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

Abstract

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.

Oxidation of 5‐hydroxymethylfurfural with a novel aryl alcohol oxidase from Mycobacterium sp. MS1601

“…Aryl alcohol oxidases in rare cases have been shown to catalyse the oxidation of aromatic aldehyde substrates to the corresponding acids (Urlacher and Koschorreck, 2021), which is proposed to proceed via the gem ‐diols formed by aldehyde hydration and hence following the mechanism similar to alcohol oxidation (Ferreira et al ., 2010). Mycsp AAO displayed very low activity against FFCA and HMFCA as observed even for other AAOs (Carro et al ., 2015; Serrano et al ., 2019; Viña‐Gonzalez et al ., 2020; Lappe et al ., 2021), which may be due to interference by the carboxylic group on the binding of the substrate to the enzyme active site.…”

“…A characteristic of these oxidoreductases is a conserved histidine in the active site that initiates the oxidation via proton subtraction from the substrate and assists in FAD re‐oxidation by the molecular oxygen. Among the subfamilies of GMC oxidoreductases are aryl alcohol oxidases (AAOs) that have been reported to oxidize HMF (Serrano et al ., 2020; Lappe et al ., 2021). Aryl‐alcohol oxidases (AAOs; EC 1.1.3.7) catalyse the oxidative dehydrogenation of primary alcohols from aliphatic unsaturated or aromatic alcohols to aldehydes using oxygen as a final electron acceptor resulting in production of hydrogen peroxide (H 2 O 2 ) (Urlacher and Koschorreck, 2021).…”

Current Advances in the Sustainable Conversion of 5‐Hydroxymethylfurfural into 2,5‐Furandicarboxylic Acid

“…Similarly, AAO from Moesziomyces antarcticus (MaAAO) was combined with unspecific peroxygenase completing the conversion of HMF into FDCA within 144 h [129] . In particular, Ma AAO oxidized HMF to DFF and FFCA, while UPO completed the conversion of FFCA into FDCA.…”

“…Similarly, AAO from Moesziomyces antarcticus (MaAAO) was combined with unspecific peroxygenase completing the conversion of HMF into FDCA within 144 h. [129] In particular, MaAAO oxidized HMF to DFF and FFCA, while UPO completed the conversion of FFCA into FDCA. H 2 O 2 produced during the oxidation of HMF to DFF and FFCA was found to have an inhibitory effect on AAO, causing a lack of activity on FFCA.…”

(Chemo)biocatalytic Upgrading of Biobased Furanic Platforms to Chemicals, Fuels, and Materials: A Comprehensive Review

“…The synthesis of FFCA is challenging, owing to its highly oxidized yet incomplete state. Indeed, biocatalysts capable of the production of this chemical remain limited, basically centered on aryl alcohol oxidases (AAOs) and the laccase–TEMPO system. − Nonetheless, their catalytic efficiencies are very low. A trienzyme system consisting of GO, ADH, and HRP and a GO variant GO M 3–5 proved to be the most promising catalysts for the synthesis of FFCA, starting from HMF and HMFCA, respectively. , …”