KR100292298B1 - 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 glutamic acid production method using the same. It is quinoline resistant 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 glutamic acid production ability and a glutamic acid using the same as a strain of Corynebacterium glutamicum KFCC 10656, which has α-naphthoquinoline resistance.

The production of glutamic acid as an aerobic process on glutamic acid metabolism pathways requires sufficient oxygenation. Therefore, proper production of oxygen is required for high concentration production of glutamic acid, and it is known that excessive by-products such as lactic acid other than glutamic acid are produced when glutamic acid is not sufficiently ventilated. The excessive production of by-products such as lactic acid results in a decrease in the production yield of glutamic acid. Therefore, even if the oxygen supply is somewhat insufficient, the development of a strain with low oxygen urine composition in which the production of organic acids or amino acids other than glutamic acid is suppressed in the metabolic pathway is difficult. It can be suggested as one method of developing glutamic acid high concentration production strain. In particular, in large-scale fermentation process where oxygen supply may be an important limiting factor, the use of a strain capable of producing glutamic acid without producing by-products such as lactic acid is more effective under limited oxygen supply. Therefore, the present inventors focus on the above, select a strain that is resistant to α-naphthoquinoline known to have a respiratory inhibitory effect as an analogue of the respiratory metabolism intermediate of the microorganism, and isolates a strain that produces a high concentration of double glutamic acid The present invention was completed by obtaining a mutant strain capable of producing glutamic acid at a high concentration even when the oxygen supply conditions are not sufficient.

An object of the present invention is to provide a mutant strain with improved glutamic acid production capacity by mutating known corynebacterium glutamicum KFCC 10656. 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 treated with a mutant-inducing agent such as ultraviolet rays, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) with the parent of Corynebacterium glutamicum KFCC 10656, α-Nattoquinoline After obtaining a mutant strain that can be grown in the added medium, the biochemical characteristics of the mutant strain were examined, and the glutathione 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.

The present invention comprises the steps of screening the mutant strains grown in the medium added to the corynebacterium glutamicum KFCC 10656 as a parent strain and the α- nattoquinoline concentrations and investigating the biochemical characteristics of the selected strains and ; 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, after treatment with mutations such as UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG), and α-naphthoquinoline Several strains were obtained to grow in the medium added by this concentration. The nutrient medium used was 1% peptone, 1% gravy, 0.25% sodium chloride, 1% yeast extract, 2% agar, pH 7.2, minimum medium 10g / L glucose, 1g / L potassium phosphate 1g / L, phosphoric acid Dibasic potassium 0.5g / L, ammonium sulfate 0.5g / L, urea 1g / L, iron sulfate 20mg / L, magnesium sulfate 0.5g / L, manganese sulfate 20mg / L, thiamine hydrochloride 200 ?? g / L, biotin 200 Μg / L, molasses 5g / L, pH was 7.0, and α-naphthoquinoline addition medium was used as the medium in which the α-naphthoquinoline 5-200mg / L concentration was added to the minimum medium.

The present inventors named the mutant strain Corynebacterium glutamicum BS2 and deposited it as KFCC-11073 on December 30, 1998 to the Korean spawn association.

Second Step: Biochemical Properties of Inventive Mutant Strains

The biochemical properties of the present invention strain Corynebacterium glutamicum BS2 isolated and separated in the first step were cultured in α-naphthoquinoline addition medium, sodium chloride addition medium and penicillin addition medium. At this time, the α-naphthoquinoline addition medium is the same as the first step, and the sodium chloride addition medium used a medium in which sodium chloride was added at a concentration of 1-1.5 mol to the minimum medium of the first step, and the penicillin addition medium was also used in the first step. A medium in which penicillin was added at a concentration of 0.01-0.1 μg / mL was used as the minimum medium. The experimental results were as shown in Table 1, Table 2 and Table 3. That is, it was highly resistant to sodium chloride, a trait that could rule out the effects of high sea osmotic pressure.

Comparison of Tolerance to α-naphthoquinoline Strain ＼ α-naphthoquinoline (㎍ / mL) 0 5 10 50 100 200 KFCC 10656 +++ +++ ++ ± - - BS2 +++ +++ +++ ++ + - [Note] +: Growth,-: Not growing (72 hours incubation at 30 ℃)

Comparison of Tolerance by Sodium Chloride Concentration Strain ＼ α-naphthoquinoline (㎍ / mL) 0 1.0 1.1 1.2 1.3 1.4 1.5 KFCC 10656 +++ ++ + - - - - BS2 +++ +++ ++ ++ + - - [Note] +: Growth,-: Not growing (96 hours incubation at 30 ℃)

Comparison of Tolerance by Penicillin Concentration Strains ＼ Penicillin (µg / mL) 0 0.01 0.02 0.03 0.04 0.05 0.07 0.1 KFCC 10656 +++ +++ +++ +++ ++ + ± - BS2 +++ +++ +++ ++ ± - - - [Note] +: Growth,-: Not growing (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 BS2 and parent strain Corynebacterium glutamicum KFCC 10656. In the fermentation method, 3 mL of the seed medium was dispensed into a 18 mm diameter test tube, inoculated using strain after autoclaving according to a conventional method, and used as a seed culture medium by incubating shaking at 18C for 18 hours. 40 mL of the fermentation broth was dispensed into a 500 mL shake flask, and autoclaved at 120 ° C. for 10 minutes to inoculate 1 mL of the seed culture solution, followed by incubation for 48 hours. Rotation speed was adjusted to 180 times per minute, temperature 30 ℃, pH 7.2. After incubation, the amount of glutamic acid in the medium was 17.1 g / L for KFCC10656 and 19.2 g / L for BS2. It was confirmed that the mutant strain Corynebacterium glutamicum BS2 of the present invention improved glutamic acid productivity by 12% in medium of 3% 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, primary seed medium and raw sugar containing 1% peptone, 0.7% yeast extract, 0.7% juice, 1% glucose, 1% sucrose, 0.25% sodium chloride, 0.3% urea and 0.001% phenol red, and pH 7.2 3%, glucose 1%, fructose 1%, magnesium sulfate 0.04%, monopotassium phosphate 0.1%, dipotassium phosphate 0.05%, ammonium sulfate 0.8%, iron sulfate 0.002%, manganese sulfate 0.002%, zinc sulfate 0.0002%, 2% seed medium and 6% sucrose, 2% glucose, 2% fructose, containing 0.0002% copper sulfate, 500 μg / L biotin, 2 mg / L thiamine hydrochloride, 0.2% soy protein hydrolyzate, 0.001% phenol red, pH 6.8 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 30µg / L, Thiamine Mutant strain corynebacter of the present invention using a fermentation broth containing 1 mg / L hydrochloride, 0.2% soy protein hydrolyzate, 0.001% phenol red, and 3% calcium carbonate and having a pH of 6.75 While cultivating help glue Tommy Com BS2 and the parent strain Corynebacterium Com KFCC 10656 compared glutamic acid-producing ability. In other words, 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 sterilized at 121 ° C. for 5 minutes, inoculated with 1 mL of the incubation solution 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. for 31 hours at 31 ° C. 8 mL of 10% aqueous urea solution was added from time to time in the middle of the culture to adjust pH and supply nitrogen. After 2 hours of fermentation, 0.04% of surfactant and 0.3U / mL of penicillin were added, followed by incubation until all of the sugar was consumed. After completion of the culture, the concentration of glutamic acid was 46.5 g / L for KFCC 10656 and 53.1 g / L for BS2.

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 crude 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 ?? g / l, potassium hydroxide 0.18% , Glutamic acid production ability was compared while culturing the present invention strain strain Corynebacterium glutamicum BS2 and parent strain Corynebacterium glutamicum KFCC 10656 using a fermentation medium containing 0.005% antifoaming agent. 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, inoculated with 30 mL of the primary seed culture incubation liquid, and then air was fed every minute. Shaking culture was performed for 18 hours at 900 rpm, 30 ℃ while feeding 0.5 liter. In the fermentation method, the fermentation broth was dispensed in 9 liters into a 30 liter test fermentation tank, autoclaved at 121 ° C. for 10 minutes, cooled and inoculated with 1.5 liters of the secondary seed culture solution, and then 2.5 liters of air per minute. It was incubated at 450 rpm, 31 ° C with feeding. Incubated under the above conditions, 0.3 U / mL of penicillin was added between the beginning and the end of the logarithmic growth period, and 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%, sterile pulmonary molasses or a mixture of waste molasses and raw sugar in a constant ratio was frequently supplied. Further culture was continued until the sum of the sugars added to the initial fermentation medium with molasses or mixed sugar became 20% of the amount of fermentation broth. After completion of the culture, the concentration of glutamic acid was 139.3 g / L for KFCC10656 and 151.4 g / L for BS2.

As described above, the mutant strain Corynebacterium glutamicum BS2 of the present invention obtained by mutating the parent strain Corynebacterium glutamicum KFCC 10656 as described through the above Examples and Comparative Examples exhibits α-naphthoquinoline resistance. It is a very useful invention in the fermentation and food industry because it has an excellent effect of producing a high yield of glutamic acid compared to the parent.

Claims (2)

Corynebacterium glutamicum BS2 (KFCC 11073) resistant to α-naphthoquinoline and capable of producing high yields of glutamic acid After autoclaving the medium containing waste sugar-wheat, glucose, starch hydrolyzate and raw sugar, inoculation was inoculated with Corynebacterium glutamicum BS2 according to claim 1, followed by shaking culture. Glutamic acid production method using Corynebacterium glutamicum BS2, characterized in that obtained.