Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Expression and Biochemical Characteristics of a Phospholipase D from Bacillus licheniformis

Bacillus licheniformis로부터 분리된 phospholipase D 유전자의 발현 및 생화학 특성

  • Kang, Han-Chul (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Yoon, Sang-Hong (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Lee, Chang-Muk (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Koo, Bon-Sung (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration)
  • 강한철 (농촌진흥청, 국립농업과학원, 기능성물질개발과) ;
  • 윤상홍 (농촌진흥청, 국립농업과학원, 기능성물질개발과) ;
  • 이창묵 (농촌진흥청, 국립농업과학원, 기능성물질개발과) ;
  • 구본성 (농촌진흥청, 국립농업과학원, 기능성물질개발과)
  • Received : 2011.04.21
  • Accepted : 2011.05.23
  • Published : 2011.06.30
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Abstract

A gene encoding a putative phospholipase D was isolated from Bacillus licheniformis and cloned into pGEM-T easy vector. The gene was expressed in E. coli BL21 (DE3) using a pET-21(a) vector containing His6 tag. Affinity purification of the recombinant phospholipase D with nickel-nitrilotriacetic acid (Ni-NTA) resin resulted major one-band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified enzyme showed a molecular weight of 44 kDa. The optimum activity of enzyme was around pH 7.0 and the enzyme was also the most stable around this condition. The optimum temperature was about $40-45^{\circ}C$ and the enzyme still showed considerable activities at wide range of temperature. Among various detergents, Triton X-100 significantly increased the enzyme activity, resulting in 181% activity of control at 0.6 mM of the detergent. Calcium ion did not significantly affect the enzyme activity, suggesting that the enzyme might be classified into $Ca^{2+}$-independent PLD.

Bacillus licheniformis로 부터 phospholipase D (PLD)로 추정되는 유전자를 PCR 기술을 이용하여 분리하여 pGEM-T easy 운반체에 cloning 하였다. 분리된 유전자는 His6가 붙은 pET-21 운반체를 이용하여 E. coli BL21 (DE3)에서 발현시켰다. 재 조합된 PLD는 nikel-nitrilotriacetic acid (Ni-NTA) resin을 갖는 column을 이용하여 affinity chromatography로 분리하였다. SDS-PAGE 분석 결과 PLD로 추정되는 단백질은 약 44 kDa의 주요 단일밴드를 나타내었다. 분리된 효소의 최적 활성도는 pH 7.0에서 나타났으며 이 조건에서 또한 효소가 제일 안정되었다. 효소활성에 미치는 최적 온도는 $40-45^{\circ}C$의 온도에서 형성되어 비교적 높은 온도를 나타내었으며 비교적 넓은 범위의 온도에서 상당히 높은 효소 활성도를 나타내었다. 여러 가지 detergent 중에서 Triton X-100을 0.6 mM까지 첨가할 경우 PLD의 효소활성도는 점진적으로 증가하여 대조구 대비 최대 181%의 효소 활성도를 나타내었다.

Keywords

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