源于博伊丁假丝酵母的甲酸脱氢酶的分离纯化与反应机制  

Purification and enzymatic catalysis mechanism of formate dehydrogenase from Candida boidinii

作　　者：彭益强[1] 方柏山[1] 

机构地区：[1]工业生物技术高等学校重点实验室华侨大学,福建厦门361021

出　　处：《化工进展》2009年第10期1808-1813,1827,共7页Chemical Industry and Engineering Progress

基　　金：国家863计划子课题(2006AA020103);国家自然科学基金(20676048)项目

摘　　要：使用两步发酵法培养博伊丁假丝酵母(Candida boidinii),菌株细胞经破碎与分离所得粗酶液经DEAE SepHaroses Fast Flow层析快速纯化获得NAD+依赖型的甲酸脱氢酶,酶的比活从0.83U/mg提高到2.67U/mg,纯化倍数和回收率分别为3.22倍和29.7%。研究了从反应物消耗到产物生成之间的酶反应历程,确定了甲酸脱氢酶的酶促反应为有序BiBi反应机制,其中NAD+是第一底物,HCOONa是第二底物,NADH是首先释放的第一产物,HCO3ˉ是随后释放的第二产物;二次作图法求解出Vmax、KiS1、KmS2、KmS1等动力学参数,确定甲酸脱氢酶有序BiBi反应速度方程为r=(2.3*10-4[S1][S2])/(1.123*10-2[S1]+1.91*10-6+[S1][S2])。NAD^＋ depended formate dehydrogenase （FateDH） from Candida boidinii was purified by diethylaminoethyl （DEAE） Sepharoses Fast Flow chromatography after the strain was cultured by two-step fermentation and crude cell extract was obtained by crushing and separation. The enzyme specific activity increased from 0.83 U/mg to 2.67 U/mg and a 3.22 fold purification was achieved with the recovery of 29.7% activity. The ordered BiBi reaction mechanism of FateDH enzymatic reaction was confirmed by the study of possible course from reactant consumption to product production. NAD^＋ was the first substrate with HCOONa as the second substrate. NADH was the first release product with HCO3^- as the subsequent release second product. The kinetics parameter including Vmax, Kis1 Kms2, Kms1 were solved by quadratic drawing and the BiBi reaction kinetic model of FateDH was established as r=2.3×10^-4[s1][s2]/1.123×10^-2[s1]＋1.91×10^-6＋[s1][s2].

关 键 词：甲酸脱氢酶 反应动力学 辅酶再生 

分 类 号：TQ923[轻工技术与工程—发酵工程]