Purification and Properties of Secondary Alcohol Oxidase from a Strain ofPseudomonas

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Abstract
An enzyme which catalyzes the oxidation of poly(vinyl alcohol) was purified to a homogeneous state from an enzyme preparation obtained from a culture broth of a strain of Pseudomonas and described previously as a poly(vinyl alcohol)-degrading enzyme. When the purified enzyme reacts on poly (vinyl alcohol), hydroxyl groups seem to be oxidized to keto groups and 1 mol of H2O2 is produced as 1 mol of O2 is consumed. Essentially no decrease is observed in the viscosity of poly(vinyl alcohol) solution. The enzyme is active not only to poly(vinyl alcohol) but also to a variety of low MW secondary alcohols and 4-heptanone is formed from 4-heptanol. A name of secondary alcohol oxidase was proposed to the enzyme. The enzyme is a single polypeptide with a MW of 50,000 with an isoelectric point at pH 10.3. Alanine and valine was identified as N[amino]- and C[carboxyl]-terminal residues, respectively. The enzyme is pink, has absorption maxima at 277, 364 and 466 nm, and contains 1 g atom of non-heme Fe/molecule. No flavin was detected. The enzyme is most active at pH 7 and at 50.degree. C, and is stable between pH 4.5 and 9 and below 50.degree. C. Among the compounds examined, Hg2+, Pb2+, Zn2+ o-phenanthroline and EDTA showed weak inhibitions of the activity. Two successive reactions, the first catalyzed by the secondary alcohol oxidase and the second by a probable hydrolase were postulated as a mechanism of bacterial degradation of poly(vinyl alcohol).