Abstract: 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 HCO3ˉ 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 .

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0.83 U/mg提高到2.67 U/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 。