RESUMO
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.
Assuntos
Formiato Desidrogenases/genética , Paracoccus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Formiato Desidrogenases/química , Formiato Desidrogenases/isolamento & purificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Paracoccus/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Two forms of NAD-dependent D-mandelate dehydrogenase (D-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different K(m) and V(max) values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.
