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Bacillus subtilis BK-17 유래 혈전용해 효소의 특성

Characterization of a Novel Fibrinolytic Enzyme Produced from Bacillus subtilis BK-17

  • 백현 (동의대학교 자연과학대 생명응용과학과) ;
  • 임학섭 ((주)천년약속 바이오연구소) ;
  • 정경태 (동의대학교 자연과학대 생명응용과학과) ;
  • 최영현 (동의대학교 한의과대학) ;
  • 최병태 (동의대학교 한의과대학) ;
  • 서민정 (동의대학교 자연과학대 생명응용과학과) ;
  • 김지은 (동의대학교 자연과학대 생명응용과학과) ;
  • 류은주 (한서대학교 미용학과) ;
  • 허만규 (동의대학교 분자생물학과) ;
  • 주우홍 (창원대학교 생물학과) ;
  • 정영기 ((주)천년약속 바이오연구소)
  • Hyun Bek (Department of Life Science and Biotechnology, Dongeui University) ;
  • Lim Hak-Seob (Institute of BIO, MILLENNIUM PROMISE CO., LTD.,) ;
  • Chung Kyung Kae (Department of Life Science and Biotechnology, Dongeui University) ;
  • Choi Yung Hyun (Collegy of Oriental Medicine, Dongeui University) ;
  • Choi Byung Tae (Collegy of Oriental Medicine, Dongeui University) ;
  • Seo Min-Jeong (Department of Life Science and Biotechnology, Dongeui University) ;
  • Kim Ji-Eun (Department of Life Science and Biotechnology, Dongeui University) ;
  • Ryu Eun-Ju (Department of Cosmetology, Hanseo University) ;
  • Huh Man Kyu (Department of Molecular Biology, Dongeui University) ;
  • Joo Woo Hong (Department of Biology, Changwon National University) ;
  • Jeong Young Kee (Institute of BIO, MILLENNIUM PROMISE CO., LTD.,)
  • 발행 : 2005.12.01

초록

마른 볏짚으로부터 fibrinolytic enzyme (BK)을 분비하는 균주를 분리하여 동정한 결과, Bacillus subtilis속으로 분류되었다. 분리된 균주(Bacillus subtilis BK-17를 배양하여 그 배양액으로부터 에탄올 침전, ion exchange, gel filtration 등의 과정을 거쳐 fibrinolytic enzyme (BK)를 분리 및 정제하였다. 이 정제된 효소를 SDS-PACE gel 전기영동한 결과 분자량은 약 31 kDa 이었다. BK의 활성 및 안정성에 대한 pH의 영향을 조사해본 결과, pH $6\∼8$범위에서 매우 높았고, 최적 pH는 7과 8이었다. 본 효소의 활성 및 안정성에 대한 최적 온도는 $50^{\circ}C$이었으며, $20^{\circ}C\∼50^{\circ}C$범위에서는 안정성이 그대로 유지되었지만 $60^{\circ}C\∼80^{\circ}C$범위에서는 현저히 감소하여 $50^{\circ}C$에 비해 $20\%\∼40\%$의 활성을 보였다. BK에 대한 활성 역시 안정성과 비슷한 양상을 보였고, 다만 $20^{\circ}C$에서 약 $62\%$의 활성을 보였고, 그 이후 $50^{\circ}C$까지 지속적으로 증가하여 $50^{\circ}C$에서 최대의 활성을 보였다. 실험한 금속이온들 중 1 mM의 $Zn^{2+}$$Ca^{2+}$에서 각각 $35\%$$23\%$의 저해활성을 보였지만 나머지 이온들에서는 의미 있는 영향이 없었다. 동일농도의 EDTA에 대해서는 $45\%$의 저해활성을 보였기 때문에 BK는 metallo enzyme으로 생각된다. Fibrinogen-rich fibrin plate 와 plasminogen-free fibrin plate에서의 분해활성을 검토해본 결과, 두 plate에서 비슷한 분해활성을 보였다. 따라서 본 효소는 plasminogen activator type 보다는 fibrin에 직접 활성을 가지는 것으로 생각된다.

A bacterium, producing a fibrinolytic enzyme, was screened from a decaying rice plant. The bacterium was identified as Bacillus subtilis by morphological, biochemical, and physiological properties and named Bacillus subtilis BK-17. The fibrinolytic enzyme (BK) was purified from supernatant of Bacillus subtilis BK-17 culture broth. The molecular weight was 31 kDa as determined by SDS-PAGE. The effect of temperature, pH, and plasminogen on the activity of the bacillokinase (BK) was analysed and the activity was compared with urokinase. The optimal temperature and pH were $50^{circ}C$ and pH 7, pH 8, respectively. The BK activity was inhibited to $45\%$, $35\%$, and $23\%$ with 1mM EDTA, $Zn^{2+}$, and $Ca^{2+}$, respectively. However, $Mg^{2+}$, $Mn^{2+}$, and $Co^{2+}$ ions did not have any significant effect on the enzyme activity The BK showed the artivity in the both plates, plasminogen-free fibrin plate and plasminogen-rich fibrin plate. The result indicates that the BK can directly act the fibrin. In comparison of fibrinolytic activity with urokinase on the fibrin plate, the BK shows about 20 folds higher activity than that of the urokinase.

키워드

참고문헌

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피인용 문헌

  1. Isolation of Bacteria with Protease Activity from Cheonggukjang and Purification of Fibrinolytic Enzyme vol.23, pp.2, 2013, https://doi.org/10.5352/JLS.2013.23.2.259