We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H2. Coupling this system to an ene reductase, we show quantitative conversion of N-ethylmaleimide to N-ethylsuccinimide. The biocatalyst system retained >50% activity after 7 h.@en

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H₂ as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25–70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH₂ or FADH₂ regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme “ene-reductases” to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min⁻¹ and total turnover numbers up to 20 200 were recorded during flavin recycling.

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