Study On The Binding Mechanism Between Alcohol Dehydrogenase And NAD~+/NADH On The Basis Of Electrochemistry And FTIR Analysis

Coenzyme plays an essential role in the catalytic reaction of oxidoreductases. However, the high-cost and recyclable difficulty of the coenzyme limit the large-scale industrial application of NAD(P)-dependent dehydrogenases. Extensive investigations have been carried out to search a route for regeneration of the coenzyme. Multi-enzyme system has been considered as one of the best strategies to facilitate the regeneration of the coenzyme. However, the transfer mechanism of coenzyme has been hitherto ignored. In this work, The ethanol dehydrogenase (ADH) from yeast was used as the model enzyme to explore the binding mechanism between ADH and NADH via some techniques listing as follows:Firstly, the reflection of solid and liquid phase of the NAD+, NADH and ADH was analyzed by the FTIR. The difference between the spectrums of binary complex of alcohol dehydrogenase (ADH) with and without NADH/NAD+was obtained in the mediums of both H2O and D2O. The changes in the FTIR spectrums of the adenine moiety of NADH/NAD+were obviously observed being binding to the ADH, which may be caused by the hydrogen bonding. The same phenomenon was also found in the nicotinamide moiety of NAD+. Meanwhile, the mutual transformation between NADH and NAD+was investigated by in situ FTIR reflection spectroscopy combined with electrochemistry analysis. It was difficult to get the NADH through the reduction of NAD+in a low voltage due to the existence of side reactions formed dipolymer of NAD+on the electrode. As the oxidative product of NADH, the NAD+was generated by0.1V, indicating that the oxidation of NADH should be triggered at the voltage lower than1V.Secondly, an alcohol dehydrogenase biosensor was constructed through castinga nano-biocomposite film on the glassy carbon electrode. This film was consisted by a multi-walled carbon nanotubes (MWCNTs)conduit, a chitosan(CHIT) binder and the ADH. The catalytic mechanism of NAD+-dependent ADH on the substrate ethanol was investigated through analyzing the sensitive chemical reaction occurred on the electrode. It was found that the NAD+did not dissociate from the ADH immediately after the catalytic reaction. Dissociation was occurred with the destruction of hydrogen-bonding between ADH and NAD+. Since the reduction of the enzymatic activity on the biosensors, it could be proposed that the dissociation of ADH and NAD+did not slow down with the constant effect of electric field.In addition, the ADH was also immobilized on Au electrode of Electrochemical Quartz Crystal Microbalance (EQCM) apparatus by cross-linked covalent method. Tentative exploration was applied to investigate the binding between ADH and NADH/NAD+with the electrochemistry analysis coupled with EQCM method. Chronoamperometry experiment was carried out by bingding1mM NADH on the enzyme modified electrode. The mass on the modified electrode decreased after oxidation of NADH with0.7V. The mass decrease was considered as the dissociation of the generated NAD+from the ADH. It proved that the affinity between ADH and NAD+was weaker than that of NADH.