KR100292299B1 - Microorganism producing glutamic acid and process for preparation glutamic acid using the same - Google Patents

Abstract

The present invention relates to a glutamic acid-producing microorganism and a method for producing glutamic acid using the same. It is resistant to ropyruvate and has an excellent effect of producing glutamic acid in high yield compared to parent strain.

Description

Glutamic acid producing microorganisms and glutamic acid production method using the same {Microorganism producing glutamic acid and process for preparation glutamic acid using the same}

The present invention relates to a glutamic acid producing microorganism and a method for producing glutamic acid using the same. More specifically, the present invention relates to a microorganism having a β-fluoropyruvate resistance and excellent glutamic acid production ability and a glutamic acid manufacturing method using the same as a variant of Corynebacterium glutamicum KFCC 10656.

Pyruvate on glutamic acid production metabolic pathways is known to be an intermediate of metabolism and to serve as an introduction material of the citric acid cycle to supply precursors of glutamic acid production. In fact, pyruvate on the glutamic acid metabolic pathway results in a precursor of amino acids produced from intermediates in the citric acid cycle. Due to its role as a metabolic intermediate, an increase in the amount of pyruvate in the metabolic pathway inhibits the activity of enzymes involved in pyruvate production by intermediate metabolite inhibition, thereby reducing the amount of pyruvate that can be supplied to the citric acid cycle. This reduces the metabolic intermediates required for amino acid production. Therefore, in order to increase the production of amino acids that receive precursors in the citric acid cycle, smooth circulation of the citric acid cycle is required and the supply of pyruvate, the starting material of the citric acid cycle, is important. However, the metabolite inhibition mechanism exists so that a certain level of pyruvate is not generated in the metabolic pathway of the general strain, so it is necessary to release this regulation mechanism so that excess pyruvate is produced in metabolism. Therefore, the present inventors have focused on the above, and modified the traits of conventional microorganisms to obtain a variant strain resistant to β-fluoropyruvate, that is, a strain in which the ability to control pyruvate production was released, and the strain strain was excessive in metabolism. The present invention was completed by confirming that pyruvate can be produced and the production amount of α-ketoglutaric acid, which is a precursor of glutamic acid production on the citric acid cycle, is finally increased to increase the production of glutamic acid.

An object of the present invention is to provide a mutant strain with improved glutamic acid production capacity by mutating known Corynebacterium glutamicum. Another object of the present invention is to provide a method for producing glutamic acid by culturing the mutant strain in a medium containing sugar to obtain glutamic acid in high yield.

The above object of the present invention is a β-fluoro by treating a mutation causing agent such as ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) with the parent of Corynebacterium glutamicum KFCC 10656 After obtaining a mutant strain that can be grown in a pyruvate-added medium, the biochemical characteristics of the mutant strain were examined, and then the glutamic acid production capacity according to the culture conditions of Corynebacterium glutamicum KFCC 10656 and the mutant strain of the present invention was used as the parent strain. Achieved by investigation.

Hereinafter, the configuration and operation of the present invention.

In the present invention, after mutating treatment with Corynebacterium glutamicum KFCC 10656 as a parent strain, the mutants growing in the medium to which β-fluoropyruvate was added were selected, and then the mutants with improved glutamic acid productivity were selected and selected. Examining the biochemical properties of one strain; After culturing the selected strain and parent strain Corynebacterium glutamicum KFCC 10656 under a variety of culture conditions, and comprises comparing and analyzing the amount of glutamic acid produced from each strain.

Hereinafter, the specific method of the present invention will be described with reference to Examples and Comparative Examples, but the scope of the present invention is not limited only to these Examples and Comparative Examples.

Example 1 Isolation of Mutant Strains of the Present Invention and Their Biochemical Properties

Step 1: Selection of Glutamic Acid-producing Mutant Strains

Corynebacterium glutamicum KFCC 10656 as a parent, β-fluoropyru after treatment with UV-induced mutations such as N-methyl-N`-nitro-N-nitrosoguanidine (NTG) Variant strains were obtained which could be grown in the medium to which bait was added by concentration. The nutrient medium used was 1% peptone, 1% gravy, 0.25% sodium chloride, 1% yeast extract, 2% agar, pH was 7.2, the minimum medium was 10g / L glucose, 1g / L potassium phosphate, 0.5 g / L dipotassium phosphate, 0.5 g / L ammonium sulfate, 1 g / L urea, 20 mg / L iron sulfate, 0.5 g / L magnesium sulfate, 20 mg / L manganese sulfate, 200 µg / L thiamine hydrochloride, 200 µg biotin / Medium containing 5 g / L of molasses and pH of 7.0 and β-fluoropyruvate addition medium used β-fluoropyruvate 5-500 mg / L medium to the minimum medium. The concentration of ropyruvate was up to 500 mg / L and the resistant strains were found to be viable at 100 mg / L β-fluoropyruvate. As a result of the flask experiment and the fermenter culture experiment on the mutant strain obtained, glutamic acid productivity was improved by about 10% compared to the existing strain. The present inventors named the mutant strain Corynebacterium glutamicum BL2 and deposited it on December 30, 1998 with the Accession No. KFCC-11074.

Second Step: Biochemical Properties of Inventive Mutant Strains

Biochemical characteristics of the present invention strain Corynebacterium glutamicum BL2 separated in the first step was as described in Table 1, Table 2 and Table 3. In other words, the strain was highly resistant to sodium chloride and resistant to sodium azide and resistant to β-fluoropyruvate and high concentrations of osmotic pressure.

Comparison of resistance to β-fluoropyruvate Strain ＼β-fluoropyruvate 0 5 10 50 100 200 500 KFCC 10656 +++ +++ ++ + - - - BL2 +++ +++ +++ ++ ++ + - [Note] +: Growth,-: Non-growth (72 hours incubation at 30 ℃)

Comparison of Tolerance by Sodium Chloride Concentration Strain Sodium Chloride (mol) 0 1.0 1.1 1.2 1.3 1.4 1.5 KFCC 10656 +++ ++ + - - - - BL2 +++ +++ ++ ++ + - - [Note] +: Growth,-: Non-growth (96 hours incubation at 30 ℃)

Comparison of resistance by sodium azide concentration Strain Samsodium Azide (µg / mL) 0 One 2 4 8 10 12 14 KFCC 10656 +++ +++ +++ +++ ± - - - BL2 +++ +++ +++ +++ +++ + + - [Note] +: Growth,-: Non-growth (72 hours incubation at 30 ℃)

Comparative Example 1: Comparison of Glutamic Acid Production Capacity of Mutant and Parent strains of the Invention According to Primary Species Culture

In this comparative example, 1% peptone, 0.5% yeast extract, 0.5% broth, 1% glucose, 0.25% sodium chloride, 0.13% urea, pH 7.2 and 3% glucose, 0.2% undiluted solution, biotin 1µg / 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 potassium phosphate 0.2%, urea 0.95%, pH 7.2 Glutamic acid production ability was compared while culturing the present invention mutant strain Corynebacterium glutamicum BL2 and parent strain Corynebacterium glutamicum KFCC 10656. The fermentation method was dispensed 3mL of the seed medium in a test tube with a diameter of 18mm, inoculated using strain after autoclaving according to a conventional method, and used as a seed culture solution by incubating shaking culture at 30 ° C. for 18 hours. 40 mL of the fermentation broth was dispensed into a 500 mL shaking flask for shaking, and sterilized by autoclaving at 121 ° C. for 10 minutes to inoculate 1 mL of the seed culture solution and incubated for 48 hours. Rotation speed was adjusted to 180 times per minute, temperature 30 ℃, pH 7.2. As a result, the accumulation amount of glutamic acid in the culture medium after completion of culture was 17.2 g / L for KFCC 10656 and 18.9 g / L for BL2. This mutant strain Corynebacterium glutamicum BL2 of the present invention was found to be about 10% improved glutamic acid productivity in a 3% medium of glucose concentration compared to the existing strain.

Comparative Example 2: Comparison of Glutamic Acid Production Capacity of Mutant and Parent strains of the Invention According to Secondary Species Culture

In this comparative example, the primary seed medium containing 1% peptone, 0.7% yeast extract, 0.7% juice, 1% glucose, 1% sucrose, 0.25% sodium chloride, 0.3% urea, 0.001% phenol red, and pH 7.2 Raw sugar 3%, glucose 1%, fructose 1%, magnesium sulfate 0.04%, potassium phosphate 0.1%, dipotassium phosphate 0.05%, ammonium sulfate 0.8%, iron sulfate 0.002%, manganese sulfate 0.002%, zinc sulfate 0.0002% , Secondary species medium containing 0.0002% copper sulfate, 500 μg / L biotin, 2 mg / L thiamine hydrochloride, 0.2% soy protein hydrolysate, 0.001% phenol red and 0.001% pH 6, sucrose 6%, glucose 2%, fructose 2 %, Magnesium sulfate 0.05%, potassium monophosphate 0.05%, potassium diphosphate 0.15%, ammonium sulfate 0.1%, iron sulfate 0.002%, manganese sulfate 0.002%, zinc sulfate 0.0002%, copper sulfate 0.0002%, biotin 30µg / L The modified strain of the present invention using a fermentation broth containing 1 mg / L thiamine hydrochloride, 0.2% soy protein hydrolyzate, 0.001% phenol red and 3% calcium carbonate and having a pH of 6.75 While cultivating tumefaciens glutamicum BL2 and the parent strain Corynebacterium glutamicum KFCC 10656 compared glutamic acid-producing ability. That is, the primary seed culture is 2.5mL of the primary seed culture medium in an 18 mm test tube, cooled by autoclaving at 121 ° C. for 5 minutes, inoculated with the strain, and incubated for 12 hours at 30 ° C. at 160 revolutions per minute. It was. In the secondary seed culture, 30 mL of the secondary seed medium was dispensed into a 250 mL shaking Erlenmeyer flask, pressurized and cooled at 121 ° C. for 5 minutes, inoculated with 1 mL of the culture medium of the primary seed culture solution, and shaken at 31 ° C. at 150 rpm for 14 hours. Incubated. In the fermentation method, 30 mL of the fermentation medium was dispensed in 250 mL Erlenmeyer flasks, 1 mL of 10% urea solution was added, and 5 mL of the secondary seed culture solution was inoculated, followed by incubation at 150 ° C. at 31 ° C. for 36 hours. 8 mL of 10% urea aqueous solution was added from time to time according to the pH in the middle of the culture, so as to adjust the pH and supply nitrogen source. After 2 hours from the start of fermentation, 0.04% of surfactant and 0.3U / mL of penicillin were added, and then cultured until all of the sugar was consumed. After incubation, the concentration of glutamic acid was 46.8 g / L for KFCC 10656 and 53.2 g / L for BL2.

Comparative Example 3: Comparison of Glutamic Acid Production Capacity of Mutant and parent strains of the Invention According to Secondary Cultivation Methods Under Different Conditions

In this comparative example, a primary seed medium containing 0.7% peptone, 0.7% yeast extract, 0.7% juice, 1% glucose, 0.25% sodium chloride, 0.13% urea, 0.1% ammonium sulfate and pH 7.5, 3% molasses and molasses 1%, magnesium sulfate 0.04%, potassium dibasic phosphate 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 Phosphorus secondary seed medium and 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 200µg / L, potassium hydroxide 0.18%, defoamer Glutamic acid production ability was compared while culturing the present invention variant strain Corynebacterium glutamicum BL2 and parent strain Corynebacterium glutamicum KFCC 10656 using a fermentation medium containing 0.005% and pH 7.5. In the primary seed culture, 40 mL of the primary seed medium was dispensed into a 500 mL shaking Erlenmeyer flask, pressurized and sterilized at 121 ° C. for 10 minutes, inoculated with the strain after cooling, and incubated at 180 ° C. per minute and shaken at 30 ° C. for 20 hours. In the secondary seed culture, the secondary seed medium was divided into 2.5 liters into a 5-liter experimental fermenter, pressurized and sterilized at 121 ° C. for 10 minutes, cooled, and inoculated with 30 mL of the primary seed culture incubation liquid, followed by air per minute. Shaking culture was performed for 18 hours at 900 rpm, 30 ℃ while feeding 0.5 liter. In the fermentation method, the fermentation broth is dispensed in 9 liters into a 30 liter test fermentation tank, pressurized and sterilized at 121 ° C. for 10 minutes, cooled, and then inoculated with a secondary seed culture solution of about 1.5 liters, followed by 2.5 liters of air per minute. It was incubated at 450 rpm, 31 ° C with feeding. Incubated under the above conditions, penicillin or surfactant was added between the beginning and end of the logarithmic growth phase, and the culture was continued to adjust the pH of the fermentation broth to pH 7.7 with 28% ammonia water. When the concentration of the residual sugar in the culture was 1.5%, a sterile waste molasses or a mixture of waste molasses and raw sugar was supplied at a constant rate. Further culture was continued until the sum of the sugars added to the initial fermentation medium with molasses or mixed sugar was 22% of the amount of fermentation broth. As a result, the concentration of glutamic acid after incubation was 139.5 g / L for KFCC10656 and 151.7 g / L for BL2.

As described above, the mutant strain Corynebacterium glutamicum BL2 of the present invention obtained by mutating the parent strain Corynebacterium glutamicum KFCC 10656 as described through the above Examples and Comparative Examples is β-fluoropyruvate resistant. It is a very useful invention in the fermentation and food industry because it has an excellent effect of producing glutamic acid in high yield compared to the parent.

Claims (2)

Corynebacterium glutamicum BL2 (KFCC 11074) resistant to β-fluoropyruvate and capable of producing high yields of glutamic acid. After autoclaving the medium containing waste sugar-wheat, glucose, starch hydrolyzate and raw sugar, the culture is inoculated by inoculation by inoculation with Corynebacterium glutamicum BL2 according to claim 1 to obtain glutamic acid from the culture. Method for preparing glutamic acid using Corynebacterium glutamicum BL2.