Lipase and alcohol oxidase cascade catalytic system for the efficient conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid.

2.5-Furandicarboxylic acid (FDCA) is considered a next-generation bio-based platform chemical that can serve as a precursor to polyethylene furanoate. This study established a dual-enzyme cascade system for FDCA production utilizing lipase from Burkholderia sp. RF7-non_BP1 (BP1-Lip) and alcohol oxidase from Colletotrichum gloeosporioides (CglAlcOx). The reaction was carried out in two steps, the molar yield of 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) reached 94.44 ± 3.02 %% after 24 h, and the molar yield of FDCA reached 100 ± 1.96 % after 84 h, with a productivity of 9.29 mg/L/h. In addition, the conversion of 50 mM HMF was successfully achieved by feeding batch substrate addition strategy. After a total reaction time of 264 h, the FDCA concentration reached 41.03 mM, with an FDCA productivity of 24.26 mg/L/h. Molecular docking analysis of BP1-Lip with HMF showed that the aldehyde group is easily oxidized and can form multiple hydrogen bonds with residues such as Asn110, Lys127, and Asn130. etc.; Lys127 enhances binding stability by localizing substrates through salt bridges and π-cation interactions; Hydrophobic residues Val131 and Tyr292 stabilize the furan ring localization and maintain the ideal conformation of the substrate; The electronic/spatial effects of hydroxymethyl and furan conformations make it difficult for HMFCA and DFF to effectively bind, inhibiting further oxidation.