KR900007946B1 - Novel microorganism for producing of glutamic acid - Google Patents

Abstract

Glutamic acid producing Brevibacterium lactofermentum KFCC 10659 (I) having osmo-tolerance and can not use glutamic acid as carbon-source or nitrogen-source 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 cong. 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 producing glutamic acid and a method for producing glutamic acid using the same, and more specifically, as a mutant of Brevibacterium lactofermentum ATCC 13869, it is possible to magnetize glutamic acid as a carbon source and a nitrogen source while having osmotic resistance. The present invention relates to a microorganism capable of producing high yield and high concentration of glutamic acid even in a medium containing waste molasses.

Conventional methods for producing glutamic acid using microorganisms include limiting the amount of biotin in the medium and using penicillin antibiotics or surfactants (cationic, anionic, nonionic surfactants) in the use of medium containing excessive amounts of biotin. Was added to produce glutamic acid.

However, such a conventional method has a problem that when molasses molasses is used as a fermentation material, waste molasses contains a large amount of impurities other than sucrose or reducing sugar, which affects productivity, and glutamic acid is added to the medium after the addition of penicillin. When accumulated, since the osmotic pressure outside the cells increases, glutamic acid producing cells are easily damaged, or there is a problem that the damage and activity of various enzymes involved in survival or production of glutamic acid are reduced.

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

The microorganism of the present invention (KFCC 10659) is a conventional method as a mutagenesis agent such as ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) using the Brevi bacterium lactofermentum ATCC 13869 as a parent After treatment in accordance with the following procedure, sodium chloride addition medium containing sodium chloride at a concentration of 1.6-2.0 molar (20 g / l glucose, 1 g potassium phosphate 1 g / l, 0.5 g / l potassium diphosphate, 0.5 g / l ammonium sulfate) 1.0 g / l, iron sulfate 20 mg / l, magnesium sulfate 0.5 g / l, manganese sulfate 20 mg / l, thiamine hydrochloride 20 µg / l, biotin 20 µg / l, molasses 5 g / l, sodium chloride 1.6-2.0 mol, pH 7.0 ) For 3-5 days. At this time, most strains cannot grow due to high concentration of sodium chloride, and strains with high osmotic resistance can be grown, so that the molar concentration of sodium chloride is gradually increased to obtain mutant strains capable of growing at 2.0 moles, and recovered after centrifugation of the mutant strains obtained therefrom. After washing, smearing the resulting colonies on the agar medium containing a concentration of 1.6-2.0 moles of sodium chloride (a medium containing 20 g / l of agar in the sodium chloride addition medium). Thus, a mutant strain capable of growing in a medium containing 2.0 mol of sodium chloride was obtained. This was again treated with the above-mentioned mutagenic agent, plated and cultured in the medium without sodium chloride in the composition of the agar medium, and then cultured with glutamate medium (10 g / l sodium glutamate, 1 g / l potassium phosphate, 0.5 g / dipotassium phosphate). l, 20 mg / l iron sulfate, 20 mg / l manganese sulfate, 200 µg / l thiamine hydrochloride, 20 µg / l biotin, 5 g / l molasses, pH 7.0) However, in glutamic acid medium, mutant strains that cannot be grown are isolated and obtained. 6. It was deposited with the Korean spawn association (KFCC 10659).

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

TABLE 1; Comparison of Tolerance to Sodium Chloride Concentration

<Note> Medium used; Agar Badge

+; Growth,-; Lack of growth

TABLE 2; Comparison of Growth in Glutamic Acid Medium

<Note> Medium used; Medium excluding agar from glutamate media

Culture method; 40 ml of the used medium was added to a 500 ml culture Erlenmeyer flask, sterilized and cooled, followed by inoculation of the strain, followed by incubation for 72 hours at 30 ° C. at 180 rpm.

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

Example 1

Used strain; Microorganism of the Invention (KFCC 10659), ATCC 13869

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 / l, pH 7.0

Secondary species medium; Lung molasses (as invert sugar) 2.0%, glucose 1%, corn steep liquor 3%, MgS 0 ₄ 0.5 g / l. Urea 0.2%, KH ₂ PO ₄ 1g / l, K ₂ HPO ₄ 0.5g / l, FeSO ₄ 10mg / l, MnSO ₄ 10mg / l, (NH ₄ ) ₂ SO ₄ 0.3%, Thiamine hydrochloride 200μg / l, Biotin 100 μg / l, Antifoam slightly, pH7.0

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 into a 250 ml shake Erlenmeyer flask with 40 ml each, and after sterilization, the strains were inoculated and incubated at 30 ° C. at 180 rpm for 20-24 hours.

Secondary species culture; Dispense the secondary seed medium into 1.2 liter each of the test fermenter with 2.6 liter capacity, inoculate 5 ml of the culture solution obtained in the primary seed culture after sterilization cooling at 121 ° C for 10 minutes, and supply 0.5-1.0 liter of air per minute. While incubated at 900rpm for 12-15 hours at 30 ℃.

Fermentation method; The fermentation broth was dispensed 1.8 liters each into a 5 liter test fermentation tank, sterilized and cooled at 121 ° C. for 10 minutes, inoculated with about 150 ml of secondary culture medium, and then supplied with 1-2.5 liters of air at 900rpm, 30- but it incubated at 37 ℃ logarithmic growth phase penicillin between the initial end from _{[OD 562 0.15-0.4 (OD 562 ×} 100)] was added 0.3-1.2μ / ml. 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 completion of the culture, the concentration of glutamic acid was 98.0 g / l for ATCC 13869 and 143.5 g / l for KFCC 0659.

Example 2

Strains used, primary and secondary seed culture medium composition and culture method were carried out in the same manner as in Example 1.

Fermentation method; Concentration of glutamic acid after completion of culture was carried out in the same manner as in Example 1 except that Tween 40, a nonionic surfactant, was added 50 µg / ml-100 µg / ml compared to the amount of fermentation broth between the beginning and the end of the logarithmic growth stage. ATCC 13869 was 101.2 g / l and KFCC 10659 was 19.7 g / l.

As described above, the microorganism of the present invention can use raw materials commonly used in fermentation of glutamic acid, that is, glucose, starch hydrolyzate (starch of corn, sweet potato, tapioca, etc.), waste molasses, and the like, especially when waste molasses is used. As described above, since it has a strong osmotic resistance, not only can the concentration of glutamic acid be increased during fermentation, but also the enzyme activity of the glutamate dehydrogenase reverse reaction mechanism is inactivated or weakened, thereby preventing the consumption of glutamic acid produced during fermentation. It was confirmed that the production concentration and yield can be improved.

Claims (2)

Brevibacterium lactoferdentum, which is a microorganism that is resistant to osmotic pressure and cannot magnetize glutamic acid to carbon and nitrogen sources, and produces glutamic acid KFCC 10659. A method for producing glutamic acid using microorganisms, comprising culturing Brevibacterium lactoferdentum KFCC 10659 in a medium containing waste molasses, glucose, starch hydrolyzate, and raw sugar.