JPS6236677B2 - - 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 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 conventionally known L-glutamic acid producing bacterium belonging to the genus Corynebacterium and found that it uses a combination of aliphatic alcohol or fatty acid and carbohydrate as a carbon source. 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 Corynebacterium and have an acetic acid requirement, and include, for example, the following strains. Corynebacterium acetoacidophyllum AJ 11601 (FERM-P 5626) (acetic acid auxotrophic strain) Corynebacterium acetoacidophyllum AJ 11602 (FERM-P 5627) (acetic acid auxotrophic strain, isocitrate lyase-deficient strain) ) Corynebacterium glutamicum AJ 11603 (FERM-P 5628) (acetic acid auxotrophic strain) AJ 11601 and AJ 11602 are ATCC 13870,
AJ 11603 was derived from ATCC 13032 as the parent strain, respectively. Other parent strains include L such as Corynebacterium acetoglutamicum ATCC 15806.
- Glutamic acid producing bacteria can also be used as parent strains. As a method for inducing mutations in the above-mentioned mutant strains, conventional methods such as ultraviolet irradiation, radiation irradiation, and mutagen treatment are used. For example, 200 μg/ml of N-methyl-
N'-nitro-N-nitrosoguanidine at 0°C.
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 ₁ 0.3g/dl, K ₂ HPO ₁ 0.1g/
dl, MgSO ₄・7H ₂ O0.01g/dl, CaCl・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・5HO1.95mg/
dl _{, (NH4)6Mo7O24 ・ 4H2O0.185mg /} dl _,
ZnSO ₄・7H ₂ O44mg/dl, MnCl ₂・4H ₂ O0.36mg/
Bacteria cultured in natural medium (peptone 1g/dl, yeast extract 1g/dl, NaCl 0.5g/dl, pH 7.0) slant for 24 hours in a medium containing dl and the amount of acetic acid shown in the table and adjusted to pH 7.0. was suspended in sterile water and inoculated, cultured for 24 hours, and 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】

[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 O1mg/dl, MnSO ₄・4H ₂ O1mg/dl, urea 0.4
g/dl, biotin 0.3 μg/dl, thiamine hydrochloride 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).
Bacterial cells are separated from the culture solution, washed and disrupted by ultrasonic waves,
The sample treated with "Cefadelux G-10" was used as an oxygen standard. Enzyme activity was measured according to the method of Shiio et al. (Journal of Biochemistry Volume 49
(262 pages, 1961) The medium for culturing these mutant strains contains the usual 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. Nutrients 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 carbohydrates and a substance that satisfies the above-mentioned oxygen demand 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, while the
A ratio of 2 to 1 for the latter is desirable. As carbohydrates,
Monosaccharides or oligosaccharides such as glucose, fructose, and diucrose, and starches containing these, hydrolysates of cellulose and the like, fruit juices, 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 culture medium in which biothione 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 1mg/
dl, MnSO ₄ 4H ₂ O 1mg/dl, soybean decomposition concentrate (as T-N) 36mg/dl and biotin 2μ
Prepare a medium containing g/l (pH 7.0), and
Pour 20ml each into a 500ml shake flask and heat at 115°C for 10 minutes.
Sterilized by heating. 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, 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】

[Table] Example 2 Corynebacterium acetoacidophyllum
L-glutamic acid fermentation was performed using ATCC13870 and AJ11602. Cane molasses (as whole sugar)
5.6g/dl, acetic acid 1.4g/dl, KH ₂ PO ₄ 0.2g/dl,
_MgSO4・_7H2O0.1g /dl, ammonium sulfate 0.05
g/dl, MnSO ₄・4H ₂ O1 mg/dl, FeSO ₄・7H ₂ O1
mg/dl, biotin 3 μg/dl, soybean hydrolysis concentrate (as total nitrogen) 36 mg/dl, and thiamine hydrochloride 200 μg/dl at 1.5 μg/dl.
The mixture was poured into a 1-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.
The cells were cultured with shaking while maintaining the temperature of 7.8. After the start of culture, polyoxyethylene sorbitan monopalmitate was added when the absorbance at 562 nm of the 26-culture dilution of the culture solution reached 0.3. In addition, when the residual acetic acid in the culture solution is 0.1 g/dl or less, add a pre-adjusted feed solution (cane molasses, 17 g/dl, ammonium acetate (acetic acid content: 15 g/dl, acetic acid 15 g/dl, acetic acid). The feed liquid was continuously supplemented with sodium (containing 13 g/dl as acetic acid) so that the residual acetic acid concentration always remained in the range of 0.1 to 0.5 g/dl. The amount of L- shown in the table
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 O1mg/dl, FeSO ₄・7H ₂ O1mg/dl,
Biotin 3μg/, 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 shaking flask and sterilized by heating at 115° C. for 10 minutes. Corynebacterium acetoacidophyllum
ATCC13870 or AJ11602 was inoculated and cultured at 31.5°C using a reciprocating shaking incubator. After the ethanol in the initial culture medium is consumed, the feed solution (glucose 30g/dl, ethanol
15g/dl, 14g/dl of ammonium sulfate (15g/dl)
The amount was added twice, and the culture was terminated after 36 hours. Table 5 shows the yield of L-glutamic acid accumulated in the fermentation broth relative to the carbon source used.

【table】

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Corynebacterium that has an acetic acid auxotrophy and an ability to produce L-glutamic acid is cultured aerobically in a liquid medium, 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 a fatty acid and a carbohydrate as a carbon source.