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 biobased 2,5-furandicarboxylic acid (FDCA), offer a promising alternative to petrochemical-derived plastics. Enzymatic conversion of 5-hydroxymethylfurfural (HMF) to FDCA via hydroxymethylfurfural oxidase (HMFO) is environmentally benign but is limited by catalyst stability. This study addresses these challenges through a high-performance biocatalytic platform based on the one-step purification and immobilization of the engineered enzyme 8BxHMFO fused with a carbohydrate-binding module (CBM3). Utilizing the high affinity of CBM3 for microcrystalline cellulose (Perloza MT100), the biocatalyst achieved a significant increase in thermal stability (T m = 53.2 °C) and enhanced resistance to oxygen interfacial inactivation. The system's efficacy was demonstrated with pure HMF (>95% conversion; 7.2 g·L-1 FDCA), and its robustness was validated using a 15% (w/w) crude extract. The biocatalyst maintained its catalytic performance in the presence of minor matrix components, exhibiting a highly competitive FDCA yield (70.6%) and titer (5.7 g·L-1). FDCA recovery was achieved with 72% efficiency using a mild ethanol-based extraction process. These findings and sustainable indicators validate the robustness of the system under realistic conditions, demonstrating its potential as a sustainable platform for FDCA production and contributing to broader PEF bioplastic adoption.