Rational Design of Laccase Mutants for Enhanced Catalytic Degradation of Benzene-Containing Pollutants: A Computational Insight into Binding Pocket Engineering

• • Fungal laccase (T.v) showed 15–23% lower binding free energy (ΔG) for benzene-containing pollutants compared to bacterial laccase (B.s), attributed to a narrow hydrophobic channel and directed hydrogen-bond network (His458–substrate bond length 0.27 nm), enabling stronger substrate retention and higher catalytic efficiency. • • Virtual mutagenesis at Ser113 to Arg/Glu/Leu achieved ΔG ≤ −7.1 kcal·mol−1 for HBT, representing a significant enhancement in binding affinity, which translates to improved degradation kinetics for recalcitrant aromatic pollutants. • • Dual-region engineering principle: core polar mutations (e.g., D112E) optimized hydrogen-bond networks, increasing catalytic efficiency by 2.3-fold; peripheral hydrophobic mutations (e.g., L387F) enhanced π-π stacking and spatial restriction, extending substrate binding half-life by 2.7-fold. • • The 'core polarization–peripheral hydrophobicity' strategy universally improved laccase family activity by an average of 2.8-fold, with activation energy reduction (ΔΔG = −3.4 kcal·mol−1) and electron transfer frequency increase (1.5–2.0-fold), offering a scalable framework for engineering oxidoreductases in environmental bioremediation.