组氨酸标签木聚糖酶的构建表达和酶特性鉴定  

Construction and Characterization of a Thermostable Xylanase Mutant with 6 × His-tag

作　　者：熊海容[1] 陈丰[1] 王亚伟[1] 张巍[1] 

机构地区：[1]中南民族大学生命科学学院,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2014年第3期27-31,共5页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：武汉市科技攻关项目(SZY13005);中央高校基本科研业务费专项基金资助项目(ZFY10006)

摘　　要：通过改变终止密码子位点，在一个耐热木聚糖酶突变株DSB的C末端添加了6聚组氨酸标签（ His-tag ），并完成了表达和酶特性鉴定．通过PCR将嗜热真菌DSM 10635来源的木聚糖酶突变体基因dsb的终止密码子序列定点突变为谷氨酸密码子序列，连接到表达载体pET-22b（＋），转化Escherichia coli．BL21（DE3），诱导表达的耐热木聚糖酶DSB在C端含有6×His-tag．表达产物经硫酸铵分级沉淀和Ni Sepharose层析纯化，获得了电泳纯重组酶DSB．结果表明：添加组氨酸标签的酶与原始酶相比，酶学特性无变化．SDS-PAGE电泳结果显示该酶分子量约为23 kDa，重组酶最适pH为6．5，最适温度为75℃，在pH 5～10具有良好的稳定性，在pH 6．5，70℃条件下处理30 min相对酶活力仍保留80％以上．A thermostable xylanase mutant DSB from Thermomyces lanuginosus was expressed in Escherichia coli with a 6 × His-tag in C terminal.DSB with terminator changed to Glu was amplified by PCR and cloned into plasmid pET-22b（＋）, then transformed into E.coli BL21 （ DE3 ） .The cultivation was purified by ammonium sulfate precipitation and Ni-nitrilotriacetate affinity chromatography .The results from SDS-PAGE showed that xylanase DSB-His was electrophoretically homogeneous , with a molecular weight of approximately 23 kDa.The xylanase DSB showed optimal activity at 75℃and pH 6.5, was highly stable at pH 5.0-10.0, and retained more than 80%activity after incubation at 70℃for 30 min.

关 键 词：耐热木聚糖酶 多聚组氨酸标签 定点突变 镍亲和层析 酶学特性 

分 类 号：R966[医药卫生—药理学]