초록
Hydrogenase is the key point of H<SUB>2</SUB>-based biotechnology. However, the O<SUB>2</SUB>-sensitivity largely hinders its applications in biofuel cells and biological H<SUB>2</SUB> production. Therefore, substantial breakthrough on understanding the molecular basis of O<SUB>2</SUB>-sensitivity and developing more O<SUB>2</SUB>-tolerant hydrogenases are urgently required. In this study, we found adding extra cysteines to the vicinity of the proximal Fe-S cluster to the NiFe active centre could largely enhance oxygen tolerance of hydrogen-evolving hydrogenase 3 from Klebsiella oxytoca HP1 (KoHyd3), through homologous sequence comparison and site-directed mutagenesis. Ratio of aerobic hydrogen yield to anaerobic hydrogen yield (RHH) of Gly47Cys (Gly47 was replaced with Cys47), Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys-Gly120Cys (double exchange) were increased by 46.99%, 42.15%, 59.19%, 44.74% and 78.72%, respectively, comparing with that of wild type. Moreover, TiO<SUB>2</SUB>-KoHyd3 (Gly47Cys, Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys-Gly120Cys) particles acted well in UV light-driven H<SUB>2</SUB> production from water. These results revealed that extra cysteines nearby Fe-S clusters had significant effects on oxygen tolerance of KoHyd3. It also provided a promising way to produce O<SUB>2</SUB>-tolerant hydrogenase as biocatalysts in biofuel cells or H<SUB>2</SUB> production by photolysis of water.