Electrochemical H₂O₂ - stat mode as reaction concept to improve the process performance of an unspecific peroxygenase

The electroenzymatic hydroxylation of 4-ethylbenzoic acid catalyzed by the recombinant unspecific peroxygenase from the fungus Agrocybe aegerita (rAaeUPO) was performed in a gas diffusion electrode (GDE)-based system. Enzyme stability and productivity are significantly affected by the way the co-substrate hydrogen peroxide (H₂O₂) is supplied. In this study, two in-situ electrogeneration modes of H₂O₂ were established and compared. Experiments under galvanostatic conditions (constant productivity of H₂O₂) were conducted at current densities spanning from 0.8 mA cm^-2 to 6.4 mA cm^-2. For comparison, experiments under H₂O₂-stat mode (constant H₂O₂ concentration) were performed. Here, four H₂O₂ concentrations between 0.06 mM and 0.28 mM were tested. A maximum H₂O₂ productivity of 5.5 µM min^-1 cm^-2 and productivity of 10.5 g L^-1 d^-1 were achieved under the galvanostatic condition at 6.4 mA cm^-2. Meanwhile, the highest total turnover number (TTN) of 710,000 mol mol^-1 and turnover frequency (TOF) of 87.5 s^-1 were obtained under the H₂O₂-stat mode at concentration limits of 0.15 mM and 0.28 mM, respectively. The most favorable outcome in terms of maximum achievable TTN, TOF and productivity was found under the H₂O₂-stat mode at concentration limit of 0.2 mM. Here, a TTN of 655,000 mol mol^-1, a TOF of 80.3 s^-1 and a productivity of 6.1 g L^-1 d^-1 were achieved. The electrochemical H₂O₂-stat mode not only offers a promising alternative reaction concept to the well-established galvanostatic mode but also enhances the process performance of unspecific peroxygenases.