KR100397317B1 - Microorganism Brevibacterium lactofermentum CJJA22 producing L-glutamine and process for producing L-glutamine by use thereof - Google Patents

Abstract

The present invention relates to a microorganism Brevibacterium lactofermentum mutant CJJA22 (KCCM-10223) producing L-glutamine and a method for producing L-glutamine using the same, Brevibacterium (L-glutamine) Brevibacterium lactofermentum, a strain of Brevibacterium sp. Strain, which is resistant to alpha-amino-n-butyric acid (α-ABA) and produces L-glutamine ) Mutant strain CJJA22 (KCCM-10223) and this microorganism were activated in fermentation broth at 30 ℃ for 16 hours and then shaken for 72 hours to treat digestive diseases, liver function enhancers, brain function enhancers, immunostimulants, gastric ulcers, alcohol The present invention relates to a method for producing L-glutamine used in medicine, cosmetics, and health foods such as addiction treatments, cosmetic moisturizers, and exercise nutrition and patient nutritional supplements.

Description

Microorganism Brevibacterium lactofermentum CJJA22 producing L-glutamine and process for producing L-glutamine by use of the L-glutamine lactofermentum CJ22

The present invention relates to a microorganism producing L-glutamine (L-glutamine) and a method for producing L-glutamine using the same, alpha-aminobutyric acid (DL-α- as a variant strain of Brevibacterium sp. The present invention relates to Brevibacterium lactopfermentum CJJA22 (KCCM-10223) which is resistant to amino-n-butyric acid (α-ABA) and produces L-glutamine, and a method for producing L-glutamine using the same.

L-Glutamine is an amino acid used in medicines, cosmetics and health foods such as digestive diseases, liver function, brain function, immune enhancer, gastric ulcer, alcoholism, cosmetic moisturizer, exercise nutrition and patient nutrition.

Production of L-glutamine by conventional microorganisms is obtained from a method of obtaining sulfaguanidine resistant strains in Japanese Patent Publication No. 53-17675, and a method obtained from resistant strains such as azaserine of Japanese Patent Publication No. 55-148094, The method using the penicillin sensitive strain of Unexamined-Japanese-Patent No. 04-088994, the method of using the tyrosine-glutamic acid (tyr-glu) resistant strain of Unexamined-Japanese-Patent No. 02-186994, etc. are mentioned.

The present inventors are expected to increase the productivity of glutamine by strains resistant to alpha-aminobutyric acid, an analog of isoleucine, one of the amino acids in the research for obtaining more productive L-glutamine producing bacteria. As a parent strain, Brevibacterium lactofermentum KFCC-10680 (Republic of Korea Patent Publication No. 91-7818), a conventional glutamine strain, was obtained as a result of obtaining a resistant strain against alpha-aminobutyric acid. The present invention was completed by the discovery that L-glutamine is produced in higher yield.

The present invention relates to a microorganism producing L-glutamine (L-glutamine) and a method for producing L-glutamine using the same, alpha-aminobutyric acid (DL-α- as a variant strain of Brevibacterium sp. The present invention relates to Brevibacterium lactopfermentum CJJA22 (KCCM-10223) which is resistant to amino-n-butyric acid (α-ABA) and produces L-glutamine, and a method for producing L-glutamine using the same.

Referring to the method of inducing a variant strain of the present invention is as follows.

Brevibacterium lactofermentum KFCC-10680 was treated with N-methyl -N'-nitro -N-nitrosoguanidine (NTG), a common mutation treatment, followed by 15 g / L of alpha-aminobutyric acid. The minimum medium (Note 1) was plated to obtain an alpha-aminobutyric acid 15 g / L resistant strain.

A more detailed description of how to induce mutations is as follows.

After culturing the activated strain for 16 hours in the activated medium (Note 2) for 14 hours in a sterile medium (Note 3) sterilized for 15 minutes at 121 ℃, 5ml of this was washed with 100mM citrate buffer (citrate buffer) After the addition of the final concentration to 200mg / L, and treated for 20 minutes, washed with 100mM phosphate buffer (phosphate buffer). The killing rate was 85% when NTG-treated strains were plated on the minimum medium (Note 1). In order to obtain the alpha-aminobutyric acid resistance mutant strain, NTG-treated strains were plated in a minimum medium (Note 1) added with an alpha-aminobutyric acid final concentration of 15 g / L, and incubated at 30 ° C. for 6 days to induce alpha-amino Butyric acid resistant mutant strains were obtained. The obtained resistant mutants were incubated for 72 hours in a glutamine-producing medium (Note 4) for agitation, followed by a 72-hour alpha-aminobutyric improvement in glutamine yield over 10% of the parent strain Brevibacterium lactofermentum KFCC-10680. Exogenous resistant strains were selected and named Brevibacterium lactofermentum CJJA22.

Applicant has assigned to the Korea Microorganism Conservation Center in Hongje-dong, Seodaemun-gu, Seoul, Korea on October 20, 2000, so that the microorganism Brevibacterium lactofermentum CJJA22 can be distributed to third parties. It was deposited in an arc.

(Note 1) Minimum badge:

Glucose 1.0%, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) 0.4%, magnesium sulfate (MgSO ₄ · 7H ₂ O) 0.04%, potassium phosphate (KH ₂ PO ₄ ) 0.1%, urea (Urea) 0.1% , Thiamine HCl 0.0001%, Biotin 200μg / L, Agar, pH7.0.

(Note 2) Activation medium:

1% Beef Extract, 1% Polypeptone, 0.5% Sodium Chloride (NaCl), 0.5% Yeast Extract, 2% Agar, pH 7.2.

(3) species:

Glucose 5%, Bacto peptone 1%, Sodium chloride (NaCl) 0.25%, Yeast Extract 1%, Biotin 3μg / L, Urea 0.4%, pH 7.0.

4) Glutamine production medium:

Glucose 4.0%, ammonium chloride (NH ₄ Cl) 3.0%, soy protein acid hydrolyzate 0.3%, calcium carbonate (CaCO ₃ ) 5%, calcium chloride (CaCl ₂ ) 0.1%, magnesium sulfate ₍ MgSO ₄ 7H ₂ O) 0.05%, Potassium phosphate (KH ₂ PO ₄ ) 0.15%, Potassium phosphate (K ₂ HPO ₄ ) 0.15%, Urea 0.3%, Thiamine (HCl) 2mg / L, Biotin 5μg / L, Ferrous Sulfate (FeSO ₄ · 7H ₂ O) 20mg / L, Manganese Sulfate (MnSO ₄ · H ₂ O) 20mg / L, Zinc Sulfate (ZnSO ₄ · 7H ₂ O) 12mg / L, pH 6.8.

The resistance to alpha-aminobutyric acid of Brevibacterium lactofermentum CJJA22, an alpha-aminobutyric acid resistance mutant with increased productivity of glutamine by mutation treatment using NTG, was as follows. Table 1

Comparison of Tolerance to Alpha-Aminobutyric Acid Strain α-ABA concentration (g / L) 0 One 5 10 15 20 KFCC-10680 +++ ++ + - - - CJJA22 +++ +++ +++ +++ ++ -

+: Growth,-: No growth, incubated at 30 ℃ for 6 days

The result of culturing the variant strain described above according to the embodiment is as follows.

Example

Example 1

Use strain: Variant Brevibacterium lactofermentum CJJA22 of the present invention

Fermentation medium:

Glucose 4.0%, ammonium chloride (NH ₄ Cl) 3.0%, soy protein acid hydrolyzate 0.3%, calcium carbonate (CaCO ₃ ) 5%, calcium chloride (CaCl ₂ ) 0.1%, magnesium sulfate ₍ MgSO ₄ 7H ₂ O) 0.05%, Potassium phosphate (KH ₂ PO ₄ ) 0.15%, Potassium phosphate (K ₂ HPO ₄ ) 0.15%, Urea 0.3%, Thiamine (HCl) 2mg / L, Biotin 5μg / L, Ferrous Sulfate (FeSO ₄ · 7H ₂ O) 20mg / L, Manganese Sulfate (MnSO ₄ · H ₂ O) 20mg / L, Zinc Sulfate (ZnSO ₄ · 7H ₂ O) 12mg / L, pH 6.8 (same as Note 4).

Fermentation Methods and Results:

Dispense 20 ml of the fermentation broth into a 250 ml Erlenmeyer flask for shaking, sterilize for 15 minutes at 121 ° C., and incubate for 16 hours in 30 ° C. activated medium (Note 2) to inoculate the activated strain (1 loopful) for 48 hours at 30 ° C. Shake culture.

The result of the fermentation broth is as follows.

KFCC-10680 (Moju) CJJA22 (variant strain) Glutamine Concentration (g / L) 12.4 14.1

Example 2

Use strain: Variant Brevibacterium lactofermentum CJJA22 of the present invention

Fermentation medium:

Glucose 10%, Ammonium chloride (NH ₄ Cl) 4.5%, Soy Protein acid hydrolyzate 0.5, Calcium carbonate (CaCO ₃ ) 5%, Calcium chloride (CaCl ₂ ) 0.1%, Magnesium sulfate ₍ MgSO _{4 2} O) 0.05%, Potassium phosphate (KH ₂ PO ₄ ) 0.15%, Potassium phosphate (K ₂ HPO ₄ ) 0.15%, Urea 0.3%, Thiamine (HCl) 2mg / L, Biotin (Biotin) 5 μg / L, Ferrous sulfate (FeSO ₄ · 7H ₂ O) 20 mg / L, Manganese sulfate (MnSO ₄ · H ₂ O) 20 mg / L, Zinc sulfate (ZnSO ₄ · 7H ₂ O) 12 mg / L, pH 6.8.

Fermentation Methods and Results:

20 ml of the fermentation broth was dispensed into a 250 ml Erlenmeyer flask for shaking, sterilized for 15 minutes at 121 ° C., and incubated for 16 hours in 30 ° C. activated medium (Note 2) to inoculate the activated strain (1 loopful) for 72 hours at 30 ° C. Shake culture.

The result of the fermentation broth is as follows.

KFCC 10680 (Moju) CJJA22 (variant strain) Glutamine Concentration (g / L) 31.1 36.5

The present invention relates to a microorganism Brevibacterium lactofermentum mutant CJJA22 (KCCM-10223) producing L-glutamine and a method for producing L-glutamine using the same, Brevibacterium (L-glutamine) Brevibacterium lactofermentum, a strain of Brevibacterium sp. Strain, which is resistant to alpha-amino-n-butyric acid (α-ABA) and produces L-glutamine ) Mutant strain CJJA22 (KCCM-10223) and a method for producing L-glutamine by activating the microorganism in a fermentation medium at 30 ℃ for 16 hours and then shaken for 72 hours. L-glutamine produced in this way can be usefully used in the treatment of digestive diseases, liver function enhancers, brain function enhancers, immune enhancers, gastric ulcers, alcoholic addictions, cosmetic moisturizers, exercise nutritions, patient nutritions, cosmetics, and health foods. will be.

Claims (2)

Brevibacterium lactofermentum mutant CJJA22 (KCCM-10223), which produces L-glutamine and is resistant to alpha-amino-n-butyric acid. The microorganism Brevibacterium lactofermentum mutant CJJA22 (KCCM-10223) according to claim 1 was activated in a fermentation broth at 30 ° C. for 16 hours and then shaken for 72 hours. Method of producing glutamine.