CAJ | 학술논문

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．
통과개변종지밀마자위점，재일개내열목취당매돌변주DSB적C말단첨가료6취조안산표첨（ His-tag ），병완성료표체화매특성감정．통과PCR장기열진균DSM 10635래원적목취당매돌변체기인dsb적종지밀마자서렬정점돌변위곡안산밀마자서렬，련접도표체재체pET-22b（＋），전화Escherichia coli．BL21（DE3），유도표체적내열목취당매DSB재C단함유6×His-tag．표체산물경류산안분급침정화Ni Sepharose층석순화，획득료전영순중조매DSB．결과표명：첨가조안산표첨적매여원시매상비，매학특성무변화．SDS-PAGE전영결과현시해매분자량약위23 kDa，중조매최괄pH위6．5，최괄온도위75℃，재pH 5～10구유량호적은정성，재pH 6．5，70℃조건하처리30 min상대매활력잉보류80％이상．
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.