Computer-aided multi-shell electrostatic remodeling of BhS7Xyl for enhanced activity and Thermostability

Xylanases with high catalytic efficiency and environmental robustness are important for lignocellulosic biomass valorization, but many enzymes are rapidly inactivated under the alkaline and high-temperature conditions used in industrial processes. In this study, a computationally guided rational-design strategy was developed to improve the catalytic performance and stability of the alkaline xylanase BhS7Xyl. Constant-pH molecular dynamics, isothermal compressibility perturbation analysis, and ECNet-assisted fitness prediction were integrated to identify alkaline-sensitive and structurally unstable residues for engineering. The triple mutant H51R/D150N/E287K showed the best overall performance, with a specific activity of 1045.29 U/mg, representing a 3.73-fold increase compared with the wild type. Its melting temperature increased from 55.82 °C to 64.58 °C, while its half-life at pH 10.0 increased from 33.96 to 95.84 min. The thermal half-life at 75 °C was extended from 10.97 to 215.42 min, corresponding to a 19.64-fold improvement. Structural analyses suggested that the improved performance of H51R/D150N/E287K was associated with a more continuous xylohexaose-binding interface, increased hydrogen-bonding contacts, additional electrostatic/polar interactions, strengthened local interaction networks and enhanced dissipation of local thermal perturbation. Under optimized hydrolysis conditions, the triple mutant produced higher levels of xylose and xylooligosaccharides from standard xylan, corn cob xylan, and hardwood pulp xylan than the wild type. These work demonstrates that multi-shell electrostatic remodeling is a useful strategy for improving the activity, alkaline tolerance, and thermal stability of xylanase for xylooligosaccharide production.