Spanou_2026_Chembiochem_27_e70506

High-free fatty acid oil cannot be converted to biodiesel through conventional chemical processes and accumulates as a waste stream in biodiesel industries. In this work, a bienzymatic system comprising Rhizopus oryzae lipase (ROL) and Fusarium oxysporum cutinase (FOC) was developed for its transesterification. Preliminary experiments, followed by response surface methodology, identified the optimal conditions of ROL/FOC ratio of 3.9:1 (mg/mg), a methanol-to-oil molar ratio of 2.6:1 applied through three stepwise additions, and a temperature of 31.8 degreesC, yielding a maximum conversion of 69.6% after 48 h. Under these conditions, FOC alone led only to ~4% conversion, while the combined ROL/FOC system outperformed ROL alone by approximately 18% after 72 h, demonstrating a potential synergistic effect between the two enzymes. To enhance process sustainability, FOC was immobilized on four carriers, with macroporous divinylbenzene and amino C6 methacrylate to perform the best, enabling the reuse over three consecutive cycles with minimal activity loss and achieving conversions up to 91% after 72 h in the second use. High efficiency was maintained upon scale-up to 100 mL, demonstrating process robustness. The synergistic action of ROL and FOC provides an effective biocatalytic route for the valorization of low-value, free fatty acid-rich lipid feedstocks into biodiesel.