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

Alessa Lappe,Katja Koschorreck,Nina Jankowski,Annemie Albrecht

doi:10.1007/s00253-021-11557-8

Alessa Lappe, Katja Koschorreck + Show 2 more

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https://doi.org/10.1007/s00253-021-11557-8

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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.

Highlights

In times of an emerging importance of a sustainable bioeconomy, biocatalytic processes have gained more and more attention as a promising alternative to chemical synthesis by utilizing enzymes with high activity and product selectivity
Activity of MaAAO in the presence of up to 40% of dimethyl sulfoxide (DMSO), 2-methyltetrahydrofuran (MeTHF), choline acetate, and choline dihydrogen phosphate towards veratryl alcohol was determined as described above
Stability of MaAAO towards up to 20% of dimethyl sulfoxide (DMSO), 40% of choline acetate, and 40% of choline dihydrogen phosphate was determined by incubating MaAAO in 50 mM potassium phosphate buffer pH 7.5 with the respective cosolvent for 24 h at 25 °C

Summary

Introduction

In times of an emerging importance of a sustainable bioeconomy, biocatalytic processes have gained more and more attention as a promising alternative to chemical synthesis by utilizing enzymes with high activity and product selectivity. Aryl-alcohol oxidases (AAOs) have emerged as promising biocatalysts. Most AAOs described so far have been found in basidiomycetous and ascomycetous fungi, enzymes with AAO activity have been identified in bacteria, insects, and gastropods as well (Ferreira et al 2015; Serrano et al 2020; Urlacher and Koschorreck 2021). AAOs typically oxidize benzylic and polyunsaturated aliphatic primary alcohols to the corresponding aldehydes via hydrogen abstraction and transfer to molecular oxygen to produce hydrogen peroxide (Guillen et al 1992). Hydrated aldehydes (gemdiols) can be further oxidized to the corresponding acids, but efficiencies are much lower (Ferreira et al 2010)

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