Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Biochemical Characterization of Cysteine(-) Mutant Alanine Racemase from Bacillus pseudomycoides

Bacillus pseudomycoides로 부터 분리된 alanine racemase 유전자의 cysteine 치환 및 생화학적 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 : 2010.09.14
  • Accepted : 2010.11.03
  • Published : 2010.12.31
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Abstract

A gene encoding an alanine racemase in B. pseudomycoides was cloned and one (Cys316) or both of two cysteines (Cys316 and Cys365) was (were) substituted with alanine. The cysteine (-) alanine racemases were expressed in E. coli BL21 (DE3) using a pET-21 vector. The expressed enzymes were purified through affinity chromatography using 6xHis ligand. The purified enzymes all showed major one bands by SDS-PAGE analysis, corresponding to 46 kDa. The cysteine (-) alanine racemases as well as the wild type enzyme showed alanine racemase activities, indicating that the enzyme is an alanine racemase and the cysteines in the enzyme may not be involved in the catalysis and/or substrate binding. Thermal stabilities of Cys (-) alanine racemases decreased considerably and half-lives were 26 (wild type), 21 (C316A) and 18 min (C316-365A), respectively at $60^{\circ}C$ pH 8.0, suggesting that cysteine is considerably contributive to the thermal stability of the alanine racemase.

B. pseudomycoides로 부터 alanine racemase 유전자를 분리한 다음 이 효소에 존재하는 두개의 cysteine을 하나(C316A) 또는 두개 모두(C316-365A) alanine으로 치환시켰다. 치환된 alanine racemamase는 pET-21 운반체에 삽입한 다음 숙주세포로서 E. coli BL21 (DE3)를 이용하여 발현시켰다. 발현된 단백질은 6XHis이 결합된 affinity chromatography를 이용하여 분리하였으며 SDS-PAGE 분석에서 모두 약 46 kDa의 주요 단일밴드를 나타내었다. Cysteine(-) 변이체의 alanine racemase가 모두 활성도를 보여 cysteine이 catalytic 또는 binding sit에 관여하지 않는 것으로 추정되었다. 변이체 효소들은 wild type에 비하여 열 안정성이 모두 떨어져 $60^{\circ}C$ pH 8.0에서의 활성도 반감시간이 각각 26(wild type), 21(C316A) 18분(C316-365A)-을 나타내었다. 이러한 결과는 cysteine이 열안정화에 상당히 기여함을 알 수 있었다. 그러나 pH 변화에 대한 안정성은 큰 차이가 없었다.

Keywords

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