大肠杆菌甘露糖异构酶(MI)基因克隆及其功能的研究.pdfVIP

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 2010 11 (M I ) 高小倩 张春晓 陈晓波 王丽丽 仪宏 ( , 0500 18 ) : 甘露糖异构酶( EC 5. 3. 1. 7, M annose isom erase, 简称M I)能够催化果糖和甘露糖之间的异构转化甘露糖加 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E scherichia coli JM 109 基因组 DNA 为模板, PCR 扩增m i基因结果表明, 所克隆的 m i基因与E. coli s r. K12 subs r. M G 1655 的M I基因序列 一致性为 999% , 氨基酸序列一致性为 99% 该基因能够在E. coli BL2 1( DE3) 中进行高效诱导表达, 诱导出5 14 kD 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 75, 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 27 : 甘露糖异构酶 克隆 原核表达 酶活 TheM annose Isomerase Gene Cloning and Function Analysis inE scherich ia coli Gao X iaoq ian Zhang Chunx iao Chen X iaobo W ang L ili Y iH ong (D epartment of B ioscience and Bioengineering,H ebei University of Science and Technology, Shij iazh ang 050018) Abstrac:t M annose isome rase ( M I, E C 5317) can ca a lyze fruc ose o m annose w hich is va luab le in m ann i ol indu s r ia l pro duc ion. T he m annose isom erase gene o fE. coli JM 109 w as successfully am plif ied by PCR reac ion and w e re cloned in o vec o r pET30a ( + ) . The resul s show ed ha he cloned m i gene had 999% iden i y w i h he m i gene ofE. coli s r. K12 subs r. M G 1655, and he a m ino acids sequence iden i y w as 99% . T he fu sed pro e in ( 514 kD ) screened by SDSPAGE ge l show ed ha i w as overexpressed in E. coli BL 21 ( DE3) w hen induced by IPTG. T he M I has he h ighes ac iv i y a 37 , pH 75. The op mi um subs ra e w as m annose compared w i h fruc ose. The ra io of Dfruc ose and Dm annose w as abou 27 w hen he reac ion reached o he ba lance. K ey w ords: M annose isom erase C lone Induced express ion Enzym e ac iv i y [ 1] ( EC 5317,