Purification, characterization, and potential applications of formate oxidase from Debaryomyces vanrijiae MH201

Abstract

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of alphaalpha, alphabeta, and betabeta subunits with apparently similar MM. The preparation acted on formate with K (m) and V (max) values of 11.7 mM and 262 micromol min(-1) mg(-1), respectively, at pH 4.5 and 25 degrees C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35 degrees C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4 degrees C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml(-1) of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml(-1) of a microbial aldehyde oxidase and 100 U ml(-1) of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.

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

“…[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.…”

“…FOD activity was measured as described previously. 6) One enzyme unit was defined as the amount of enzyme that produces 1 mmole hydrogen peroxide per min at pH 4.5 at 30 C, when the concentration of formate is 5 mM. To determine the optimum pH, the FOD activities at 30 C were measured at various pH levels.…”

“…D. vanrijiae MH201 was cultivated, and FOD was purified from the cell-free extract, as described previously. 6) Rabbit anti-serum against purified yeast FOD was prepared by Keari. (Osaka, Japan).…”

“…These results indicate that FOD AO was composed of two identical subunits, as are the D. vanrijiae FOD and the FOD from A. nomius IRI013. 6,7) UV-Visible spectra The purified FOD AO showed a UV-visible spectrum with maxima at 368, 440, and 478 nm and with shoulders at 512 and 577 nm (Fig. 1B).…”

“…vanrijiae MH201 produces as little as three FOD isozymes, with different pI values. 6) Recently we reported the cloning and expression of three FOD genes, FOD1, FOD2, and FOD3, from the yeast using Escherichia coli. 8) The amino acid sequences deduced from their nucleotide sequences (BAF9891, BAF9892, and BAF9893) suggested that the yeast FOD isozymes have a conserved motif of the FAD binding site, xhxhGxGxxGxxxhxxh(x) 6{9 hxhE (x is any residue and h is a hydrophobic residue) and are to be classified to the glucose-methanol-choline oxidoreductase family.…”