청국장에서 분리한 Bacillus subtilis GS-2에 의한 Poly(γ-Glutamic Acid) 생산의 최적 배양조건

Abstract

ABSTRACT A bacterium strain GS-2 isolated from the Korean traditional seasoning food, Chungkookjang and was determined to produce large amounts of γ-PGA with high productivity when provided with simple nutrients (L-glutamic acid 2.0%, glucose 1.0%, NH 4 Cl 0.5%, KH 2 PO 4 0.05%, MgSO 4 ․ 7H 2 O 0.01%, pH 7.0). In this study, the culture medium for this strain was optimized for the production of γ-PGA. The Bacillus subtilis GS-2 required supplementation with L-glutamic acid and other nutrients for maximal production of γ-PGA. The optimal culture conditions for γ-PGA production were a 48 hr culture time, a temperature of 33℃ and initial pH of 6.5 by rotary shaking (220 rpm). A maximum γ-PGA production of 31.0 g/l was obtained with L-glutamic acid (30 g/ l ), sucrose (the main carbon source, 30 g/l), NH 4 Cl (the main nitrogen source, 2.5 g/l), KH 2 PO 4 (1.5 g/l) and MgSO 4 ․ 7H 2 O (0.15 g/l) in the culture medium. REFERENCES Ashiuchi M, Kamei T, Baek DH, Shin SY, Sung MH, Soda K, Yagi T, Misono H. 2001. Isolation of Bacillus subtilis (Chungkookjang), a poly-γ-glutamate producer with high genetic competence. Appl Microbiol Biotechnol 57:764-769Back LM, Park LY, Park KS, Lee SH. 2008. Effect of starter cultures on the fermentative characteristics of Cheonggukjang. Kor J Food Sci Technol 40:400-405Bang BH, Jeong EJ, Rhee MS, Kim YM, Yi DH. 2011. Isolation of Bacillus subtilis GS-2 producing γ-PGA from Chungkukjang bean paste and identification of γ-PGA. J Appl Biol Chem 54:1-6Borbely M, Nagasaki Y, Borbely J, Fan K, Bhogle A, Sevoian M. 1994. Biosynthesis and chemical modification of poly (-γ-glutamic acid). Polymer Bull 32:127-132Bovarnick M. 1942. The formation of extracellular D(-)glutamic acid polypeptide by Bacillus subtilis. J Bio Chem 145:415- 424Bradford MM. 1976. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254Cheng C, Asada Y, Asada T. 1989. Production of γ-poly- glutamic acid by Bacillus subtilis A 35 under denitrifynig conditions. Agric Biol Chem 53:2369-2375Choi HJ, Yang R, Kunioka M. 1995. Synthesis and charac-terization of pH-sensitive and biodegradable hydrogels pre-pared by γ-irradiation using microbial poly(γ-glutamic acid) and poly(e- lysine). J Appl Polym Sci 58:807-814Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956. Colorimetric method for determination of sugar and related substances. Anal Che 28:350-356Francis F, Sabu A, Madhavan Nampoothiri K, Ramachandran S, Ghosh S, Szakacs G, Pandey A. 2003. Use of response surface methodology for optimizing process parameters for the production of α-amylase by Aspergillus oryzae. Biochem Eng J 15:107-115Goto A, Kunioka M. 1992. Biosynthesis and hydrolysis of poly (-γ-glutamic acid) from Bacillus subtilis IFO 3335. Biosci Biotechnol Biochem 56:1031-1035Hara T, Ueda S. 1982. Regulation of polyglutamate production in Bacillus subtilis(natto); transformation of high PGA pro-ductivity. Agric Biol Chem 46: 2275-2281Hasebe K, Inagaki M. 1999. Preparation composition for external use containing gamma-polyglutamic acid an vegetable extract in combination. JP. Patent 11240827Ito Y, Tanaka T, Asada Y. 1996. Glutamic acid independent pro-duction of poly(-γ-glutamic acid) by Bacillus subtilis TAM- 4. Biosci Biotechnol Biochem 60:1239-1242Kambourova M, Tangney M, Priest FG. 2001. Regulation of poly glutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis. Appl Environ Microbiol 67:1004-1007Kubota H, Matsunobu T, Uotani K, Takebe H, Satoh A, Tanaka T, Tanguchi M. 1993. Production of poly(γ-glutamic acid) by Bacillus subtilis F-2-01. Biosci Biotechnol Biochem 57: 1212-1213Leonard CG, Housewright RD, Thorne CR. 1958. Effects of some metallic ions on glutamyl polypeptide synthesis by Bacillus subtilis. J Bacteriol 76:499-503Markland P, Amidon GL, Yang VC. 1999. Modified polypeptides