NMN转移酶和乙醇脱氢酶共固定化及其动力学特性

Co-immobilization of NMN Transferase and Alcohol Dehydrogenase and Its Kinetics

采用磁性纳米颗粒对烟酰胺单核苷酸(NMN)转移酶和乙醇脱氢酶进行共价共固定,用以生产NADH(烟酰胺腺嘌呤二核苷酸),并对共固定化条件、共固定化双酶酶学性质及其动力学进行了考察。结果显示:双酶最佳共固定化反应条件为:NMN转移酶和乙醇脱氢酶添加量分别为6.5U/mg和10.3U/mg,体系pH控制在5.0~7.0,25℃固定化2 h,NADH产率可达到87%,与游离酶的催化产率73%相比提高了14%。所得共固定化双酶在连续使用11次后,剩余酶活仍保留在61.1%左右,表明共固定化酶具有较好的操作稳定性。双酶动力学方程推导与验证结果表明:共固定化双酶反应体系符合米氏方程,其动力学反应速率取决于固定化乙醇脱氢酶的反应速率。

Nicotinamide mononucleotide(NMN)transferase and ethanol dehydrogenase were covalently immobilized with magnetic nanoparticles to produce nicotinamide adenine dinucleotide(NADH).The conditions of co-immobilization,the properties and kinetics of co-immobilized enzyme were investigated.The optimized immobilization conditions were obtained as follows:addition amount of NMN transferase6.5 U/mg,addition amount of alcohol dehydrogenase 10.3 U/mg,system pH being controlled at 5.0~7.0,immobilization temperature 25℃and immobilization time 2 h.Under these conditions,NADH yield reached 87%,which was an improvement of 14%than that catalyzed by free enzyme.After 11 reuses of the co-immobilized double enzyme,the residual enzyme activity still remained about 61.1%,which showed that the co-immobilized double enzyme had a good operational stabilization.The deduction and validation of the kinetic equation of two enzymes revealed that the reaction system of co-immobilized double enzymes was accorded with Michaelis equation,and its kinetic reaction rate depended on the reaction rate of immobilized ethanol dehydrogenase.