Purification and Characterization of Formate Dehydrogenase

Anaylobacter aguaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated. The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa. The specific activity was 9.5 ulmg, and the enzyme was optimum at pH 6.3 and 500C, most stabte at pH 7.0, and stable at 50"C or lovver. The apparent K. yalues for formate and NAD' were 2.4 and O.057mM, respectiye]y. The enzyme was specific to formate and was inhibited by SH reage"ts and heavy metal ions. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 43,895; this gene was highly expressed in E. coli cells. The FDH had high identity to other FDHs, i.e. , those ef Ilseudomonas, Mycobacterium, Morcvcella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.