Biochemical Characterization of an Extracellular Protease in Serratia proteamaculans Isolated from a Spider

무당거미에서 분리한 Serratia proteamaculans에서 분비되는 단백질분해효소의 생화학적 특성

  • Lee Kieun (Department of Biology, Chungnam National University) ;
  • Kim Chul-Hee (Department of Biology, Chungnam National University) ;
  • Kwon Hyun-Jung (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kwak Jangyul (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Shin Dong-Ha (Insect Biotech Co., Ltd.) ;
  • Park Doo-Sang (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Bae Kyung-Sook (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Park Ho-Yong (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology)
  • 이기은 (충남대학교 생물학과) ;
  • 김철희 (충남대학교 생물학과) ;
  • 권현정 (한국생명공학연구원 곤충자원연구실) ;
  • 곽장열 (한국생명공학연구원 곤충자원연구실) ;
  • 신동하 ((주)인섹트바이오텍) ;
  • 박두상 (한국생명공학연구원 곤충자원연구실) ;
  • 배경숙 (한국생명공학연구원 곤충자원연구실) ;
  • 박호용 (한국생명공학연구원 곤충자원연구실)
  • Published : 2004.12.01


Serratia proteamaculans isolated from the midgut of a spider formed big halos around the bacterial colonies, indicating that the bacterial strain produces an extracellular protease. Activity staining of the extracellular pro­tein fractions using zymogram also demonstrated that the major protein with an estimated molecular mass of 52 kDa contained a high proteolytic activity. The protease was purified to near electrophoretic homogeneity from the culture supernatant after filtration and ion exchange and size exclusion chromatography. The purified enzyme had a relatively high proteolytic activity between pH 6.0 and 10.0 and at broad temperature range. The proteolytic activity of the enzyme was not inhibited by phenylmethylsulfonyl fluoride but strongly inhibited by 1, 10-phenanthroline and EDTA. The activity also was dependent on the presence of $Ca^{++}\;and\;Zn^{++}$ ions. These observations indicate that the enzyme is a metalloprotease.

거미의 중장에서 분리한 장내 세균인 Serratia proteamaculans는 우유 단백질 배지상에서 투명환을 형성하는 것으로 보아 세포 외로 분비되는 단백질 분해효소를 생산함을 알 수 있었다. Zymogram을 사용한 단백질 분획의 활성 염색 실험에서 세포 외로 분비된 분자량 52 KD의 단백질이 높은 단백질분해 활성을 가진 것으로 추정되었다. 이 단백질 분해효소의 배양 상등액을 여과, 이온교환, 크로마토그래피 등의 방법을 사용하여 순수 정제하였다. 정제된 단백질 분해 효소는 pH 6.0과 10.0사이와 넓은 온도범위에서 상대적으로 높은 활성을 나타내었다. 1,10-phenanthroline과 EDTA등의 단백질분해효소 저해제를 처리하였을 때 단백질 분해 활성이 강하게 억제되며 $Zn^{2+}$이나 $Ca^{2+}$ 이온의 존재에 의해 단백질 분해효소의 활성이 증가되는 것으로 보아 이 효소가 금속성 단백질 분해효소임을 알 수 있었다.



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