*5.3. Enzyme Cascades*

Typically, a biocatalytic cascade reaction is a reaction system in which two or more transformations are performed simultaneously [179]. Through biocatalytic cascades, the isolation of reaction intermediates is circumvented, saving time and the use of reagents. The synthesis of chemicals with unstable intermediates is feasible as the isolation of the intermediates is not necessary [180]. Enzyme cascades can enhance the current density as the oxidation of the substrate in a sequential manner enables more electrons to be extracted [34]. Dong et al. designed a biphasic bioelectrocatalytic system that used a reaction cascade to prepare (R)-ethyl-4-cyano-3-hydroxybutyrate from ethyl 4-chloroacetoacetate using alcohol dehydrogenase and halohydrin dehalogenase (Figure 12). Ethyl 4-chloroacetoacetate was reduced to (S)-4-chloro-3-hydroxybutanoate using alcohol dehydrogenase which was then converted to its R enantiomer using halohydrin dehalogenase. Efficient NADH regeneration was performed using diaphorase with the redox polymer cobaltocene-modified poly-(allylamine) as a mediator on the electrode surface [181]. In total, 85% of substrate was converted into (R)-ethyl-4-cyano-3-hydroxybutyrate, an 8.8 higher yield than that achieved with a single-phase system.

**Figure 12.** Schematic diagram of the biphasic bioelectrocatalytic reaction cascade for the synthesis of (R)-ethyl-4-cyano-3-hydroxybutyrate. Reprinted from [181] Copyright (2020) with permission from American Chemical Society.

Abdellaoui et al. combined aldehyde deformylating oxygenase, immobilised on the surface of an electrode, with NAD+-dependent alcohol dehydrogenase to create an enzymatic cascade reaction for the conversion of fatty alcohols to aldehydes followed by the decarbonylation to produce alkenes (Figure 13) [182].

**Figure 13.** Schematic diagram of a bioelectrochemical enzymatic cascade for the preparation of alkenes [182].

Kuk et al. constructed a photoelectrochemical system for the production of methanol by a multienzyme cascade (a three-dehydrogenase cascade system) and the efficient regeneration of the NADH via visible-light assistance (Figure 14) [183].

Chen et al. developed an enzyme cascade to produce chiral amines from nitrogen. NH<sup>3</sup> was produced from N<sup>2</sup> via nitrogenase and then used, with L-alanine dehydrogenase, to produce alanine from pyruvate. The desired chiral amines were produced via the ω-transaminase transfer of an amino group from alanine. Pyruvate was generated as a by-product and converted to alanine using L-alanine dehydrogenase. The system was used for the successful amination of the substrates, 4-phenyl-2 butanone, 4-methyl methoxy phenyl acetone, phenoxyacetone, 2-pentanone, methoxyacetone and 2-octanone with an enantiomeric excess >99% (Figure 15) [52].

ω

**Figure 14.** Schematic diagram of the light-assisted synthesis of methanol from CO<sup>2</sup> via an enzyme cascade (formaldehyde dehydrogenase, formate dehydrogenase, and alcohol dehydrogenase) [183].

**Figure 15.** A schematic of bioelectrochemical asymmetric amination [52].

In a sequential catalytic cascade, noble metal catalysts can also be used. For example, the biocatalytic reduction of nitrite to ammonia is slow in comparison with metal catalysts [184]. Duca et al. fabricated a hybrid electrochemical cascade that combined nitrate reductase and Pt nanoparticles on a carbon electrode surface. The reduction of nitrate to nitrite and nitrite to ammonia was carried out by the Pt nanoparticles and nitrate reductase, respectively [185].
