Abstract

The coupled formate dehydrogenase (FDH) and glycerol dehydrogenase (GlyDH) system enables the simultaneous production of formate and 1,3-dihydroxyacetone (DHA) from CO2 and glycerol, with internal NAD(H) cofactor recycling, mimicking metabolic pathways. However, mismatched reaction rates between FDH and GlyDH limit overall efficiency. In this study, we investigated the individual and co-immobilization of FDH and GlyDH on mesoporous silica and evaluated the addition of electrochemical NADH regeneration to improve cofactor balance. Biocatalysts were characterized in terms of immobilization efficiency, activity retention, and stability across temperature and pH ranges. We demonstrate that the optimal FDH-to-GlyDH ratio depends on whether electrochemical regeneration is applied: without it, a lower FDH/GlyDH ratio favors formate production due to GlyDH's faster kinetics; with it, a higher ratio enhances early-stage formate synthesis by alleviating NADH limitations. The system achieved a maximum DHA concentration of 17 mM (FDH/GlyDH = 1:8, no electrochemical regeneration) and a maximum formate concentration of 2.75 mM (FDH/GlyDH = 2.3:1, with electrochemical regeneration). These results demonstrate that combining enzyme immobilization with electrochemical cofactor regeneration can significantly enhance CO2 bioconversion, offering a promising strategy not only for carbon capture and valorization but also for optimizing other NAD(H)-dependent multienzymatic systems.