Enhancement In The Thermotolerance Of Xylanase (AoXyn11A) By N-terminus Replacement And Site-directed Mutagenesis

Recently, with the exploitation of hemicellulose resources,xylanases as environmentally effective biocatalysts, have been widely used in animal feed, food, pulp and paper, medical and pharmaceutical, textile, biofuel and so on. Xylanases can randomly catalyze the hydrolysis of internal β-1,4-D-xylosidic linkages of xylans with producing xylooligosaccharides and small amounts of xylose. Although a lot of xylanases have been cloned and expressed, most of them are mesophilic xylanases. Their applications are limited by their poor thermostability in the high-temperature environment of industry. Thus, the modification of mesophilic enzymes becomes meaningful. In our previous study, a mesophilic glycoside hydrolase(GH) family 11 xylanase of Aspergillus oryzae(AoXyn11A) was cloned and expressed in Pichia pastoris GS115, with high specific activity, broad p H stability and strong tolerance towards metal ions, but poor thermostability. This work amied to enhance the thermotolerance of Ao Xyn11 A and explore the thermotolerant mechanism by N-terminus replacement and site-directed mutagenesis technologies.Based on sequence aligment of Ao Xyn11 A and the same family thermostable xylanase p XYL11 from Thermobifida fusca, molecular dynamics simulation and comparison of B-factor values, a hybrid xylanase ATX11A was constructed by substituting a segment S1~V41 at N-terminus of Ao Xyn11 A with the corresponding one from A1~T42 of p XYL11. The results indicated that the temperature optimum(Topt) of the hybrid xylanase ATX11A was 65℃, which was 15℃ higher than that of Ao Xyn11 A. Its half-life at 60℃(t1/260) was 55 min, which was 41.3-fold longer than that of AoXyn11A(t1/260=1.3 min). Based on multiple aligment between Ao Xyn11 A and other five thermostable GH 11 family xylanases(including p XYL11), another mutant xylanase ATX11AM was designed by mutating 96NPGSG100 into RPT in “cord” of ATX11A. Consistent with the ATX11A, ATX11AM also displayed the highest activity at 65℃, while, its t1/260 was extended from 55 min to 83 min. Its t1/265 was 31 min, which was 1.8 times of ATX11AM. In addition, the melting temperatures(Tm) of ATX11A and ATX11AM were 72.7 and 77.9℃, which were 12.5 and 17.7℃ higher than that of AoXyn11A(Tm=60.2℃), respectively.Based on the primary and three-dimensional(3-D) structures analysis, two salt bridges(H10-D11 and D11-K49) and two N-glycosylations(N5-E-T7 and N34-Y-S36) were introduced into ATX11A resulted from N-terminus repalcement. In order to explore the thermotolerant mechanism of ATX11A, the two salt bridges were removed from ATX11A by site-directed mutagenesis(D11N). Analytical results showed that the Topt of the mutant xylanase ATX11AD11 N dropped to 60℃. After being incubated at 55 and 60℃ for 60 min, ATX11A retained 79.4% and 49.9% of its original activity while ATX11AD11 N retained 42.3% and 17.8% of its original activity respectively. It was confirmed that the N-terminal salt bridge is one of the factors affecting the thermotolerance of ATX11A. Then, two N-glycosylations were removed from ATX11A by site-directed mutagenesis(N5S and N34S). Consistent with the ATX11A, the mutant xylanase ATX11AS also displayed the highest activity at 65℃. After being incubated at 55 and 60℃ for 60 min, the changes of residual activities resulted from the mutagenesis were not obvious. That is, the N-glycosylation has no effect on the thermotolerance of ATX11A.