Purification and Characterization of Formate Dehydrogenase fromAncylobacter aquaticusStrain KNK607M, and Cloning of the Gene

Abstract

Ancylobacter aquaticus 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 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower. The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively. The enzyme was specific to formate and was inhibited by SH reagents 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 of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.

Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7

“…Fresh Blastocystis isolates, though, have been demonstrated to cause significant cytopathology of Chinese hamster ovary cells (CHO), adenocarcinoma HT-29 cells and rat intestinal epithelia (IEC-6) cells in culture (Puthia et al, 2006;Walderich et al, 1998). Metronidazole, paromomycin and ketoconazole have been used as therapeutic agents with mixed success (Zierdt et al, 1983;Dunn & Boreham, 1991;Haresh et al, 1999;Nanba et al, 2003). Although there have been some advances made concerning the morphology and cytochemistry of the organism, little is known of the biochemistry.…”

Expression inEscherichia coliof an Unnamed Protein Gene fromAspergillus oryzaeRIB40 and Cofactor Analyses of the Gene Product as Formate Oxidase

“…There are many reports on characterization of FDH from various microorganisms and higher plants and on the cloning and expression of their genes. [1][2][3][4][5] On the other hand, only two FODs have been purified from Debaryomyces vanrijiae MH201 and Aspergillus nomius IRI013 and characterized. [6][7][8] These FODs show similar UV-visible spectra, suggesting the presence of similar cofactors in their molecules.…”

Purification and Characterization of an α-Haloketone-resistant Formate Dehydrogenase fromThiobacillussp. Strain KNK65MA, and Cloning of the Gene

“…The use of NAD-dependent FDH, [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] which catalyzes the oxidation of formate to carbon dioxide with the reduction of NAD ＋ to NADH, is suitable for practical use in the regeneration of the cofactor, because no by-products from this regeneration reaction accumulate in the reaction mixture; therefore, the product is easily isolated, the amount of waste is very low, and the thermodynamic equilibrium of the reaction is favorable. Various NAD-dependent FDHs from plants, 10,11) methylotrophic yeasts, [12][13][14][15][16] and bacteria [17][18][19][20][21][22][23][24] have been investigated.…”