Candida sp. BT001의 xylose reductase의 정제 및 성질

Purification of xylose reductase from Candida sp. BT001 and characterization of its properties

D-xylose의 통성 발효성 효모, Candida sp. BT001로부터 D-xylose를 xylitol로의 전환을 촉매하는 효소, xylose reductase(alditol: $MADP^+$ 1-oxidoreductase, EC 1.1.1.21)를 salt fractionation, ion exchange, gel filtration과 affinity chromatography를 거쳐 정제하여 그 성질을 조사하였다. 정제된 xylose reductase는 보효소 NADPH 및 NADH에 모두 특이성을 나타내었으며, 또한 각각의 보효소에 대해 활성을 지니는 효소는 따로 분리되지 않았다. Specific activity는 NADPH에 대해 11.78 U/mg, MADH에 대해 6.01 U/mg이었으며, NADH/NADPH의 활성비는 0.51이었다. 정제된 xylose reductase의 분자량은 SDS-PAGE상에서 31,000, gel filtration상에서 61,000으로 2개의 subunit로 구성된 효소로 추정하였다. 정제된 xylose reductase의 D-xylose와 NADPH 및 NADH에 대한 Km값은 각각 $94.2{\times}10^{-3}M,\;0.011{\times}10^{-3}M$ 및 $0.032{\times}10^{-3}M$ 이었다. Aldose들에 대한 xylose reductase의 활성은 L-arabinose, D-xylose순으로 높았다. 최적 효소 반응의 pH 및 반응 온도는 각각 6.2와 $45^{\circ}C$이었으며, 이 효소는 $30^{\circ}C$에서 20분간 안정하였다.

Xylose reductase (alditol: $NADP^+$ 1-oxidoreductase, EC 1.1.1.21) from the xylose-fermenting yeast, Candida sp. BT001, was purified via salt fractionation, ion-exchange, gel filtration and affinity chromatography, and its properties were characterized. The enzyme from the yeast was active with both NADPH and NADH as coenzyme. The xylose reductase activity with NADH was approximately 51% of that with NADPH and the specific activities of purified enzyme with NADPH and NADH were 11.78 U/mg and 6.01 U/mg, respectively. Molecular weight of the purified enzyme was 31,000 on SDS-PAGE and 61,000 on gel filtration. The Km for D-xylose, NADPH, and NADH was $94.2{\times}10^{-3}M,\;0.011{\times}10^{-3}M\;and \;0.032{\times}10^{-3}M$, respectively. The purified xylose reductase had relatively higher substrate affinity for L-arabinose than other aldoses tested. The optimal pH was 6.2 and the optimal reaction temperature was $45^{\circ}C$. The thermal stability of the enzyme was for 20 minutes at $30^{\circ}C$.