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 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.
Kondo
Tetsuya
T
Food Research Center, Aichi Industrial Technology Institute, Aichi Prefectural Government, 2-1-1 Shinpukuji-cho, Nishi-ku, Nagoya, 451-0083, Japan. PXF00674@nifty.com
Morikawa
Yutaka
Y
Hayashi
Naohiro
N
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D000438:Alcohols / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004783:Environmental Microbiology; D004795:Enzyme Stability; D005415:Flavins / Q000302:isolation & purification; D005420:Flavoproteins / Q000737:chemistry / Q000302:isolation & purification; D005557:Formaldehyde / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010143:Paecilomyces / Q000201:enzymology* / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature
D000438:Alcohols; D005415:Flavins; D005420:Flavoproteins; D005557:Formaldehyde; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase