酿酒酵母3-磷酸甘油脱氢酶的诱导、提纯和性质

在YEPD培养基中添加NaCl,可以诱导酿酒酵母(Saccharomyces cerevisiae)细胞内3-磷酸甘油脱氢酶的形成,当NaCl浓度达5％时,酶比活从0.05U/mg提高0.5U/mg;若再限制培养墓中葡萄糖浓度在100mg/L以下,酶比活可达到0.89U/mg。酶比活与培养基中的NaCl浓度的函数关系式为:Sa=0.129C~3-0.038C~2+0.034C+0.063(0≤C≤5％)。粗酶液经Sephadex G-25凝胶过滤,Blue Sepharose亲和层析以及Mono Q离子交换等步骤,提纯123.6倍,得纯酶液。经SDS-凝胶电泳测得分子量为45000±2000。酶的最适温度为51℃,最适pH值为6.8。保温30分钟的半失活温度(t_(1/2))为41℃。NADH和DHAP的Km(mmol/L)值分别为:0.017和0.134。

INDUCED FORMATION, PURIFICATION AND PROPERTIES OF GLYCEROL-3-PHOSPHATE DEHYDROGENASE FROM SACCHAROMYCES CEREVISIAE

The formation of glycerol - 3 - phosphate dehydrogenase (EC. 1.1.1. 8; G3P DHG) of Saccharomyces cerevisiae in vivo was induced by addition of NaCl into YEPD medium. When NaCl concentration reached 5% in the medium, the specific activity of enzyme was increased from 0.05U /mg to 0.5 U /mg . Limiting the glucose concentration in the medium to below 100 mg /L, the specific activity reached 0. 89U / mg. The function between the specific activity (Sa) and NaCl concentration (C) is Sa = 0.129C3-0.038C2+0.034C +0.063 (0≤C≤5%). G3P DHG was purified 123.6-fold by gel filtration on Sephadex G-25, affinity chromatography on blue Sepharose and ion-exchange chromatography on Mono Q. Its molecular weight was determined to be 45 000 ±2 000 dalton by SDS-PAGE. The enzyme showed optimal activity at pH 6.8 and 51 ℃ . The temperature for loss of half activity (t1/2) in 30min was 41 ℃ . Km values obtained were 0. 017 ( mmol / L) and 0. 134(mmol/L) for NADH and DHAP respectively.