Electrobiosynthesis of 25-Hydroxy-Vitamin D ₃ by Molybdenum-Containing Steroid C25 Dehydrogenase Coupled to Antimony Tin Oxide Thin-Film Electrodes

Vitamin D3 (VitD3) deficiency is linked to numerous health conditions, increasing the demand for its bioavailable metabolite, 25-hydroxy-VitD3 (25OHVitD3). While chemical synthesis remains the standard production method, enzymatic approaches using steroid C25 dehydrogenases (S25DH) from Sterolibacterium denitrificans provide a highly selective, viable alternative. Herein, we present an S25DH-based bioelectrosynthesis approach, where optimizing electron transfer (ET) between S25DH and the working electrode is essential for achieving efficient bioelectrosynthesis. This study uses 8% antimony-doped tin oxide (ATO) thin-film electrodes to investigate S25DH immobilization dynamics, adsorption/desorption behavior, and electrocatalytic performance using in situ ATR-IR spectroelectrochemistry. Our findings indicate that S25DH binds to ATO surfaces via electrostatic and coordinative interactions, with surface-exposed histidine clusters playing a key role in stable immobilization. Adsorption kinetics reveal a transition from rapid monolayer formation to slower multilayer growth, with 30–45 min identified as the optimal immobilization period for balancing enzyme coverage and orientation. Direct electron transfer (DET) was observed between the S25DH and the ATO electrode, a behavior not previously reported, highlighting ATO’s potential as a platform for S25DH-based bioelectrosynthesis. These insights pave the way for scaling ATO-based bioelectrocatalytic systems, including three-dimensional porous electrodes for enhanced enzyme loading and current densities. This work establishes a foundation for optimizing enzyme-electrode interactions in bioelectrosynthesis, offering a promising route toward high-yield 25OHVitD3 production.