密苏里游动放线菌葡萄糖异构酶基因xylA的克隆及其表达条件的优化  

Cloning of Glucose Isomerase Gene from Actinoplanes missouriensis and Its Expression in Escherichia coli

作　　者：王贺[1,2] 杨瑞金[2] 华霄[2] 钱婷婷[1] 张文斌[2] 蒋孝燕[2] 

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学食品学院,江苏无锡214122

出　　处：《食品科学》2010年第15期171-176,共6页Food Science

基　　金：国家"863"计划项目(2006AA10Z336);江南大学食品科学与技术国家重点实验室目标导向资助项目(SKLF-MB-200804);江南大学博士研究生科学研究基金项目;以乳糖异构化为目标的葡萄糖异构酶定向改造研究(SKLF-TS-200903)

摘　　要：为克隆、表达密苏里游动放线菌葡萄糖异构酶(GI)基因xylA,并对其诱导表达条件进行初步优化。采用Slowdown PCR方法克隆得到密苏里游动放线菌(Actinoplanes missouriensis)CICIM B0118(A)的葡萄糖异构酶基因xylA,构建pET-28a(+)-xylA表达载体,并转化至E.coliBL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并对其表达产物进行SDS-PAGE电泳。结果表明,融合蛋白分子质量约为45kD。在诱导时间9h、0.6mmol/LIPTG、30℃和OD600nm值为0.8的最适培养条件下,酶比活力最高达到62.42U/mL。The xylA gene encoded glucose isomerase（GI） was amplified from genomic DNA of A.missouriensis CICIM B0118（A） by Slowdown PCR and subcloned into the expression vector pET-28a（＋） to obtain an N-terminus His-tagged fusion expression plasmid pET-28a（＋）-xylA.pET-28a（＋）-xylA was then transformed into E.coli BL21（DE3）.The recombinant protein was actively expressed in E.coli BL21（DE3） in the presence of isopropy-β-D-thiogalactoside（IPTG）.SDS-PAGE analysis showed that the partially purified recombinant protein exhibited a major band with an apparent molecular weight of 45 kD in,which was consistent with the molecular weight calculated from the amino acid sequence.When the induction conditions were time 9h,IPTG 0.6mmol/L,temperature 30℃ and OD600nm 0.8,the enzyme activity of recombinant GI were 62.42 U/mL.These results can offer an experimental basis for the further investigation of directed evolution of GI into an artificial enzyme with the ability to isomerize lactose into lactulose.

关 键 词：密苏里游动放线菌 葡萄糖异构酶 表达 SLOWDOWN PCR 

分 类 号：Q786[生物学—分子生物学]