MICROORGANISMS PRODUCING L-GLUTAMINE AND PROCESSES FOR PRODUCING L-GLUTAMINE USING THE SAME

Abstract: Disclosed are novel microorganisms, Brevibacterium lactofermentum CJJA21 (Accession No. KCCM-10222) resistant to sodium azide and Brevibacterium lactofermentum CJJA22 (Accession No. KCCM-10223) resistant to alpha -aminobutyric acid, both of which are capable of producing L-glutamine in a higher yield than the known strains, and processes for producing L-glutamine using the same.

Description: MICROORGANISMS PRODUCING L-GLUTAMINE AND PROCESSES FOR PRODUCING L-GLUTAMINE USING THE SAME TECHNICAL FIELD The present invention relates to novel microorganisms producing L-glutamine and to processes for producing L-glutamine using the same. More specifically, the invention relates to Brevibacterium lactqfermentum CJJA21 (KCCM-10222) resistant to sodium azide and Brevibacterium lactqfermentum CJJA22 (KCCM- 10223) resistant to D,L-α- amino-n-butyric acid: α-ABA), both of which are capable of producing L-glutamine in a higher yield than the known strains, and to processes for producing L-glutamine using the same. BACKGROUND ART L-glutamine is an amino acid widely used as medicines such as therapeutic agents of gastroenterologic disorders, potentiators of liver and brain functions, immuno- enhancement agents, and therapeutic agents of gastric ulcer and alcoholism, etc., cosmetics such as moisturizers, etc., and health foods such as sports nutrients and nutrients for patients, etc. According to the prior arts, L-glutamine was obtained from sulfaguanidine- resistant strains (Japanese Patent, Laid-Open No. Sho53-17675), azaserine-resistant strains (Japanese Patent Laid-Open No. Sho55-148094, penicillin-sensitive strains (Japanese Patent Laid-Open No. Hei04-088994), tyrosine-glutamic acid (tyr-glu)-resistant strains (Japanese Patent Laid-Open No. Hei02-186994) and the like. DISCLOSURE OF THE INVENTION The present inventors performed extensive studies to develop novel strains, which are capable of producing L-glutamine in a high productivity. We contemplated that strains resistant to sodium azide, a respiratory inhibitor, or to α-aminobutyric acid, an analogue of an amino acid, isoleucine, would have the increased productivity of L- glutamine. Thus, we screened sodium azide or α-aminobutyric acid-resistant strains from an original strain, Brevibacterium lactofermentum KFCC-10680 (Korean Patent Publication No. 91-7818). As a result, we identified that the sodium azide- or α- aminobutyric acid-resistant strains produce L-glutamine in a higher yield than the known strains and thus, completed the present invention. The present invention provides microorganisms producing L-glutamine and processes for producing L-glutamine using the same. The microorganisms according to the present invention are Brevibacterium lactqfermentum CJJA21 (KCCM- 10222) having a resistance to sodium azide and Brevibacterium lactofermentum CJJA22 (KCCM- 10223) having a resistance to α-aminobutyric acid, both of which produce L-glutamine in a high yield. Further, the processes for producing L-glutamine according to the present invention is characterized by the activation of Brevibacterium lactofermentum CJJA21 or Brevibacterium lactofermentum CJJA22 followed by the cultivation of the activated strains. In the present invention, mutants were induced by the following methods. Brevibacterium lactofermentum KFCC-10680 was treated with N-methyl-N'-nitro-N- nitrosoguanidine (NTG), a conventional mutagen and then, spread on a minimal medium (Medium 1) containing 500 mg/1 of sodium azide thereby to obtain strains having a resistance to 500 mg/1 of sodium azide More specifically, Brevibacterium lactofermentum KFCC-10680, which had been previously activated by cultivation on an activation medium (Medium 2) for 16 hours, was cultivated for 14 hours on a seed , medium (Medium 3) sterilized at 121 °C for 15 minutes. Then, 5 ml of the culture medium was washed with 100 mM citrate buffer and thereto was added NTG at a final concentration of 200 mg/1. After 20 minutes, the medium was washed with 100 mM phosphate buffer. The strains treated with NTG were spread on a minimal medium (Medium 1) and the death rate was measured. As a result, the death rate was 85%. In order to obtain sodium azide-resistant mutants, the NTG-treated strains were spread on a minimal medium (Medium 1) containing sodium azide at a final concentration of 500 mg/1 and then, cultivated at 30 °C for 6 days to obtain sodium azide-resistant strains. The obtained resistant mutants were cultivated in a shaking Erlenmeyer flask containing a glutamine production medium (Medium 4) for 72 hours thereby to select a sodium azide- resistant strain producing L-glutamine in a 10% or more higher yield than the original strain, Brevibacterium lactofermentum KFCC-10680. The obtained strain was designated as CJJA21. Brevibacterium lactofermentum CJJA21 was deposited under the Budapest Treaty to the Korean Culture Center of Microorganisms whose address is Hongje-dong, Seodaemun-gu, Seoul, on October 20, 2000, with the Accession No. KCCM-10222. In addition, Brevibacterium lactofermentum KFCC-10680 was activated and cultivated in the substantially same manner as above. It was subsequently treated with NTG in the substantially same manner as above. In order to obtain α-aminobutyric acid- resistant mutants, the NTG-treated strains were spread on a minimal medium (Medium 1) containing α-aminobutyric acid at a final concentration of 15 g/1 and then, cultivated at 30 °C for 6 days to obtain α-aminobutyric acid-resistant strains. The obtained resistant mutants were cultivated in a shaking Erlenmeyer flask containing a glutamine production medium (Medium 4) for 72 hours thereby to select a α-aminobutyric acid-resistant strain producing L-glutamine in a 10% or more higher yield than the original strain, Brevibacterium lactofermentum KFCC-10680. The obtained strain was designated as CJJA22. Brevibacterium lactofermentum CJJA22 was deposited under the Budapest Treaty to the Korean Culture Center of Microorganisms whose address is Hongje-dong, Seodaemun-gu, Seoul, on October 20, 2000, with the Accession No. KCCM- 10223. Culture media employed in the present invention have the following compositions: Medium 1: Minimal medium Glucose 1.0%, Ammonium Sulfate ((NH 4 ) 2 SO 4 ) 0.4%, Magnesium Sulfate (MgSO 4 -7H 2 O) 0.04%, Potassium Dihydrogen Phosphate (KH 2 PO 4 ) 0.1%, Urea 0.1%, ThiamineΗCl 0.0001%, Biotin 200 μg/1, Agar, pH 7.0 Medium 2: Activation medium Beef extract 1%, Polypeptone 1%, Sodium Chloride (NaCl) 0.5%, Yeast Extract 0.5%, Agar 2%, pH 7.2 Medium 3 : Seed medium Glucose 5%, Bactopeptone 1%, Sodium Chloride (NaCl) 0.25%, Yeast Extract 1%, Biotin 3 μg/1, Urea 0.4%, pH 7.0 Medium 4: Glutamine production medium Glucose 4.0%, Ammonium Chloride (NH 4 C1) 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 Dihydrogen Phosphate (KH 2 PO 4 ) 0.15%, Potassium Monohydrogen Phosphate (K 2 HPO 4 ) 0.15%, Urea 0.3%, Thiamine-HCl 2 mg/1, Biotin 5 μg/1, Ferric Sulfate (FeSO 4 -7H 2 O) 20 mg/1, Manganese Sulfate (MnSO 4 -H 2 O) 20 mg/1, Zinc Sulfate (ZnSO 4 -7H 2 O) 12 mg/1, pH 6.8 Sodium azide-resistance of Brevibacterium lactofermentum CJJA21 is shown in the following Table 1-1. Table 1-1 +; growth, -: no growth, cultivation at 30 °C for 6 days α-Aminobutyric acid-resistance of Brevibacterium lactofermentum CJJA22 is shown in the following Table 1-2. Table 1-2 +: growth, -: no growth, cultivation at 30 °C for 6 days BEST MODE FOR CARRYING OUT THE INVENTION This invention will be better understood from the following examples. However, one skilled in the art will readily appreciate the specific materials and results described are merely illustrative of, and are not intended to, nor should be intended to, limit the invention as described more fully in the claims which follows thereafter. Example 1: * Strain: Brevibacterium lactofermentum CJJA21 Fermentation Medium: Glucose 4.0%, Ammonium Chloride (NH 4 C1) 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 Dihydrogen Phosphate (KH 2 PO 4 ) 0.15%, Potassium Monohydrogen Phosphate (K 2 HPO 4 ) 0.15%, Urea 0.3%, Thiamine-HCl 2 mg/1, Biotin 5 μg/1, Ferric Sulfate (FeSO 4 -7H 2 O) 20 mg/1, Manganese Sulfate (MnSO 4 -H 2 O) 20 mg/1, Zinc Sulfate (ZnSO 4 -7H 2 O) 12 mg/1, pH 6.8 (the same as Medium 4) Fermentation procedure and result: To a shaking Erlenmeyer flask of 250 ml was injected 20 ml of the fermentation medium. The medium was sterilized at 121 °C for 15 minutes. Thereto was inoculated 1 loopful of strains activated by the cultivation on an activation medium (Medium 2) at 30 °C for 16 hours and then, cultivated while shaking at 30 °C for 48 hours. The composition of the fermented broth was as follows. Table 2 Example 2: Strain: Brevibacterium lactofermentum CJJA21 Fermentation medium: Glucose 10%, Ammonium Chloride (NH 4 C1) 4.5%, Soy Protein Acid Hydrolyzate 0.5%, Calcium Carbonate (CaCO 3 ) 5%, Calcium Chloride (CaCl 2 ) 0.1%, Magnesium Sulfate (MgSO 4 -7H 2 O) 0.05%, Potassium Dihydrogen Phosphate (KH 2 PO 4 ) 0.15%, Potassium Monohydrogen Phosphate (K 2 HPO 4 ) 0.15%, Urea 0.3%, Thiamin (Thiamine-HCl) 2 mg/1, Biotin 5 μg/1, Ferric Sulfate (FeSO 4 -7H 2 O) 20 mg/1, Manganese Sulfate (MnSO 4 -H 2 O) 20 mg/1, Zinc Sulfate (ZnSO 4 -7H 2 O) 12 mg/1, pH 6.8 Fermentation procedure and result: To a shaking Erlenmeyer flask of 250 ml was injected 20 ml of the fermentation medium. The medium was sterilized at 121 °C for 15 minutes. Thereto was inoculated 1 loopful of strains activated by the cultivation on an activation medium (Medium 2) at 30 °C for 16 hours and then, cultivated while shaking at 30 °C for 72 hours. The composition of the fermented broth was as follows. Table 3 As shown in Table 3, Brevibacterium lactofermentum CJJA21 of the present invention produced L-glutamine in a 10% or more higher yield than the original strain, Brevibacterium lactofermentum KFCC-10680. Example 3: Brevibacterium lactofermentum CJJA22 was cultivated according to the substantially same method as in Example 1. The composition of the fermented broth was as follows. Table 4 Example 4: Brevibacterium lactofermentum CJJA22 was cultivated according to the substantially same method as in Example 2. The composition of the fermented broth was as follows. Table 4 INDUSTRIAL APPLICABILITY According to the present invention, L-glutamine can be obtained in a higher yield than the prior arts. The obtained L-glutamine is useful as medicines such as therapeutic agents of gastroenterologic disorders, potentiators of liver and brain functions, immuno- enhancement agents, therapeutic agents of gastric ulcer and alcoholism, etc., cosmetics such as moisturizers, etc., and health foods such as sports nutrients and nutrients for patients, etc. BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNAΗONAL FORM To. CHEΠJEDANG RECEIPT IN THE CASE OF AN ORIGINAL 500 5-GA NAMDAEMUN-RO issued pursuant to Rule 7. 1 by the SEOUL, CHUNG-KU INTERNATIONAL DEPOSITARY AUTHORITY KOREA, REPUBLIC OF identified at the bottom of this page I . IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR : INTERNAΗONAL DEPOSITARY AUTHORITY: Brevibacterium lactofermentum CJJA21 KCCM-10222 H. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was accompanied by: G a scientific description D a proposed taxono ic designation (Mark with a cross where applicable) in. RECEIPT AND ACCEPTANCE This International Depositaiy Authouity accepts the microorganism identified under I above, which was received by it on Oct. 20. 2000. (date of the original deposit) 1 IV. INTERNATIONAL DEPOSITARY AUTHORITY Name : Korean Culture Center of Microorganisms Signature (s) of person (s) having the power to represent the International De αsitrøs,- ^ . Address 361-221, Yurim B/D Authority of Hongje-1-dong, Seodaemun-gu SEOUL 120-091 Date: Oct Republic of Korea 1 Where Rule 6. 4 (d) applies, such date is the date on which the status of international depositary authority was acquired where a deposit made outside the Budapest Treaty after the acquisition of the status of i nternational depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authouity. Form BP/4 Sole page BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM To. CHE-LJEDANG RECEIPT IN THE CASE OF AN ORIGINAL 500 5-GA NAMDAEMUN-RO issued pursuant to Rule 7. 1 by the SEOUL, CHUNG-KU INTERNATIONAL DEPOSITARY AUTHORITY KOREA, REPUBLIC OF identified at the bottom of this page 1 Where Rule 6. 4 (d) applies, such date is the date on which the status of international depositary authority was acquired where a deposit made outside the Budapest Treaty after the acquisition of the status of i nternational depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authouity. Form BP/4 Sole page

Claims: WHAT IS CLAIMED IS: 1. Brevibacterium lactofermentum CJJA21 characterized by having a resistance to sodium azide and producing L-glutamine (Accession No. KCCM- 10222). 2. Brevibacterium lactofermentum CJJA22 characterized by having a resistance to α-aminobutyric acid and producing L-glutamine (Accession No. KCCM-10223). 3. A process for producing L-glutamine characterized by the activation of Brevibacterium lactofermentum CJJA21 according to Claim 1 or Brevibacterium lactofermentum CJJA22 according to Claim 2 followed by the cultivation of the activated strains.

Inventors: JEONG SANG-CHEOL, KIM CHEON-JU, LEE DONG-WOO, LEE KEUN-CHUL, PARK SUNG-SIK, SUH SEUNG-HYUN, JEONG, SANG-CHEOL, KIM, CHEON-JU, LEE, DONG-WOO, LEE, KEUN-CHUL, PARK, SUNG-SIK, SUH, SEUNG-HYUN
Applicants: CHEIL JEDANG CORP, JEONG SANG CHEOL, KIM CHEON JU, LEE DONG WOO, LEE KEUN CHUL, PARK SUNG SIK, SUH SEUNG HYUN, CHEIL JEDANG CORPORATION, JEONG, SANG-CHEOL, KIM, CHEON-JU, LEE, DONG-WOO, LEE, KEUN-CHUL, PARK, SUNG-SIK, SUH, SEUNG-HYUN
Application year: 2001
Main classification: C12N1
IPC codes: C12N1, C12P13, C12R1