Improved purification of Candida boidinii formate dehydrogenase.
Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD+/2 mM Na2SO3). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).
Labrou
N E
NE
Department of Agricultural Biotechnology, Agricultural University of Athens, Greece. lambrou@auadec.aua.gr
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D005560:Formate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight
D005560:Formate Dehydrogenases