Cascade reactions with two non-physiological partners for NAD(P)H regeneration via renewable hydrogen.

Abstract

An attractive application of hydrogenases, combined with the availability of cheap and renewable hydrogen (i.e., from solar and wind powered electrolysis or from recycled wastes), is the production of high-value electron-rich intermediates such as reduced nicotinamide adenine dinucleotides. Here, the capability of a very robust and oxygen-resilient [FeFe]-hydrogenase (CbA5H) from Clostridium beijerinckii SM10, previously identified in our group, combined with a reductase (BMR) from Bacillus megaterium (now reclassified as Priestia megaterium) was tested. The system shows a good stability and it was demonstrated to reach up to 28±2nmol NADPH regenerated s-1mg of hydrogenase-1 (i.e., 1.68±0.12 Umg-1, TOF: 126±9min-1) and 0.46±0.04nmol NADH regenerated s-1mg of hydrogenase-1 (i.e., 0.028±0.002 Umg-1, TOF: 2.1±0.2min-1), meaning up to 74mg of NADPH and 1.23mg of NADH produced per hour by a system involving 1mg of CbA5H. The TOF is comparable with similar systems based on hydrogen as regenerating molecule for NADPH, but the system is first of its kind as for the [FeFe]-hydrogenase and the non-physiological partners used. As a proof of concept a cascade reaction involving CbA5H, BMR and a mutant BVMO from Acinetobacter radioresistens able to oxidize indole is presented. The data show how the cascade can be exploited for indigo production and multiple reaction cycles can be sustained using the regenerated NADPH.