二硫键对提高木聚糖酶AoXyn11A热稳定性的作用  

Effect of Disulfide Bridge on Thermostability Iimprovement of the Xylanase AoXyn11A

作　　者：刘晓彤[1] 邬敏辰[2] 殷欣[3] 汪俊卿[3] 

机构地区：[1]江南大学药学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2014年第10期1038-1043,共6页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31101229)

摘　　要：为提高米曲霉11家族中温木聚糖酶Ao Xyn11A的热稳定性,将Ao Xyn11A与源于Thermomyces lanuginosus的耐热木聚糖酶Xyn A进行三维结构比对分析,发现Xyn A的α-螺旋与β9-折叠之间存在一个二硫键;基于分子动力学模拟预测结果,利用定点突变技术在Ao Xyn11A的相应位置引入二硫键(Cys108-Cys152),获得突变酶Ao Xyn11AT;分别将Ao Xyn11A及其突变酶基因在大肠杆菌BL21(DE3)中表达,经纯化,获得重组Ao Xyn11A和Ao Xyn11AT,并分析比较温度对其酶活性的影响。结果显示,重组Ao Xyn11AT的最适温度为60℃,比重组Ao Xyn11A提高了5℃;其在50℃下的半衰期t1/250为71 min,较重组Ao Xyn11A(t1/250=25 min)提高了近2倍。研究表明二硫键对提高Ao Xyn11A的热稳定性有重要作用。To improve the thermostability of the family 11 xylanase AoXyn11 A from Aspergillus oryzae,homology alignment and analysis of the three dimensional structures between thermostable xylanase Xyn A from Thermomyces lanuginosus and Ao Xyn11 A were carried out. Resultantly,a disulfide bridge was located between the α-helix and β9 sheet of Xyn A while none in AoXyn11 A.Based on the prediction by molecular dynamics simulation,a disulfide bridge（Cys^108-Cys^152） was introduced into the corresponding region（α-helix region） of AoXyn11 A by site-directed mutagenesis. The mutant xylanase,AoXyn11 AT,was obtained. Then,the AoXyn11A- and AoXyn11AT-encoding genes were separately expressed in Escherichia coli BL21（DE3）. After purification,the effects of temperature on xylanase activity of the recombinant AoXyn11 A and AoXyn11 ATwere analyzed and compared. The temperature optimum（Topt） of the recombinant AoXyn11 ATwas 60 ℃,which was 5 ℃ higher than that of the recombinant AoXyn11 A. The half-life of the recombinant Ao Xyn11 ATat 50 ℃（t1/2^50） was 71 min,which was about 3-fold than that（25min） of the recombinant Ao Xyn11 A. This study demonstrated that the disulfide bridge played an important role in thermostability improvement of the xylanase Ao Xyn11 A.

关 键 词：木聚糖酶 二硫键 定点突变 耐热性 

分 类 号：Q55[生物学—生物化学]