乳糖诱导对重组大肠杆菌表达CGTase的影响  

Expression of cyclodextrin glycosyltransferase( CGTase) gene in recombinant Escherichia coli induced by lactose

作　　者：林晓栩 林新坚[2] 邱宏端[1] 陈龙军[2] 陈济琛[2] 

机构地区：[1]福州大学生物科学与工程学院,福建福州350108 [2]福建省农业科学院土壤肥料研究所,福建福州350003

出　　处：《食品工业科技》2015年第17期114-120,126,共8页Science and Technology of Food Industry

基　　金：国家公益性农业科研专项(201303094-05);福建省财政厅社会公益研究项目(2060302);福建省科技计划项目-省属公益类科研院所基本科研专项(2009R10002-2);福建省星火计划项目(2012S0061);2013年院创新团队编号CXTD-2-1318

摘　　要：以重组环糊精葡萄糖基转移酶(CGTase)表达菌株BL21(DE3)作为研究对象,比较IPTG和乳糖诱导对重组大肠杆菌表达CGTase量的影响,确定优化诱导方式。以Geobacillus CHB1的基因组DNA为模板,采用PCR技术克隆CGTase基因;利用分子操作技术构建携带有大肠杆菌Omp A信号肽的分泌型表达质粒p EASY-E2-Omp A-CGT,重组质粒热激转化至Escherichia coli BL21中进行表达;在摇瓶发酵条件下,分别以IPTG和乳糖作为诱导剂,通过SDSPAGE分析和测定胞外酶活,确定优化诱导方式。CGTase基因在Escherichia coli BL21中实现表达,且具有一定量的重组CGTase分泌至胞外;乳糖诱导的蛋白表达量优于IPTG诱导,不易形成包涵体;乳糖最佳的诱导起始菌浓度OD600为1.4,诱导浓度、温度分别为0.5%和25℃,分批流加乳糖5次和一次性加入,重组CGTase表达量无明显差异;经初步优化,胞外酶活达19.87 U/m L。Geobacillus CHB1 CGTase基因在大肠杆菌中成功实现胞外表达,乳糖可替代IPTG诱导重组CGTase。The optimizied expression of the cloned Cyclodextrin glycosyltransferase（ CGTase） Gene in Escherichia coli strain BL21（ DE3） induced by lactose was reported. By comparing the influence of lactose and IPTG on the expression amount of the cloned CGTase,the induction mode was optimized.The genomic DNA of Geobacillus sp.CHB1 was used as template and the CGTase gene was amplified by the PCR technology. The secretory plasmid p EASY- E2- Omp A- cgt which contained the signal peptide Omp A of E.coli was constructed. The recombinant plasmid was transformed into E.coli BL21 and then induced to express.The expression level of the target protein in the shake- flask cultures induced by IPTG and lactose were analyzed by SDS- PAGE and meanwhile the extracellular enzyme activities were measured.The factors including inducer concentration,induction time,induction temperature and induction method were optimized. The CGTase gene was successfully expressed in E.coli BL2 l,and a certain amount of recombinant CGTase were secreted into the culture medium.The expression amount of the cloned CGTase induced by lactose was more than that induced by IPTG and the amount of inclusion body was less than that induced by IPTG.The optimal conditions for extracellular expression were as follows： 0.5% lactose,initial concentration of E.coli with OD600= 1.4 and the temperature with 25 ℃. There was no significant difference in the expression amount between addition of lactose in batch and one time. Under this condition,the extracellular activity reached 19.87 U / m L.The extracellular expression of CGTase gene from Geobacillus sp.CHB1 in E.coli BL2 l was achieved.Lactose could be used as an inducer instead of IPTG for the expression of recombinant CGTase.

关 键 词：GEOBACILLUS CHB1 环糊精葡萄糖基转移酶 乳糖 IPTG 诱导表达 

分 类 号：TS201[轻工技术与工程—食品科学]