Li, Yifei; Liu, Meixia; Zheng, Qian; Zhai, Zhenyu; Ren, Shujing; Sun, Junsong; Zhang, Yi-Heng P. Job

ACS SUSTAINABLE CHEMISTRY & ENGINEERING

2025, 19,

Li, Yifei; Liu, Meixia; Zheng, Qian; Zhai, Zhenyu; Ren, Shujing; Sun, Junsong; Zhang, Yi-Heng P. Job

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 x His tag through affinity chromatography. The purified recombinant TkSH (rTkSH) showed a maximum activity (H-2 oxidation using benzyl viologen) of 1066 U/mg at 80 degrees 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 degrees C. The half-life times of thermal deactivation of rTkSH were 4800 +/- 170 min at 80 degrees C and 45 +/- 4 min at 90 degrees 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 H-2 and gluconate from NADH under anaerobic conditions, replacing NADH oxidation mediated by NADH oxidase under aerobic conditions.