JPS6236675B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-glutamic acid by fermentation. L-glutamic acid is produced by Brevibacterium spp.
Alternatively, it is produced by a fermentation method using microorganisms belonging to the genus Corynebacterium. The present inventors derived a mutant strain of a previously known L-glutamic acid producing bacterium of the genus Brevibacterium that was endowed with acetic acid auxotrophy. In cases where this mutant strain produces L-glutamic acid in a higher yield than the previously known L-glutamic acid producing bacteria,
-I learned that glutamic acid is produced and accumulated. The mutant strains used in the present invention belong to the genus Brevibacterium and have an acetic acid requirement, and include, for example, the following strains. Brevibacterium lactofermentum AJ 11515 (FERMP 5335) (acetic acid auxotrophic strain)
Brevibacterium lactofermentum AJ 11516 (FERMP 5336) (acetic acid auxotrophic, isoecitrate lyase reduced strain) Brevibacterium flavum AJ 11517 (FERMP 5337) (acetic acid auxotrophic strain) AJ11515 and AJ11516 are ATCC13869,
AJ11517 was derived from ATCC14067 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 ATCC 14066 and Brevibacterium deivaricata ATCC 14020. To induce 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 of N-methyl-N'-nitro-N-nitrosoguanidine ℃
There are methods such as processing for 20 minutes. The growth rates of the above-mentioned exemplified bacterial strains with respect to the concentration of acetic acid added are shown in Table 1 below. The growth rate of each strain was tested as follows. Glucose 0.5g/dl, urea 0.15g/dl, ammonium sulfate
0.15g/dl, KH ₂ PO ₄ 0.3g/dl, K ₂ HPO ₄ 0.1g/
dl, MgSO ₄・7H ₂ O0.01g/dl, CaCl ₂・2H ₂ O0.1
mg/dl, thiamine hydrochloride 10 μg/dl, biotin 3 μg/dl, Na ₂ B ₄ O ₇・10H _{2 O} 0.44 mg/dl,
FeCl ₂・6H ₂ O4.85mg/dl, CuSO ₄・5H ₂ O1.95
mg/dl, (NH ₄ ) ₆ MO ₇ O ₂₄・4H ₂ O0.185 mg/dl,
ZnSO ₄・7H ₂ O44mg/dl, MnCl ₂・4H ₂ O0.3mg/dl
Bacteria were cultured for 24 hours in a natural medium (peptone 1 g/dl, yeast extract 1 g/dl, NaCl 0.5 g/dl, pH 7.0) on a slant in a medium containing the amount of acetic acid shown in the table and adjusted to pH 7.0. Suspend and inoculate with sterile water,
After culturing for 24 hours, the growth value was measured by turbidity.

[Table] Also, isocitrate lyase exemplified above (hereinafter
Table 2 shows the ICL activity of the reduced strains (abbreviated as ICL). ICL activity was assayed as follows.

[Table] Glucose 2.5g/dl, ammonium acetate 0.8
g/dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.1
g/dl, FeSO ₄・7H ₂ O0.1g/dl, FeSO ₄・
7H ₂ O1 mg/dl, MnSO ₄・4H ₂ O1 mg/dl, urea 0.4
g/dl, biotin 0.3 μg/dl, thiamine/hydroacetic acid 20 μg/dl, and soybean decomposition concentrate (T-N
A medium containing 48 mg/dl (pH 7.0) was prepared, and 20 ml of each was placed in a 500 ml shake flask and sterilized by heating at 115°C for 10 minutes. This medium was inoculated with the test bacterial strain and incubated at 31.5°C using a reciprocating shaking incubator.
The cells were cultured until early logarithmic growth (10 to 16 hours).
Isolate the bacterial cells from the culture solution, wash them, and crush them with ultrasonic waves.
The enzyme treated with Cephaderax G-10 was used as an enzyme preparation. Enzyme activity was measured according to the method of Shiio et al. (Journal of Biochemistry 49
(Vol. 262, 1961) The medium for culturing these mutant strains is a normal medium that contains substances that satisfy the acetic acid requirement, as well as carbon sources, nitrogen sources, inorganic ions, and, if necessary, organic micronutrients. It is a medium for Nutrient sources that should satisfy the requirement for acetic acid include lower fatty acids such as acetic acid and propionic acid, higher fatty acids such as palmitic acid and stearic acid, ethanol,
There are aliphatic alcohols such as propanol. As a carbon source, in addition to carbohydrates, substances that satisfy the above-mentioned requirement for acetic acid can also be used. In particular, more favorable results can be obtained when a carbohydrate and a substance that satisfies the above-mentioned requirement for acetic acid are used together as carbon sources. When used in combination, the amount of the substance required to satisfy carbohydrate and acetic acid requirements varies depending on the type of carbon source, but the former is usually 3 to 2 by weight;
A ratio of 2 to 1 for the latter is desirable. As carbohydrates,
Monosaccharides or oligosaccharides such as glucose, fructose, and sucrose, and starch containing these, hydrolysates of cellulose and the like, fruit juice, cane molasses, sugar beet molasses, soybean whey, and the like can be used. As nitrogen sources, for example, ammonium salts, ammonia water, 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. Ru. 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 best carried out under aerobic conditions, with a culture temperature of 24-37℃, during cultivation
It is best to control the pH between 6 and 9, and use inorganic or organic acidic or alkaline substances to adjust the pH.
Furthermore, urea, calcium carbonate, ammonia gas, etc. can be used. Collection of L-glutamic acid from the fermentation liquor is carried out by appropriately combining the ion exchange resin method and other known methods. Example 1 Glucose 2.3g/dl, ammonium acetate 1
g/dl, sodium acetate 1g/dl, KH ₂ PO ₄ 0.1
g/dl, MgSO ₄ 7H ₂ O0.1g/dl, thiamine.
Hydrochloride 20μg/dl, urea 0.6g/dl, FeSO ₄ .
Prepare a medium containing 7H ₂ O 1 mg/dl, MnSO ₄ 4H ₂ O 1 mg/dl, soybean decomposition concentrate (as T-N) 36 mg/dl and biotin 2 μg/(pH 7.0),
Pour 20ml of each into a 500ml shaking flask and heat to 115°C.
Sterilized by heating 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 incubator. During cultivation, the pH of the culture solution was maintained at 6.5 to 8.0 using 45 g/dl urea or 2NH ₂ SO ₄ . The culture was completed after 36 hours, and the L accumulated in the culture solution
- The yield of glutamic acid versus substrate was determined. The results are shown in Table 3 below.

[Table] Example 2 Brevibacterium lactofermentum
ATCC13869 and AJ11516, cane molasses (as total sugar) 5.6g/dl, acetic acid 1.4g/dl, KH ₂ PO ₄ 0.2
g/dl, MgSO ₄・7H ₂ O0.1g/dl, ammonium sulfate 0.05g/dl, MnSO ₄・4H ₂ O1mg/dl,
FeSO ₄ 7H ₂ O 1 mg/dl, biotin 3 μg/dl, soybean hydrolysis concentrate (as total nitrogen) 36 mg/dl,
300 ml of a medium containing 200 μg/l of thiamine hydrochloride was poured into a 1.5 volume jar fermenter and sterilized at 120° C. for 15 minutes. This medium was inoculated with the pre-cultured bacteria, and the pH was adjusted to 31.5°C.
Culture was carried out with shaking while maintaining the temperature of 7.8. After the start of culture, polyoxyethylene sorbitan monopartate was added when the absorbance at 562 nm of the 26-fold dilution of the culture solution reached 0.3. Also, when the residual acetic acid in the culture solution is 0.1 g/dl or less, add the previously prepared feed solution (cane molasses, 17 g/dl, ammonium acetate (as acetic acid content) 15 g/dl, acetic acid 15 g/dl, The feed liquid was continuously replenished with sodium acetate (containing 13 g/dl as acetic acid content) so that the residual acetic acid concentration always remained in the range of 0.1 to 0.5 g/dl. After 48 hours of culture, the amount of L- shown in Table 4 was as follows.
Glutamic acid was produced and accumulated in the culture medium.

[Table] Example 3 Glucose 1g/dl, Ethanol 0.5g/dl,
KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.1g/dl,
MnSO ₄・4H ₂ O1 mg/dl, FeSO ₄・7H ₂ O1 mg/dl,
Biotin 3μg/dl, ammonium sulfate 1.0g/
dl, soybean hydrolysis concentrate (as total nitrogen) 96mg/
A medium containing 200 μg/dl of thiamine hydrochloride was prepared, and 30 ml of the medium was dispensed into a 500 ml shake flask and sterilized by heating at 115° C. for 10 minutes. Brevibacterium lactofermentum
ATCC13869 or AJ11516 was inoculated and cultured at 31.5°C using a reciprocating shaking incubator. Once the ethanol in the initial culture medium has been consumed, the feed liquid (glucose 30g/dl, ethanol
15g/dl, ammonium sulfate 15g/dl)
dl amount was added twice, and the culture was terminated in 36 hours. Table 5 shows the yield of L-glutamic acid accumulated in the fermentation broth relative to the carbon source used.

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Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Brevibacterium, having an acetic acid auxotrophy, and an ability to produce L-glutamic acid is cultivated aerobically in a liquid culture, and the L-glutamic acid produced and accumulated in the medium is - A method for producing L-glutamic acid by a fermentation method, which comprises collecting glutamic acid. 2. The production method according to claim 1, wherein the liquid medium contains an aliphatic alcohol or fatty acid as a carbon source, and a carbohydrate.