KR900007948B1 - 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 that produces glutamic acid and a method for producing glutamic acid using the same, and more specifically, as a mutant of Brevibacterium flaboom ATCC 14067, while having an osmotic resistance, the glutamic acid cannot be magnetized as a carbon source and a nitrogen source. The present invention relates to a microorganism capable of producing glutamic acid in high yield and high concentration 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, in the conventional method, when waste molasses is used as a fermentation raw material, waste molasses contains a large amount of impurities other than each other or reducing sugars, which affects productivity, and glutamic acid after penicillin is added to the medium. 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 10661) is based on the Brevi bacterium flavum ATCC 14067 as a parent strain in the conventional method as a mutation inducing agent such as UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) After treatment, sodium chloride added medium containing 1.6-2.0 moles of sodium chloride (20 g / l glucose, 1 g / l potassium phosphate, 0.5 g / l potassium diphosphate, 0.5 g / l ammonium sulfate, urea 1.0 g / l, iron sulfate 20 mg / l, magnesium sulfate 0.5 g / l, manganese sulfate 20 mg / l, thiamine hydrochloride 200 µg / l, biotin 20 µg / l, molasses 5 g / l, sodium chloride 1.6-2.0 mol, pH7.0 ) For 3-5 days

Incubated. 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 a mutant strain capable of growing even at 2.0 mol, and centrifugation of the mutant strain obtained therefrom. After collecting and washing, the agar medium containing 1.6-2.0 molar sodium chloride (agar containing 20 g / l of agar in the sodium chloride addition medium) was smeared, and the resulting colony was purely separated, and then the resultant was removed from the sodium chloride addition medium. As a result, a mutant strain capable of growing even in a medium containing 2.0 mol of sodium chloride was obtained. This was again treated with the above-mentioned mutagenic agent, plated and incubated in a 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, iron sulfate 20mg / l, manganese sulfate 20mg / l, thiamine hydrochloride 200µg / l, biotin 20µg / l, molasses 5g / l, pH7.0) Mutant strains that grew but could not grow in glutamic acid medium were isolated and deposited with the Korean spawn association as of January 1, 1988 (KFCC 10661).

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; Badges except KEPCO in glutamate medium composition

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

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

Example 1

Used strain; Invention Microorganism (KFCC 10661), ATCC 14067

Primary species medium; Waste molasses 0.5%, glucose 2.0%, MgSO ₄ 0.5g / l, urea 0.1%, FeSO ₄ 20

mg / l, MnSO ₄ 20 mg / l, K ₂ HPO ₄ 0.5 g / l, KH ₂ PO ₄ 1 g / l, (NH ₄ ) ₂ SO ₄ 0.5%. Thiamine Hydrochloride 200µg / l, Biotin 20µg / l, pH7.0

Secondary species medium; Waste molasses (as invert sugar) 2.0%, glucose 1%, corn steep liquor 3%, MgSO

₄ 0.5g / 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%, Thiamin Hydrochloride 200µg / l, Biotin 100µg / l, Defoamer, 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, pH7.0

Primary species culture; 1 tea seedling medium was put into a 250ml shaking Erlenmeyer flask of 40ml each and then sterilized and strain cultured for 20-24 hours at 30 ℃ 180 rpm.

Secondary species culture; After dispensing 1.2 liters each into a 2.6 liter test fermenter, sterilizing and cooling at 121 ° C for 10 minutes, inoculating 5 ml of the culture solution obtained in the primary culture and feeding 0.5-1.0 liters of air per minute. Incubated at 900 rpm for 12-15 hours.

Fermentation method; The fermentation broth was dispensed in 1.8 liter aliquots into a 5 liter test fermentation tank, sterilized and cooled at 121 ° C. for 10 minutes, inoculated with approximately 150 ml of the secondary culture, and then fed with 1-2.5 liters of meat each minute at 900 rpm, 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 incubation, the concentration of glutamic acid was 94.9 g / l for ATCC 14067 and 134.2 g / l for KFCC 10661.

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; As a result of the same procedure as in Example 1, except that Tween 40, a nonionic surfactant, was added at a concentration of 50 µg / ml-100 µg / ml relative to the amount of fermentation broth between the beginning and the end of the logarithmic growth phase. ATCC 14067 was 102.4 g / l and KFCC 10661 was 137.7 g / l.

As described above, the microorganism of the present invention can use raw materials normally used in fermentation of glutamic acid, that is, glucose, starch hydrolysate (starch such as corn, sweet potato, tapioca), 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 enzymatic activity of 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 plaboom KFCC 10661, which is a microorganism having resistance to infiltration pressure and unable to magnetize glutamic acid as a carbon and nitrogen source, and produces glutamic acid. A method for producing glutamic acid using microorganisms, comprising culturing Brevibacterium boom KFCC 10661 in a medium containing waste molasses, glucose, starch hydrolyzate and raw sugar.