Abstract
This chapter describes the assay method, purification, and properties of alcohol oxidase isolated from Candida boidinii . The enzyme activity is measured by determining the color produced from H 2 O 2 in a coupled reaction utilizing peroxidase and 2,2'-azino-bis(3-ethylbenzthiazoline 6-sulfonate). The reaction rate is measured by the increase in absorbance at 420 nm. Alcohol oxidase is assayed by determining the rate of oxygen consumption in an oxygen electrode cell, or by measuring the formaldehyde. The steps involved in the purification of the enzyme are (1) the preparation of crude extract, (2) streptomycin sulfate precipitation, (3) diethylaminoethyl (DEAE)-cellulose column chromatography, and (4) Sephadex G-200 gel filtration. All operations are carried out in a cold room at 4°C. The molecular weight of the alcohol oxidase is determined by the sedimentation equilibrium method and is found to be about 600,000. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that this enzyme is an octamer composed of eight probably identical subunits that are noncovalently associated. Alcohol oxidase is a common mechanism for the oxidation of methanol in methanol-utilizing yeasts.
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