A Fungal Ascorbate Oxidase with Unexpected Laccase Activity

© 2020 by the authorsAscorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.V.B. gratefully acknowledges the financial support of the NÖ Forschungs- und Bildungsges.m.b.H. (NFB) and the provincial government of Lower Austria through the Science Calls (Project ID: SC16-019). Next Generation Bioproduction is funded by BMVIT, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria und Vienna Business Agency in the framework of COMET—Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG. Supported by BOKU Vienna Open Access Publishing Fund.Peer reviewe