Analytical Science and Technology (분석과학)

The Korean Society of Analytical Sciences (한국분석과학회)
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The Expression and Functional Analysis of Recombinant Alcohol Dehydrogenase

재조합 alcohol dehydrogenase의 발현 및 기능분석

  • Kong, Kwang-Hoon (Department of Chemistry, Chung-ang University) ;
  • Shim, Eun-Jung (Department of Chemistry, Chung-ang University) ;
  • Park, Hee-Joong (Department of Chemistry, Chung-ang University) ;
  • Kim, Eun-Ho (Department of Chemistry, Chung-ang University) ;
  • Cho, Sung-Hye (Department of Chemistry, Chung-ang University) ;
  • Park, Sung-Woo (Forensic Science Department, National Institute of Scientific Investigation) ;
  • Kim, Young-Mann (Advanced Analysis Center, Korea Institute of Science & Technology)
  • 공광훈 (중앙대학교 자연과학대학 화학과) ;
  • 심은정 (중앙대학교 자연과학대학 화학과) ;
  • 박희중 (중앙대학교 자연과학대학 화학과) ;
  • 김은호 (중앙대학교 자연과학대학 화학과) ;
  • 조성희 (중앙대학교 자연과학대학 화학과) ;
  • 박성우 (국립과학수사연구소 법과학부) ;
  • 김영만 (한국과학기술연구원 특성분석센터)
  • Received : 1999.04.29
  • Published : 1999.12.25
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Abstract

The alcohol dehydrogenase (ADH) gene from Bacillus stearothermopilus was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pGEX-KG, and expressed it as a fusion protein with glutathione S-transferase (GST) in E. coli. The recombinant ADH was produced by induction with 1 mM isopropyl-${\beta}$-D-thiogalactopyranoside at $37^{\circ}C$ and purified by glutathione affinity chromatography. The recombinant ADH exhibited high substrate specificity for ethanol. The activity of the recombinant ADH proceeded optimally at pH 9.0 and $70^{\circ}C$. The recombinant ADH was highly stable against high temperature. This thermostable alcohol dehydrogenase can be used for the enzymatic determination of alcohol and for the industrial production of alcohol.

Bacillus stearothermopilus의 염색체 DNA로부터 polymerase chain reaction 법을 이용하여 ADH의 구조유전자를 증폭시킨 후, 발현 벡터 pGEX-KC에 삽입시켜 glutathion S-tansferase와 융합 단백질로 대장균에서 대랑 발현시켰다. 재조합 ADH는 $37^{\circ}C$에서 1 mM의 isopropyl-${\beta}$-D-thiogalactopyranoside로 단백질의 발현을 유도시켰으며, 발현된 단백질은 glutathione affinity 컬럼을 이용하여 정제하였다. 재조합 ADH는 에탄올에 높은 기질특이성을 나타내었으며 최적 pH와 온도는 각각 pH 9.0과 $70^{\circ}C$이었다. 또한 이 재조합 ADH는 본래의 효소보다 열에 안정하였다. 이 효소는 알코을 측정을 위한 효소학적 방법과 알코올의 공업적 생산에 이용될 수 있다.

Keywords

Acknowledgement

Supported by : 과기부

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