Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus.

The GH11 xylanase XynA from L2001 has favorable hydrolytic properties. However, its poor thermal stability hinders its widespread application in industry. In this study, mutants Mut1 and Mut2 were constructed by rationally combining the mutations YHDGYF, AP/SP, and GP. The residual enzyme activity of these combinational mutants was more than 85% when incubated at 80 and 90 °C for 12 h, and thus are the most thermotolerant xylanases known to date. The reduced flexibility of the N-terminus, increased overall rigidity, as well as the surface net charge of Mut1 and Mut2 may be partially responsible for the improved thermal stability. In addition, the specific activity and catalytic efficiency of Mut1 and Mut2 were improved compared with those of wild-type XynA. The broader catalytic cleft and enhanced flexibility of the "thumb" of Mut1 and Mut2 may be partially responsible for the improved specific activity and catalytic efficiency.