Purification and Properties of Secondary Alcohol Oxidase with an Acidic Isoelectric Point

Abstract

An enzyme which catalyzes the oxidation of poly(vinyl alcohol) (PVA) has been purified from a fraction adsorbed to DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a culture broth when the culture was grown in a minimal medium where PVA served as a sole source of carbon and energy. The enzyme was separated from a coexisting oxidized PVA hydrolase by dye-ligand chromatography on Matrex Gel Blue A. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoreses in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 40,000 and has an isoelectric point of 4.5. The amino acid composition of the enzyme has been determined and found to have no histidine. The N- and C-terminal amino acid residues are both alanine. The enzyme solution is pink and shows absorption maxima at 276, 364, and 469 nm. One atom of non-heme iron has been detected per molecule in the enzyme.

The enzyme catalyzes the oxidation of PVA and also of various low molecular weight secondary alcohols to the corresponding ketones with the production of H₂0₂ and the consumption of 0₂. The molar ratio of these ketones, H₂0₂ and 0₂ is 1:1:1. The most effective electron acceptor is 0₂, while 2,6-dichlorophenolindophenol and nitro blue tetrazolium also serve as the acceptor with efficiencies to 0₂ of about 31 and 16%, respectively. The enzyme is, therefore, considered to be a secondary alcohol oxidase.

The enzyme is most active at pH 7.0 and at 45°C and is stable between pH 5.0 and 9.0 and at temperatures below 45°C. The activity is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to a secondary alcohol oxidase previously isolated from another fraction adsorbed on SP-Sephadex at pH 7.0 of the PVA-degrading enzyme activities [Agric. Biol. Chem., 43, 1225 (1979)]. The relations between these two secondary alcohol oxidases are discussed.