Immobilization and electrochemical activation synergistically enhance activity, stability and solvent tolerance of an...

Unspecific peroxygenases (UPOs) are promising biocatalysts for selective oxyfunctionalization but are limited by low stability and oxidative inactivation by H₂O₂. Here, electroreductive activation (ERA), based on electrochemical O₂ reduction to generate low levels of reactive oxygen species, is combined with site-specific immobilization on Ni-NTA-modified surfaces to enhance the performance of Daldinia caldariorum UPO (DcaUPO). ERA enables controlled in situ ROS generation, mitigating enzyme deactivation while supporting formation of the catalytically active oxoferryl intermediate. Immobilization preserves the active-site structure, shifts the resting-state equilibrium toward the high-spin form, increases thermal stability 19-fold, upshifts the optimal pH, and improves tolerance to acetonitrile. Maximal activity is achieved when DcaUPO is immobilized on electrically disconnected surfaces, minimizing electrochemical stress. Under optimized ERA conditions, DcaUPO reaches k_cat = 52 s^-1 and over 163,000 turnovers, representing 650- and 6040-fold improvements over conventional H₂O₂-driven activation.