大肠杆菌甘露糖异构酶(MI)基因克隆及功能研究  

The Mannose Isomerase Gene Cloning and Function Analysis in Escherichia coli

作　　者：高小倩[1] 张春晓[1] 陈晓波[1] 王丽丽[1] 仪宏[1] 

机构地区：[1]河北科技大学生物科学与工程学院,石家庄050018

出　　处：《生物技术通报》2010年第11期219-222,237,共5页Biotechnology Bulletin

基　　金：国家科技支撑计划项目(2007BAI26B01)

摘　　要：甘露糖异构酶(EC5.3.1.7,Mannose isomerase,简称MI)能够催化果糖和甘露糖之间的异构转化。甘露糖加氢催化形成甘露醇,因此甘露糖异构酶的研究为工业生产甘露糖和甘露醇提供了新的可能途径。本研究以Escherichia coliJM109基因组DNA为模板,PCR扩增mi基因。结果表明,所克隆的mi基因与E.colistr.K-12substr.MG1655的MI基因序列一致性为99.9%,氨基酸序列一致性为99%。该基因能够在E.coliBL21(DE3)中进行高效诱导表达,诱导出51.4kD的特异性融合蛋白。酶学研究表明,该酶最适反应温度为37℃,最适pH为7.5,甘露糖为最佳反应底物,反应平衡时果糖与甘露糖的比值约为2.7。Mannose isomerase（MI,EC 5.3.1.7）can catalyze fructose to mannose which is valuable in mannitol industrial production.The mannose isomerase gene of E.coli JM109 was successfully amplified by PCR reaction and were cloned into vector pET-30a（＋）.The results showed that the cloned mi gene had 99.9% identity with the mi gene of E.coli str.K-12 substr.MG1655,and the amino acids sequence identity was 99%.The fused protein（51.4 kD）screened by SDS-PAGE gel showed that it was overexpressed in E.coli BL21（DE3）when induced by IPTG.The MI has the highest activity at 37℃,pH 7.5.The optimum substrate was mannose compared with fructose.The ratio of D-fructose and D-mannose was about 2.7 when the reaction reached to the balance.

关 键 词：甘露糖异构酶 克隆 原核表达 酶活 

分 类 号：Q78[生物学—分子生物学]