Structure-informed engineering of a laccase with enhanced catalytic activity and thermostability for facilitating lignocellulosic biomass saccharification.

Lignocellulose is an abundant renewable feedstock for biofuels and value-added bioproducts, yet its efficient bioconversion is hindered by the recalcitrant lignin barrier. While laccases show great potential for lignin modification and delignification, their limited thermostability restricts their application in high-temperature lignocellulose biorefinery processes. To address this constraint, an integrated computational engineering strategy was employed to rationally improve the thermostability and catalytic performance of a laccase from Bacillus aryabhattai TCCC 11368. The optimal variant, S281E/N387D, obtained through the combination of FireProt, PROSS, and supercharge-based engineering, exhibited enhanced thermal stability and catalytic efficiency. Its half-life at 60 °C increased 3.7-fold to 330  min compared with the wild type, accompanied by a 35.4% improvement in catalytic efficiency (kcat/Km). Structural analysis suggested that the improved performance may result from enhanced hydrogen-bond networks, strengthened electrostatic interactions, improved hydrophobic packing, and optimized substrate-binding interactions, which collectively contribute to its enhanced capability in lignocellulosic biomass saccharification. Under optimized conditions, enzymatic treatment of wheat straw using the S281E/N387D variant combined with cellulase yielded 8.63 mg/mL reducing sugars, representing a 10.86% increase over the wild-type laccase treatment. This study provides an effective computational framework for developing robust laccases and demonstrates their potential for improving lignocellulosic biomass conversion in biorefinery applications.