黑曲霉木聚糖酶XynB耐热性的定向改造及表达、纯化  

Expression and purification of thermostable xylanase from Aspergillus niger based on rational design

作　　者：余英鹏[1] 姜腾飞[1] 李茜[1] 马晓瑞[1] 宋济君 刘博[1] 乔代蓉[1] 曹毅[1] 徐辉[1] 

机构地区：[1]四川大学生命科学学院微生物与代谢工程四川省重点实验室,成都610065

出　　处：《应用与环境生物学报》2014年第6期1082-1085,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家"十二五"科技支撑计划项目(2013BAD10B01;2014BAD02B02);国家自然科学基金项目(31171447);四川省科技支撑项目(2013HH0007;2013GZX0161-3;2013GZ0058)资助~~

摘　　要：酶活性高的木聚糖酶在工业中具有巨大应用潜力,但大部分野生木聚糖酶不能耐受工业生产过程中的极端环境比如高温,因此很有必要通过分子生物学的方法改造木聚糖酶.本文通过分子动力学模拟和定点突变的方法以提高黑曲霉(SCTCC400264)来源的木聚糖酶Xyn B的耐热性.利用定点突变技术获得突变载体p ET32a-Xyn B-T79V并转化大肠杆菌Escherichia coli BL21(DE3),经IPTG诱导成功表达木聚糖酶Xyn B-T79V.在镍亲和层析纯化蛋白后测定了突变酶的耐热性.突变体Xyn B-T79V在30、40、50℃处理120 min后酶活几乎没有损失,当处理温度升高到60-80℃时,酶活开始下降.温度升到80℃时,残余酶活仍有37%,而此时野生型Xyn B的活性几乎完全丧失.突变体Xyn B-T79V半衰期t1/2提高了8.7 min.本研究获得的突变酶Xyn B-T79V耐热性显著提高,为工业应用提供了新的酶资源.Xylanases have drawn much attention because of their considerable potential in industrial applications. However, most wild-type xylanases do not possess the characteristics to suit extreme conditions such as high temperature in the industrial process. It is worthwhile and necessary to design xylanases by molecular approaches. The xylanase （XynB） from Aspergillus niger （SCTCC400264） possesses high activity but displays low thermostability. In this work, an attempt was made to improve the thermostability of xylanase （XynB） from A. niger （SCTCC400264） by molecular dynamics simulation and site-directed mutagenesis. The recombinant pET32a-XynB-T79V was constructed by site-directed mutagenesis and then transformed into Escherichia coli BL21 （DE3）. Xylanase XynB-T79V was successfully expressed by the addition of IPTG. The enzyme was purified by using nickel-affinity chromatography and the thermal stability of mutant xylanase determined. After treatment at 30, 40 and 50 ℃ for 2h, respectively, the activity of the enzyme XynB-T79V was almost as high as before. The activity of mutant enzyme decreased after incubation at 60, 70 and 80 ℃. When the temperature rose to 80 ℃, the enzyme XynB- T79V had a remaining enzyme activity of 37% while the wild-type XynB completely lost its activity. The half-life of XynB- T79V was 8.7 min longer than that of XynB. This work obtained a mutant enzyme XynB-T79V with a significantly improved thermostability, providing a potential enzyme source for industrial processes.

关 键 词：木聚糖酶 耐热性 分子动力学模拟 定点突变 理性设计 

分 类 号：Q936[生物学—微生物学]