Purification and characterization of alcohol oxidase from Paecilomyces variotii isolated as a formaldehyde-resistant fungus

• DOI: 10.1007/s00253-007-1237-9
• Publication Date: 2007-11-05T14:00:03Z
• Authors: Tetsuya Kondo; Yutaka Morikawa; Naohiro Hayashi
• Keywords: 0301 basic medicine; Flavoproteins; Temperature; Hydrogen-Ion Concentration; 6. Clean water; Substrate Specificity; Molecular Weight; Alcohol Oxidoreductases; Kinetics; 03 medical and health sciences; Sequence Analysis, Protein; Spectrophotometry; Alcohols; Flavins; Formaldehyde; Enzyme Stability; Chromatography, Gel; Environmental Microbiology; Electrophoresis, Polyacrylamide Gel; Paecilomyces

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6-10. The enzyme was stable in the pH range of pH 5-10. The enzyme retained full activity after incubation at 50 degrees C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K (m) values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l(-1), respectively.