D-甘露糖异构酶的克隆表达及酶法制备D-甘露糖  

Cloning and Expression of D-Mannose Isomerase from Escherichia coli BL21 and its Application for D-Mannose Production

作　　者：胡兴 张涛[1] 沐万孟[1] 缪铭[1] 江波[1] HU Xing;ZHANG Tao;MU Wanmeng;MIAO Ming;JIANG Bo(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China;School of Food Science and Technology,Jiangnan University,Wuxi 214122,China)

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

出　　处：《食品与生物技术学报》2019年第4期58-63,共6页Journal of Food Science and Biotechnology

基　　金：国家863计划项目(2013AA102102);国家自然科学基金项目(31230057)

摘　　要：将大肠杆菌BL21中的D-甘露糖异构酶(MIase)基因yihS克隆到表达载体pET-28a(+)中,并转入大肠杆菌BL21(DE3)感受态细胞中表达。重组菌株经IPTG诱导培养6 h后MIase发酵酶活可达4.2 U/mL。重组MIase生产D-甘露糖时不需要金属离子的参与。该酶在40℃和pH 7.5条件下表现出催化D-果糖的最高活性,转化率为25%左右;通过研究底物浓度对酶活性的影响,得出该酶的活性不受底物浓度的抑制,以D-果糖为底物时,动力学参数Km为123.32mmol/L,Vmax为113.64μmol/(min·mg),催化效率kcat/Km为0.691 (s·mmol/L)。The gene encoding D-mannose isomerase(MIase) from Escherichia coli(E. coli) BL21 was cloned into expression vector pET-28a(+),and then the recombinant plasmid was transformed into the strain E. coli BL21(DE3). Efficient MIase intracellular expression by the recombinant E. coli BL21 was achieved with an activity of 4.2 U/mL(D-mannose forming) after IPTG induction for 6 h. The enzyme was identified as a metal-independent protein. The enzyme activity reached the maximum with a conversion rate of 25% at 40 ℃ and pH 7.5. Using D-fructose as the substrate,a Km value of 123.32 mmol/L,Vmax value of 113.64 μmol/(min·mg) and catalytic efficiency kcat/Km value of 0.691 s·mmol/L were estimated,respectively.

关 键 词：D-甘露糖 D-甘露糖异构酶 表达 生物制备 

分 类 号：Q814.1[生物学—生物工程]