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

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°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.