Enhancement in the Thermotolerance of Xylanase（AoXyn11A）by N-Terminus Replacement

Abstract:

To enhance the thermotolerance of AoXyn11A，a glycoside hydrolase family（GHF） 11 mesophilic xylanase from Aspergillus oryzae，its N-terminal region was substituted with the corresponding one of the same family thermostable xylanase pXYL11 from Thermobifida fusca NTU22. Based on the rational design of xylanase thermotolerance，an ATX11A-encoding gene（ATx11A） was constructed by replacing the 5′-end DNA fragment of AoXyn11A gene（Aoxyn11A） with the corresponding one of the synthesized pXYL11 gene（xyn11PM） using the megaprimer PCR technique. Aoxyn11A and ATx11A were expressed in Pichia pastoris GS115，respectively，and the temperature characteristics of the expressed recombinant products，AoXyn11A and ATX11A，were analyzed. The results indicated that the temperature optimum（Topt） of hybrid xylanase ATX11A was 65 ℃，which was 15 ℃ higher than that of AoXyn11A. Its half-life at 60 ℃（t1/260） was 55 min，which was 41.3-fold longer than that of AoXyn11A. When incubated at 55 ℃，ATX11A retained more than 60% of its original activity for 3 h，while AoXyn11A entirely lost its activity only for 15 min. In this work，the temperature characteristics of AoXyn11A were significantly improved by N-terminus replacement.