变形假单胞菌2-酮基葡萄糖酸差向异构酶基因的原核表达  

Prokaryotic Expression of a 2-Ketogluconate Epimerase Gene from Pseudomonas plecoglossicida

作　　者：孙文敬[1,2] 杨荔 栾方[1] 何小用 崔凤杰[1,2] 王大明[2,3] 钱静亚[1,2] 齐向辉[1] SUN Wenjing;YANG Li;LUAN Fang;HE Xiaoyong;CUI Fengjie;WANG Daming;QIAN Jingya;QI Xianghui(School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China;Parchn Sodium Isovitamin C Co. Ltd., Dexing 334221, China;School of Biotechnology, Jiangnan University, Wuxi 214122, China)

机构地区：[1]江苏大学食品与生物工程学院,江苏镇江212013 [2]百勤异VC钠有限公司,江西德兴334221 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《食品科学》2018年第2期66-72,共7页Food Science

基　　金：国家自然科学基金面上项目(31571885);国家高技术研究发展计划(863计划)项目(2012AA022103);江西省科技计划项目(赣知发[2015]64号);江西省创新团队建设计划项目(20142BCB24024);江西省科技平台建设计划项目(2010DTZ01900);德兴市科技计划项目(德科发[2015]44号)

摘　　要：采用降落聚合酶链式反应(touchdown polymerase chain reaction,TD-PCR)技术,从2-酮基葡萄糖酸工业生产菌株变形假单胞菌JUIM01中克隆了编码2-酮基葡萄糖酸差向异构酶的基因kgu E。将目的基因与质粒pET-28a(+)重组后转入宿主表达菌中,获得了重组菌株大肠杆菌BL21(DE3)/pET-28a(+)-kgu E。在异丙基-β-D-硫代半乳糖苷的诱导下,该重组菌株表达了一个分子质量约为30.5 ku的融合蛋白,且该蛋白的Western-blot鉴定结果显示为阳性。生物信息学分析结果表明,该蛋白是一种由256个氨基酸残基组成的分子质量为28.5 ku的亲水性蛋白,与恶臭假单胞菌的2-酮基葡萄糖酸差向异构酶在氨基酸序列上的一致性达78%,定位于细胞质中,其二级结构中α-螺旋、延伸链和无规则卷曲所占的比例分别为40.62%、17.19%和42.19%。本研究结果为变形假单胞菌2-酮基葡萄糖酸差向异构酶的功能研究提供了一定理论支持。The complete nucleotide sequence of the gene（kguE） encoding 2-ketogluconate epimerase was cloned by touchdown polymerase chain reaction（TD-PCR） from an industrial 2-ketogluconic acid producer,Pseudomonas plecoglossicida JUIM01.A recombinant vector was constructed by ligating the restriction enzymes digested products of the target gene to pET-28 a（＋） vector and then transferred into the expression host E.coli BL21（DE3）.A specific fusion protein with molecular weight of about 30.5 ku was expressed in the recombinant strain E.coli BL21（DE3）/pET-28 a（＋）-kgu E after isopropyl β-D-1-thiogalactopyranoside（IPTG） induction with a positive Western-blot result.Bioinformatic analysis showed that the protein was predicted to be a hydrophilic protein with molecular weight of 28.5 ku located in the cytoplasm,sharing 78% amino acid sequence identity with the 2-ketogluconate epimerase from P.putida.The predicted secondary structure consisted of 40.62% of α-helix,17.19% of extended strand and 42.19% of random coil.This study is expected to provide a basis for further elucidating the function of 2-ketogluconate epimerase in P.plecoglossicida.

关 键 词：变形假单胞菌 2-酮基葡萄糖酸差向异构酶 kgu E基因 克隆 表达 生物信息学 

分 类 号：Q936[生物学—微生物学]