Semi-Rational Design of Lignin Peroxidase for Enhanced Activity and Application in Wheat Straw Lignin Degradation

Abstract

Lignin peroxidase (LiP) is a key bottleneck in wheat straw lignin degradation due to its limited catalytic efficiency. In this study, a lignin peroxidase from Phanerochaete chrysosporium (PcLiP; GenBank accession no. X15599.1) was engineered using a semirational design strategy. Seven candidate residues were identified by molecular docking, and alanine scanning yielded three mutants (H38A, E320A, and T267A) with over 80% increased activity. Subsequent saturation mutagenesis at T267 identified T267Q as a more active variant. The triple mutant PcLiP-L4 (H38A/T267Q/E320A) exhibited the highest activity, corresponding to a 2.9-fold increase over the wild type. Enhanced catalytic performance was confirmed by ABTS oxidation and alkali lignin degradation assays. After expression in Pichia pastoris, PcLiP-L4 achieved a wheat straw lignin degradation rate of 12.6%, which was 3.26-fold that of the control. Molecular dynamics simulations indicate improved substrate binding stability and reduced binding free energy, contributing to enhanced catalytic efficiency.