NAD(+)-dependent formate dehydrogenase (FDH) enzyme catalyses the NAD(P)(+)-dependent interconversion of formate anion to carbon dioxide coupled with the conversion of NAD(P)(+) to NAD(P)H. NADH is widely used as cofactor by enzymes utilized in the biocatalytic synthesis of chiral pharmaceutical intermediates and chemical compounds. For this reason, FDH is used in the enzymatic synthesis of optically active prochiral carbonyl compounds in order to regenerate the NADH. For wider applicability in industrial usage of FDH, there is a demand for higher stability of the enzyme. We report here the overexpression in Escherichia coli and subsequent characterization of FDH from thermophilic fungus Chaetomium thermophilum (CtFDH). CtFDH was found to be more acidic than the typical FDHs from various sources, having its optimum at pH 5, and it also showed moderate thermostability at pH 5. The modelling of the enzyme and formate in the active site indicated that the enzyme functions with metal-independent hydride shift in the same way as the other NAD(+)-dependent FDHs of this enzyme group. Dimer interface properties were also analyzed. CtFDH shows potential being a template for engineering higher thermostability for applications in acidic conditions. (C) 2015 Elsevier B.V. All rights reserved.