2,5-DKG还原酶I的分离纯化与性质研究  

Studies on Purification and Characterization of 2,5-DKG Reductase I from ER97

作　　者：李越[1] 陈策实[1] 尹光琳[1] 

机构地区：[1]中国科学院上海生物工程研究中心,上海200233

出　　处：《微生物学通报》2005年第6期63-67,共5页Microbiology China

基　　金：国家自然科学基金资助项目(No.39770021)

摘　　要：欧文氏菌ER97高效表达了从棒状杆菌SCB3058克隆的2,5-二酮基-D-葡萄糖酸(2,5-DKG)还原酶I基因,5 L罐发酵后,收集菌体破碎,将胞内可溶性的蛋白通过硫酸铵分级沉淀、DEAE-Sepharose CL-6B离子交换柱层析和Phenyl Sepharose CL-4B疏水柱层析后分离纯化到了2,5-DKG还原酶I,纯化了5倍,得率27%,比活力为3,418 U/mg。测定了该酶的一些特性参数:分子量为34 kD,等电点为6.0,它以NADPH为辅酶,将2,5-DKG还原为2-酮基-L-古龙酸(2-KLG),对NADPH和2,5-DKG底物的Km值分别是0.29mmol/L和14.7 mmol/L,1 mmol/L Cu2+、Zn2+等有强烈抑制作用,EDTA和巯基乙醇对该酶没有抑制作用,酶的最适pH为7.0,最适反应温度为40℃。2, 5-DKG reductase I was purified from eell-free extracts of a recombinant, ER97 by a procedure involving ammonium sulfate precipitation and successive column chromatography on DEAE-Sepharose CL-6B and Phenyl Sepharose CL-4B with 5 fold purification, 27 % recovery and 3, 418 U/mg specific activity. The molecular weight of the enzyme estimated by SDS-PAGE was 34kD. The isoelectric point was estimated to be 6.0 by PAG-IEF. The optimum pH was 7.0 and the optimum temperature was about 40℃. The enzyme can catalyze the stereospecific NADPH-dependent reduction of 2, 5-DKG to 2-KLG. The michaelis-menten constant （Km） for 2, 5-DKG and NADPH were 0.29 mmoL/L and 14.7 mmoL/L respectively. The enzyme is specific for NADPH and 2, 5-DKG, 1 mmol/L Cu^2＋ or Zn^2＋ could highly inhibited the enzyme activity. EDTA and β-Mereaptoethanol have no effect on the enzyme activity.

关 键 词：欧文氏菌ER97 2 5-DKG还原酶Ⅰ 纯化与性质 

分 类 号：Q936[生物学—微生物学]