JPH055477B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Fields) L-glutamic acid is widely used as a seasoning, and also as a raw material for pharmaceuticals, a raw material for surfactants, etc. The present invention improves the method for producing L-glutamic acid by fermentation. (Prior Art) L-glutamic acid is an amino acid that is produced in large quantities, and various improvements have been made to the manufacturing method. (Problems to be Solved by the Present Invention) The problems to be solved by the present invention are to develop an industrially inexpensive method for producing L-glutamic acid. (Means for Solving the Problems) As a result of intensive research to improve the method for producing L-glutamic acid by fermentation, the present inventors found that The present inventors succeeded in isolating a strain with excellent L-glutamic acid production ability from mutant strains resistant to aspartate analogs or asparagine analogs, and completed the present invention. Aspartic acid analogs as used in the present invention refer to those that inhibit the growth of microorganisms belonging to the genus Brevibacterium or Corynebacterium, but this inhibition is partially or completely canceled by the coexistence of aspartic acid. say. For example, fluoroaspartic acid, β-aspartic acid hydrazide, aspartic acid hydroxamate, α-methylaspartic acid, β-methylaspartic acid,
cysteine sulfinic acid, difluorosuccinic acid,
There are Hadashijin, etc. The growth inhibition of microorganisms belonging to the genus Brevibacterium or Corynebacterium caused by the aspartic acid analog described above can be partially or completely canceled by the coexistence of asparagine. The gist of the present invention is to culture a mutant strain belonging to the genus Brevibacterium or Corynebacterium that is resistant to aspartate analogs or asparagine analogs and has the ability to produce L-glutamic acid. This is a method for producing L-glutamic acid using a fermentation method, which is characterized by producing and accumulating glutamic acid and collecting it. The present invention will be explained in more detail below. Bacterial strains resistant to aspartate analogs used in the present invention include, for example, the following strains. Brevibacterium lactofermentum AJ12247 (FERM-P8364, FERM BP-1167) Fluoroaspartate-resistant Brevibacterium flavum AJ12246 (FERM-P8363) Fluoroaspartate-resistant Corynebacterium glutamicum AJ12248 (FERM-P8365) Fluoroasparagine Acid Tolerance These strains are mutated from Brevibacterium lactofermentum ATCC13869, Brevibacterium flavum ATCC14067, and Corynebacterium glutamicum ATCC13032, respectively, as parent strains. Others such as Brevibacterium divaricatum
ATCC14020, Brevibacterium saccharoliticum
ATCC14066, and Corynebacterium acetophyllum
acetoacidophilum) ATCC13870 etc. as a parent strain, mutant strains can be created in the same way. As a method for inducing mutations in the above-mentioned mutant strains, conventional methods such as ultraviolet irradiation, X-ray irradiation, radiation irradiation, and mutagenic agent treatment are used.
-Nitro-N'-methyl-N-nitrosoguanidine is used at 30°C for 20 minutes. Experiments regarding fluoroaspartic acid resistance of the above-mentioned exemplified bacterial strains were conducted by the method described below. Glucose 5g/L, Urea 1.5g/L, Ammonium sulfate 1.5
g/L, KH ₂ PO ₄ 3g/L, K ₂ HPO ₄ 1g/L,
MgSO ₄・7H ₂ O0.1g/L, CaCl ₂・2H ₂ O1.0mg/
L, thiamine hydrochloride 100μg/L, biotin 30μ
g/L, _{Na2B4O7 ・ 10H2O4.4mg} /L, _{FeCl3 ・}
6H ₂ O48.5mg/L, CuSO ₄・_5H2O19.5mg /L,
(NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O1.85mg/L, ZnSO ₄・7H ₂
The amount of fluoroaspartic acid shown in Table 1 was added to a medium containing 440 mg/L of O and 3.6 mg/L of MnCl ₂ 4H ₂ O, and the bacterial strains shown in Table 1 were inoculated at a temperature of 31.5°C.
Shaking culture was performed for 96 hours. Growth in fluoroaspartate-free medium
Table 1 shows the relative growth rate when taken as 100.

[Table] In order to produce and accumulate L-glutamic acid using the aspartate analog of the present invention or a strain resistant to asparagine analog, a conventional method used for L-glutamic acid fermentation may be used. 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 a limiting amount of biotin or a biotin active substance below the appropriate amount for growth is used, and when using a biotin-excessive raw material such as blackstrap molasses as a carbon source, penicillin G, F, K, O, V, X is used. This is carried out by a conventional method such as adding a surfactant consisting of penicillins such as sucrose monopalmitate, polyoxyethylene sorbitan monopalmitate, higher fatty acids such as palmitic acid, or derivatives thereof as a biotin inhibitor. Regarding the culture conditions, the temperature is 30-40℃.
It is carried out by a conventional method such as carried out under aerobic conditions at a temperature of 6 to 8 degrees Celsius and a pH of 6 to 8. Examples using the mutant strain of the present invention that is resistant to fluoroaspartic acid 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
Prepare a medium containing 10 μg/ml of O, 8 μg/ml of MnSO _{4 4} H ₂ O, 5 μg/ml of soybean protein acid hydrolyzate (“taste liquid”), 100 μg/ml of thiamine hydrochloride, and 2.5 μg/ml of biotin, and add 20 ml of each medium. The mixture was placed in a 500 ml shaking flask and sterilized by heating at 115°C for 10 minutes. This medium was inoculated with the following bacterial strains and was shaken back and forth.
Culture was performed at 31.5°C. During culturing, keep the culture solution at pH6.5.
A urea solution with a concentration of 450 mg/ml was added little by little to maintain the concentration between 0 and 8.0. Fermentation was completed in 30 hours, and the yield of L-glutamic acid accumulated in the fermentation liquid relative to sugar was measured. The results are shown in Table 2.

【table】

[Table] Example 2 Cane molasses as sugar 100mg/ml, KH ₂ PO ₄ 1
A medium having a composition of 1 mg/ml, MgSO ₄ .7H ₂ O 1 mg/ml, and 100 μg/ml of thiamine hydrochloride was prepared (PH 7.0), and 30 ml of the medium 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-fold dilution of the 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. Cultivating a mutant strain belonging to the genus Brevibacterium or Corynebacterium that is resistant to aspartate analogs or asparagine analogs and has the ability to produce L-glutamic acid, and produces and accumulates L-glutamic acid in the culture solution. 1. A method for producing L-glutamic acid, which comprises the steps of: