Surface Engineering of Glutamic Acid Residues to Enhance the Thermostability of GH10 Xylanases.

Abstract

Thermostability is critical for the industrial applications of xylanase, including paper production, animal feed, and lignocellulosic biomass conversion. Here, we report that the rational introduction of surface-exposed glutamic acid (Glu) residues significantly enhances the thermostability of GH10 xylanases. Engineered variants of XT6, BhS7Xyl, and FXYN exhibited prolonged half-lives that were elevated by 2.5-, 1.5-, and 3-fold relative to their respective wild-type enzymes. The stabilization arises from strengthened conformational rigidity due to the formation of numerous new salt bridges. This strategy was further validated in two novel xylanases of Xyn466 and Xyn486 from Cellulomonas bogoriensis 69B4T. In contrast to disulfide bond engineering and ΔΔG-based engineering, surface Glu modification provides superior stabilization (Xyn466-9QE of 5.9-fold and Xyn486-11QE of 9.7-fold increased half-life at 60 °C) with lower mutational load. Our results provide a more efficient strategy with a higher success rate and lower activity trade-off for improving the thermostability of GH10 xylanases.