组氨酸标签木聚糖酶的构建表达和酶特性鉴定

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 5¹0具有良好的稳定性,在pH 6.5,70℃条件下处理30min相对酶活力仍保留80%以上.

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

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.