Significantly enhanced thermostability and inhibitory resistance of Aspergillus usamii xylanase by modifying its N-terminus and thumb regions

To improve the inhibitory insensitivity towards Aspergillus usamii E001 GH11F xylanase (Auxyn11A) towards xylanase inhibitor protein (XIP-I), and Triticum aestivum xylanase inhibitor (TAXI-I) the site-directed mutagenesis was conducted based on the computer-aided redesign. Firstly, two xylanase inhibitory proteins-encoding xip-I and taxi-I were synthesized and expressed in Pichia pastoris SMD1168, namely SyXIP-I and SyTAXI-I. The highest inhibitory activities of SyXIP-I and SyTAXI-I on Auxyn11A occurred at molar ratio (xylanase:inhibitor) 10:1, 40 °C for 30 min, and 100:1, 30 °C for 30 min, respectively. Secondly, nine variants of a Auxyn11A-encoding gene (Auxyn11A) were constructed as designed theoretically and expressed in E. coli BL21(DE3), respectively. One mutant, Auxyn11ASDSS exhibited the highest xylanase activity and showed 100