Purification and Properties of Thermostable L - Iactate Dehydrogenase Produced by Escherichia coli

국문 초록 (Abstract)

Bacillus stearothermophilus의 유산탈수소 효소에 대한 4.3kb인 유전자를 subcloning하여 대장균에서 생산된 효소의 특성을 조사하였다. 본 효소는 25%의 수율로 200배 정제되었으며, 열처리, OEAE-Sephadex A-50, NAD^+ -Sepharose CL-4B affinity chromatography, gel filteration을 거쳐서 정제되었다. Gel filteration에 의해 정제된 효소의 분자량은 약 140,000이었으며, SDS-polyacrylamide 전기영동 결과, 분자량 35,000의 단일 band를 나타내어 4개의 subunit로 구성된 효소임을 알 수 있었다. 정제효소에 대한 pyruvate 환원 반응과 lactate 산화 반응에 있어서의 최적 pH는 각각 5.5와 8.5이었고, pH 안정성은 pyruvate 환원 반응에 있어서 5에서 7 사이였다. 효소활성에 대한 최적온도 및 열안정성은 pyruvate 환원 반응에서 60℃, lactate 산화 반응에서 70℃와 75℃이었다. 본 효소는 FeSO4와 pCMB에 의해 강한 저해 반응을 나타내었으며, sodium oxalate는 경쟁적 저해제로 작용하였다. 또한 fructose 1,6-bisphosphate에 의한 효소활성 촉진은 0.8mM에서 최대의 활성을 나타내었다.

다국어 초록 (Multilingual Abstract)

The 4.3-kb gene coding for L-Iactate dehydrogenase of Bacillus stearothermophilus has been subcloned and expressed in E. coli cells. The enzyme was purified 200-fold with 25% yield by heat treatment, DEAE-Sephadex, and NAD^+ -Sepharose CL-4B affinity chromatography followed by gel filtration through Sephadex G-200. The molecular weight of the purified enzyme was estimated to be about 35,000 and 140,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of four identical subunits. The enzyme for pyruvate reduction and lactate oxidation was stable at 60 and 75℃ for 30min, and the optimal temperatures for both reactions were 60 and 70℃, respectively. The enzyme had an optimal pH at 5.5 and 8.5 in pyruvate reduction and lactate oxidation, respectively. The pH stability of enzyme for pyruvate reduction was stable between pH 5 and 7. More than 90% of enzyme activity was lost at 1mM FeSO₄ and p-chloromercuribonzoate. The maximal activation of the enzyme was obtained with 0.8mM fructose 1,6-bisphosphate.

목차 (Table of Contents)

• Abatract
  
• INTRODUCTION
  
• MATERIALS AND METHODS
  
• RESULTS AND DISCUSSION
  
• REFERENCES
  
• 요약