KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Fibrinolytic Activity and Characterization of Bacillus licheniformis HK-12 Isolated from Chungkook-Jang

청국장에서 분리한 세균인 Bacillus licheniformis HK-12의 혈전용해활성 및 특징

  • Sohn, Byung-Hee (Department of Biotechnology, Soonchunhyang University) ;
  • Song, Yu-Jin (Department of Biotechnology, Soonchunhyang University) ;
  • Oh, Kye-Heon (Department of Biotechnology, Soonchunhyang University)
  • 손병희 (순천향대학교 생명공학과) ;
  • 송유진 (순천향대학교 생명공학과) ;
  • 오계헌 (순천향대학교 생명공학과)
  • Published : 2008.06.30
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Abstract

The aim of this work was to investigate the fibrinolytic activity and characterization of Bacillus licheniformis HK-12, which produces the fibrinolytic enzyme excreted from naturally fermented Chungkook-Jang. Initially, the physiological and biochemical characteristics of strain HK-12 was examined. Both physiological analysis using BIOLOG system and phylogenetic analysis using 16S rRNA sequencing were performed to identify the strain, and the strain could be assigned to Bacillus licheniformis, designated as B. lichenformis HK-12, and registered in GenBank as [EU288193]. Phylogenetic analysis of B. licheniformis HK-12 was plotted based on 16S rRNA sequence comparisons. During the incubation period of B. licheniformis HK-12, the changes of bacterial growth, fibrinolytic activity, and pH were monitored. As the results, after 36 hours of incubation, the maximum fibinolytic activity was about 2.25 times than that of plasmin used as standard. Optimal conditions on the growth of B. licheniformis HK-12 associated with the fibrinolytic activity was initial pH 7.0 and 40$^{\circ}C$, respectively.

이 연구의 목적은 자연 천연한 청국장에서 분비되는 혈전용해효소를 생산하는 Bacillus Iicheniformis HK-12의 혈전용해활성과 특성을 조사하기 위하여 실시한 것이다. 먼저 균주 HK-12의 생리생화학적 특성에 대하여 조사하였다. BIOLOG GP2 MicroPlate system과 16S rRNA 염기서열 분석을 통하여 균주를 동정하였고, 그 결과 Bacillus licheniformis로 확인되었고, B. licheniformis HK-12로 명명하였으며, 이 균주는 GenBank에 [Eu288193]로 등재하였다. 16S rRNA 염기서열 분석에 근거하여, B. licheniformis HK-12의 계통수를 작성하였다. B. licheniformis HK-12의 배양기간동안 세균의 생장, 혈전용해활성, pH의 변화를 모니터링하였다. 36시간배양 후, 배양의 최대 혈전용해활성은 대조군으로서 플라스민과 비교하여 약 2.25배로 나타났다. 혈전용해활성과 관련하여, B. licheniformis HK-12의 생장에서 최적 pH와 온도는 각각 초기 pH 7.0과 40$^{\circ}C$로 나타났다.

Keywords

References

  1. Fujita, M., K. Hong, Y. Ito, R. Fujii, K. Kariya, and S. Nishimuro (1995), Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat. BioI. Pharm. Bull. 18, 1387-1391 https://doi.org/10.1248/bpb.18.1387
  2. Jeong, Y. K., W. S. Yang, J. O. Kang, I. S. Kong, and J. O. Kim (1995), Fibrinolysis of fermented Kimchi. J. Life Sci. 5, 203-207
  3. Sumi, H., H. Hamada, H. Tsushima, H. Mihara, and H. Muraki (1987), A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a typical and popular soybean food of the Japanese diet. Experientia. 43, 1110-1111 https://doi.org/10.1007/BF01956052
  4. Sumi, H., N. Nakajima, and C. Yatagai (1995), A unique strong fibrinolytic enzyme (katsuwokinase) in skipjack Shiokara, a Japanese traditional fermented food. Comp. Thromb. Haemost. 112, 543-547
  5. Fenton, J. W., F. A. Ofosu, D. G. Moon, and J. M. Maraganore (1991), Thrombin structure and function: why thrombin is the primary target for antithrombotics. Blood Coagul. Fibrin. 2, 69-75 https://doi.org/10.1097/00001721-199102000-00011
  6. Kim, S. H., J. L. Yang, and Y. S. Song (1999), Physiological functions of Chongkukjang. Food Ind. Nutr. 4, 40-46
  7. Lee, J. 0., S. D. Ha, A. J. Kim, C. S. Yuh, I. S. Bang, and S. H. Park (2005), Industrial application and physiological functions of Chongkukjang. Food Sci. Ind. 38, 69-78
  8. Hiroshi, S. and H. Kadota (1976), Intracelluler protease of Bacillus subtilis. Agric. BioI. Chem. 40, 1047-1049 https://doi.org/10.1271/bbb1961.40.1047
  9. Mantsala, H. P. and H. Zalkin (1980), Extracelluler and membrane-bound protease from Bacillus subtilis. J. Bacteriol. 141, 493-501
  10. Prestidge, L., V. Gage, and J. Spizizen (1971), Protease activities during the course of sporulation in Bacillus subtilis. J. Bacteriol. 107, 815-823
  11. Strongin, A. Y., L. S. Izotova, Z. T. Abramov, D. I. Gorodetsky, L. H. Ermakova, L. A. Baratova, L. P. Belyanova, and V. M. Stepanov (1978), Intracelluler serine protese of Bacillus subtilis: sequence homology with extracelluler subtilisins. J. Bacteriol. 133, 1401-1411
  12. Kim, W., K. Choi, Y. Kim, H. Park, J. Choi, Y. Lee, H. Oh, I. Kwon, and S. Lee (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
  13. Collins, C.H. and P.M. Lyne (1984), Microbiological method. 5th ed., Butterworths, London
  14. Benson, H. J (1998), Microbiological applications: laboratory manual in general microbiology. 7th ed., p155-163, McGraw-Hill, Inc., SF. USA.
  15. 오계헌, 강형일, 소재성, 송홍규, 이병욱 (2005), 최신 미생물실험. 신광문화사. 109-139
  16. Astrup, T., and S. Mullertz (1952), The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 40, 346-351 https://doi.org/10.1016/0003-9861(52)90121-5
  17. Chung, Y. J., (1999) Isolation and characterization of a bacterium with a fibrinolytic activity. Kor. J. Biotechnol. Bioeng. 14, 103-108
  18. Lee S.K., S. Heo, D. H. Bae, and K. H. Choi (1998), Medium optimization for fibrinolytic enzyme production by Bacillus subtilis KCK-7 isolated by Korean traditional chungkookjang. kor J. Appl. Microbial Biotechnol. 26, 226-231
  19. Choi, M. Y. (2003), Screening of fibrinolytic enzyme producing from microorganisms and optimum conditions of enzyme production. J. Kor. Soc. Food Sci. Nutr. 32, 976-980 https://doi.org/10.3746/jkfn.2003.32.7.976
  20. Lee, J. Y., N. S. Paek, and Y. M. Kim (2005), Medium optimization for fibrinolytic enzyme production by Bacillus subtilis MG410 isolated. Kor. J. Food & Nutr. 18, 39-47
  21. Choi, W. A., J. W. Lee, K. H. Lee, and S. Park (1998), Effect of environmental and nutritional conditions on fibrinolytic enzyme production from Bacillus subtilis BK-17 in flask culture. Kor. J. Biotechhnol. Bioeng. 13, 491-496
  22. Seo, J. H., and S. P. Lee (2004), Optimization of the production of fibrinolytic enzyme from BacilLis firmus NA-1 in fermented soybeans. J. Food. Sci. Nutr. 9, 14-20 https://doi.org/10.3746/jfn.2004.9.1.014
  23. Kil, J. O., G. N. Kim, and I. S. Park (1998), Production and characterization of fibrinolytic enzyme: optimal condition for production of the enzyme from Bacillus sp. KP-6408 isolated from chungkook-jang. J. Kor. Soc. Food Sci. Nutr. 27, 51-56