Abstract

This work evaluates the potential of formate dehydrogenase from the methylotrophic yeastspaceKomagataella phaffiispace(KpFDH) for the valorization of CO₂spacereduction into formate. The catalytic performance of the soluble form was benchmarked against the model enzyme ofspaceCandida boidiniispace(CbFDH). Both enzymes exhibited very similar optimal pH and temperature profiles. Structural bioinformatics revealed a root-mean-square deviation (RMSD) of 0.334space & Aring;, confirming the high conservation of catalytic residues between both yeast-derived enzymes.spaceKpFDH was immobilized on amino-functionalized mesoporous silica (MSU-F-NH₂). Successful protein immobilization was confirmed by Fourier-Transform Infrared (FT-IR) Spectroscopy, photoluminescence analysis, and catalytic activity measurements, achieving complete protein loading (4spacemgspaceg(-1)) with 76% retained activity. During batch CO₂spacereduction without cofactor regeneration, both configurations (using immobilized and free enzyme) yielded 2.4spacemM of formate within 7spaceh. Both setups achieved a competitive volumetric space-time yield (STY) of 0.357spacemMspaceh(-1)space(0.343spacemMspaceh(-1)spacefor the free form). Owing to the exceptionally low enzyme dosages deployed (0.3spaceU), the platforms demonstrated outstanding active-site efficiencies: the free enzyme achieved a turnover number (TON) of 42,283 and a turnover frequency (TOF) of 6040.4spaceh(-1), while the immobilizedspaceKpFDH_MSU-F system maintained a high TON (5497) and TOF (785.3spaceh(-1)). Notably, the immobilized system enabled four consecutive reuse cycles retaining 71% residual activity. These findings positionspaceKpFDH as a viable candidate for sustainable process design, leveraging the natural methanol utilization pathway ofspaceK. phaffiispaceto bypass costly downstream purification steps.