Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Cloning and Characterization of a Gene for Fibrinolytic Enzyme from Bacillus subtilis BB-1 Isolated from Black Bean Chung-kuk

흑두로 제조한 청국에서 분리된 Bacillus subtillus BB-1으로 부터 혈전용해효소 유전자 크로닝 및 특성규명

  • Lee Young-Hoon (Department of Microbiological Engineering, Jinju National University) ;
  • Lee Sung-Ho (Environmental Biotechnology National Core Research center, Gyeongsang National University) ;
  • Jeon Ju-Mi (Environmental Biotechnology National Core Research center, Gyeongsang National University) ;
  • Kim Hong-Chul (Department of Microbiological Engineering, Jinju National University) ;
  • Cho Yong-Un (Department of Microbiological Engineering, Jinju National University) ;
  • Park Ki-Hoon (Department of Applied Life Science, Gyeongsang National University) ;
  • Choi Young-Ju (Department of Food and Nutrition, Silla University) ;
  • Gal Sang-Wan (Department of Microbiological Engineering, Jinju National University)
  • 이영훈 (진주산업대학교 미생물공학과) ;
  • 이성호 (경상대학교 환경생명국가 핵심연구센터) ;
  • 전주미 (경상대학교 환경생명국가 핵심연구센터) ;
  • 김홍출 (진주산업대학교 미생물공학과) ;
  • 조용운 (진주산업대학교 미생물공학과) ;
  • 박기훈 (경상대학교 응용생명과학부) ;
  • 최영주 (신라대학교 식품영양학부) ;
  • 갈상완 (진주산업대학교 미생물공학과)
  • Published : 2005.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

A bacterium producing five fibrinolytic isozymes was isolated from black bean chung kuk. The bacterium was identified as Bacillus subtilis BB-1 by 16s rDNA sequence homology search. A gene out of five fibrinolytic genes in the Bacillus subtilis BB-1 was cloned by shot-gun method. A Cla I DNA fragment of B. subtilis BB-1 chromosome was cloned in to pBluescript II SK(-) and showed the fibrinolytic activity to bacterial cells. The Cla I DNA fragment was sequenced and the sequences did not show homology with gene for protease or fibrinolytic enzyme genes in other organisms. The Cla I DNA fragment was reduced to 2,142 bp by activity-guided PCR cloning method. The optimum pH and temperature of the enzyme were 5.0 and $35^{\circ}C$, respectively. Substrate specificity of the fibrinolytic enzyme was detected in skim milk, casein, gelatin and blood agar plates. The activity of the enzyme was not detected with these substrates. Taken together, this enzyme is a new fibrinolytic enzyme and may be used to prevent thrombosis and arteriosclerosis.

흑두로 제조한 청국으로부터 혈전용해력이 우수한 균을 선발하여 동정하였으며, 그를 Bacillus subtilis BB-1로 명명하였다. 이 균은 혈전용해효소 isozyme을 적어도 5개이상 생성하는 균주로 확인되었다. 이 균으로부터 크로모좀을 분리하여 shot gun법으로 혈전용해효소 유전자를 크로닝하였으며, 이 유전자를 BSF-1이라 명명하였다. 이 유전자는 714개의 아미노산을 암호화하고 있으며 기존에 밝혀진 혈전용해효소 유전자와 상동성은 검출되지 않은 새로운 혈전용해효소 유전자였다. 혈전용해효소활성 최적 pH 및 온도는 5.0과 $35^{\circ}C$였다. 기질특이성은 적혈구 배지 또는 skim milk, gelatin등에 전혀 분해활성이 없었다. 이는 혈전만을 특이적으로 분해하는 기질특이성을 보였으며, 혈전분해효소로서의 이용가능성이 충분한 것으로 판단된다.

Keywords

References

  1. Astrup, T and S. Hullertz. 1952. The fibrin plate method for estimating fibrinolytic activity. The National Danish Society against Rheumatic Disease. 346-351
  2. Astrup, T. and Stermdorff, I. 1956. The plasminogen activator in animal tissue. ACTA physiol. scand., 363 250
  3. Bok-Nan Kim, Jong-Dai Kim, Seung-shi Ham, Yong-Sorn Choi and Sang-Young Lee. 1995. Effects of spice added natto supplementation on the Lipid Metabolism in Rats. J. Korean Soc. Food Nutr., 24(1), 121-126
  4. Bullock, W.O., J. M. Fernandez and J. M. Short. 1987. XL1blue: A high efficiency plasmid transforming recA Escherichia coli strain with beta galactosidase selation. Bio Techiques 5, 376
  5. Birhbion, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res. 7, 1513-1523 https://doi.org/10.1093/nar/7.6.1513
  6. Chung KH and Kim DS. 1992. Fibrinolytic and cogulation activies of korean Snake Venoms. Kar Biachem J, 25, 699-701
  7. DaKa MD and Semba CP. 1995. Thrombolytic therapy in venous occlusive disease. J Vasc Intero Radiol, 6, 73-77 https://doi.org/10.1016/S1051-0443(95)71252-0
  8. Fletcher, A. P and Johnson, A. J. 1975. Methods employed for purification of streptokinase., Par. Soc. Exp. Bial. Med., 94, 233
  9. Fujita, M, Hong, K., lto, Y., Misawa, S., Takeuchi, N., Kartya, K and Nishimuro, S. 1995. Transport of nattokinase across the rat intestinal tract. BioI. Pharm Bull. 18, 1194-1196 https://doi.org/10.1248/bpb.18.1194
  10. Fujita, M., Nomura, K., Hong, K., lto, Y., Asada, A and Nishimuro, S. 1993. Purification and characterization of a strong fibrinolytic enzyme(nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem. Biophys. Res. Comm. 197, 1340-1347 https://doi.org/10.1006/bbrc.1993.2624
  11. Hanahan, D. 1985. Techniques for transformation of E. coli, pp. 109-135. In D. M. Glover(ed.), DNA cloning, vol. I. IRL press, Oxford
  12. Kim YT, Kim WK and Oh HS. 1996. Purification and characterization of a fibrinolytic enzyme producrd from Bacillus sp. strain CK11-4 screened from chungkookjang. Appl Envitronm Microbiolo. 2482-2488
  13. Kim SH. 1998. New trends of studying on potential activities of Doen-jang. Korea Soybean digest, 15(1), 8-15
  14. Kim HK, Kim GT, Kim DK, Choi WA, Park SH, Jeong YK and Kang IS. 1997. Purification abd characterization of a novel fibrinolytic enzyme from Bacillus sp. KA38 originated from fermenter fish. J of Ferment and Bioengin, 84(4), 307-312 https://doi.org/10.1016/S0922-338X(97)89249-5
  15. Kyu-chum Youn, Dong-Ho Kim, Jang-Ok Kim, Byung-Jun Park, Hong-Sun Yook, Jae-Min Cho and Myung-Woo Byun. 2002, Quality characteristics of the chung kook jang fermented by the mixed culture of Bacillus natto and B. licheniformis. J. Korean Soc. Food Sci. Nutr. 31(2), 204-210 https://doi.org/10.3746/jkfn.2002.31.2.204
  16. Mihara H, Nakajima N and Sumi H. 1993. Characterization of potent fibrinolytic enzyme in earthworm, Lumbicus rubellus. Biosci. Biotech. Biochem, 57(10), 1730
  17. Mihara H, Sumi H, Yonata T, Mizumoto H, Ike da R, Seiki M and Maruyama M. 1991. A novel fibrinolytic enzyme extracted from the earthqorm, Lumbricus rubellus. Jpn J Physiolo, 41, 461 https://doi.org/10.2170/jjphysiol.41.461
  18. Maniatis, T., E. F. Fritsch and J. Sambrook. 1989. Molecular cloning A Laboratory Manual. Cold spring Harbor Laboratory, New York
  19. Nakajima, N., Taya, N and Sumi, H. 1993. Potent fibrinolytic enzyme from the lysate of katspwonus pelamis digestive tract (shiokara) : purification and characterization. Biosci. Biotech, Biochem. 57, 1604-1605 https://doi.org/10.1271/bbb.57.1604
  20. Nakamura, T., Yamagata, Y and lchishima, E. 1992. Nucleotide sequence of the subtilisin NTA gene, aprN, of Bacillus subtilis(natto). Biasci. Biotech. Biochem. 56, 1869-1871 https://doi.org/10.1271/bbb.56.1869
  21. Nack-Shick Choi, Kab-Seog Yoon, Jin-Young Lee, KyoungYoen Han and Seung-Ho Kim. 2001. Comparison of three Substrates(casein, fibrin, and gelatin) in Zymographic Gel. J. Biochemistry and Molecular Biology, 34(6), 531-536
  22. Park YD, Kim JW, Min BG, Seo JW and Jeong JM. 1998. Rapid purification and biochemical characteristics of Lumbrokinase III from earthworm for use as a fibrinolytic agent. Biotechnol Lett, 20(2), 169-172 https://doi.org/10.1023/A:1005384625974
  23. Plug, J and kieldgarrd, O. 1957. Urokinase an activator from buman urine. I. lsalation and proper ties. Biochem. Biophys., ACTA., 61, 624
  24. Sambrook, Fritsh, Maniatis. Molecular cloning. 3rd ed. p13.1-p13.101, 6.36-p6.38
  25. Sang-Wan Gal, Yong-Un Cho. 1997. 'Simple DNA elution from agarose gels. Korean Biotechnol. Bioprocess Eng. 2, 62-63 https://doi.org/10.1007/BF02932466
  26. Sumi, H., Seiki, M., Morimato, N., Tsushima, H., Maruyama, M and Mihara, H. 1985. Plasma fibrinolysis after intraduodenal administration of urokinase in rats. Enzyme 33, 121-127
  27. Sumi, H., Hamada, H., Tsushima, H., Mihara, H and Muraki, H. 1987. A novel fibrinolytic enzyme(nattokinase) in the vegetable cheese Natto. a typical and popular soybean food in the japan diet. Experientia. 43, 1110-1111 https://doi.org/10.1007/BF01956052
  28. Toki, N., Sumi, H., Saski, K., Boreisha, I and Robbins, K. C. 1995. Transport of urokinase across the intestinal tract of normal Uman Subjects with Stimulation of synthesis and release of urokinase-type proteins. J. din Ivest., 75, 1212
  29. Yong-Kee Jeong, Woong-Suk Yang, Jeong-Ok Kang, In-Soo Kong, Jeong-Ok Kim. 1995. Fibrinolysis of fermented kimchi. Korean. J. Life Science, 5(4), 203-210
  30. Yoo CK, Seo WS, Lee CS and Kang SM. 1998. Purification and characterization of fibrinolytic enzyme extracted by Bacillus subtilis K-54 isolated from chunggukgang. Kor. J. Appl. Microbiol. Biotechnol. 26, 507-514

Cited by

  1. Antioxidative activity and Angiotensin Converting Enzyme Inhibitory activity of Fermented Medical Plants (DeulBit) and Its Modulatory Effects of Nitric Oxide Production vol.53, pp.2, 2010, https://doi.org/10.3839/jabc.2010.017