Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Cloning and Biochemical Characterization of Aspartate Aminotransferase from Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae로 부터 aspartate aminotransferase 유전자의 분리 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-Mook (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)
  • Published : 2009.09.30
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Abstract

The gene encoding a putative aspartate aminotransferase in Xanthomonas oryzae pv. oryzae (Xoo) was cloned using PCR technique. The gene was ligated with pET-21(a) vector containing His6 tag and expressed in E. coli BL21(DE3). Affinity purification of the recombinant aspartate aminotransferase with Ni-NTA resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 43 kDa, as expected. The enzyme was the most active toward L-aspartate as an amino donor, indicating that the purified enzyme is one of aspartate aminotrans-ferases exist in Xoo. Optimal activity of the enzyme was observed at around pH 7.5 and stability was much higher at alkaline pH rather than acidic pH values. The enzyme was considerably activated by the presence of manganese ion, showing about 157% of control activity at 1.0 mM.

Xoo로 부터 aspartate aminotransferase로 추정되는 유전자를 분리한 다음 발현시켜 생화학 특성을 조사하였다. 분리된 유전자는 His6 pET-21(a) 운반체에 삽입시켰으며 E. coli BL21(DE3)에서 발현시켰다. 재조합된 Asp-AT는 affinity chromatography를 이용하여 분리하였으며 SDS-PAGE분석에서 43kDa의 단일 밴드를 나타내었다. 분리된 효소는 amino donor 로서 L-aspartate에 대하여 효소활성도가 가장 높았고, L-leucine 및 L-cysteine에 대하여서도 상당한 활성도를 나타내었다. 효소의 최적 pH는 약 7.5 근처에서 나타났고 효소의 안정성은 산성조건 보다는 알칼리 조건에서 훨씬 높았다. 최적 온도는 약 $35-40^{\circ}C$로 나타났고 $55^{\circ}C$에서 20분간 열처리한 이후의 잔여 활성도는 약 78%로 나타났다. 여러 중금속 중에서 망간 이온에 의해 효소활성이 촉진되었다.

Keywords

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