KR100317902B1 - A microorganism producing glutamic acid and a method for producing glutamic acid using said microorganism - Google Patents

Abstract

The present invention is directed to mutations such as UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG) as a parent strain of Corynebacterium glutamicum KFCC-10656, which produces glutamic acid. The present invention relates to a mutant strain having a change in the metabolic pathway of the strain by modifying the trait of the parent strain by treating it according to a conventional method to have mercaptopurine resistance, thereby improving glutamic acid production ability as compared to the parent strain. The present invention also relates to a method for producing glutamic acid using microorganisms, wherein the mutant strain is cultured in a medium containing waste molasses, glucose, starch hydrolyzate, raw sugar, and the like to extract glutamic acid from the culture.

Description

A microorganism producing glutamic acid and a method for producing glutamic acid using said microorganism}

The present invention relates to a microorganism that produces glutamic acid and a method for preparing glutamic acid using the same, and more specifically, has a mercaptopurine resistance as a mutant strain of Corynebacterium glutamicum KFCC-10656. Accordingly, the present invention relates to a microorganism having improved production of glutamic acid, and a method for preparing glutamic acid using the same, by changes in metabolic pathways.

Microorganisms use high-energy phosphate compounds such as ATP and GTP as energy storage materials in energy metabolism. These substances are synthesized during energy metabolism, consumed as needed to generate energy, and play an important role in the metabolism of microorganisms. Therefore, by enhancing the production of energy storage materials such as ATP, GTP, it is expected that the energy metabolism of the microorganism is strengthened and the activity of the cells is also enhanced. Increasing energy metabolic activity was thought to improve the overall flow of the metabolism process and thus affect the production of metabolites. Adenine and guanine, which are basic substances of high-energy phosphoric acid compounds such as ATP and GTP, are purine compounds. Therefore, mercaptopurine, which is a purine analogue, can enhance the production of purine-based energy storage materials ATP and GTP. It was thought. Strains resistant to the purine analog, mercaptopurine, were expected to exhibit enhanced ATP and GTP production fluxes, and this enhanced energy metabolism could facilitate the overall metabolic flow and increase the production of glutamate, the desired product. Expected.

The present inventors have completed the present invention by modifying the traits of conventional microorganisms to make mercaptopurine resistant to obtain a mutant strain with improved glutamic acid productivity compared to the existing strain.

The microorganism SM5 of the present invention is a parent strain of Corynebacterium glutamicum KFCC-10656, which is used as a mutation causing agent such as UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG), etc. After treatment according to a conventional method, medium in which mercaptopurine was added at different concentrations [glucose 10 g / l, 1 g of potassium phosphate, 0.5 g / l of potassium diphosphate, 0.5 g / l of ammonium sulfate, Consisting of urea 1 g / l, iron sulfate 20 mg / l, magnesium sulfate 0.5 g / l, manganese sulfate 20 mg / l, thiamine hydrochloride 200 μg / l, biotin 200 μg / l, molasses 5 g / l, pH 7.0 Medium is a strain selected from among the mutant strains that can be grown in a medium with mercaptopurine 50-1000 mg / l added to the minimum medium. At this time, the concentration of mercaptopurine in the medium used was up to 1000 mg / l, and it was confirmed that the resistant strain that was successfully separated can be grown at the concentration of 400 mg / l mercaptopurine.

Flask test and fermenter culture test of the mutant strains obtained here proved about 9% improvement in glutamic acid productivity compared to the existing strain.The strain was named SM5 and was designated as KFCC-11112 on November 2, 1999. It was deposited in the spawn association.

The nutrient medium used for the strain of the present invention is composed of peptone 1, gravy 1, sodium chloride 0.25, yeast extract 1, agar 2, pH 7.2.

The biochemical properties of the novel mutant strain SM5 isolated from the present invention are as described in Table 1 and Table 2. According to these contents, the microorganism of the present invention can also be grown in a medium containing 400 mg / l mercaptopurine. In addition, this strain was shown to have a high resistance to α-naphthoquinoline, which means that the oxygen demand of the strain is lowered, and it is expected to show excellent fermentation ability compared to the existing strain even under conditions of insufficient oxygen supply. I think.

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

Comparison of resistance to mercaptopurine Strain Mercaptopurine (mg / l) 0 50 100 200 400 700 1000 KFCC-10656 +++ ++ + - - - - SM5 +++ +++ ++ + + - -

(Note) +; Growth,-; Not grown, incubated for 72 hours at 30 ° C.

resistance to α-naphthoquinoline Strain α-naphthoquinoline (μg / ml) 0 10 30 50 100 150 200 KFCC-10656 +++ +++ ++ + - - - SM5 +++ +++ +++ ++ + + -

(Note) +; Growth,-; Not grown, incubated for 72 hours at 30 ° C.

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

Example 1

Use strain: Corynebacterium glutamicum KFCC-10656 and microorganism SM5 of the present invention (KFCC-11112).

Species medium: peptone 1, yeast extract 0.5, gravy 0.5, glucose 1, sodium chloride 0.25, urea 0.13, pH 7.2.

Fermentation medium: glucose 3, liquid 0.2, biotin 1 μg / l, waste molasses 0.05, ammonium sulfate 0.1, iron sulfate 0.002, manganese sulfate 0.002, magnesium sulfate 0.05, thiamine hydrochloride 200 μg / l, potassium phosphate 0.2, urea 0.95 , pH 7.2.

Fermentation method: 3 ml of the seed medium was dispensed into a test tube with a diameter of 18 mm, inoculated using strain after autoclaving according to a conventional method, and shaken for 18 hours at 30 ° C. to use as a seed culture solution. 40 ml of the fermentation broth was dispensed into a 500 ml shake flask for pressure and sterilized under pressure at 121 ° C. for 10 minutes to inoculate 1 ml of the seed culture solution and incubated for 48 hours. The rotation speed was adjusted to 180 times per minute, 30 ° C., pH 7.2. After incubation, the amount of glutamic acid in the medium was 16.5 g / l for KFCC10656 and 17.8 g / l for SM5.

As a result of the present embodiment, the microorganism of the present invention was confirmed that the productivity of glutamic acid is improved by about 8 at a glucose concentration of 3 compared to the existing strain.

Example 2

Strains used: same as Example 1

Primary species: peptone 1, yeast extract 0.7, gravy 0.7, glucose 1, sucrose 1, sodium chloride 0.25, urea 0.3, phenol red 0.001, pH 7.2.

Secondary species: raw sugar 3, glucose 1, fructose 1, magnesium sulfate 0.04, potassium monophosphate 0.1, dipotassium phosphate 0.05, ammonium sulfate 0.8, iron sulfate 0.002, manganese sulfate 0.002, zinc sulfate 0.0002, copper sulfate 0.0002, biotin 500 μg / l, thiamine hydrochloride 2 mg / l, soy protein hydrolyzate 0.2, phenol red 0.001, pH 6.8.

Fermentation medium: Sucrose 6, Glucose 2, Fructose 2, Magnesium sulfate 0.05, Potassium phosphate 0.05, Dipotassium phosphate 0.15, Ammonium sulfate 0.1, Iron sulfate 0.002, Manganese sulfate 0.002, Zinc sulfate 0.0002, Copper sulfate 0.0002, Biotin 30 ㎍ / l, thiamine hydrochloride 1 mg / l, soy protein hydrolyzate 0.2, phenol red 0.001, calcium carbonate 3, pH 6.75.

Primary seed culture: 2.5 ml of the primary seed culture medium was dispensed into an 18 mm test tube, sterilized by autoclaving at 121 ° C. for 5 minutes, inoculated with the used strain, and incubated for 12 hours at 30 ° C. at 160 revolutions per minute. .

Secondary seed culture: 30 ml of secondary seed medium is dispensed into a 250 ml shaking Erlenmeyer flask, pressurized and sterilized at 121 ° C. for 5 minutes, cooled, inoculated 1 ml of the primary seed culture solution, and then 150 rpm at 31 ° C. Shaking culture for 14 hours.

Fermentation method: The fermentation broth was dispensed 30 ml each in a 250 ml Erlenmeyer flask, 1 ml of 10 urea solution was added, 5 ml of the secondary seed culture solution was inoculated, and then cultured for 36 hours at 150 rpm at 31 ° C. 10 ml of aqueous solution of urea was added from time to time depending on the pH in the middle of the culture, and pH adjustment and nitrogen supply were performed. After 2 hours from the start of fermentation, 0.04 of surfactant and 0.3 U / ml of penicillin were added and cultured until all of the sugar was consumed.

The concentration of glutamic acid after incubation was 45.2 g / l for KFCC-10656 and 50.6 g / l for SM5.

Example 3

Primary species: peptone 0.7, yeast extract 0.7, gravy 0.7, glucose 1, sodium chloride 0.25, urea 0.13, ammonium sulfate 0.1, pH 7.5.

Secondary species: raw sugar 3, molasses 1, magnesium sulfate 0.04, potassium diphosphate 0.1, ammonium sulfate 0.3, iron sulfate 0.001, manganese sulfate 0.001, biotin 500 μg / l, thiamine hydrochloride 2 mg / l, urea 0.1, pH 7.1.

Fermentation medium: raw sugar 1.7, molasses 6.8, phosphoric acid 0.13, magnesium sulfate 0.04, iron sulfate 0.001, manganese sulfate 0.001, fine liquid 0.1, thiamine hydrochloride 200 μg / l, potassium hydroxide 0.18, antifoam 0.005, pH 7.5.

Primary seed culture: 40 ml of the primary seed medium was dispensed into a 500 ml shake Erlenmeyer flask, pressurized and sterilized at 121 ° C. for 10 minutes, inoculated with the strain after cooling, and incubated at 180 ° C. per minute for 20 hours at 30 ° C.

Secondary seed culture: Dispense the secondary seed medium into 2.5 liter each in a 5 liter experimental fermenter, pressurize and sterilize at 121 ℃ for 10 minutes, cool, inoculate 30 ml of the primary seed culture culture solution, and air Shaking culture was performed for 18 hours at 900 rpm, 30 ℃ while feeding 0.5 liter per minute.

Fermentation method: The fermentation broth was dispensed by 9 liters into a 30 liter test fermentation tank, pressurized and sterilized at 121 ° C. for 10 minutes, cooled, and inoculated with about 1.5 liters of the secondary seed culture solution, followed by 2.5 liters of air per minute. Incubation was performed at 450 rpm and 31 ° C. while feeding.

Cultivation was carried out under the above conditions, and then cultured with penicillin or a surfactant added between the beginning and the end of the logarithmic phase, and the pH of the fermentation broth during the culture was continuously adjusted to pH 7.7 with 28 ammonia water. When the concentration of the residual sugar in the culture was 1.5, sugars which were mixed with sterilized waste molasses or waste molasses and raw sugar in a constant ratio were frequently supplied. Incubation was continued until the sum of added molasses or mixed sugar and sugar added to the initial fermentation broth was 22 relative to the amount of fermentation broth. After incubation, the concentration of glutamic acid was 108.6 g / l for KFCC-10656 and 118.4 g / l for SM5.

Corynebacterium glutamicum SM5 according to the present invention can be produced in high yield by culturing in a medium containing glutamate, glucose, starch hydrolyzate and raw sugar as a strain having improved glutamic acid production ability.

Claims (4)

Microorganism SM5 (KFCC-11112), which produces glutamic acid as a variant of Corynebacterium glutamicum . The microorganism according to claim 1, which is capable of growing at a mercaptopurine concentration of 400 mg / l or more. The microorganism of claim 1 or 2, wherein said microorganism is viable at a concentration of at least 150 µg / l of α-naphthoquinoline. A method for producing glutamic acid, comprising extracting glutamic acid from a culture by culturing Corynebacterium glutamicum SM5 in a medium containing waste molasses, glucose, starch hydrolyzate and raw sugar.