Screening of Fibrinolytic Enzyme Producing from Microorganisms in Korean Fermented Soybean Paste and Optimum Conditions of Enzyme Production.

된장 유래 혈전분해효소 생산균주의 분리 및 최적 효소생산 조건 탐색

  • Ok Min (Department of Functional Food Consulting, Sorabol college) ;
  • Choi Young-Su (Department of Biotechnology, Dong-A University)
  • 옥민 (서라벌대학 기능식품컨설팅과) ;
  • 조영수 (동아대학교 응용생명공학부)
  • Published : 2005.12.01

Abstract

This study was investigated to find out optimal medium maximizing the production of fibrinolytic enzyme by Bacillus sp. isolated from Korean fermented soybean paste, which could hydrolyze the fibrin produced through the blood coagulation mechanism in human body. Among carbon sources tested, galactose was most effective for the enzyme production, and the level of the concentration for the optimal enzyme production was $4\%$(w/v). For nitrogen sources tested, malt extract was most effective for the enzyme production, and level of the concentration for optimal enzyme production was $4\%$(w/v). For mineral sources tested, $K_2HPO_4$ was most effective for enzyme production. The enzyme was maximally produced by cultivating the organism at the liquid medium of the initial pH 6 and temperature of $40^{\circ}C$.

전통발효식품인 된장으로부터 높은 활성의 fibrinolytic enzyme를 생산하는 미생물을 수십 종 분리하였다. 산업적으로 우수한 균종을 선별하기 위하여 분리된 미생물중 fibrinolytic enzyme활성과 성장 속도면에서 가장 우수한 균주를 선별하여 하였으며, 형태학적, 생화학적 및 생리학적 특성을 조사한 후 Bacillus sp.로 동정되었다. Fibrinolytic enzyme생산을 위한 최적 배양조건은 초기 pH $6\~8$일 때 상대활성도가 $80\%$이상을 나타내었고, 배양 12시간째에 가장 높은 활성도를 나타내었다. Fibrin plate를 이용한 혈전용해능을 확인한 결과 높은 혈전용해능을 가지고 있었다. 탄소원으로 galactose를 $4\%$ 첨가 하였을 때 가장 우수하였으며, 질소원은 malt extract를 $4\%$ 첨가 하였을 때 가장 우수하였다. 무기염은 $K_2HPO_4$를 첨가하였을 때 가장 우수한 활성을 나타내었다.

Keywords

References

  1. Abe, T., Kazama, M., Kinoshita, T. Naito, I. Ogushi, T., Yoshimura, Y., Teruya, J. and Shimizu, N. (1982) Shift of fibrinolysis system at oral administration of urokinase in human subjects; Double blind cross over study, Blood and Vessels(in Japanese), 13, 472-479 https://doi.org/10.2491/jjsth1970.13.472
  2. Collen, D., Lijnen, H.R. and Gold, H.K. (1991) Towards better thrombolytic therapy, Progress in Cardiovascular Diseases 34, 101-112 https://doi.org/10.1016/0033-0620(91)90008-A
  3. Sumi, H., Toki, N., Sasaki, K. and Robbins, K.C. (1980) Oral administration of urokinase, Thromb. Res., 20, 711-714 https://doi.org/10.1016/0049-3848(80)90161-9
  4. Sumi, H., Maruyama, M., Yoneta, T. and Mihara, H. (1983) Activation of plasma fibrinolysis after intrarectal administration of high molecular weight urokinase and its derivative, Acta Haematol., 70, 289-295 https://doi.org/10.1159/000206755
  5. Sasaki, K., Moriyama, S., Sumi, H., Toki, N. and Robbins, K. C. (1985) The transport of 125I-labeled human high molecular weight urokinase across the intestinal tract in a dog model with stimulation of synthesis and/or release of plasminogen activators, Blood, 66, 67-75
  6. Toki, N., Sumi, H, Sasaki, K., Boreish, I. and Robbins, K.C. (1985) Transport of urokinase across the intestinal tract of normal human subjects with stimulation of synthesis and/or release of urokinase-type proteins, J. Clin. Invest., 75, 1212-1222 https://doi.org/10.1172/JCI111818
  7. Reed, G.L., Lin, F.F., Parhaml-seren, B. and Kussie, P. (1995) Identification of plasminogen binding region in streptokinase that is necessary for the creation of streptokinase plasminogen activator complex. Biochem., 34, 10266-10271 https://doi.org/10.1021/bi00032a021
  8. Mihara, H., Sumi, H., Yoneta, T., Mizumoto, H., Ikeda, R,. Seiki, M. and Maruyama, M. (1991) A novel fibrinolytic enzyme extracted from the earthworm, Lumbricus rubellus. Jap. J. Physiol., 41, 461-472 https://doi.org/10.2170/jjphysiol.41.461
  9. Nakajima, N., Mihara, H. and Sumi, H. (1993) Characterization of potent fibrinolytic enzyme in earthworm, Lumbricus rubellus. Biosci. Biotech. Biochem., 57, 1726-1730 https://doi.org/10.1271/bbb.57.1726
  10. Nakajima, N., Taya, N. and Sumi, H. (1993) Potent fibrinolytic enzyme from the lysate of Katsuwonus pelamis digestive tract(Shiokara) : Purification and characterization, Biosci. Biotech. Biochem., 57, 1604-1605 https://doi.org/10.1271/bbb.57.1604
  11. Samy, A. and Aassar, E.I. (1995) Production and properties of fibrinolytic enzyme in solid state cultures of fusarium pallidoroseum, Biotech. Lett., 17, 943-948 https://doi.org/10.1007/BF00127431
  12. Fletcher, A.P. and Johnson, A.J. (1995) Methods employed for purification of streptokinase, Proc. Soc. Exp. Bio. Med., 94, 233
  13. Guntelber, A.V. and Ottesen, M. (1952) Preparation of crystals containing the plakalbumin forming enzyme from Bacillus subtilis. Nature(London), 170, 802 https://doi.org/10.1038/170802a0
  14. Hagihara, B., Matrubaraa, H., Nakai, M. and Okunuki, K. (1958) Crystalline bacterial proteinase. 1. Preparation of crystalline proteinase of Bacillus subtilis. J. Biochem., 45, 185-194 https://doi.org/10.1093/oxfordjournals.jbchem.a126856
  15. Horikoshi, K.(1966) Production of alkaline enzymes by alkalophilic microorganism. part 1. alkaline protease produced by Bacillus No.221. Agr. Biol. Chem., 35, 1407-1414
  16. Hwang, S.Y. (1995) Purification and characterization of an extracellular serine protease from Bacillus sp. strain KUN-17. Kor. J. Appl. Microbiol. Biotechnol., 23, 53-59
  17. Kil, J.O., Kim, G.N. and Park, I.S. (1998) Production and characterization of fibrinoloytic enzyme : Optimal condition for Production of the Enzyme from Bacillus sp. KP-6408 Isolated from Chungkook-jang. J. Korean Soc. Food Sci. Nutr., 27, 51-56
  18. Kim, H.K., Kim, G.T., Kim, D.K., Choi, W.A., Park, S.H., Jeong, Y.K. and Kong, I.S. (1997) Purification and charaterization of a novel fibrinolytic Enzyme from Bacillus sp. KA38 Originated from Fermented Fish. J. Ferment. Bioeng., 84, 307-312 https://doi.org/10.1016/S0922-338X(97)89249-5
  19. Kim, S.J., Yoon J.H., Lee, M.S. and Kim, H.B. (1997) Isolation and charaterization of Bacillus cereus secreting protease from korean Soybean Paste. Kor. J. Microbiol., 33, 136-141
  20. Kim, W.K., Choi, K.H., Kim, Y.T., Park, H.H., Choi, J.Y., Lee, Y.S., Oh, H.I., Kwon, I.B. and Lee, S.Y. (1996) Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. Strain CK 11-4 Screened from chungkook - jang, Appl. Environ. Microbiol., 62, 2482-2488
  21. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) : Protein mesurement with the Folin phenol regent. J. Bio. Chem., 193, 265
  22. Lee, S.K., Heo, S., Bae, D.H. and Choi, K.H. (1998) Medium optimization for fibrinolytic enzyme production by Bacillus subtilis KCK-7 isolated from korean Traditional Chungkookjang. Kor. J. Appl. Microbiol. Biotechnol., 26, 226-231
  23. Ok, M., Seo, W.S., Cha, J.Y. and Cho, Y.S. (2001) Isolation and characterization of Bacillus sp. WRD-2 extracellular protease from soil. J. Korean Soc. Agri. Chem. Biotechnol., 44, 246-25
  24. Koo, J.H., Choi, I.J., Nam, H.S., Lee, H.J., Shin, Z.I. and Oh, T.K. (1997) Medium optimization for production of thermostable alkaline protease from Bacillus licheniformis NS70. Kor. J. Appl. Microbiol. Biotechnol., 25, 207-211
  25. Choi, K.J. (1995) Separation of Bacillus sp. and changes of $NH_2-N,\;NH_3-N$, and protease activity in chonggukchang meju adding with mugwort extract. Kon-Kuk Univ. Master's Degree Thesis
  26. Lee, E.G., Park, E.H., Lee, H.H. and Hyun, H.H. (1996) Isolation and characterization of pseudomonas sp. producing alkaline protease. Kor. J. Microbiol., 22, 289-297
  27. Kim, Y.T. (1995) Characteristics of fibrinolytic enzyme produced by Bacillus sp. isolated from Chungkookjang. Sejong Univ. Doctoral Degree Thesis
  28. Kalebina, T.S., Galina, N.R., Selyakh, I.O., Khodova, O.M. and Kulaev, I.S. (1998) Serine protease from Bacillus brevis. Appl. Microbiol. Biotech., 28, 531-533
  29. Kim, H.K., Kim, K.H., Lee, J.K., Kim, Y.O., Nam H.S. and Oh, T.K (1995) Characterization of a thermostable protease from thermophilic Bacillus amyloliquefaciens NS 15-4. Kor. J. Appl. Microbiol. Biotechnol., 23, 322-328
  30. Horikoshi, K. (1971) Production of alkaline enzyme by alkalophilic microorganism. Agric. Biol. Chem., 35, 1783-1791 https://doi.org/10.1271/bbb1961.35.1783
  31. Zlotnik, H., Schramm, V.L. and Buckley, H.R. (1984) Purification and partial characterization of a Norcardia brasiliensis extracellular protease. J. Bacteriol., 157, 627-631
  32. Kembhavi, A.A., Kukalni, A. and Pant, A. (1993) Salt tolerant and thermostable alkaline protease from Bacillus sp. NCIM No. 64. Appl. Biochem Biotech., 38, 83-89 https://doi.org/10.1007/BF02916414