Integrated Flow Enzymatic Immobilization and Esterification for Synthesizing 5‑Hydroxymethylfurfural Oleate

Enzyme immobilization can be carried out in either batch or flow mode. Developing flow-through enzymatic reactions requires integrating upstream flow bioprocesses, such as flow immobilization, to minimize downtime, reduce labor requirements, and support the development of sustainable bioprocesses. In this study, Burkholderia cepacia lipase (BCL) was immobilized on Immobead 150 P, and flow esterification of 5-hydroxymethylfurfural (HMF) with oleic acid was performed to synthesize 5-hydroxymethylfurfural oleate. For batch immobilization, the optimal enzyme loading was determined to be 7 mg protein per gram of support, yielding an activity yield (IY%) of 99.9% and a protein yield (IP%) of 98.6%. In flow mode, the best immobilization results were achieved at a flow rate of 0.05 mL/min, yielding an IY of 98% and an IP of 80% within 130 min. The resulting biocatalyst exhibited high operational stability, maintaining activity after five reaction cycles. Batch synthesis of 5-hydroxymethylfurfural oleate achieved conversions of 97% and 45% using 30 mM and 50 mM HMF, respectively, over 24 h. In flow esterification of HMF and oleic acid, conversions of 84% (30 mM HMF) and 75% (50 mM HMF) were obtained at a flow rate of 0.05 mL/min. The space-time yield (STY) was higher in flow bioreactors, with values of 4.7 mM/min and 5.3 mM/min at the same flow rate for 30 mM and 50 mM HMF, respectively. This work demonstrates the integration of two flow processeslipase immobilization and HMF esterification with oleic acidhighlighting the significant potential for process scale-up.