Rational Design of Flexible Regions in a GH10 Xylanase from Paecilomyces aerugineus to Improve Its Thermostability, Catalytic Efficiency, and XOS Production from Corn Stover

Rational design was performed to enhance the thermostability and catalytic efficiency of a GH family 10 xylanase (PaXyn10A) from Paecilomyces aerugineus. By identification of flexible regions and avoidance of functionally conflicting sites via molecular dynamics simulations and sequence alignment, the mutant PaXyn10A D68F/S95A was obtained. The optimum temperature of PaXyn10AD68F/S95A was increased by 5 °C, and the thermal inactivation half-lives at 50 °C was increased by 11.5 times when compared to that of PaXyn10A. Meanwhile, PaXyn10AD68F/S95A showed a 2.1-fold increase in specific activity as well as a 3.9-fold increase in catalytic efficiency. Under optimal conditions, an XOS yield of 64.27% from corn stover was obtained via the combination of PaXyn10AD68F/S95A hydrolysis and steam explosion. These findings demonstrate that computational-simulation-guided rigidification of flexible regions via rational point mutagenesis is an effective strategy to enhance both the thermostability and catalytic efficiency of GH10 xylanases.