Computer-Aided Virtual Saturation Mutagenesis Improves the Lignocellulose-Degrading Performance of an Aspergillus niger LPMO

Lytic polysaccharide monooxygenases (LPMOs) are promising enzymes for lignocellulose degradation; however, wild-type LPMOs often exhibit limited catalytic activity and stability. In this study, computer-aided virtual saturation mutagenesis was applied to AnLPMO15g from Aspergillus niger, and eight potentially beneficial mutants (S197H, S197F, E185V, E185L, E185M, E185I, Q108M, and A249P) were identified based on predicted changes in unfolding free energy (∆∆G). Six mutants demonstrated enhanced activity in a 2,6-dimethoxyphenol (2,6-DMP) oxidation assay, which serves as a proxy for peroxidase-like activity. The E185V mutant exhibited a 45% increase over the wild type. The triple mutant E185V/Q108M/A249P further increased the catalytic efficiency by 56%. Notably, when combined with cellulase, E185V/Q108M/A249P enabled a 202.5% increase in reducing sugars from wheat straw, achieving a synergy degree of 1.83, highlighting its potential to improve agricultural residue conversion. Molecular dynamics simulation suggested that the E185V/Q108M/A249P triple mutant induced flexible conformational changes in six residues, which may improve substrate binding affinity. This study presents an effective strategy for engineering AA9 family LPMOs to enhance catalytic performance, facilitating efficient and cost-effective degradation of lignocellulosic biomass with implications for sustainable agricultural waste management and circular bioeconomy.