Dual Enhancement\nof Thermostability and Activity of\nXylanase through Computer-Aided Rational Design

In the realm of enzyme engineering, the dual enhancement\nof thermostability\nand activity remains a challenge. Herein, we employed a computer-aided\napproach integrating folding free energy calculations and evolutionary\nanalysis to engineer Paecilomyces thermophila xylanase into a hyperthermophilic enzyme for application in the\npaper and pulp industry. Through the computational rational design,\nXynM9 with superior thermostability and enhanced activity was designed.\nIts optimal reaction temperature increases by 10 °C to 85 °C,\nits <i>T</i><sub>m</sub> increases by 10 °C to 93 °C,\nand its half-life increases 11-fold to 5.8 h. Additionally, its catalytic\nefficiency improves by 57% to 3926 s<sup>–1</sup> mM<sup>–1</sup>. Molecular dynamics simulations revealed that XynM9 is stabilized\nby more hydrogen bonds and salt bridges than wild-type xylanase. The\nmutant’s narrower catalytic cleft enhances the substrate-binding\naffinity, thus improving the catalytic efficiency. In harsh conditions\nat 80 °C and pH 10, using XynM9 significantly reduced both hemicellulose\nand lignin, which makes it a good candidate for use in the paper and\npulp process. Our study presents an accurate and efficient strategy\nfor the dual enhancement of enzyme properties, guiding further improvement\nof computational tools for protein stabilization.