Purification, characterization, and potential applications of formate oxidase from Debaryomyces vanrijiae MH201.
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.
Uchida
H
H
Applied Chemistry and Biotechnology, Faculty of Engineering, University of Fukui, 9-1, Bunkyo 3-Chome, Fukui-Shi, Japan. uchida@acbio2.acbio.fukui-u.ac.jp
Hojyo
M
M
Fujii
Y
Y
Maeda
Y
Y
Kajimura
R
R
Yamanaka
H
H
Sakurai
A
A
Sakakibara
M
M
Aisaka
K
K
D042931:Aldehyde Oxidase / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005557:Formaldehyde / Q000378:metabolism; D005561:Formates / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D004718:Saccharomycetales / Q000201:enzymology* / Q000302:isolation & purification; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D005561:Formates; D005656:Fungal Proteins; D020033:Protein Isoforms; D021122:Protein Subunits; C030544:formic acid; D002245:Carbon Dioxide; D005557:Formaldehyde; D010088:Oxidoreductases; D002374:Catalase; D042931:Aldehyde Oxidase