Furans bioconversion by semi-crude culture supernatants of Pleurotus pulmonarius enriched in aryl alcohol oxidase activity

Aryl alcohol oxidases (AAOs) are flavin adenine dinucleotide (FAD)–dependent fungal oxidases, acting as accessory oxidases during lignocellulose degradation. Their catalytic cycle involves oxidation of primary alcohols to the corresponding aldehydes, with concomitant production of H 2 O 2 . Although they are valuable catalysts, their application is hampered by poor heterologous expression. In the present work, the AAO-containing culture supernatant of the white-rot fungus Pleurotus pulmonarius was partially purified with anion-exchange chromatography, to remove side enzymes and obtain an AAO-enriched, semi-crude preparation. Two fractions with elevated AAO content were obtained, with specific activity of 5 and 1.4 U mg -1 using veratryl alcohol as the substrate, for Fractions 1 and 2 respectively. These were characterized in terms of optimum activity conditions and oxidase-substrate specificity. Proteomic analysis revealed their differential composition. Both fractions were applied to the bioconversion of 5-(hydroxymethyl)furfural (HMF) towards the oxidation products 2,5-diformylfuran (DFF), 5-formyl-2-furan carboxylic acid (FFCA) and 2,5-furan dicarboxylic acid (FDCA), achieving yields up to 65% total products. The main oxidation products were DFF and FFCA, while FDCA was produced in traces. Overall, our results support the feasibility of using semi-crude preparations, enriched in the desired enzyme, for the production of high-added value products in the frame of Circular Bioeconomy.