Bacillus subtilis MG410에 의한 Fibrin 분해효소 생산배지의 최적화

Medium Optimization for Fibrinolytic Enzyme Production by Bacillus subtilis MG410 Isolated

  • 이주연 (한경대학교 식품생물공학과 및 식품생물 산업연구소) ;
  • 백남수 ((주)메디오젠) ;
  • 김영만 (한경대학교 식품생물공학과 및 식품생물 산업연구소)
  • Lee Ju-Youn (Dept. of Food Science & Biotechnology, Food Science & Biotechnology Research Center, Hankyong National University) ;
  • Paek Nam-Soo (Mediogen, Co. Ltd) ;
  • Kim Young-Man (Dept. of Food Science & Biotechnology, Food Science & Biotechnology Research Center, Hankyong National University)
  • 발행 : 2005.03.01

초록

청국장으로부터 분리한 균을 이용하여 fibrin을 분해 효소활성이 우수한 균주 Bacillus subtilis MG410을 분리한 균주를 이용하여 fibrinolytic enzyme의 생성을 위한 최적 배지 조건을 확립하고자 하였다. 최적 배지 조성은 탄소원 cellobiose 0.5% 첨가시 control보다 균의 성장은 0.5배, 효소 활성은 9배 정도 높은 효과를 보였고, 단당류인 glucose를 첨가시에는 균의 성장은 다소 높았으나 효소활성은 높지 않은 수치를 나타냈다. 질소원으로 soybean meal, soybean peptone을 각각 첨가시 기본배지에서 보다 균의 성장과 효소활성이 높게 나타났고, bacto peptone은 균의 성장은 낮았지만 효소 활성은 높게 나타났다. 그 중 soybean meal을 첨가시에 가장 높은 균의 성장과 효소활성이 나타났다. 질소원 soybean meal 2%를 첨가시 control보다 균의 성장은 6배, 효소 활성은 0.5배 높은 효과를 보였다. 무기질원으로는 Na₂HPO₄·12H₂O을 첨가시 효소생산이 가장 높게 나타났으며, CaCl₂, K₂HPO₄, KH₂PO₄ 첨가시에는 효소 생산에 높은 효과를 보였으나 CuSO₄·5H₂O, CoCl₂·6H₂O 첨가시에는 효소생산 및 균체 생육이 강력히 억제되었다. pH에서는 pH 5가 control보다는 균의 성장은 5배, 효소 활성은 10배 이상 높은 효과를 보였다. 따라서 최적 배지 조성은 탄소원 cellobiose 0.5%, 질소원 soybean meal 2%, 무기질원 Na₂HPO₄·12H₂O 0.02%, pH 5로 조성되었다. 이 조건하에서 배양 37℃, 48시간, 150 rpm에서 효소 생성이 가장 높은 것으로 나타났다.

Using the bacteria isolated from Chungkookjang, Bacillus sublilis MG410 which is excellent in fibrinolytic enzyme activity was isolated. In increase the high production of fibrinolytic enzyme from Bacillus sublilis MG410, the effect of various carbon sources, nitrogen sources, inorganic sources, the initial pH of medium were investigated. The most effective carbon and nitrogen sources were founded cellobiose 0.5%(w/v) and soybean meal 2%(w/v) respectively. None of inorganic sources examined had any detectable stimulating effect on fibrinolytic enzyme production except Na₂HPO₄·12H₂O. The initial optimum pH for fibrinolytic enzyme production ranged from 5∼6 and agitation speed was effect at 150rpm. In jar fermentor experiments under optimal culture conditions, the activity of fibrinolytic enzyme reached about 5.050 unit after 48hours.

키워드

참고문헌

  1. Chang, HS. Study on the alkaline prease from Xanthomonas sp. YL-37. Kon-Kuk Univ, Doctoral Degree Thesis. J 997
  2. Harlan, JM and Harker, LA. Haemostasis, thrombosis and thromboembolic disorder. Med. Clin. North Am. 65:855-857. 1981 https://doi.org/10.1016/S0025-7125(16)31502-4
  3. Choi, KJ, Separation of Bacillus sp. and changes of NH2-N, and protease activity in Chonggukchang rmejiu adding with mufwort (Artemisia asiatica N) extract. Kon-Kuk Univ. Master's Degree Thesis. 1995
  4. Electricwala, AR, Sawyer, T, Powell, JC and Atkinson, T. Isolation of thrombin inhibitor from the jeech Hirudinaria manillensis. Blood Coagul. Fibrin. 2:83-85. 1991 https://doi.org/10.1097/00001721-199102000-00013
  5. Lapentina, EG, Geep, B, Read, NG and Moncada, S. Adhesion of human platelet to collagen in the preaence of prmstacyclin, inometacin and compoihund BW775C. Thromb. Res. 37:325-330. 1986
  6. Horikishi, K Producrion of alkaline enxyme by aikalophilic micriirganism, Agric. Biol. Chem. 35: 1783-1791. 1971 https://doi.org/10.1271/bbb1961.35.1783
  7. Gomex, CC, Simoncarballo, R, Coma, AC, Sanchex de Leon, T, Montero, DE and Rodrignez, PR. The relatoionship between lipid peroxidation and platelets aggregation in atherosclerotic patients. Angiologia 43:241-246. 1991
  8. Kalebina, TS, Galina, NR, Selyakh, LO, Khodova, OM and Kulaev, IS. Serine proteinase from Bacillus brevis. Appl. Microbiol. Biotech. 28:531-533. 1988
  9. Kembhavi, AA, Knkalni, A and Pant, A. Salt to lerant and thermostable alkaline protease from Bacillus sp. NCIM No. 64. Appl. Biochem. Biotech. 38:83-89. 1993 https://doi.org/10.1007/BF02916414
  10. Kim, HK, Kim, KH, Lee, JK, Kim, YO, Nam HS and Oh, TK. Characterization of a thermostable protease from thermophilic Bacillus amyloliquefaciens NS 15-4. Kor. J. Appl. Microbiol Biotechnol. 23:322-328. 1995
  11. Kim, KJ, Ryu MK and Kim, SS. Chungkookjang koji fermentation with rice straw. Kor. J. Food Sci. Technol. 14:301-308. 1982
  12. Kim, YT. Characteristics of fibrinolytic enzyme peoduced by Bacillus sp. isolated from Chungkookiang, Sejong Univ, Doctoral Degree Thesis. 1995
  13. Koo, JH, Choi, IJ, Nam, HS, Lee, HJ, Shin, ZI and Oh, TK. Modium optimization for production of thermostable alkaline proteases from Bacillus licheniformis NS70. Kor. J. Appl. Microbiol. Biotechnol. 25:207-221. 1997
  14. Krstenansky, JL, Owen, TJ, Yates, MT and Man, SJT. The C-terminal binding domain of hirullin P18 antithrombin activity and comparison to hirudin peptides. FEBS Letters 269:425-4310. 1990 https://doi.org/10.1016/0014-5793(90)81208-6
  15. Kwon, OJ, Kim, JK and Chung, YG. The characteristics of bacteria isolated from ordinary Korean soy sauce soybean paste. Kor. Agr. Chem. Soc. 29:422428. 1986
  16. Lee, EO, Park, EH, Lee HH and Hyun, HH. Isolation and characterization of Pseudomonas sp. producing alkaline protease. Kor. J. Microbiol. 22: 289-297. 1990
  17. Lee, KH, Lee, HJ and Chung, MK. Studies on Chungkookjang(Part 1) on the changes of soybean protein in manufacturing Chungkookjang. Kor. Agr. Chrm. Soc. 14:191-196. 1971
  18. Lee, SK. Sohn, JH, Choi, ES and Rhee, SK. Screening and purification of anticoagulant protein from Korean lecches. Kor. J. Biochem. 26:228-234. 1993
  19. Lee, SK, Heo, S and Joo, HK. Isolation and identification of fibrinolytic bacteric from Korean traditional Chungkookjang. Kor. Agr. Chem. Soc. 41 (inpress). 1998
  20. Kol, JO, Kim, HK and Park, I. Production and characterization of fibrinolytic enzyme: Optimal condition for production of the enzyme from Bacillus sp. KP-6408 isolated from Chungkookjang. L. Korean Soc. Food Sci. Nutr. 27:51-56. 1997
  21. Markwardt, F. The isolation and chemical characterization of hirudin. Hoppe-Seylers Z. Physiol. Chem. 308:147-152. 1957 https://doi.org/10.1515/bchm2.1957.308.1.147
  22. Mihars, H, Sumi, H, Yoneta, T, Mizumoto, H, Ikeda, R, Seiki, M and Maruyama, M. A novel fibrinolytic enzyme extracted from the earthworm, Lumbricus fubellus. Jap. J. Physiol. 41 :461-468. 1991 https://doi.org/10.2170/jjphysiol.41.461
  23. Nakajima, N, Mihara, H and Surni. Characterization of potent fibrinolytic enzymes in earthworm. Lumbricus rubellus. Biosci. Biotech. Biothem. 57: 1730: 1737. 1993
  24. Ranhman, RN, ZA, Kamaruzaman, CNR, Yunus, WM, MB, ZW and Salleh, AB. Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1. Appl. Microbiol Biotechnol. 40:822- 827. 1994 https://doi.org/10.1007/BF00173982
  25. Santos, JA, LJ, Cabral, MS and Cooney, CL. Recovery of alkaline protease by membrane filtration. Bioprocess Eng. 7:205-211. 1992 https://doi.org/10.1007/BF00369547
  26. Schinner, F and Maregensin, R. Production and properties of an extracellulae metalloprotease from a psychophilic Pseudomonas fluorescens J. Biotech. 24:207-210. 1994 https://doi.org/10.1016/0168-1656(92)90125-S
  27. Teiner, SD, Knecht, VR and Gruetter, M. Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography. J. Chromatograpy 530:273-279. 1990 https://doi.org/10.1016/S0378-4347(00)82331-2
  28. Sumi, H. Nattokinase and health-development' of natto. Bioindustry 7:725-730. 1990
  29. Sumi, H, Hamada, H, Tsushima, H, Mihara, H and Muraki, H. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Nattor; a typical and popular soybean food in the Japanese diet. Experimentia 43:1110-1111. 1987 https://doi.org/10.1007/BF01956052
  30. Takami, H, Akjba, T and Horikoshi, K. Production of extremly thermostable alkaline prutease from Bacillus sp. No. AH-101. Appl. Microbiol. Biotech. 30:120-124. 1989
  31. Lee, SK, Heo, S, Bae, DH and Choi, KH. Medium Optimization for Fibrinolytic Enzyme Production by Bacillus subtilis KCK-7 Isolated from Korean Traditional Chungkookjang. Kor. J. Appl. Microbiol. Biotechnol 26:226-232. 1998
  32. Kim, WK, Kim, CY, Park, H, Choi, J, Lee, Y, Kwon, HI and Lee, S. Purification and characterizztion of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl. Environ. Microbiol. 62:2482-2488. 1996