KR900007939B1 - Novel microorganism for producing of glutamic acid - Google Patents

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Description

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

The present invention relates to a microorganism producing glutamic acid and a method for producing glutamic acid using the same, and more specifically, as a variant of Brevibacterium flavia ATCC 14067, it is highly susceptible to penicillin antibiotics and magnetizes glutamic acid as a carbon source and a nitrogen source. The present invention relates to a microorganism capable of producing high yield and high concentration of glutamic acid when cultured in a medium containing glucose, starch hydrolyzate and waste molasses as its main component.

Conventional methods for producing glutamic acid using microorganisms include biotin in the medium without glucose or starch hydrolyzate, or limited amounts of biotin in the medium, instead of expensive glucose and starch hydrolysates. In the case of using waste molasses containing excessive amounts of biotin as a raw material, glutamic acid was produced by adding a surfactant (cation, anion, nonionic) or penicillin antibiotic.

However, there is an excess of biotin in waste molasses (sugar cane molasses, sugar beet molasses), which is advantageous in terms of production cost. In addition, the concentration of biotin varies depending on the type of molasses and the place of production. In accordance with the addition time and amount of the penicillin antibiotic or surfactant is changed, thereby there was a problem affecting the productivity.

In addition, in the actual fermentation of glutamic acid, as the fermentation proceeds, the yield of glutamic acid production in the latter part of the fermentation is markedly lowered by the accumulation of glutamic acid in the medium. Is found.

This phenomenon is considered to be a decrease in productivity due to the consumption of parasitic glutamic acid by the reaction of glutamic acid to alpha ketoglutarate when glutamate dihydrogenase, a key enzyme, is added in the latter part of fermentation.

Therefore, the present inventors have completed the present invention by removing the above-mentioned causes and obtaining a microorganism capable of producing glutamic acid at a high yield and high concentration at all times even if using any raw material or production method.

In other words, the microorganism of the present invention partially discharges the glutamic acid produced intracellularly due to partial deficiency of cell wall synthesis ability, so that glutamic acid is accumulated in the cell at a high concentration, or the glutamic acid is excessively produced and accumulated in the cell. Higher sensitivity to penicillin antibiotics and / or fermentation to remove possible dysfunction in vivo may result in higher concentrations of glutamic acid in the cell, resulting in higher concentrations of glutamic acid in the cell. Hydrogenase is a microorganism having a characteristic that can not magnetize glutamic acid as a carbon source and a nitrogen source in order to alleviate the dysfunction that does not produce glutamic acid by acting in reverse to produce alpha ketoglutarate or parallel to each other.

The microorganism of the present invention (KFCC 10652) is a conventional method as a mutagenesis agent such as ultraviolet irradiation, N-methyl-N'-nitro-N-nitroso-guanidine (NTG) with the Brevi bacterium flame ATCC 14067 as a parent Complex 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 1 g / l, (NH ₄ ) ₂ SO ₄ 0.5%, thiamine hydrochloride 200 μg / l, biotin μ20 g / l, agar 20 g / l, pH 7.0) to form a colony, and then penicillin is added by concentration Susceptibility to penicillin by transplanting into penicillin supplemented medium (1% broth, 1% peptone, 0.5% yeast extract, 1.0% glucose, 0.25% NaCl, 20g / l agar, added to 0.01-0.4μ / ml penicillin) After obtaining a high primary mutant strain, it was again treated with the above-described modifying agent to form a composite medium and to form a colony, and then glutamic acid with only glutamic acid as a carbon source and a nitrogen source. (Glutamic acid sodium salt, _{10g / l, KH 2 PO 4} 1g / l, K 2 HPO 4 0.5g / l, FeSO 4 20mg / l, MnSO 4, 20mg / l, MgSO 4 0.05g / l, thiamine hydrochloride 200μg / l , Biotin 20μg / l, pH 7.0, agar 20g / l) to obtain a non-growing mutant strain was deposited on June 6, 1988 to the Korean spawn association (Kee Ki KFCC 10652).

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

Table 1 Comparison of sensitivity to penicillin antibiotics

<Note> +; Growth,-; No growth, incubated at 30 ° C for 48 hours.

Table 2 Growth Comparison in Glutamic Acid Medium

<Note> Glutamic acid in the composition of the medium except agar, 40 ml of the medium used in a culture flask for the culture of 500ml capacity, and after sterilization, strains were incubated for 72 hours at 30 ℃ 180 rpm shaking culture.

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

Example 1

Used strain; Microorganism of the Invention (KFCC 10652), ATCC 14067.

Primary species medium; Waste molasses 0.5%, glucose 9.0%, 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%, corn steep liquor 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, (NH ₄ ) ₂ SO ₄ 0.3%, thiamine hydrochloride 200 μg / l, biotin 100 μg / l, pH 7.0.

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

Primary species culture; The primary seed medium was placed in a 250 ml shake Erlenmeyer flask with 40 ml each, followed by sterilization of the strains used and shaken at 180 rpm for 20-24 hours at 30 ° C.

Secondary species culture; Dispense 1.2 liters of each secondary medium into a 2.6 liter test fermenter, sterilize at 121 ° C for 10 minutes, inoculate 5 ml of the culture solution obtained in the primary culture after cooling, and supply 900-1.0 liters of air per minute. Incubated for 12-25 hours at 30 ℃.

Fermentation method; The fermentation broth was dispensed in 1.8 liter test fermentation tanks of 5 liters, sterilized and cooled at 121 ° C for 10 minutes, inoculated with approximately 150ml of secondary culture medium, and then supplied with 1-2.5 liters of air at 900rpm, 30- Incubation was carried out at 37 ° C., but 0.3-1.2 μ / ml of penicillin was added between the beginning and the end of the logarithmic growth phase [OD ₅₆₂ 0.15-0.4 (OD ₅₆₂ × 100)]. When the residual sugar concentration in the culture reached 0.5-1.5%, sterilized waste molasses or mixed sugars containing a certain ratio of waste molasses and raw sugar were frequently supplied. 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. The pH of the culture was adjusted to 7.8 using ammonia water. After incubation, the concentration of glutamic acid was 96.6 g / l for ATCC 14067 and 140.8 g / l for KFCC 10652.

As described above, the microorganism of the present invention is a raw material used in fermentation of glutamic acid, that is, glucose, starch hydrolyzate (starch such as corn, sweet potato, tapioca, etc.), waste molasses (sugar cane molasses, sugar beet molasses) and the like. It can be used to produce high concentration and high yield of glutamic acid by using any raw materials, as well as deactivation or very weakening of enzymatic activity of glutamate dehydrogenase and reverse reaction mechanism to improve glutamic acid production concentration and production yield at the end of fermentation. There is a characteristic that can be made.

Claims (2)

Brevi bacterium flame KFCC 10652, which is a microorganism which cannot grow under conditions of penicillin antibiotic concentration of 0.03 µ / ml or more and cannot use glutamic acid as a carbon and nitrogen source, and produces glutamic acid. A method for producing glutamic acid using microorganisms, the method comprising culturing Brevi bacterium flavum KFCC 10652 in a medium containing waste molasses, glucose, starch hydrolyzate and raw sugar to obtain glutamic acid from the culture.