JPH0349690A - Preparation of l-glutamic acid - Google Patents

Abstract

PURPOSE:To contrive to inexpensively prepare the subject compound in an improved yield by culturing a microorganism which belongs to the genus Brevibacterium, etc., resistant against prumycin (derivative) and having an activity to produce L-glutamic acid. CONSTITUTION:Brevibacterium lactofermentum stain ATCC 13869 is subjected to a mutation treatment with approximately 2000 mug/ml of nitrosoguanidine, etc., at approximately 0 deg.C for approximately 20min to prepare a prumycin- resistant Brevibacterium or Corynebacterium microorganism such as Brevibacterium lactofermentum strain AJ 12475. The microorganism is aerobically cultured in a medium containing waste syrup, urea, etc., and, if necessary, biotin action-suppressing substance such as penicillin at 24-37 deg.C at a pH of 6-9. The culture product is purified with an ion exchange resin, etc., to prepare L-glutamic acid.

Description

[Detailed Description of the Invention] [Industrial Application Field] L-glutamic acid is an important amino acid for seasonings and raw materials for amino acid infusions, and the present invention improves the method for producing this L-glutamic acid by fermentation. It is. [Prior Art] Methods using wild strains of the genus Brevibacterium or Corynebacterium, strains resistant to glutamic acid analogs, strains resistant to respiratory inhibitors such as ketomalonic acid and fluoroacetic acid, strains resistant to esculetin, etc. are known.

(Problems to be Solved by the Invention) An object of the present invention is to improve the fermentation yield in order to reduce the manufacturing cost of L-glutamic acid.

[Means for solving problems]

The present invention was made to solve the above-mentioned problems, and it has been made to make microorganisms belonging to the genus Brevibacterium or Corynebacterium, which are conventionally known as L-glutamic acid producing bacteria, resistant to purmycin or derivatives thereof. It has been found that the yield of L-glutamic acid is improved by breeding a mutant strain having the following. Purmycin is known as an antibiotic produced by bacteria of the genus Bacillus.

Examples of strains of the genus Brevibacterium or Corynebacterium that are resistant to purmycin and derivatives thereof used in the present invention include the following strains.

Y AJ 1247B (FERM- These strains are mutated from Brevibacterium lactofermentum^TCC 13869, Brevibacterium flavum ATCC 14067, and Corynebacterium glutamicum ATCC 13032, respectively, using parent strains.

As a method for inducing mutations in the above-mentioned mutant strain, conventional methods such as ultraviolet irradiation, radiation irradiation, and treatment with mutagenic agents are used. For example, 2000 μg/n+j! O with nitrosoguanidine
There are methods such as processing at ℃ for 20 minutes. Table 1 shows the experimental results regarding the drug resistance of the above-mentioned exemplified bacterial strains. The following method is used to produce L-glutamic acid using these strains. As a medium, sugar juice from cane, sugar beet or waste I! Dense, starchy raw material hydrolyzed substances, etc. I! Culture is performed using a liquid medium containing non-carbohydrate raw materials such as raw materials, acetic acid, ethyl alcohol, and paraffin. Nitrogen sources include ammonium salts, aqueous ammonia, urea, and CSL used in normal L-glutamic acid fermentation.
, proteolytic amino acids, etc. are used. In addition, inorganic salts such as phosphates and magnesium salts, micronutrients such as thiamine and biotin, etc. are used as appropriate. It is added to the soil. Cultivation is best carried out under aerobic conditions, with a culture temperature of 24°C.
During cultivation at ~37°C, the pH should be controlled at 6-9.To adjust the pH, inorganic or organic acidic or alkaline substances, as well as urea calcium carbonate, ammonia gas, etc. can be used. Collection of L-glutamic acid from the fermentation liquor is carried out by a combination of the ion exchange resin method and other known methods.

The growth rate of each strain in Table 1 was tested as follows. Glucose 0.5g/a, urea 0.15g/a, ammonium sulfate 0.
15 g/a, KHzPO*0. 3g
/ +I4-κ2 liver 04Q, Ig/dl, MgSO
,・711!0 0.01g/d1, CaC 1 z
・2Hg0 0. ' 1 rrg/a, Siayone Hydrochloride 10 r/dl, Biotin 3 r/dl,
NazB407' 10ftg00.44trz/
a, FeCl t ・6H20 4. 8 5 m
g/ilJ, CLISO4 ・5Hi0 1.9 5
mg/dl, (NI44) tMQ1ozm ・41
1200. 1 8 5+ngldl, ZnSO4 ・
7H20 4 4 nv/dI, MnCl14Ht0
0.3611W/d1 and the indicated tffi] A natural medium (peptone 1g/dI, yeast extract Ig/〃, NaCj) was added to a medium containing purmycin and adjusted to pH 7.0.
! 0.5g/dJ, pl1 7. 0 > Bacteria cultured in a slant for 24 hours were suspended in sterile water and inoculated.
After culturing for hours, growth values were measured in suspension.

4. Example-1 Glucose 10 g/dl, KHZPO4 0
．． 1 g/J, MgSO4.7FlzO o.
1 g/dl, thiamine hydrochloride 20r/d1, soybean decomposition concentrate (as T-N) 36mg/dl, ammonium sulfate 2.0g/d1, FeSOn'7H20 1vw/d
l, MnSO4・4Hz0 1mg/d! A medium having a composition of 3 r/1 and biotin 3 r/1 (pH 7.0) was prepared, and 20 mji of each was placed in a 500 ml shake flask and sterilized by heating at 115°C for 10 minutes. 31. Inoculate each bacteria into this medium and use a reciprocating shaking culture machine.
Culture was performed at 5°C.

During cultivation, the culture solution should be kept at pH 6.5-8. 5g/d to keep it at 0! of calcium carbonate was added. 24 again
2.5 g/dl of ammonium sulfate was added at 46 hours, and the fermentation was completed in 46 hours, and the yield of glutamic acid accumulated in the fermentation liquid relative to sugar was determined.

The results are shown in Table 2.

Table 2 Example-2 Beet molasses was used as reducing sugar at 100 μ/value, KH. P
Prepare a medium with a composition of O4 1■/dI (pH 7.0), and add 30 ml of it to 50 On! ! The mixture was dispensed into shaker flasks and sterilized by heating at 115°C for 10 minutes. Each bacteria was inoculated into this medium, and the culture medium was incubated with a reciprocating shaking culture machine for 31 days.
．． Culture was performed at 5°C. During cultivation, the culture solution should be kept at pH 6.
In order to maintain the temperature between 5 and 8.0, 400 μ/〃 of urea water was added as appropriate. When the specified growth is reached after inoculating the bacteria, add 4■/+++j of polyoxysorbicne monopal main night! The fermentation was completed in 30 hours, and the yield of L-glutanic acid accumulated in the fermentation liquid relative to sugar was determined.

The results are shown in Table 3.

Table 3

Claims (1)

[Claims] It belongs to the genus Brevibacterium or Corynebacterium and is resistant to purmycin and its derivatives.
1. A method for producing L-glutamic acid, which comprises culturing a microorganism capable of producing L-glutamic acid and collecting L-glutamic acid produced and accumulated in the culture solution.