JPS6236676B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-glutamic acid using a fermentation method. As a result of intensive research aimed at improving the production method of L-glutamic acid by fermentation, the present inventors discovered that Brevibacterium genus or Corynebacterium spp.
A strain with excellent L-glutamic acid production ability among mutant strains resistant to 2-heptyl-4-hydroxyquinoline-N-oxide (hereinafter referred to as "HOQNO") derived from L-glutamic acid producing bacteria of the genus We succeeded in isolating and cultivating this, leading to the completion of the present invention. That is, the gist of the present invention belongs to the genus Brevibacterium or Corynebacterium, and
A method of producing L-glutamic acid by a fermentation method characterized by culturing a mutant strain that is resistant to This is the manufacturing method. The present invention will be explained in more detail below. Bacterial strains resistant to HOQNO used in the present invention include, for example, the following strains. Brevibacterium lactofermentum AJ11563 (FERM-P 5471) Brevibacterium flavum AJ11564 (FERM-P 5472) Corynebacterium glutamicum AJ11565 (FERM-P 5473) These strains are Brevibacterium lactofermentum, respectively. fermentum ATCC13859, Brevibacterium flavum ATCC14067, and Corynebacterium glutamicum ATCC13032 as parent strains. Others such as Brevibacterium divaricatum
ATCC14020, Brevibacterium saccharoliticum
ATCC14066, and Corynebacterium acetophyllum
acetoacidophilum) ATCC 13870 or the like as a parent strain, mutant strains can be similarly created. Conventional methods such as ultraviolet irradiation, X-ray irradiation, radiation irradiation, and treatment with mutagens are used to induce mutations in the above mutant strains.
-Nitro-N'-methyl-N-nitrosoguanidi at 30°C for 20 minutes. Table 1 shows the experimental results regarding the HOQNO resistance of the above-mentioned exemplary strains.

[Table] To produce and accumulate L-glutamic acid using the strain resistant to HOQNO of the present invention, L
- It may be carried out using a conventional method used for glutamic acid fermentation. That is, as a medium to be used, a conventional medium containing a conventional carbon source, nitrogen source, inorganic ions, and other nutrients is used. As a carbon source, for example, sugar cane, sugar juice from sugar beet or blackstrap molasses,
Carbohydrate raw materials such as starch and hydrolysates, or organic acids such as acetic acid are used. As the nitrogen source, for example, ammonium salts, ammonia water, urea, etc., which are used in ordinary L-glutamic acid fermentation, are used, and other inorganic ions such as phosphate ions and magnesium ions are used as appropriate. Regarding biotin, a medium containing biotin or a biotin active substance in a limiting amount below the appropriate amount for growth is used, and when using biotin-excess raw materials such as blackstrap molasses as a carbon source, penicillins G, F, K, O, and V are used. , X, etc., or a surfactant consisting of a higher fatty acid such as sucrose monopalmitate, polyoxyethylene sorbitan monopalmitate, palmitic acid, or a derivative thereof, as a biotin inhibitor. . As for the culture conditions, the temperature is 30~
It is carried out by a conventional method such as carried out under aerobic conditions at 40°C and pH within the range of 6 to 8. Examples using the mutant strain of the present invention having resistance to HOQNO are shown below. Example 1 Glucose 36 mg/ml, urea 2 mg/ml,
KH ₂ PO ₄ 1mg/ml, MgSO ₄・7aq0.4mg/ml,
FeSO ₄・7H ₂ O 10 μg/ml, MnSO ₄・4H ₂ O 8 μ
g/ml, soy protein acid hydrolyzate (“flavor liquid”) 5μ
Prepare a medium containing 100 μg/ml of thiamine hydrochloride and 2.5 μg/ml of biotin and add 20 ml of it.
Each sample was placed in a 500 ml shaking flask and sterilized by heating at 115°C for 10 minutes. The following bacterial strains were inoculated into this medium and cultured at 31.5°C with reciprocal shaking. During cultivation, keep the culture solution at pH 6.5 to 8.0.
A urea solution with a concentration of mg/ml was added in small portions. 30
Fermentation was completed in time, and the yield of L-glutamic acid accumulated in the fermentation liquid relative to sugar was measured. The second result is
Shown in the table.

[Table] Example 2 Cane molasses, 100mg/ml as sugar, KH ₂ PO ₄ 1
_{Prepare a} medium with the composition of
(pH 7.0), 30 ml of each was dispensed into 500 ml shaking flasks and heat sterilized. The following bacterial strains were inoculated into this medium and cultured at 31.5°C using a reciprocating shaker. 400 to keep the culture solution at pH 6.5-8.0 during cultivation.
A urea solution with a concentration of mg/ml was added in small portions. Polyoxyethylene sorbitan monopalmitate was added when the absorbance at 562 mμ of the 26-diluted medium reached 0.30. Fermentation was completed in 36 hours, and the yield of L-glutamic acid accumulated in the fermentation liquid relative to sugar was measured. The results are shown in Table 3.

【table】

Claims (1)

[Claims] 1. A mutant strain belonging to the genus Brevibacterium or Corynebacterium that is resistant to 2-heptyl-4-hydroxyquinoline-N-oxide and has the ability to produce L-glutamic acid is cultured, and the mutant strain is added to the culture solution. L-glutamic acid is produced by a fermentation method characterized by producing and accumulating L-glutamic acid and collecting it.
Method for producing glutamic acid.