JPS6143038B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to a method for producing L-glutamic acid by fermentation. L-glutamic acid is produced by a fermentation method using microorganisms belonging to the genus Brevibacterium or Corynebacterium. The present inventors developed isocitrate lyase (4.1.3.1Ls-isocitrate lyase) from a conventionally known L-glutamic acid producing bacterium of the genus Brevobacterium.
They induced a mutant strain with reduced levels of L-glutamic acid (glyoxalate-lyase) and found that this mutant strain produced and accumulated L-glutamic acid at a higher yield than previously known L-glutamic acid producing bacteria. The mutant strain used in the present invention belongs to the genus Brevibacterium and is a mutant strain with reduced isocitrate lyase. Specific examples include the following mutant strains. Brevibacterium lactofermentum AJ 11516 FERM-P 5336 (acetic acid auxotrophy, isocitrate lyase reduced strain) Brevibacterium flavum AJ 11518 FERM-P 5338 (isocitrate lyase reduced strain) AJ 11516 is ATCC 13869, AJ 11518 is
ATCC 14067 was derived as the parent strain, respectively. Other parent strains that can be used include L-glutamic acid producing bacteria of the genus Brevibacterium, such as Brevibacterium satucaroliticum ATCC14066 and Brevibacterium devaricatam ATCC14020. "Reduced isocitrate lyase" as used in the present invention naturally includes an isocitrate lyase-deficient strain in which the isocitrate lyase activity has decreased to the point where it is undetectable. As methods for inducing mutations in the above mutant strains, conventional methods such as ultraviolet irradiation, radiation irradiation, and treatment with mutagenic agents are used. For example, 200 μg/ml N-methyl-N ¹ -nitro-N-nitrosoguanidine 0℃
There are methods such as processing for 20 minutes. Table 1 shows the results of measuring the isocitrate lyase activity of these mutant strains in comparison with the parent strain.

[Table] Isocitrate lyase activity was assayed as follows. Glucose 2.5g/dl, ammonium acetate 0.8g/dl
dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.1g/dl,
FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/dl, urea 0.4g/dl, biotin 0.3μg/dl, thiamine hydrochloride 20μg/dl and soybean decomposition concentrate (as T-N) 48mg/dl Adjust the medium containing (PH7.0),
Pour 20ml of each into a 500ml shaking flask and heat to 115°C.
sterilized by heating for 10 minutes. The test strain was inoculated into this medium and cultured at 31.5°C in a reciprocating shaking incubator until the early stage of logarithmic growth (10 to 16 hours). Bacterial cells were separated from the culture solution, washed, disrupted by ultrasonication, and treated with Sephadex G-10, and used as an enzyme preparation. The yeast strain activity was measured according to the method of Shiio et al. (Journal of Biochemistry 49
vol., p. 262, 1961) The method for producing and accumulating L-glutamic acid using these strains is the same as the conventional method used for culturing L-glutamic acid-producing microorganisms. That is, as the carbon source, for example, sugar juice or blackstrap molasses from sugar beet or sugar beet, carbohydrate raw materials such as starchy raw material hydrolysates, acetic acid, ethyl alcohol, etc. are used. As nitrogen sources, for example, ammonium salts, aqueous ammonia, ammonia gas, urea, etc. used in normal L-glutamic acid fermentation are used, and inorganic ions such as phosphates and magnesium salts are added to the medium as necessary. be done. Additionally, micronutrients such as thiamine and biotin are used as appropriate. Furthermore, a biotin action inhibitor such as polyoxyethylene sorbitan monopalmitate or penicillin is added to the medium in which biotin is present in excess by a conventional method. Cultivation is preferably carried out under aerobic conditions, with a culture temperature of 24 to 37°C and a pH of 6 to 9 during cultivation. To adjust the pH, use inorganic or organic acidic or alkaline substances, and Urea, calcium carbonate, alumonia gas, etc. can be used. L from fermentation liquid
- Glutamic acid is collected by appropriately combining the ion exchange resin method and other known methods. Example 1 Glucose 2.3g/dl, ammonium acetate 1g/dl
dl, sodium acetate 1g/dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O0.1g/dl, thiamine hydrochloride 20μ
g/dl, urea 0.6g/dl, FeSO ₄・7H ₂ O1mg/dl,
MnSO ₄・4H ₂ O1mg/dl, soybean decomposition concentrate (T-N
) 36 mg/dl and biotin 2 μg/(PH
Prepare a medium containing 7.0) and add 20 ml of it to each
The mixture was placed in a 500 ml shaking flask and sterilized by heating at 115°C for 10 minutes. Each of the bacteria shown in the table below was inoculated into this medium and cultured at 31.5°C in a reciprocating shaking incubator. During cultivation, 45 g/dl urea or 2N H ₂ SO ₄ was used to maintain the pH of the culture solution at 6.5 to 8.0. After 36 hours, the culture was terminated, and the substrate yield of L-glutamic acid accumulated in the culture solution was determined. The results are shown in Table 2 below.

[Table] Example 2 Glucose 10g/dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O0.1g/dl, thiamine hydrochloride 20μ
g/dl, soybean decomposition concentrate (as T-N) 36mg/
dl, ammonium sulfate 2.0g/dl, FeSO ₄・7H ₂ O1mg/dl
MnSO ₄ 4H ₂ O 1 mg/dl and bitin 3 μg/dl,
(PH7.0) was prepared, and 20 ml of each was placed in a 500 ml shaking flask and sterilized by heating at 115°C for 10 minutes. Each of the bacteria shown in the table below was inoculated into this medium and cultured at 31.5°C using a reciprocating shaking culture solution. During cultivation, 5 g/dl of calcium carbonate was added to maintain the pH of the culture solution at 6.5 to 8.0. Also
At 24 hours, 2.5 g/dl of ammonium sulfate was added, the fermentation was completed in 46 hours, and the yield of L-glutamic acid accumulated in the fermentation liquid relative to sugar was determined. The results are shown in Table 3 below.

【table】

Claims (1)

[Claims] 1 Belongs to the genus Brevibacterium and produces isocitrate lyase (4.1.3.1 Ls-isocitrate
1. A method for producing L-glutamic acid by a fermentation method, which comprises culturing a mutant strain with reduced levels of glyoxalatelyase and collecting L-glutamic acid produced and accumulated in a medium.