Eun-Hie Koh  Soo-Mee Song  Sun-Young Kim

Vol. 39,  No. 6, pp. 459-465, Dec.  1995

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효모 acetolactate synthase를 분리 정제하여 기본적인 생화학적 성질에 대한 연구를 수행하였다. 효모를 0.5% glucose, 51 mM K2HPO4, 22 mM KH2PO4, 8 mM(NH4)2SO4, 0.4 mM MgSO4를 포함하는 최소배지에서 37℃로 18시간 동안 배양하였다. 배양된 세포들을 원심분리법으로 수확해 0.1 mM TPP, 0.5 mM DTT, 1 μM FAD와 1 mM MgCl2를 포함하는 20mM phosphate 완충용액(pH 7.0)에 현탁시켜 하룻밤 동안 방치하였다. 효모를 파쇄한 후 이것의 10,000×g 상층액을 모아 ammonium sulfate 분별 침전법과 DEAE-Se-phacel 그리고 leucine-agarose chromatography법을 이용하여 부분 정제하였다. 단백질의 농도, 시간, 온도, pH, 기질농도 등의 영향을 조사하였으며 측정한 결과 최적온도는 50℃이고, pH 8.0∼8.5 사이에서 최고 값을 나타냈다. Km 과 Vmax값은 각각 8.4mM과 17.9nmol/mg/min으로 얻어졌다. 효소의 안정성은 ethylene glycol과 glycerol의 존재하에서 크게 향상됨이 관찰되었다. Feedback inhibition 연구 결과 Val에 의해 가장 많은 영향을 받았고 Leu에는 거의 영향을 받지 않았다. Acetolactate Synthase (ALS) was partially purified from the yeast and its basic biochemical studies were carried out. Yeast was grown in the minimum media containing 0.5% glucose, 51 mM K2HPO4, 22 mM KH2PO4, 8 mM (NH4)2SO4, 0.4 mM MgSO4 for 18 hours at 37 ℃. The cell was ruptured in the buffer (20 mM phosphate buffer pH 7.0, 0.1 mM TPP, 0.5 mM DTT, 1 μM FAD, and 1 mM MgCl2) following an overnight suspension. The supernatant fraction was collected from 10,000×g and the enzyme was further purified by ammonium sulfate fractionation, DEAE-Sephacel chromatography and leucine-agarose chromatography. The enzyme activity was measured under the various conditions by the function of protein concentration, time, temperature, pH, and substrate. The optimum temperature was found to be 50℃, optimum pH 8.0∼8.5. The kinetic parameters, Km and Vmax were 8.4 mM and 17.9 nmol/mg/min respectively. Stability of the enzyme was studied with ethylene glycol and glycerol added to the enzyme solution. Both ethylene glycol and glycerol improved the enzyme stability up to 50%. The study of feedback inhibition showed that valine was a strong inhibitor while leucine was a weak inhibitor.