Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Biochemical Characteristics of a Bacteria (Bacillus pseudomycoides) Alanine Racemase Expressed in Escherichia coli

Bacillus pseudomycoides로 부터 분리된 alanine racemase 유전자의 발현 및 생화학 특성

  • Kang, Han-Chul (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Kim, Na-Hyun (Department of Functional Bio-material, National Academy of Agricultural Science, Rural Development Administration) ;
  • Jeong, Yu-Jeong (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)
  • 강한철 (농촌진흥청, 국립농업과학원, 기능성물질개발과) ;
  • 김나현 (농촌진흥청, 국립농업과학원, 기능성물질개발과) ;
  • 정유정 (농촌진흥청, 국립농업과학원, 기능성물질개발과) ;
  • 윤상홍 (농촌진흥청, 국립농업과학원, 기능성물질개발과) ;
  • 이창묵 (농촌진흥청, 국립농업과학원, 기능성물질개발과)
  • Received : 2010.07.12
  • Accepted : 2010.09.01
  • Published : 2010.09.30
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Abstract

A gene encoding a putative alanine racemase in B. pseudomycoides was cloned and expressed in Escherichia coli BL21(DE3) using a pET-21 vector harbouring 6xHistidine tag. Affinity purification of the recombinant alanine racemase with a nickel resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 46 kDa. The enzyme was the most active toward L-alanine and secondly D-alanine, implying that the enzyme is an alanine racemase. D-cysteine significantly inhibited the enzyme activity and also L-cysteine to a lesser extent. The enzyme was considerably activated by addition of pyridoxal-5'-phosphate (PLP), showing that 73% increase in activity was observed at 0.3 mM, compared to control. The enzyme was the most active at pH 9.0 and more stable at alkaline pHs than acidic pHs.

B. pseudomycoides로 부터 alanine racemase로 추정되는 유전자를 분리한 다음 6xHistidine 이 결합된 pET-21 운반체를 이용하여 E. coli BL21(DE3)에서 발현시키고 생화학 특성을 조사하였다. 재조합된 alanine racemase는 affinity chromatography를 이용하여 분리하였으며 SDS-PAGE 분석에서 약 46 kDa의 단일밴드를 나타내었다. 분리된 효소는 여러 아미노산 중에서 L-alanine에 대하여 가장 높은 활성도를 보이고 D-alanine에 대하여 두번째로 높은 활성도를 보였다. 따라서 분리된 효소는 alanine racemase로 판단되었다. 분리된 효소는 D-cysteine에 하여 상당히 저해가 되었다. 효소의 최적 활성도는 pH 9.0 근처에서 관찰되었고 산성의 조건보다는 알칼리의 조건에서 보다 안정하였다. 보효소인 PLP 0.3mM의 첨가에 의해 효소의 활성도는 약 70% 가량 증대되었다.

Keywords

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