KR900007941B1 - Novel microorganism for producing of glutamic acid - Google Patents

Abstract

Brevibacterium lactofermentum KFCC 10654 (I) having osmotolerance and producing glutamic acid in the waste syrup or culture medium contg. 98 g/l biotin concn. without penicillin or surfactant. (I) is prepd. by treating the original strain Brevibacterium lactofermentum ATCC 13869 with UV or NTG. Prodn. of glutamic acid comprises culturing (I) in a medium contg. waste syrup, glucose, starch hydrolyte, etc., and obtaining glutamic acid from the culture broth.

Description

Microorganisms Producing Glutamic Acid and Methods for Preparing Glutamic Acid Using the Same

The present invention relates to a microorganism that produces glutamic acid and a method for producing glutamic acid using the same, and more specifically, as a variant of Brevibacterium lactofermentum ATCC 13869, the penicillin system has an osmotic resistance and an excessive amount of biotin in the medium. The present invention relates to a microorganism that produces glutamic acid in high concentration and high yield without adding or adding an antibiotic or a surfactant (cation, anion, nonionic surfactant) between the beginning and the end of the fermentation or the logarithmic growth stage.

In the industrial method for producing glutamic acid using microorganisms, glucose, starch hydrolyzate and waste molasses have been used as fermentation raw materials. However, excessive amounts of biotin (0.3-1.5 μg / Glutamic acid was produced by adding penicillin antibiotics or surfactants at the beginning to the end of fermentation or during the logarithmic growth phase by using low-cost waste molasses present in g).

In the conventional method, the addition of penicillin-based antibiotics and the like is well known, and since the substance inhibits cell wall synthesis, the ability to form crosslinks of the peptidoglycan chain, which is a component of the cell wall, becomes unstable. This is because the glutamic acid produced in the cell is secreted easily through the cell wall.

Generally, microorganisms belonging to the genus Brevibacterium or Corynebacterium that produce glutamic acid require biotin for growth, but when biotin is present in the fermentation broth, biotin promotes cell membrane synthesis. It is necessary to limit the concentration of biotin in the fermentation broth because it is difficult to produce glutamic acid. However, when using molasses, biotin is excessively contained and glutamic acid cannot be produced by the aforementioned method. Production was possible only by the addition of penicillin antibiotics or surfactants between the beginning and end of the growth phase.

Therefore, the present inventors have completed the present invention by acquiring a novel microorganism having an osmotic resistance while not affecting the production of glutamic acid regardless of the concentration of biotin in the fermentation broth to remove the disadvantages of the conventional method described above.

Osmotic resistance, which is one of the characteristics of the microorganism of the present invention, is an environment in which salty salts are highly salted, that is, an environment in which the osmotic difference between the inside and the outside of the cell is high (the concentration of sodium chloride in the medium is high, or In the environment with high osmotic pressure of cells, the Na-K pump of the cell system can be increased by increasing the specific amino acid pool (Amino Acid pool, mainly glutamic acid or proline pool) in the cell to maintain the osmotic equilibrium inside and outside the cell. As a result of studying and examining various ecological phenomena, it was induced to produce intracellular glutamic acid to maintain the osmotic equilibrium caused by glutamate pool by inducing sodium chloride resistant strains.

That is, the microorganism (KFCC 10654) of the present invention is usually used as a mutagenesis agent such as UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), with Brevibacterium lactofermentum ATCC 13869 as a parent. Sodium chloride supplemented medium containing 1.6-2.0 molar concentrations of sodium chloride that most strains cannot grow by treatment with phosphorus (20 g / l per glucose, 1 g / l potassium phosphate, 0.5 g / l potassium phosphate, Ammonium sulfate 0.5g / l, urea 1.0g / l, iron sulfate 20mg / l, magnesium sulfate 0.5g / l, manganese sulfate 20mg / l, thiamine hydrochloride 200μg / l, biotin 20μg / l, molasses 5g / l, sodium chloride 1.6 -2.0 moles, pH 7.0) for 3-5 days, and after centrifuging the culture medium, the cells that can be grown even when the concentration of sodium chloride is 2.0 moles are recovered and washed, and agar medium containing 1.6-2.0 molar concentrations of sodium chloride is used. To form colonies by smearing in (Medium added with 20 g / l of agar in the medium of sodium chloride) Pure colony of the formed colony was confirmed again in the sodium chloride addition medium to obtain a primary mutant strain that can be grown even at a sodium chloride concentration of 1.6-2.0 mol, and then treated with the above-mentioned mutagenesis agent, the composition of the medium is 30 g / l , 1 g / l potassium phosphate, 0.5 g / l potassium phosphate, 0.5 g / l ammonium sulfate, 3.0 g / l urea, 20 μg / l iron sulfate, 0.5 g / l magnesium sulfate, 20 mg / l manganese sulfate, Glutamic acid will always be produced regardless of the concentration of biotin in a medium with 200 μg / l thiamine hydrochloride, 5 g / l molasses, 2 g / l soybean hydrolyzate, 0-200 μg / l biotin, pH 7.0 Mutant strains were obtained and deposited with the Korean spawn association as of January 1, 1988 (Accession No. KFCC 10654).

The characteristics of the microorganism of the present invention are as described in the following table.

[Table 1] Comparison of Resistance to Sodium Chloride

<Note> Medium used; Agar Badge

+; Growth,-; Inability to grow

[Table 2] Comparison of glutamic acid production according to biotin concentration

<Note> the medium was confirmed in the production of glutamic acid by using the soybean meal gas decomposition product added medium seed culture medium is used a medium which removes the joseongjung sodium chloride in sodium chloride added to the medium, were inoculated with cultured strain centrifugal when approximately 4.0 OD ₅₆₂ After separation, the cells were recovered and washed two to three times with physiological saline to completely remove the remaining biotin in the medium, and used as a seed.

The production of glutamic acid was determined by dispensing 40 ml of soybean meal hydrolysate medium into a 250 ml Erlenmeyer flask, sterilized and cooled, and incubated at rpm 220 and 30 ° C for 72 hours.

The present invention is explained in more detail in the following examples.

Example 1

Used strain; Microorganism of the Invention (KFCC 10654), ATCC 13869

Species medium; Waste molasses 0.5%, glucose 2.0%, MgSO ₄ 0.5g / l, urea 0.1%, FeSO ₄ 20mg / l, K ₂ HPO ₄ 0.5g / l, KH ₂ PO ₄ 1g / l, (NH ₄ ) ₂ SO ₄ 0.5%, thiamine hydrochloride 200 μg / l, biotin 20 μg / 1, pH 7.0.

Fermentation medium; Waste molasses (as invert sugar) 4%, KH ₂ PO ₄ 1 g / l, K ₂ HPO ₄ 0.5 g / l, (NH ₄ ) ₂ SO ₄ 0.5 g / l, FeSO ₄ 20 mg / l, MgSO ₄ 0.5 g / l, MnSO ₄ 20 mg / l, urea 30 g / l, thiamine hydrochloride 200 μg / l, soybean extract 2 g / l, pH 7.0.

Species culture; 40 ml of each seed medium was dispensed into a 250 ml culture Erlenmeyer flask, and after sterilization and cooling, the strain was inoculated and cultured for about 16-20 hours (30 ° C., 180 rpm) to be used as a spawn seed.

Fermentation method; The fermentation broth was dispensed by dispensing 40 ml each into a 250 ml Erlenmeyer flask, sterilized (121 ° C., 10 minutes), cooled, and seeded with 1 ml each and cultured for about 72 hours (32 ° C., 220 rpm). The accumulation amount of glutamic acid in the culture completed solution is shown in Table 3.

TABLE 3

The molasses used in this example was a sugar cane molasses from the Philippines, and the biotin content was 1.4 μg per 1 g of molasses, and the biotin concentration in the fermentation medium was 98 μL.

As a result of this example, it was confirmed that the microorganism of the present invention can produce a high concentration of glutamic acid even without adding penicillin antibiotic or surfactant even if the concentration of biotin in the fermentation broth is excessive.

Example 2

Used strain; Same as Example 1

Primary species medium; Waste molasses 0.5%, glucose 2.0%, MgSO ₄ 0.5g / l, urea 0.1%, FeSO ₄ 20mg / l, MnSO ₄ 20mg / l, K ₂ HPO ₄ 0.5g / l, KH ₂ PO ₄ 1g / l, ( NH ₄ ) ₂ SO ₄ 0.5%, thiamine hydrochloride 200 μg / l, biotin 20 μg / 1, pH 7.0.

Secondary species medium; Lung molasses (as invert sugar) 2.0%, glucose 1%, cornsipsticker 3%, MgSO ₄ 0.5 g / l, urea 0.2%, KH ₂ PO ₄ 1 g / l, K ₂ HPO ₄ 0.5 g / l, FeSO ₄ 10 mg / l, MnSO ₄ 10 mg / l, MnSO ₄ 20 mg / l, (NH ₄ ) ₂ SO ₄ 0.3%, thiamine hydrochloride 200 μg / l, biotin 100 μg / l, antifoam slightly, pH 7.0.

Fermentation medium; Waste molasses (as invert sugar) 8%, NH ₄ H ₂ PO ₄ 0.15%, FeSO ₄ 10 mg / l, MnSO ₄ 10 mg / l, corn steep liquor 3%, (NH ₄ ) ₂ SO ₄ 0.3%, thiamine hydrochloride 200 μg / l, antifoam slightly, pH 7.0.

Primary species culture; The primary seed medium was put in a 40ml Erlenmeyer flask and inoculated with the strain after sterilization and cultured at 180 rpm and 30 ° C for 20-24 hours.

Secondary species culture; After dispensing 1.2 liters into a 2.6 liter test fermentation tank, sterilized for 10 minutes at 121 ° C, inoculating 5 ml of the culture solution obtained in the primary culture, and supplying 0.5-1.0 liters of air per minute. Shake culture was carried out at 900rpm, 30 ℃ for 12-15 hours.

Fermentation method; 1.8 liters of the fermentation broth was dispensed in a 5-liter test fermenter, sterilized and cooled at 121 ° C. for 1 minute, inoculated with about 150 ml of the secondary culture medium, and then supplied with 1-2.5 liters of air at 900 rpm for 30 minutes. Shake culture at -37 ℃.

Incubate under the above conditions and add 0.3-1.2μ / ml of penicillin between the early and the late stage of logarithmic growth [OD ₅₆₂ 0.15-0.4 (OD ₅₆₂ × 100)], and keep the culture medium at pH 7.8 with ammonia water. Adjusted.

When the concentration of residual sugar in the culture was 0.5-1.5%, sterile lung molasses or a mixture of waste molasses and raw sugars was supplied at a constant rate.

Culture was continued until the sum of added molasses or mixed sugar and sugar added to the initial fermentation broth was 19% of the fermentation broth. After completion of the culture, the concentration of glutamic acid was 103.7 g / l for ATCC 13869 and 137.8 g / l for KFCC 10654.

Example 3

Strains used, primary and secondary seed media, fermentation broth, primary and secondary seed cultures were carried out in the same manner as in Example 2.

Fermentation method; When OD was about 0.1-0.3 (OD ₅₆₂ × 100), fermentation was carried out in the same manner as in Example 2 except that Tween 40, a nonionic surfactant, was added to 50-100 μg / ml relative to the fermentation broth. The concentration of glutamic acid in the finished solution was 106.8 g / l for ATCC 13869 and 138.3 g / l for KFCC 10654.

Claims (2)

Brevibacterium lactofermentum KFCC 10654, characterized by producing glutamic acid with osmotic resistance and in the presence of 98 μg / l biotin concentration in waste sugar and fermentation broth without the addition of penicillin antibiotics or surfactants. A method for producing glutamic acid using microorganisms, comprising culturing Brevibacterium lactofermentum KFCC 10654 in a medium containing waste molasses, glucose, starch hydrolysate, and raw sugar.