Characterization of a thermotolerant aryl-alcohol oxidase from Moesziomyces antarcticus oxidizing 5-hydroxymethyl-2-furancarboxylic acid.

The development of enzymatic processes for the environmentally friendly production of 2,5-furandicarboxylic acid (FDCA), a renewable precursor for bioplastics, from 5-hydroxymethylfurfural (HMF) has gained increasing attention over the last years. Aryl-alcohol oxidases (AAOs) catalyze the oxidation of HMF to 5-formyl-2-furancarboxylic acid (FFCA) through 2,5-diformylfuran (DFF) and have thus been applied in enzymatic reaction cascades for the production of FDCA. AAOs are flavoproteins that oxidize a broad range of benzylic and aliphatic allylic primary alcohols to the corresponding aldehydes, and in some cases further to acids, while reducing molecular oxygen to hydrogen peroxide. These promising biocatalysts can also be used for the synthesis of flavors, fragrances, and chemical building blocks, but their industrial applicability suffers from low production yield in natural and heterologous hosts. Here we report on heterologous expression of a new aryl-alcohol oxidase, MaAAO, from Moesziomyces antarcticus at high yields in the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). Fed-batch fermentation of recombinant P. pastoris yielded around 750 mg of active enzyme per liter of culture. Purified MaAAO was highly stable at pH 2-9 and exhibited high thermal stability with almost 95% residual activity after 48 h at 57.5 °C. MaAAO accepts a broad range of benzylic primary alcohols, aliphatic allylic alcohols, and furan derivatives like HMF as substrates and some oxidation products thereof like piperonal or perillaldehyde serve as building blocks for pharmaceuticals or show health-promoting effects. Besides this, MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FFCA, which has not been shown for any other AAO so far. Combining MaAAO with an unspecific peroxygenase oxidizing HMFCA to FFCA in one pot resulted in complete conversion of HMF to FDCA within 144 h. MaAAO is thus a promising biocatalyst for the production of precursors for bioplastics and bioactive compounds. KEY POINTS: • MaAAO from M. antarcticus was expressed in P. pastoris at 750 mg/l. • MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). • Complete conversion of HMF to 2,5-furandicarboxylic acid by combining MaAAO and UPO.