Abstract
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 $K_2HPO_4$, 22 mM $KH_2PO_4$, 8 mM $(NH_4)2SO_4,\;0.4\;m M\;MgSO_4$ for 18 hours at 37 $^{\circ}C$. The cell was ruptured in the buffer (20 mM phosphate buffer pH 7.0, 0.1 mM TPP, 0.5 mM DTT, 1 ${\mu}M$ FAD, and 1 mM MgCl_2$) following an overnight suspension. The supernatant fraction was collected from $10,000{\times}g$ 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$^{\circ}C$, optimum pH 8.0∼8.5. The kinetic parameters, $K_m\;and\;V_{max}$ 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.
효모 acetolactate synthase를 분리 정제하여 기본적인 생화학적 성질에 대한 연구를 수행하였다. 효모를 0.5% glucose, 51 mM $K_2HPO_4$, 22 mM $KH_2PO_4$, 8mM$(NH_4)2SO_4,\;0.4\;m M\;MgSO_4$를 포함하는 최소 배지에서 37$^{\circ}C$로 18시간 동안 배양하였다. 배양된 세포들을 원심분리법으로 수학해 0.1 mM TPP, 0.5 mM DTT, 1${\mu}M$ FAD와 1mM MgCl_2$를 포함하는 20 mM phosphate 완충용액(pH 7.0)에 현탁시켜 하룻밤 동안 방치하였다. 효모를 파쇄한 후 이것의 $10,000{\times}g$ 상충액을 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$^{\circ}C$이고, pH 8.0∼8.5 사이에서 최고 값을 나타냈다. $K_m$과 $V_{max}$값은 각각 8.4 mM과 17.9 nmol/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.