청국장으로부터 고 비활성 세포외 Protease 생산 세균의 분리 및 동정
Isolation from Chungkookjang and Characterization of a Bacterium Producing an Extracellular Protease of High Specific Activity
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우리나라 전통발효식품의 하나인 청국장으로부터 고 비활성 세포외 protease 생산능이 우수한 균주를 분리하고 그 특성을 조사하였다. 분리된 균주 중 D14 균주 배양 상징액의 protease 활성이 15.2 U/㎖로서 가장 강하였으며 비활성 또한 40.0 U/㎎ protein으로서 가장 강하게 나타났다. 이 균주의 특성을 조사한 후 Bergey's Manual of Systematic Bacteriology에 준하여 Bacillus subtilis로 동정하였다. 균주 D14의 16S rDNA 염기서열을 분석하여 B. subtilis subsp. subtilis NCIB 3610 균주와 99.9%의 상동성을 가짐을 확인하였다. 또한 16S rDNA의 염기서열을 토대로 계통분석을 통하여 다른 Bacillus sp.의 균주들보다 NCIB 3610 균주와 유전적으로 매우 가까운 관계에 있음을 확인하였다. Soluble starch는 사용한 탄소원 중 가장 높은 수준의 protease 생산능을 보였으나 단당류, 이당류 등은 모두 효소 생산능에 대하여 저해효과를 나타내었다. 특히 fructose를 첨가한 경우에는 12.7%, glucose를 첨가한 경우에는 35.9% 수준의 효소 생산능을 나타내었다. 질소원의 경우는 soytone의 첨가구에서 대조구에 비하여 약 61.4% 수준의 효소활성을 나타내어 저해효과가 가장 높았으며 다른 질소원은 효소 생산에 크게 영향을 미치지 않았다.
Abstract
Several extracellular protease-producing bacteria were isolated from Chungkookjang, a traditional Korean food of fermented soybeans, on skim milk agar plates. Among these bacteria, strain D14 exhibited the highest production (15.2 U/㎖) and specific activity (40.0 U/㎎ protein) of extracellular protease activity as assessed on growth in a protease induction medium composed of 1% (w/v) soluble starch, 1.5% (w/v) skim milk, 0.5% (w/v) yeast extract, and 2% (w/v) NaCl. The bacterium was identified as Bacillus subtilis based on morphological and physiological characteristics and 16S rDNA sequence. A BLAST search of 16S rDNA sequences revealed that the isolate was most closely related to Bacillus subtilis subsp. subtilis strain NCIB 3610. The 16S rDNA sequence homology was 99.9%. Our isolate produced the highest level of protease when grown in a protease induction medium containing 1% (w/v) sorbitol and 0.5% (w/v) yeast extract. Fructose and glucose reduced enzyme production to 12.7% and 35.9%, respectively, of the level seen when the strain was grown in medium containing soluble starch. Soytone also reduced enzyme production to 61.4% of the level noted when the strain was grown in medium containing yeast extract.
참고문헌 (45)
- National Rural Living Science Institute, R.D.A, "Food Composition Table 5", pp.324~325 (1996)
- Kim, S.Y., "Studies on the changes of protein, peptide and amino acid during preparation", Korean J. Agric. Chem., 8, pp.11~20 (1967)
- Kim, J.G., "Fatty acid composition and electrophoretic patterns of protein of Korean soy beans", Korean J. Food Sci. Technol., 20, pp.263~271 (1988)
- Landers, R.E., "Vegetable oils: Effect of processing storage and use on nutritional values", J. Am. Oil Chem., 58, pp.255~259 (1981)
- Onishi, R., "Studies on a viscous substance of the natto", Nippon Kasei Gakkaishi, 10, pp.871~875 (1987)
- Lee, Y.L., "A study on the production of viscous substance during Chungkookjang fermentation", J. Korean Soc. Agric. Chem. Soc., 35, pp.202~209 (1992)
- Ashiuchi, M., "Isolation of Bacillus subtilis (chungkookjang), a poly-gamma- glutamate producer with high genetic competence", Appl. Microbiol. Biotechnol., 57, pp.764~769 (2001)
- Lee, B.Y., "Physico-chemical properties of viscous substance extracted from Chungkookjang", Korean J. Food Sci. Technol., 23, pp.500~506 (1991)
- Lee, J.O., "Industrial application and physiological function of Chongkukjang", Food Sci. Ind., 38, pp.69~78 (2005)
- Heo, S., "and identification of fibrinolythic bacteria from Korean traditional Chungkookjang", Agric. Chem. Biotechnol., 41, pp.119~124 (1998)
- Kim, W.K., "Purification and characterization of fibrinolytic enzyme produced from Bacillus sp. strains CK11-4 screened from chungkookjang", Appl. Environ. Microbiol., 62, pp.2482~2488 (1996)
- Kim, N.Y., "Antioxidant and antigenotoxic activities of Korean fermented soybean", Food Chem. Toxicol., 46, pp.1184~1189 (2008)
- Yoon, K.D., "Inhibitory effect of soybean and fermented soybean products on the chemically induced mutagenesis", Korean J. Appl. Microbiol. Biotechnol., 24, pp.525~528 (1996)
- Yong Hwan Choi, "Anti-Inflammatory Activity of the Ethanol Extract of Chungkukjang, Korean Fermented Bean: 5-Lipoxygenase Inhibition", Journal of Medicinal Food, 11(3), pp.539~543 (2008), 한국식품영양과학회
- Dong-Ju Kim, "Beneficial Effect of Chungkukjang on Regulating Blood Glucose and Pancreatic β-Cell Functions in C75BL", KsJ-db, Journal of Medicinal Food, pp.2008, 11(2)[DOI]
- Kwon, D.Y., "Antidiabetic effects of fermented soybean products on type 2 diabetes", Nutr. Res., 30, pp.1~13 (2010)
- Kim, J.I., "Antidiabetic effects of soybean and chungkookjang", Korean Soybean Digest., 20, pp.44~52 (2009)
- 양정례, "자발성 고혈압 흰쥐에서 찐콩과 청국장 분말의 혈압 및 지질대사 개선 효과", 한국식품영양과학회지, 32(6), pp.899~905 (2003), 한국식품영양과학회
- Choi, Y.B., "Isoflavone content in Korean fermented and unfermented soybean foods", Korean J. Food Sci. Technol., 30, pp.745~750 (1998)
- Naim, M., "Antioxidative and antihemolytic activity of soybean isoflavones", J. Agric. Food Chem., 22, pp.806~810 (1976)
- Messina, M., "Increasing use of soyfoods and their potential role in cancer prevention", J. Am. Diet Assoc., 91, pp.836~841 (1991)
- 연규춘, "Bacillus natto와 B. licheniformis의 혼합 Starter로 제조된 청국장의 품질특성", 한국식품영양과학회지, 31(2), pp.204~210 (2002), 한국식품영양과학회
- Lee, O.K., "Industrial application and physiological functions of Chongkukjang", Food Sci. Ind., 38, pp.69~78 (2005)
- Godfrey, T., "Industrial enzymology : The application of enzyme in industry", pp.127~172 (1983), The Nature's Press Co.
- Peckman, E.V., "Aspergillus protease", Biochemistry, 5, pp.321~325 (1951)
- Masaaki, Y., "Purification and properties of acid protease from Monascus sp. No. 3405", Agric. Biol. Chem., 48, pp.1637~1645 (1984)
- Kageyama, K., "Studies on Aspergillus oryzae strain for sake brewing", J. Ferment. Technol., 33, pp.53~57 (1955)
- Nunokawa, Y., "A study of the rice Koji protease", J. Soc. Brew., 53, pp.930~933 (1961)
- 권윤정, "Bacillus subtilis K-54의 단백질 분해효소 처리에 의한 양모와 견의 품질개선효과", 한국의류산업학회지, 8(2), pp.239~244 (2006), 한국의류산업학회
- Bruckner, R., "Multiple active forms of a novel serine protease from B. subtilis", Mol. Gen. Genet., 221, pp.486~490 (1990)
- Molière, N., "Chaperone-protease systems in regulation and protein quality control in Bacillus subtilis", Res. Microbiol., 160, pp.637~644 (2009)
- 박찬수, "대두단백질 분해균주 Bacillus subtilis EB464의 선발 및 분해 특성", 한국미생물·생명공학회지, 30(3), pp.210~215 (2002), 한국미생물·생명공학회
- 안용선, "전통 청국장으로부터 protease 분비능이 우수한 Bacillus sp.균주의 분리 동정 및 발효 특성", 한국식품과학회지, 38(1), pp.82~87 (2006), 한국식품과학회
- Kim, D.Y., "Purification and characterization of fibrinolytic enzyme produced by Bacillus subtilis K7 isolated from Korean traditional soy sauce", J. Korean Soc. Agric. Chem. Biotechnol., 46, pp.176~182 (2003)
- 김관필, "토양으로부터 Protease 생산 세균의 분리 및 특성", 한국식품영양과학회지, 31(5), pp.754~759 (2002), 한국식품영양과학회
- Cha, Y.J., "Characterization of the strong proteolytic bacteria isolated from low salt fermented anchovy and of protease produced by that strain", Bull. Korean Fish. Soc., 21, pp.71~79 (1988)
- 정희경, "해양심층수 및 Protease 고생산성 Bacillus subtilis DH3으로 제조한 된장(Doenjang)의 품질특성", 한국식품저장유통학회지, 16(3), pp.348~354 (2009)
- Sung-Tae Jung, "Isolation and Identification of Bacillus sp. with High Protease andAmylase Activity from Sunchang Traditional Kochujang", Food Science and Biotechnology, 17(3), pp.519~526 (2008), 한국식품과학회
- Murray, R.G.E., "Bergey's Mannual of Systematic Bacteriology, 2", 1104~1139 (1986)
- Sheehan, D., "Theory and practice of histotechnology", pp.330~331 (1980), Battelle Press
- Knutton, S., "Electron microscopical methods in adhesion", Method. Enzymol., 253(145)통권 : 158 (1995)
- Hayashi, H., "Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal Bifidobacterium spp. in health subjects", Microbiol. Immunol., 48, pp.1~6 (2004)
- Sambrook, J., "Molecular cloning, a laboratory manual", pp.8.18~8.24 (2001), Cold Spring Harbor Laboratory Press
- Bradford, M.M., "A rapid and sensitive method for the quantitation of microorganism quantities of protein utilizing the principle of protein-dye binding", Anal. Biochem., 72, pp.248~254 (1964)
- Hull, M.E., "Studies on milk proteins Ⅱ. Colorimetric determination of the partial hydrolysis of the proteins in milk", J. Dairy Sci., 30, pp.881~884 (1974)