Heterologous production of extreme alkaline thermostable NAD+-dependent formate dehydrogenase with wide-range pH activity from Myceliophthora thermophila

Abstract NAD + -dependent formate dehydrogenase(s) (EC 1.2.1.2, FDH) catalyzes the interconversion of formate anion to carbon dioxide coupled with the conversion of NAD + or NADH. FDHs attract significant attention in biotechnology due to their potential applications in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, a new FDH from thermophilic fungus Myceliophthora thermophile ( Mt FDH) was characterized. The gene of the enzyme was synthesised, cloned, expressed in E. coli , as 6His-tagged protein, and purified to homogeneity by metal chelate affinity chromatography. Kinetic analysis suggested that Mt FDH exhibits higher catalytic efficiency on NaHCO 3 compared to formate. Notable, recombinant Mt FDH displays a pH optimum for the conversion of formate anion to carbon dioxide at extreme alkaline pH (pH 10.5). Thermal stability analysis showed that the enzyme displays good thermostability with T m 48 °C. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the D-specific 2-hydroxy acid dehydrogenases family. The active-site residues are well conserved compared to other homologous FDHs. The results of the present work provide new knowledge on the structure, function and diversity of FDHs and indicate that Mt FDH possess a huge potential for CO 2 reduction or NADH generation and under extreme alkaline conditions.