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The enzyme CbA5H, a [FeFe]‐hydrogenase from Clostridium beijerinckii, has previously been shown to survive exposure to oxygen, making it a promising candidate for biotechnological applications. Thus far [NiFe]‐hydrogenases are typically considered for such applications, due to the superior O2‐tolerance and therefore simplified enzyme handling. However, methods for production of [FeFe]‐hydrogenases are generally more successful than for other classes of hydrogenases, therefore in this work we focus on demonstrating scalable CbA5H production, and report results with active enzyme prepared in bioreactors (up to 10 L) with >20‐fold improvement in purified enzyme yield. We then go on to confirm excellent H2/H+‐cycling activity of the air‐purified protein, highlighting that CbA5H can be prepared and isolated without the need for complex and expensive infrastructure. Next, we demonstrate good stability of the air‐purified CbA5H both in solution assays, and as a heterogenous catalyst system when immobilized on a carbon support. Finally, we successfully implement this enzyme within previously demonstrated biotechnologies for flavin and NADH recycling, highlighting its relevance in chemical synthesis, and we demonstrate production of an important API precursor, 3‐quinuclidinol at >0.4 g scale in standard benchtop hydrogenation infrastructure, with >100,000 CbA5H turnovers over 18 operational hours. [ABSTRACT FROM AUTHOR]

Hydrogenase-mediated reduction of flavin mononucleotide by H2 is exploited to enable cleaner application of nitroreductase enzymes for reduction of aromatic nitro functional groups. This turns the overall reaction into a biocatalytic hydrogenation. Use of flavincontaining nitroreductases in industrial biotechnology typically relies upon NADH or NADPH as reductant, together with glucose dehydrogenase and glucose as a regeneration system for the reduced nicotinamide cofactor, with 3 equivalents of the carbon-intensive glucose required for a single 6-electron nitro to amine conversion. We show here that reduced flavin mononucleotide is an alternative reductant for nitroreductases, and by combining this with H2-driven recycling of reduced flavin, we avoid glucose, thereby enabling atom-efficient biocatalytic nitro reductions. We compare this biocatalytic system, via green chemistry metrics, to existing strategies for biocatalytic nitro-group reductions, particularly with respect to replacing glucose with H2 gas.We take steps towards demonstrating industrial viability: we report an overexpression system for E. coli hydrogenase 1, giving a 12-fold improvement in enzyme yield; we show a reaction in which the hydrogenase exhibits > 26,000 enzyme turnovers; and we demonstrate reasonable solvent tolerance of the hydrogenase and flavin reduction system which would enable reaction intensification. [ABSTRACT FROM AUTHOR]

This practitioner protocol describes the synthesis of a family of deuterated nicotinamide cofactors: [4S‐2H]NADH, [4R‐2H]NADH, [4‐2H2]NADH and [4‐2H]NAD+. The application of a recently developed H2‐driven heterogeneous biocatalyst enables the cofactors to be prepared with high (>90%) 2H‐incorporation with 2H2O as the only isotope source. [ABSTRACT FROM AUTHOR]

The O2‐tolerant NAD+‐reducing hydrogenase (SH) from Ralstonia eutropha (Cupriavidus necator) has already been applied in vitro and in vivo for H2‐driven NADH recycling in coupled enzymatic reactions with various NADH‐dependent oxidoreductases. To expand the scope for application in NADPH‐dependent biocatalysis, we introduced changes in the NAD+‐binding pocket of the enzyme by rational mutagenesis, and generated a variant with significantly higher affinity for NADP+ than for the natural substrate NAD+, while retaining native O2‐tolerance. The applicability of the SH variant in H2‐driven NADPH supply was demonstrated by the full conversion of 2‐methyl‐1‐pyrroline into a single enantiomer of 2‐methylpyrrolidine catalysed by a stereoselective imine reductase. In an even more challenging reaction, the SH supported a cytochrome P450 monooxygenase for the oxidation of octane under safe H2/O2 mixtures. Thus, the re‐designed SH represents a versatile platform for atom‐efficient, H2‐driven cofactor recycling in biotransformations involving NADPH‐dependent oxidoreductases. [ABSTRACT FROM AUTHOR]

The present study considers the ways in which redox enzyme modules are coupled in living cells for linking reductive and oxidative half-reactions, and then reviews examples in which this concept can be exploited technologically in applications of coupled enzyme pairs. We discuss many examples in which enzymes are interfaced with electronically conductive particles to build up heterogeneous catalytic systems in an approach which could be termed synthetic biochemistry. We focus on reactions involving the H+/H2 redox couple catalysed by NiFe hydrogenase moieties in conjunction with other biocatalysed reactions to assemble systems directed towards synthesis of specialised chemicals, chemical building blocks or bio-derived fuel molecules. We review our work in which this approach is applied in designing enzyme-modified particles for H2-driven recycling of the nicotinamide cofactor NADH to provide a clean cofactor source for applications of NADH-dependent enzymes in chemical synthesis, presenting a combination of published and new work on these systems. We also consider related photobiocatalytic approaches for light-driven production of chemicals or H2 as a fuel. We emphasise the techniques available for understanding detailed catalytic properties of the enzymes responsible for individual redox half-reactions, and the importance of a fundamental understanding of the enzyme characteristics in enabling effective applications of redox biocatalysis. [ABSTRACT FROM AUTHOR]

We describe the implementation of a system of immobilised enzymes for H2-driven NADH recycling coupled to a selective biotransformation to enable H2-driven biocatalysis in flow. This approach represents a platform that can be optimised for a wide range of hydrogenation steps and is shown here for enantioselective ketone reduction and reductive amination. [ABSTRACT FROM AUTHOR]

Direct electrochemical methods have been productive in revealing mechanistic details of catalysis by a range of metalloenzymes including hydrogenases and carbon and nitrogen cycling enzymes. In this approach, termed protein film electrochemistry, the protein is attached or adsorbed on the electrode surface and exchanges electrons directly, providing precise control over redox states or catalysis and avoiding diffusion-limited electron transfer. The ‘edge’ surface of pyrolytic graphite has proved to be a particularly good surface for adsorption of proteins in electroactive conformations. We now describe development of an approach that combines the precise control achieved in direct electrochemical measurements at a graphite electrode with surface infrared (IR) spectroscopic analysis of chemistry occurring at metallocentres in proteins. Hydrogenases are of particular interest: their unusual organo-metallic active sites – iron or nickel-iron centres coordinated by CO and CN−– give rise to IR ν(CO) and ν(CN) bands that are detected readily because these ligands are strong vibrational oscillators and are sensitive to changes in electron density and coordination at the metals. Small diatomic species also bind as exogenous ligands (as substrate, product, activator or inhibitor) to a range of other important metalloproteins, and understanding their reactivity and binding selectivity is critical in building up a multidimensional picture of enzyme chemistry and evolutionary history. The surface IR spectroelectrochemical approach we describe is based around Attenuated Total Reflectance (ATR) mode sampling of a film of pyrolytic graphite particles modified with a protein of interest. The particle network extends the electrode into three-dimensional space, providing sufficient adsorbed protein for spectroscopic analysis under precise electrochemical control. This strategy should open up new opportunities for detection of redox-dependent chemistry at metal centres in proteins, including short-lived catalytic intermediates and time-resolved details of catalysis and inhibition. [ABSTRACT FROM AUTHOR]

Heterogeneous biocatalytic hydrogenation is an attractive strategy for clean, enantioselective C[double bond, length as m-dash]X reduction. This approach relies on enzymes powered by H 2 -driven NADH recycling. Commercially available carbon-supported metal (metal/C) catalysts are investigated here for direct H 2 -driven NAD + reduction. Selected metal/C catalysts are then used for H 2 oxidation with electrons transferred via the conductive carbon support material to an adsorbed enzyme for NAD + reduction. These chemo-bio catalysts show improved activity and selectivity for generating bioactive NADH under ambient reaction conditions compared to metal/C catalysts. The metal/C catalysts and carbon support materials (all activated carbon or carbon black) are characterised to probe which properties potentially influence catalyst activity. The optimised chemo-bio catalysts are then used to supply NADH to an alcohol dehydrogenase for enantioselective (>99% ee) ketone reductions, leading to high cofactor turnover numbers and Pd and NAD + reductase activities of 441 h -1 and 2347 h -1 , respectively. This method demonstrates a new way of combining chemo- and biocatalysis on carbon supports, highlighted here for selective hydrogenation reactions., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

This practitioner protocol describes the synthesis of a family of deuterated nicotinamide cofactors: [4S- 2 H]NADH, [4R- 2 H]NADH, [4- 2 H 2 ]NADH and [4- 2 H]NAD + . The application of a recently developed H 2 -driven heterogeneous biocatalyst enables the cofactors to be prepared with high (>90%) 2 H-incorporation with 2 H 2 O as the only isotope source., (© 2021 The Authors. Journal of Labelled Compounds and Radiopharmaceuticals published by John Wiley & Sons Ltd.)

We describe the use of carbon as a versatile support for H 2 -driven redox biocatalysis for NADH-dependent CX bond reductions in batch and flow reactions. In each case, carbon is providing an electronic link between enzymes for H 2 oxidation and reduction of the biological cofactor NAD + as well as a support for a multi-enzyme biocatalysis system. Carbon nanopowders offer high surface areas for enzyme immobilization and good dispersion in aqueous solution for heterogeneous batch reactions. Difficulties in handling multi-wall carbon nanotubes in aqueous solution are overcome by growing them on quartz tubes to form carbon nanotube column reactors, and we show that these facilitate simple translation of H , as well as a support for a multi-enzyme biocatalysis system. Carbon nanopowders offer high surface areas for enzyme immobilization and good dispersion in aqueous solution for heterogeneous batch reactions. Difficulties in handling multi-wall carbon nanotubes in aqueous solution are overcome by growing them on quartz tubes to form carbon nanotube column reactors, and we show that these facilitate simple translation of H 2 -driven biocatalysis into flow processes. Using this flow reactor design, high conversions (90%) and total enzyme turnover numbers up to 54,000 could be achieved. Use of an entirely heterogeneous biocatalysis system simplifies recovery and re-use of the enzymes; combined with highly atom-efficient cofactor recycling, this means that high product purity can be achieved. We demonstrate these methods as platform approaches for overcoming challenges with NADH-dependent biocatalysis., (© 2020 Elsevier Inc. All rights reserved.)

We describe the implementation of a system of immobilised enzymes for H 2 -driven NADH recycling coupled to a selective biotransformation to enable H 2 -driven biocatalysis in flow. This approach represents a platform that can be optimised for a wide range of hydrogenation steps and is shown here for enantioselective ketone reduction and reductive amination.

Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase., (This journal is © The Royal Society of Chemistry 2012)