Regeneration of the NADH Cofactor by a Rhodium Complex Immobilized on Multi-Walled Carbon Nanotubes

Abstract

The regeneration of the enzymatic cofactor nicotinamide adenine dinucleotide (NADH) by rhodium-based catalysts such as [Rh(Cp * )(bpy)Cl] + (Cp * = pentamethylcyclopentadienyl, bpy = 2,2 -bipyridine) and derivatives have previously been studied extensively in solution. In this work, we report a synthetic route of a rhodium complex with a pyrene-substituted phenanthroline ligand (pyr-Rh). The immobilization of the pyr-Rh complex was accomplished on multi-walled carbon nanotubes (MWCNTs) via π-π stacking to obtain effective and durable indirect electrochemical regeneration of NADH. Cyclic voltammetry and amperometry were used to demonstrate the electrochemical activity of the surface-confined pyr-Rh complex. The loading quantity of the pyr-Rh complex was found to be 47 ± 2 nmol/mg of MWCNTs. The reusability of the electrodes modified with the pyr-Rh complex was investigated and an average turnover frequency of 3.6 ± 0.1 s −1 over ten cycles in the presence of 2 mM nicotinamide adenine dinucleotide (NAD + ) was observed. Lastly, malate dehydrogenase (MDH), a NADH-dependent enzyme, was evaluated in the presence of the immobilized pyr-Rh complex to confirm the catalyst's capability to regenerate biologically active NADH for biocatalysis. In biological systems, the cofactor nicotinamide adenine dinucleotide (NAD + ) and its reduced form (NADH) are of significance in assisting oxidoreductase enzymes to catalyze redox reactions.1 The efficient in situ regeneration of NADH from NAD + is valuable in biocatalysis due to the stoichiometric amount required for NADHdependent enzymes along with its relatively high cost.1 It was reported that the combination of three NAD + -dependent dehydrogenases (formate dehydrogenase, formaldehyde dehydrogenase and alcohol dehydrogenase) is capable of generating methanol from CO 2 by reversing the original enzymatic reaction direction. [2][3][4][5] In this enzyme cascade, one molecule of methanol is produced at the cost of three equivalents of NADH. It is environmentally beneficial to convert CO 2 into useful biofuel by enzymatic reactions, but the high cost of stoichiometric NADH consumption makes this process impractical. Therefore, a regeneration system is necessary. Galarneau and coworkers also investigated the application of this three NADH-dependent dehydrogenases cascade and the use of the enzymatic regeneration of NADH by phosphite dehydrogenase (PTDH). 6 In comparison to enzyme systems which do not incorporate a NADH regeneration system, their polyenzymatic system had a higher activity in generating methanol from carbon dioxide.6 Practical cofactor regeneration is necessary for maintaining stable concentrations of NAD + /NADH, which provides a driving force for the desired enzymatic reactions. The reduced NADH cofactor can be regenerated enzymatically, 7-9 chemically, 10 photochemically 11-13 and electrochemically. 14,15 Two methods are commonly used when electrochemically converting NAD + to NADH: direct and indirect regeneration. In the direct regeneration of NADH, the cofactor N...

Direct enzymatic bioelectrocatalysis: differentiating between myth and reality

“…Most commonly, CO 2 reduction has been explored with the aim of producing formate or alkanes/alcohols, although NAD þ reduction to NADH is frequently attempted where NAD-dependent enzymes can support reductive reactions [22][23][24]; Vincent and co-workers [25] have also demonstrated that the coimmobilization of hydrogenase and NAD þ -reducing proteins onto conductive particles can facilitate H 2 -driven NADH production. Recent research has also explored the possibility of reducing dinitrogen (N 2 ) to an important chemical commodity, ammonia (NH 3 ), while simultaneously producing electrical energy [26].…”

Strategies for Bioelectrochemical CO2 Reduction

“…It has been reportedt hat the combination of the three NADH-dependent enzymes, formate dehydrogenase (FDH), formaldehyde dehydrogenase (FLDH), and alcohol dehydrogenase (ADH) enables the production of methanol from CO 2 in am etabolically reversedr eactionc ascade. [33] To increaset he efficiency of this cascade, efforts have been made to drive the equilibrium toward CO 2 reduction using electrochemical NADH regeneration systems. [34] Lee and colleagues have screened the various protein homologs to determinet he variants with the highest activities in the reductive direction; [35] the results of this work are certaint oi nform further efforts involving this enzyme cascade.…”

A Simple Procedure to Fabricate Paper Biosensor and Its Applicability—NADH/NAD+ Redox System

“…One strategy employed to overcome these difficulties (fouling and overvoltage and side reactions) was the use of mediator-modified electrodes, where the mediators are used to shuttle electrons from NADH to the electrode surface and allow electron transport between them [5,[9][10][11][12][13][14][15][16]. Some mediators (electrocatalysts) were immobilized on the electrode surface by covalent attachment, electrochemical polymerization, incorporation in carbon paste, adsorption, self-assembly and via entrapment in polymeric matrices [4,[17][18][19]. Another strategy is modification of the electrode surface with a polymeric substance, using electrodes modified with carbon nanotubes, nanofibers or using enzymatic methods that follow bioelectrocatalytic reaction [2,4,13,15,16,[20][21][22][23].…”