ /ml. -Nitro-N-nitrosoguanidine was added and kept at 30°C for 20 minutes. Then, centrifuge to collect the bacterial cells, 1/30M
After thorough washing with phosphate buffer, the cells were inoculated into a medium with the following composition and cultured at 31.5°C for 4 to 10 days. Medium composition (PH7.0) Component content Glucose 1.0g/dl Urea 0.2 _〃 KH ₂ PO ₄ 0.1 〃 MgSO ₄・7H ₂ O 0.1 〃 FeSO ₄・7H ₂ O 0.002 〃 MnSO ₄・7H 2 O 0.002 〃 Biotin 100μg/thiamine hydrochloride 100μg/AHV 0.1g/dl Agar 2.0 Among the strains grown on agar medium, Brevibacterium flavum AJ12144FERM-P7642 (2-TA resistant, AHV resistant) and Corynebacterium glutamicum AJ12145FERM-P7643 (2-TA resistant,
AHV resistance) was obtained. The AHV resistance of the thus obtained mutant strain was compared with that of the parent strain. Glucose 0.5g/dl, urea 0.15g/dl, ammonium sulfate
0.15g/dl, KH ₂ PO ₄ 0.3g/dl, K ₂ HPO ₄ 0.1
g/dl, MgSO ₄・7H ₂ O0.01g/dl, CaCl ₂・
2H ₂ O 0.1mg/dl, biotin 100μg/, thiamine hydrochloride 100μg/, FeSO ₄・7H ₂ O 0.002g/
Natural medium (peptone ₁ g/dl, yeast extract 1 _g /dl,
(NaCl 0.5 g/dl, PH 7.0) Bacterial cells cultured in a slant for 24 hours were suspended in sterilized water and inoculated, cultured for 24 hours, and the growth rate was measured by turbidity.

【table】

[Effect]

The medium used for culturing such mutant strains is a normal medium containing a carbon source, a nitrogen source, inorganic ions, substances that should satisfy the above-mentioned requirements, and other organic micronutrients such as vitamins as necessary. . Preferred carbon sources include carbohydrates such as glucose and sucrose, organic acids such as acetic acid, and preferred nitrogen sources include aqueous ammonia, ammonia gas, and ammonium salts. As inorganic ions, potassium ions, sodium ions, magnesium ions, phosphate ions, and others are appropriately added to the medium as necessary. It is preferable to culture under aerobic conditions, and the culture medium should be
More favorable results can be obtained by controlling the pH to 4 to 8 and the temperature to 25 to 37°C. Thus, if the culture is continued for 1 to 7 days, a significant amount of L will be present in the medium.
- Arginine is produced and accumulated. The method for collecting L-arginine from the culture solution is as follows:
It can be collected using conventional methods such as using ion exchange resin. This will be explained below using examples. Example Glucose 10g/dl, (NH ₄ ) ₂ SO ₄ 7g/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,
Thiamine/HCl 100μg/, biotin 100μg/
A medium containing 60 mg/dl of soybean protein acid hydrolyzate (as total nitrogen) and 5 g/dl of calcium carbonate (separately sterilized) was adjusted to pH 7.0, and 20 ml of it was placed in a 500 ml shoulder flask and sterilized by heating. A loopful of the bacterial strains shown in Table 1 was inoculated into this, and the mixture was shaken for 4 days while being kept at 31.5°C. In the culture solution of each strain, L-arginine was produced and accumulated in the amounts shown in Table 2.

[Table] AJ12144 was cultured using the above method to obtain culture solution 1, which was centrifuged to remove bacterial cells and the supernatant was collected using weakly acidic ion exchange resin "Amberlite" C-50.
(NH ₄ ⁺ form). After washing the resin with water, 2N
L-arginine was eluted with NH ₄ OH, the eluate was concentrated, and crude crystals of L-arginine were obtained.
24.0g was obtained.

Claims (1)

[Claims] 1 A microorganism belonging to the genus Brevibacterium or Corynebacterium and having resistance to α-amino-β-hydroxyvaleric acid and capable of producing L-arginine was cultured in a liquid medium, and produced and accumulated in the medium. A method for producing L-arginine, which comprises collecting L-arginine.