Cloning, Nucleotide Sequencing, and Expression in<i>Escherichia coli</i>of the Gene for Formate Dehydrogenase of<i>Paracoccus</i>sp. 12-A, a Formate-assimilating Bacterium

Abstract

The gene for the NAD-dependent formate dehydrogenase (FDH) of Paracoccus sp. 12-A, a formate-assimilating bacterium, was cloned through screening of the genomic library with activity staining. The FDH gene included an open reading frame of 1,200 base pairs, and encoded a protein of 43,757 Da, which had high amino acid sequence identity with known FDHs, in particular, with bacterial enzymes such as those of Moraxella sp. (86.5%) and Pseudomonas sp. 101 (83.5%). The gene was highly expressed in Escherichia coli cells using an expression plasmid with the pUC ori and tac promoter. The recombinant enzyme was somewhat inactive in the stage of the cell-free extract, but its activity markedly increased with purification, in particular, with the step of heat-treatment at 50 degrees C. The purified enzyme showed essentially the same properties as the enzyme from the original Paracoccus cells.