An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity

• An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase side-activity.
• Several AOX are known from fungi, but so far no AOX has been published which is able to oxidize glycerol.
• The present AOX from P. chrysosporium is the first AOX accepting both, small monovalent alcohols and glycerol.
• The green oxidant hydrogen peroxide could be generated from industrial side-streams of glycerol.

An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75 kDa subunits. The open reading frame of 1,956 bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50 °C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide.