Coproduction of Biohydrogen and Gluconate by an Enzyme Cocktail Composed of an Artificial Electron Transfer Chain and a Recombinant Thermostable [NiFe]-Hydrogenase

A cytoplasmic [NiFe]-hydrogenase from the anaerobic hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk soluble hydrogenase, TkSH), whose physiological substrate is NADP(H), is an oxygen-sensitive enzyme consisting of four subunits (HyhBGSL). The recombinant production of oxygen-sensitive complex enzymes (e.g., hydrogenase, nitrogenase, and CO2 reductase) remains a large technical challenge. Here, we overexpressed TkSH in T. kodakarensis KOD1 by a factor of 470 and purified the enzyme by adding a 12 × His tag through affinity chromatography. The purified recombinant TkSH (rTkSH) showed a maximum activity (H2 oxidation using benzyl viologen) of 1066 U/mg at 80 °C, comparable to the activity of the native TkSH, and had almost twice the specific activity of the Pyrococcus furiosus soluble hydrogenase I at 80 °C. The half-life times of thermal deactivation of rTkSH were 4800 ± 170 min at 80 °C and 45 ± 4 min at 90 °C. Additionally, we designed a novel enzyme cocktail containing three thermostable enzymes (i.e., NAD-based glucose dehydrogenase, diaphorase, and rTkSH) and an abiotic electron mediator. These three enzymes, along with BV and NAD+, can form an artificial electron transfer chain from glucose to hydrogen. This work also demonstrated the use of this recombinant enzyme for the coproduction of H2 and gluconate from NADH under anaerobic conditions, replacing NADH oxidation mediated by NADH oxidase under aerobic conditions.