Purification and Properties of Oxidized Poly(vinyl alcohol) Hydrolase with an Acidic Isoelectric Point

Abstract

An enzyme which catalyzes the degradation of polyvinyl alcohol) (PVA) oxidized by secondary alcohol oxidase, in which hydroxyl groups of PVA are partially converted to carbonyl groups, has been purified from a fraction adsorbed on DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a minimal medium containing PVA as a sole source of carbon and energy. The purified enzyme was electrophoretically homogeneous in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 36,000 and has an isoelectric point of 5.1. The N- and C-terminal amino acid residues are both alanine. The enzyme is most active at pH 6.5 and at 40°C and is stable between pH 6.0 and 9.0 and at temperatures below 45°C. The enzyme is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme was active on oxidized PVA, but not on PVA and on various low molecular weight carbonyl compounds examined. The enzyme reaction on oxidized PVA resulted in a rapid decrease in viscosity, a fall of pH, and production of carboxylic acids. The enzyme, therefore, is considered to be an oxidized PVA hydrolase.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to an oxidized PVA hydrolase previously purified from another fraction adsorbed on SP-Sephadex at pH 7.0 from the PVA-degrading enzyme activities [Agric. Biol. Chem., 45, 63 (1981)]. The relations between these two oxidized PVA hydrolases are discussed.