Engineering an acid–base dyad into the peroxygenase-enabled mutant CYP199A4T252E yielded four in silico-designed double mutants, of which CYP199A4F182R/T252E showed the best dyad-like geometry and was characterized further. It delivered 10-fold higher initial H2O2-driven O-demethylation activity than wild type and CYP199A4T252E, alongside reduced catalase activity and improved peroxide utilization. However, it was more prone to H2O2-induced heme bleaching and rapid inactivation under standard dosing; slow, controlled H2O2 feeding sustained catalysis for hours. Overall, adding a second basic residue boosts per-oxy-gen-ase-like activity but reduces oxidative robustness, underscoring the trade-off between efficiency and peroxide tolerance and guiding future engineering of robust P450 peroxygenases.

Halophenols (HPs) cause serious problems for the health of living beings and environment due to their toxigenicity, mutagenicity and carcinogenicity. Enzymes have recently attracted significant attention as an eco-friendly and sustainable approach for the environmental remediation of pollutants. In this study, the recombinant unspecific peroxygenase from Agrocybe aegerita (r Aae UPO, recombinantly expressed in Komagataella pastoris known as Pichia pastoris ) was used to degrade five representative HPs (2-Chlorophenol (2-CP), 3-Chlorophenol (3-CP), 4-Chlorophenol (4-CP), 4-Bromophenol (4-BP), and 3-Iodophenol (3-IP)) in the batch and fed-batch systems. r Aae UPO (5 µM) completely removed up to 10 mM HPs from the fed-batch system in 48 h, while the almost complete removal of 2.5 mM 4-CP and 4-BP in batch systems occurred within 72 h. The enzyme was more effective upon slow, continuous fed with H₂O₂ concentrations (2 mM/h) than supplying stoichiometric H₂O₂ from the beginning. The activity of r Aae UPO towards HPs was: 3-IP > 2-CP > 3-CP > 4-BP > 4-CP. These results were also confirmed by molecular docking results. r Aae UPO-catalyzed primary degradation of HPs occurred via catechol formation followed by polymerization. Toxicity assays using E. coli DH5α demonstrated a significant reduction in toxicity after enzymatic degradation of HPs. This study revealed that r Aae UPO is an efficient biocatalyst capable of effectively degrading HPs, showing great potential for environmental bioremediation applications.