Study on the Methyl Viologen-Mediated Electrochemical Reduction of Coenzyme NAD+ in the Triton X-100-Stabilized Methyl tert -Butyl Ether-in-H2O Microemulsion

Abstract An oil-in-water microemulsion is an excellent candidate medium for the electrochemical regeneration of the coenzyme NADH and its subsequent coupled conversion application due to its good electrical conductivity and solubilization capacity for hydrophobic substrates. In this work, the reduced methyl viologen (MV+•)-mediated electrochemical reduction of coenzyme NAD+ was studied in a Triton X-100 (TX-100)-stabilized methyl tert-butyl ether (MTBE)-in-water microemulsion. The effects of the surfactant concentration and the oil content on the electrochemical behavior of the MV2+/MV+• couple and the activity of diaphorase (DH) were systematically investigated. The results show that in the microemulsion, the couple exhibits excellent reversibility. Compared with the aqueous buffer system, the couple in the microemulsion system has a more positive formal potential and a lower redox peak current, depending on the levels of both the surfactant and the oil. The positive shift of the formal potential is attributed to the stabilizing effect of the surfactant aggregates on the relatively hydrophobic MV+• species, and the decrease of the redox currents is ascribed to the decrease of the electroactive area of the electrode due to the adsorption of the surfactant and the oil as well as the decrease of the diffusion coefficients of MV species associated with or solubilized in the surfactant aggregates. The activity of DH in the microemulsion system also depends on the TX-100 concentration and the MTBE content. Increases in both [TX-100] and [MTBE] result in a decrease in DH activity. This phenomenon could be explained based on the [TX-100]/[MTBE]-dependent changes in the local acidity and the local concentration of the substrate DCPIP in the microemulsions. Based on cyclic voltammetry, the MV+•-mediated electroreduction of NAD+ in the TX-100-stabilized MTBE/H2O (0.1 M PB, pH = 7.0) microemulsion was studied. For the microemulsion system containing 0.5 mM MV2+, 5 mM NAD+, and 26.6 μM DH, when a scan rate of 2 mV s–1 was applied, a typical steady-state S-shaped CV curve was obtained. UV–vis spectroscopy confirms that the NADH generated by the mediated reaction is bioactive. By coupling the MV+•-mediated NADH electrochemical regeneration reaction with the horse liver alcohol dehydrogenase (HLADH)-catalyzed reduction of the hydrophobic substrate 4-phenyl-2-butanone, the enantiomerically pure (S)-4-phenyl-2-butanol can be selectively obtained. After 12 h, the yield of the product in the microemulsion is approximately 10 times that in the DMSO-buffer mixed homogeneous system. This difference in yield is caused by the differences in enzyme activity and stability, as well as in the substrate local concentration and reactivity in the two systems. The present study indicates that the TX-100-stabilized MTBE-in-water microemulsion is a suitable medium for the electrochemical regeneration of NADH and the electroenzymatic conversion of water-insoluble substrates catalyzed by NADH-dependent oxidoreductases.