Rational Design of Laccase Mutants for Enhanced Catalytic Degradation of Benzene-Containing Pollutants: A Computational Insight into Binding Pocket Engineering
Laccases are promising biocatalysts for environmental remediation, yet their application is hindered by the instability and limited substrate affinity of wild-type enzymes. Here, we report a computational strategy integrating homology modeling, molecular docking, and virtual mutagenesis to engineer high-performance laccase mutants targeting benzene-containing pollutants. Structural analysis revealed that fungal laccase (Trametes versicolor, T.v) exhibited stronger binding affinities for aniline, o-phenylenediamine, and 1-hydroxybenzotriazole (HBT) compared to bacterial laccase (Bacillus subtilis, B.s), attributed to optimized hydrophobic and hydrogen-bond interactions within the substrate-binding pocket. Virtual mutagenesis identified critical residues (e.g., Ser113, Leu459) regulating substrate stability. Notably, mutations at Ser113 to Arg/Glu/Leu significantly enhanced binding energy (ΔG ≤ −7.1 kcal·mol−1 for HBT) by narrowing the pocket exit and reinforcing hydrophobic constraints. Mechanistically, polar mutations in the pocket interior promoted hydrogen bonding, while hydrophobic substitutions at peripheral residues restricted substrate dissociation. Our findings establish a dual-region engineering principle—enhancing hydrogen bonds internally and hydrophobicity externally—to optimize laccase activity. This work provides a generalizable framework for the rational design of oxidoreductases in pollutant degradation.