생명과학회지 (Journal of Life Science)

  • 제23권2호
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  • Pages.259-266
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  • 2013
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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청국장으로부터 혈전용해 활성이 우수한 균주 분리 및 혈전용해효소정제

Isolation of Bacteria with Protease Activity from Cheonggukjang and Purification of Fibrinolytic Enzyme

  • 투고 : 2013.01.09
  • 심사 : 2013.02.13
  • 발행 : 2013.02.28
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초록

혈전용해효소를 생산하는 균주 분리를 위해서 우선 한국, 일본 등지에서 모은 21개의 청국장 시료를 준비하였고 총 268개의 균주를 분리하였다. 이 중에서 1% skim milk가 포함된 nutrient agar 배지에서 protease를 생산하는 bacteria를 분리하였고, 이 결과로 22개의 균주가 분리되었다. 균주들은 apiweb을 통해 생화학적 특성에 근거하여 동정하였다. 또한 세균동정을 위해 16S rRNA 염기서열 분석을 수행하였다. 분리된 대부분의 균주는 Bacillus subtilis와 Bacillus amyloliquefaciens였다. 혈전용해효소의 활성은 fibrin plate 방법에 의해 측정되었고 A2-14, A2-20, C1-05, C1-09, F2-01로 명명된 다섯 균주가 선택되었다. 이중에서 A2-20 균주는 강한 혈전용해 활성을 보였고 동정결과 Bacillus amyloliquefaciens에 가까웠다. A2-20 균주에 의해 생산되는 혈전용해효소는 균 상등액을 이용한 gel filtration과 ion exchange chromatography를 거쳐 부분정제 되었다. 부분 정제된 효소의 최적 pH는 7.0이었고, 최적 온도는 $35^{\circ}C$였다. 정제된 단백질의 분석은 SDS-PAGE와 zymography로 이루어졌다. 이와 더불어 혈전용해효소의 유전자적 분석도 수행되었으며 A2-20 균주가 생산하는 혈전용해효소의 부분적인 염기서열과 유전적 상동성을 보이는 서열을 규명하였다.

To isolate the fibrinolytic enzyme, 268 strains from 21 samples were morphologically isolated from Cheonggukjang collected from Korea and Japan. Among the 268 strains, protease-producing bacteria were isolated in nutrient agar medium including 1% skimmed milk. As a result of this, 22 strains were isolated. Apiweb site was used to identify these strains based on their biochemical properties. In addition, 16S rRNA sequencing was performed to identify the strain. Most of the identified strains were Bacillus subtilis and B. amyloliquefaciens. Fibrinolytic enzyme activity was measured with the fibrin plate method. Five strains were finally selected: A2-14, A2-20, C1-05, C1-09, and F2-01. Of those five strains, the A2-20 strain, which is close to B. amyloliquefaciens, showed the strongest fibrinolytic activity. The fibrinolytic enzyme produced by the A2-20 strain was partially purified from culture supernatant by gel filtration and ion exchange chromatography. The optimal pH and temperature values of the partially purified enzyme were 7.0 and $35^{\circ}C$, respectively. Purified protein analysis was carried out with SDS-PAGE and zymography. A genetic analysis was also conducted by PCR based on the consensus sequence of fibrinolytic enzyme. Corresponding genes with a partial sequence of the A2-20 strain were identified.

키워드

참고문헌

  1. Astrup, T. and Mullertz, S. 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
  2. Back, L. M., Park, L. Y., Park, K. S. and Lee, S. H. 2008. Effect of starter cultures on the fermentative characteristics of cheonggukjang. Korean J Food Sci Technol 40, 400-405.
  3. Baek, H., Lim, H. S., Chung, K. T., Choi, Y. H., Choi, B. T., Seo, M. J., Kim, J. E., Ryu, E. J., Huh, M. K., Joo, W. H. and Jeong, Y. K. 2005. Characterization of a novel fibrinolytic enzyme produced from Bacillus subtilis BK-17. J Life Sci 15, 987-993. https://doi.org/10.5352/JLS.2005.15.6.987
  4. Baruah, D. B., Dash, R. N., Chaudhari, M. R. and Kadam, S. S. 2006. Plasminogen activators: A comparison. Vascul Pharmacol 44, 1-9. https://doi.org/10.1016/j.vph.2005.09.003
  5. Cho, Y. J., Cha, W. S., Bok, S. K., Kim, M. U., Chun, S. S. and Choi, U. K. 2000. Production and separation of anti-hypertensive peptide during chunggugjang fermentation with Bacillus subtilis CH-1023. J Korean Soc Agric Chem Biotechnol 43, 247-252.
  6. Choi, N. S., Chung, D. M., Ryu, C. R., Yoon, K. S., Maeng, P. J. and Kim, S. H. 2006. Identification of three extracellular proteases from Bacillus subtilis KCTC 3014. J Microbiol Biotechnol 16, 457-464.
  7. Jo, H. D., Lee, H. A., Jeong, S. J. and Kim, J. H. 2011. Purification and characterization of a major fibrinolytic enzyme from Bacillus amyloliquefaciens MJ5-41 isolated from Meju. J Microbiol Biotechnol 21, 1166-1173. https://doi.org/10.4014/jmb.1106.06008
  8. Kim, I. J., Kim, H. K., Chung, J. H., Jeong, Y. K. and Ryu, C. H. 2002. Study of functional chungkukjang contain fibrinolytic enzyme. J Life Sci 12, 357-362. https://doi.org/10.5352/JLS.2002.12.3.357
  9. Kim, S. H. and Choi, N. S. 2000. Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang. Biosci Biotechnol Biochem 64, 1722-1725. https://doi.org/10.1271/bbb.64.1722
  10. Kim, S. H., Choi, N. S. and Lee, W. Y. 1998. Fibrin Zymography: A direct analysis of fibrinolytic enzymes on gels. Anal Biochem 263, 305-600.
  11. 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.
  12. Kwon, G. H., Lee, H. A., Park, J. Y., Kim, J. S., Lim, J., Park, C. S., Kwon, D. Y., Kim, Y. S. and Kim, J. H. 2009. Development of a RAPD-PCR method for identification of Bacillus species isolated from cheonggukjang. Int J Food Microbiol 29, 282-287.
  13. Lee, J. J., Cho, C. H., Kim, J. Y., Kee, D. S. and Kim, H. B. 2001. Antioxidant activity of substances extracted by alcohol from chungkukjang powder. Korean J Microbiol 37, 177-181.
  14. Oh, J. H., Lee, B. J., Paik, H. R., Jung, S. C., Baik, K. S. and Choi, S. K. 2009. Isolation of bacteria from chunggukjang prepared by rice straw and Identification of protease secreted. J Life Sci 19, 397-402. https://doi.org/10.5352/JLS.2009.19.3.397
  15. Peng, Y., Huang, Q., Zhang, R. H. and Zhang, Y. Z. 2003. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douche, a traditional Chinese soybean food. Comp Biochem Physiol 134B, 45-52.
  16. 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 Japanese diet. Experientia 43, 1110-1111. https://doi.org/10.1007/BF01956052
  17. Wei, H., Wei, L., Frenkel, K., Bowen, R. and Barnes, S. 1993. Inhibition of tumor promoter-induced hydrogen peroxide formation in vitro and in vivo by genistein. Nutr Cancer 20, 1-12. https://doi.org/10.1080/01635589309514265
  18. Wei, X., Luo, M., Xu, L., Zhang, Y., Lin, X., Kong, P. and Liu, H. 2011. Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas. J Agric Food Chem 59, 3957-3963. https://doi.org/10.1021/jf1049535
  19. Weisburg, W. G., Barns, S. M., Pelletier, D. A. and Lane, D. J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 679-703.

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