Development of H₂O₂-Driven Peroxygenase-Type P450BM3 for Efficient Substrate Removal of Lignin-Derived Phenolics

Lignin-derived aromatic compounds (LDACs) are persistent pollutants in pulp and paper effluents, hindering agro-industrial biomass valorization. We developed H₂O₂-driven peroxygenase-type P450BM3 system that efficiently removes LDACs using H₂O₂ as the sole oxidant, eliminating the need for NADPH or redox-partner cofactors. Mechanistically guided mutations at F87, T268, A328, and V78 generated highly efficient mutants. The F87A/T268D/A328H mutant is an affinity-optimized mutant with exceptionally low apparent K_m values of 0.26 and 0.61 mM for EU and ISO, respectively, corresponding to apparent catalytic efficiencies (k_cat/K_m) of 1.38 × 10⁴ and 1.61 × 10⁴ M^-1 s^-1. The F87A/T268D/A328F/V78A mutant, a turnover-optimized mutant, exhibits the highest apparent activity toward CA (1.47 × 10⁴ M^-1 s^-1). UPLC-MS was employed to propose potential qualitative substrate transformation pathways for substrates. The optimized enzyme removed over 95% of LDACs in the simplified simulated wastewater. This DFSM/NADPH-free biocatalytic platform provides a low-cost, sustainable strategy for LDACs remediation and a reference for lignin-containing substrate removal.