Immobilized hydroxymethylfurfural oxidase enables robust biocatalytic production of 2,5-furandicarboxylic acid from crude 5-hydroxymethylfurfural

Bioplastics such as poly(ethylene 2,5-furandicarboxylate) (PEF), synthesized from bio based 2,5-furandicarboxylic acid (FDCA), offer a promising alternative to conventional petrochemical-derived plastics. The enzymatic conversion of 5-hydroxymethylfurfural (HMF) to FDCA using engineered variants of hydroxymethylfurfural oxidase (HMFO) represents an environmentally benign approach, as the enzyme catalyzes all three oxidation steps required for FDCA synthesis. This study presents an intensified biocatalytic system for the sustainable production of FDCA, employing the engineered enzyme 8BxHMFO fused with a carbohydrate-binding module (CBM3) and immobilized on microcrystalline cellulose (Perloza MT100) to avoid oxygen interfacial inactivation. The system was first optimized using pure HMF, achieving high conversion rates (>95%), a maximum FDCA yield of 7.2 g/L. Reactions were performed using a crude extract rich in HMF (15% wt/wt). Despite the presence of impurities, the immobilized enzyme maintained high conversion efficiency, with only a 30% reduction in FDCA yield compared to pure substrate. FDCA recovery was achieved with 72% efficiency using a mild, ethanol-based extraction process. These findings, along with an evaluation of sustainable indicators, validate the robustness of the immobilized HMFO system under realistic conditions and demonstrate its potential as a sustainable platform for FDCA production, contributing to the broader adoption of PEF bioplastics.