Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Cloning and Expression of Alkaline Amylase Gene of Alkalophic Bacillus sp. AL-8 and Enzyme Properties in E. coli

호알카리성 Bacillus sp. AL-8의 알카리성 아밀라제 유전자의 대장균에의 클로닝과 발현된 아밀라제의 특징

  • Bae, Moo (Department of Biology, College of Natural Science, Ewha Womans University) ;
  • Hwang, Jae-Won (Department of Biology, College of Natural Science, Ewha Womans University) ;
  • Park, Sin-Hye (Department of Biology, College of Natural Science, Ewha Womans University)
  • 배무 (이화여자학교 자연과학대학 생물학과) ;
  • 황재원 (이화여자학교 자연과학대학 생물학과) ;
  • 박신혜 (이화여자학교 자연과학대학 생물학과)
  • Published : 1987.12.01
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Abstract

The gene coding for alkaline amylase of alkalophilic Bacillus sp. AL-8 was cloned and expressed in Escherichia coli which was lack of amylase activity. For the cloning of the alkaline amylase gene, the chromosomal DNA and plasmid vector pBR322 were cleaved at the site of EcoRI and the gene was cloned. The selection of the transformants carrying the amylase gene was based on the their antibiotics resistance and amylase activity of the transformants. The recombinant plasmids pJW8 and pJW200 containing 5.8Kb and 3.0Kb EcoRI inserts respectively were proved to can the alkaline amylase gene. Alkaline amylase expressed in E. coli was characterized. The enzyme was proved to be stable at the range of alkaline pH.

알카리성 amylase를 내는 Bacillus sp. AL-8의 알카리성 amylase 유전자를 amylase를 생성하지 않는 Escherichia coli HB101에 pBR322를 vector로 하여 형질전환하였다. E. coli도 알카리성 amylase를 생성하여 5$0^{\circ}C$에서 최적 활성온도를 가지며 5$0^{\circ}C$까지 열안정성을 갖고, pH10에서 최적 활성 pH를 나타내는 동시에 pH8-10에서 안정하였다. 알카리성 amylase 유전자가 E. coli에 형질전환되어 donor 세포와 같은 효소 성질을 갖고 있으며 pBR322로 삽입된 유전자는 pJW8에서 5.8kb이며 pJW200에서는 3.0kb로 E. coli에서 안정하게 발현되었다.

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