枯草芽孢杆菌6-磷酸葡萄糖脱氢酶的纯化及性质

Purification and characterization of G6PD From B.Subtilis

枯草芽孢杆菌通过超声破壁,(NH4)2SO4分段盐析,DEAE SepharoseFF离子交换层析,Blue SepharoseCL 6B亲和层析,SephadexG 200凝胶过滤等纯化步骤,分离出6 磷酸葡萄糖脱氢酶。经PAGE和SDS PAGE检测为单一蛋白区带,比活1 7375IU/mg,纯化倍数72 7,收率13 6%。凝胶过滤法测定表观分子量220KD,SDS PAGE测定亚基分子量50 5KD,可见该酶是由四个相同亚基构成的四聚体。测定最适pH、温度分别为8 5,37℃,底物G6P的Km值0 177mmol·L-1。考察了部分金属离子及EDPA对该酶活性、紫外、荧光光谱的影响。

G6PD was isolated and purified from B.subtilis by a procedure including cell sonication,salting out with(NH4)2SO4,ionexchange chromatographing with DEAESepharose FF,affinity chromatographing with BlueSepharose CL6B and gel filtration with Sephadex G200.The purified G6PD showed a single protein band in PAGE and SDSPAGE determination,respectively.The purified enzyme had a specific activity of 17375IU/mg,a 727fold purification was obtained with a recovery of 136%.The apparent molecular weight of G6PD,determined by gel filtration,was 220kD.The subunit molecular weight was 505kD as determined by SDSPAGE,suggesting that the native enzyme was a tetramer consisting of four identical subunits.The optimal pH of G6PD was 85 and the optimal temperature was 37℃.The Km value of G6P on G6PD was 0177mmol·L-1.The effect of some metal ions and EDTA on enzyme activity,ultraviolet absorption spectra and fluorescent spectra were also investigated.