*2.1. Mediated Electron Transfer*

⁺ − ⁺ The redox properties of the mediator and of the enzymes need to match in order to have an efficient rate of electron transfer. The reduction potential of the mediator (E◦ mediator) should be more positive than the redox potential of the prosthetic group of the enzyme (E◦ enzyme) for oxidation to occur, while for reduction, E◦ mediator should be more negative than E◦ enzyme. The mediator should undergo fast electron transfer both at the surface of the electrode and with the active site of the enzyme [34,35]. Properties such as mediator stability, selectivity and the electrochemical reversibility of the redox couple also need to be considered [33]. Approximately 90% of oxidoreductases utilised nicotinamide coenzymes to catalyse reactions. For example, alcohol dehydrogenase (ADH) uses the cofactor nicotinamide adenine dinucleotide (NAD+) to catalyse the oxidation of substrates such as cyclohexanol [36], primary and secondary alcohols [37] and methionine [38]. Due to the high cost of NAD(P)+, it is important to use an efficient regeneration system. The direct regeneration of nicotinamide coenzymes at unmodified electrode surfaces requires the use of high overpotentials that can result in the formation of dimers of NAD(P)<sup>+</sup> that are enzymatically inactive. Mediators such as methylene green, methylene blue [39–42], 2,2-azino-bis-(3-ethyl-benzo-thiazoline-6-sulfonic acid (ABTS) [43], naphthoquinone [44], ferrocenes [45,46], and viologens [47] are widely used to regenerate NAD+, etc. For example; an indirect NAD(P)<sup>+</sup> regeneration system utilised ABTS to oxidise alcohols with a turnover of 1200 h−<sup>1</sup> [48]. A number of enzymes such as dihydrolipoamide dehydrogenase (DLD), ferredoxin-NADP<sup>+</sup> reductase (FNR) have been used to regenerate NAD [49–51]. Chen et al. used reduced methyl viologen (MV•+) and diaphorase for effective NADH regeneration in the production of NH<sup>3</sup> and for asymmetric amination [52]. The mediated electrochemical regeneration of NADH through methyl viologen was also used in the enzymatic reduction of ketones [53]. Badalyan et al. used the negatively charged viologen derivative [(SPr)2V • ]¯ as an effective mediator for nitrogenase electrocatalysis [54]. Tosstorff et al. used three different mediators, cobalt sepulchrate, safranin T, and [Cp\*Rh(bpy)(H2O)]2<sup>+</sup> in an NADH regeneration system that was combined with old yellow enzyme for the asymmetric reduction of C=C [55].

Redox polymers are used to wire enzymes and also act as an immobilisation matrix facilitating electron transfer between the electrode and enzyme by transferring electrons within the polymer matrix. Redox polymers possess a polymeric backbone with attached redox mediators [56,57]. Early reports focused on redox polymers based on osmium complexes with polymer backbones comprised of poly(vinyl imidazole)s and poly(vinyl pyridine)s [58,59]. A range of other redox polymers based on poly(vinylalcohol), poly(vinyl imidazole), poly(vinyl pyridine) and poly(ethylenimine) modified with redox species such as ferrocene [60], cobaltocene [61], viologen [62] and quinone [44] have been described. For example, Alkotaini et al. used a redox polymer N-benzyl-N′ -propyl-4,4′ -bipyridiniummodified linear polyethylenimine benzylpropylviologen (BPV-LPEI) and diaphorase for effective cofactor regeneration for the bioelectrosynthesis of the bioplastic, polyhydroxybutyrate (PHB) [62]. Szczesny et al. used a viologen-modified redox polymer for the electrical wiring of W-dependent formate dehydrogenase to reduce CO<sup>2</sup> gas to formate [63], whereas Yuan et al. described the electrochemical regeneration of NADH using a cobaltocene-modified poly(allylamine) redox polymer and diaphorase. The system produced 1,4-NADH with high yields close to 100% and turnover frequencies between 2091 and 3680 h−<sup>1</sup> at different temperatures. The system was coupled with ADH to produce methanol and propanol (Figure 2) [57]. ′ ′ −

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**Figure 2.** A schematic of the designed system for the electrochemical regeneration of NADH [57].
