Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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High-Level Expression of A Bacillus subtilis Mannanase Gene in Escherichia coli.

대장균에서 Bacillus subtilis의 Mannanase 유전자 과잉발현

  • 권민아 (우송대학교, 응용식품ㆍ영양학부) ;
  • 손지영 (우송대학교, 응용식품ㆍ영양학부) ;
  • 윤기홍 (우송대학교, 응용식품ㆍ영양학부, 생물소재응용연구센터)
  • Published : 2004.09.01
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Abstract

The gene coding for mannanase from Bacillus subtilis WL-7, a number of glycosyl hydrolase family 26, was hyperexpressed in Escherichia coli. Two recombinant plasmids, pE7MAN and pENS7, were constructed by introducing the complete mannanase gene and the mature mannanase gene lacking N-terminal signal peptide region into a expression vector pET24a(＋), respectively. The level of mannanase produced by E. coli BL21 (DE3) carrying pENS7, which included the mature mannanase gene, was considerably higher than that by E. coli BL21 (DE3)/pE7MAN. Almost mannanase produced by the recombinant E. coli carrying pENS7 at growth temperature of $37^{\circ}C$ existed as inactive enzyme of insoluble form. Growth at temperature below $31^{\circ}C$ increased the soluble fraction of mannanase having catalytic activity in the recombinant E. coli cells. The highest productivity of active mannanase was observed in cell-free extract of the recombinant E. coli grown at growth temperature ranging from $25^{\circ}C$ to $28^{\circ}C$, while mannanase activity per soluble protein of the cell-free extract was highest in the cells grown at $^31{\circ}C$.

Glycosyl hydrolase family 26에 속하는 Bacillus subtilis WL-7 mannanase를 코드하는 유전자를 대장균에서 과잉 발현하였다. 아미노 말단의 signal peptide를 포함하거나 포함하지 않은 mannanase 유전자를 각각 pET24a(＋)에 도입하여 재조합 플라스미드 pETMAN과 pENS7를 제조하였다. 이들 플라스미드를 함유하는 Escherichia coli BL21(DE3)에서 mannanase를 발현시킨 결과 signal peptide가 제거된 mannanase유전자의 발현량이 매우 높았다. 그러나 배양온도 $37^{\circ}C$에서 pENS7를 함유한 재조합 대장균에서 과잉 발현된 mannanase는 대부분이 불활성 형태로 존재하였으며, 배양온도를 $31^{\circ}C$이하로 하였을 때 수용화 형태의 효소량이 증가하면서 효소활성이 높아졌다. IPTG에 의해 발현된 재조합 대장균의 균체파쇄 상등액 중에 존재하는 mannanase 활성은 배양온도 $25^{\circ}C$～28$^{\circ}C$에서 가장 높았으며, 전체 단백질량을 기준으로 볼 때는 배양온도 $31^{\circ}C$에서 비활성이 가장 높은 것으로 확인되었다.

Keywords

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