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

Area of Science:

• Biotechnology
• Enzyme Engineering
• Biomass Valorization

Background:

• Industrial xylanase applications require enzymes with high catalytic efficiency and stability under alkaline, high-temperature conditions.
• Many xylanases are rapidly inactivated in such environments, limiting their use in lignocellulosic biomass processing.

Purpose of the Study:

• To improve the catalytic performance and stability of the alkaline xylanase BhS7Xyl using a computationally guided rational-design strategy.
• To identify and engineer alkaline-sensitive and structurally unstable residues to enhance enzyme function.

Main Methods:

• Integrated constant-pH molecular dynamics, isothermal compressibility perturbation analysis, and ECNet-assisted fitness prediction.
• Employed a triple mutation strategy (H51R/D150N/E287K) based on computational predictions.

Main Results:

• The triple mutant H51R/D150N/E287K exhibited a 3.73-fold increase in specific activity and enhanced thermal and alkaline stability.
• Melting temperature increased by 8.76°C, half-life at pH 10.0 increased from 33.96 to 95.84 min, and thermal half-life at 75°C extended by 19.64-fold.
• The mutant demonstrated superior production of xylose and xylooligosaccharides from various xylan sources compared to the wild type.

Conclusions:

• Multi-shell electrostatic remodeling is an effective strategy for enhancing xylanase activity, alkaline tolerance, and thermal stability.
• The engineered xylanase shows significant potential for industrial applications in xylooligosaccharide production from lignocellulosic biomass.