解淀粉芽孢杆菌α-淀粉酶基因的克隆与表达  

Cloning and expression of α-amylase from Bacillus amyloliquefaciens

作　　者：李尔汉 彭思露 李汉昕 徐艳红[1] 王筱兰[1] 朱笃[1,2] 杨慧林[1,2] 

机构地区：[1]江西师范大学生命科学学院,江西省亚热带植物资源保护与利用重点实验室,江西南昌330022 [2]江西科技师范大学生命科学学院,江西省有机功能分子重点实验室,江西南昌330013

出　　处：《生物技术》2014年第6期13-18,共6页Biotechnology

基　　金：国家"十二五"科技支撑项目("鄱阳湖流域重要珍稀濒危植物资源可持续利用研究与示范";No.2011BAC13B04);江西省教育厅青年科学基金项目("基于全基因组测序的转谷氨酰胺酶酶原激活途径研究";No.GJJ13211);江西省亚热带植物资源保护与利用重点实验室开放基金("蛇足石杉内生真菌Shiraia sp.Slf14中石杉碱甲生物合成途径研究";No.YRD201405)资助

摘　　要：[目的]实现解淀粉芽孢杆菌α-淀粉酶在大肠杆菌中的高效表达,建立有效的透析复性方法,获得有活性的重组淀粉酶。[方法]以解淀粉芽孢杆菌DSM 7基因组DNA为模板,PCR扩增获得无信号肽的α-淀粉酶结构基因,克隆至p ET-22b(+),转化E.coli BL21(DE3),IPTG诱导表达,SDS-PAGE检测重组蛋白表达情况,采用透析法进行包涵体复性并检测酶活。[结果]成功表达重组蛋白,相对分子量约为54.8k Da,成功复性包涵体,复性效率为22.78%,重组α-淀粉酶酶活力为102.4 U/m L。[结论]实现了解淀粉芽孢杆菌α-淀粉酶在大肠杆菌中的高效表达,包涵体经透析法成功复性,获得具有催化活性的重组淀粉酶。[Objective]Achieved α -amylase from efficiently expression in E. coil,established an effective dialysis renaturation method, and obtain catalytically active recombinant amylase. [ Methods ] An α - amylase gene devoid of signal peptide sequence from Bacillus amyloliquefaciens DSM 7 was gained by PCR and cloned into expression vector pET -22b（ ＋ ）. The right plasmid was transformed into E-. coli BL21 （DE3） ,induced expression by IPTG and SDS -PAGE electrophoresis to detected recombinant protein expression situation. The inclusion body was refolded using a dialysis renaturation process and subsequently determinated its α -amylase activity. [ Results] The re- combinant protein was successfully expressed, and the molecular weight was about of 54. 8 kDa. The inclusion body proteins refolded back to the bioactive native conformation, the efficiency of renaturation is 22. 78%. Through measuring, the amylase activity was 102.4 U/mL [ Conclusion] Amylase from Bacillus amyloliquefaciens DSM 7 expressed in E. coli in inclusion body form. Through dialysis, the inclusion bodies refolded back to the bioactive native conformation, showed amylase activity.

关 键 词：解淀粉芽孢杆菌 Α-淀粉酶 诱导表达 包涵体 透析复性 

分 类 号：Q786[生物学—分子生物学]