생명과학회지 (Journal of Life Science)

  • 제16권2호
  • /
  • Pages.274-281
  • /
  • 2006
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

Bacillus subtilis BB-1으로부터 나토키나아제 유전자 크로닝 및 대량발현

Cloning and High Expression of Nattokinase Gene from Bacillus subtilis BB-1

  • 이영훈 (진주산업대학교 미생물공학과) ;
  • 이성호 (경상대학교 생물학과) ;
  • 박기훈 (경상대학교 응용생명과학부) ;
  • 최영주 (신라대학교 식품영양학부) ;
  • 정영기 (동의대학교 미생물학과) ;
  • 갈상완 (진주산업대학교 미생물공학과)
  • Lee Young-Hoon (Department of Microbiological Engineering, Jinju National University) ;
  • Lee Sung-Ho (Department of Biology, Gyeongsang Natinal University) ;
  • Park Ki-Hoon (Department of Applied Life Science, Gyeongsang National University) ;
  • Choi Young-Ju (Department of Food and Nutrition, Silla University) ;
  • Jeong Yong-Kee (Department of Life science and Biotechnology, Dong Eui University) ;
  • Gal Sang-Wan (Department of Microbiological Engineering, Jinju National University)
  • 발행 : 2006.04.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

흑두청국으로부터 분리된 혈전용해력이 우수한 Bacillus subtilis BB-1(KFCC 11344P)으로부터 혈전용해효소 유전자를 PCR법에 의해 크로닝하였고 이를 BCF-1으로 명명하였다. BCF-1의 DNA 염기서열결정 결과 1,145 bp 크기의 혈전용해 효소로, 일본의 natto로부터 분리된 nattokinase 유전자와 99%의 상동성을 보임을 확인하였다. 혈전용해효소 유전자의 발현을 위하여 Bacillus 발현계인 Bacillus-E. coli의 shuttle vector인 pEB vector에 크로닝 하고 host로서 B. subtilis 168에 형질전환시켜 대량 발현시켰다. 생산된 혈전용해효소의 최 적활성 pH와 온도는 7.0과 $35^{\circ}C$로 확인되었다, 기질에 대한 분해양상을 조사한 결과 fibrin에서만 특이적으로 강한 분해가 일어났으며, skim milk에서 아주 약한 분해능을 보였으나 blood agar, gelatin, casein에서는 전혀 분해능을 보이지 않았다. 특히 blood agar plate에서 분해능이 없는 것으로 보아 혈액 내에서의 적혈구 파괴현상과 같은 부작용에 대한 위험을 배제할 수 있을 것으로 사료된다. BCF-1에 의해 생산된 혈전용해효소는 fibrin 특이적으로 활성을 나타냄을 확인할 수 있으며, 이는 임상적이나 산업적으로 적용하였을 때 부작용에 대한 위험적인 문제는 배제될 수 있으리라 생각된다.

A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

키워드

참고문헌

  1. Astrup, T. and I. Stermdorff. 1956. The plasminogen activator in animal tissue. ACTA physiol. scand. 363, 250
  2. Astrup. T., and S. Mullertz. 1952. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys. 40, 346 https://doi.org/10.1016/0003-9861(52)90121-5
  3. Electricwala, A., R. T. Sawyer, J. C. Powell and T. Atkinson. 1991. Isolation of thrombin inhibitor from the leech Hirudinaria manillensis. Blood Coagulation and Fibrinolysis 2, 83-91 https://doi.org/10.1097/00001721-199102000-00013
  4. Fletcher, A. P. and A. J. Johnson 1975. Methods employed for purification of streptokinase. Por. Soc. Exp. Biol. Med. 94, 233
  5. Jeong, Y. K. W. S. Yang, J. O. Kang, I. S. Kong, J. O. Kim. 1995. Fibrinolysis of fermented kimchi. Korean. J. Life Science 5(4), 203-210
  6. Kalu, D., E. J. Masoro, B. P. Yu, R. R. Hardin and B. W Hallis. 1988. Modulation of age-related hyperparthyroid and senile bone loss in Fischer rats by soy protein and food restriction. Endocrinology 122, 1847 https://doi.org/10.1210/endo-122-5-1847
  7. Kim, B. N., J. D. Kim, S. S. Ham, Y. S. Choi and S. Y. Lee. 1995. Effects of spice added natto supplementation on the lipid metabolism in rats. J. Korean Soc. Food Nutr. 24(1), 121-126
  8. Kim, B. N. and S. Y. Lee. 1995. Nattokinase ${\gamma}-GTP$ protease activity and sensory evaluation of natto added with spice. J. Korean Soc. Food Nutr. 24(2), 228-233
  9. Kim, D. H. G. Y. Lee, N. M. Kim, J. S. Lee. 2003. Physiological functionality of various extracts from danmemil and legumes. Korean J. Food Nutr. 16(4), 347-352
  10. Kim, H. K. G. T. Kim, D. K. Kim, W. A. Choi, S. H. Park, Y. K. Jeong and I. S. Kang. 1997. Purification abd characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermenter fish. J. Ferment. Bioengin. 84(4), 307-312 https://doi.org/10.1016/S0922-338X(97)89249-5
  11. Kim, J. S. and K. W. Hong. 2000. Optimum conditions for electro-transformation of Bacillus subtilis 168. Food Engineering Progress 4(3), 162-166
  12. Kim, K. J., M. K. Ryu and S. S. Kim. 1992. Chung kook Jang Koji fermentations with rice straw. Kor. J. Food Sci. Technol. 14, 301-308
  13. Kim S. H. 1998. New trends of studying on potential activities of Doen-jang. Korea Soybean Digest. 15(1), 8-15
  14. Kim Y. T. W. K. Kim and H. S. Oh. 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK11-4 screened from chungkookjang. Appl. Environ. Microbiol. 62(7), 2482-2488
  15. Krstensky, J. L., T. J. Owen, M. T. Yates and S. J. T. Mao 1990. The C-terminal binding domain of hirullin P19 Antithrombin activity and comparison to hirudin peptides. FEMS Letters 269, 425-431
  16. Liu, J. G., J. M. Xing, Ruishen, C. L. Yang, H. E. Liu. 2004. Reverse micelles extraction from fermentation broth. Biochemical Engineering Journal 21, 273-278 https://doi.org/10.1016/j.bej.2004.07.008
  17. Lee, S. K., J. H. Sohn, E. S. choi and S. K. Rhee. 1993. Screening and purification of anticoagulant proteins from korean leeches. Kor. J. Biochem. 26(3), 228-234
  18. Lee. S. K. S. Heo, D. H. Bae and H. H. Choi. 1998. Medium optimization for fibrinolytic enzyme production by Bacillus subtilis KCK-7 Isolated from korean traditional chung kookjang. Kor. J. Appl. Microbiol. Biotechnol. 26(3), 226-231
  19. Lijnen, H. R., Van Hoef, and D. Goolen. 1992. Interaction of staphylokinase with different molecular form of plasminogen. Biochem. Biophys. ACTA 144, 1118
  20. Mitsugu, F., N. Keiichi, K. S. Hong, I. Yae, A. Aki and N. Satoshi. 1993. Purification and characterization of a strong fibrinolytic enzyme (Nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochemical and Biophysical Research Communications 197(3), 1340-1347 https://doi.org/10.1006/bbrc.1993.2624
  21. Plug, J and O. kieldgarrd. 1957. Urokinase an activator from buman urine. I. Isalation and proper ties. Biochem. Biophys., ACTA 61, 624
  22. Sumi, H., H. Hamada, H. Mihara, K. Nakanishi and H. Hiratani. 1990. Enhancement of the fibrinolytic in plasma by oral administration of nattokinase. Acta Haemat. 84, 139 https://doi.org/10.1159/000205051
  23. Sumi H., H. Hamada, H. Tsushima and H. Mihara. 1998. A novel strong fibrinolytic enzyme (Nattokinase) in the vegetable cheese 'NATTO'. Fibrinolysis 2(1), 67
  24. Toki, N., H. Sumi, K. Saski, I. Boreisha and K. C. Robbins. 1995. Transport of urokinase across the intestinal tract of normal uman subjects with stimulation of synthesis and release of urokinase-type proteins. J. Clin. Invest. 75, 1212 https://doi.org/10.1172/JCI111818
  25. Towbin H, T. Staehelin, J. Gordon. 1979. Electrophoresis transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354
  26. Yoo, C. K., W. S. Seo, C. S. Lee and S. M. Kang. 1998. Purification and characterization of fibrinolytic enzyme excreted by Bacillus subtilis K-54 isolated from Chung Guk Jang. Kor. J. Appl. Microbiol. Biotechnol. 26(6), 507-514
  27. Yoo, J. Y. 1997. Present status of industries and reseach activities of korean fermented soybean products. The Microorganism and Industry 23, 13-30
  28. Yudkin, J. 1957. Diet and coronary thrombosis; Hypothesis and fact. Lancet. 2, 155
  29. Zhang, Z. I. Z. Y. Zuo, Z. G. Liu, K. C. Tsai, A. F. Liu, G. I. Zou. 2005. Construction of a 3D model of nattokinase, a novel fibrinolytic enzyme Bacillus natto A novel mucleophilic catalyticmechanism for nattokinase. J. Molecular Graphics and Modelling 23, 373-380 https://doi.org/10.1016/j.jmgm.2004.10.002