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-28 a(+),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,Vmaxvalue of 113.64 μmol/(min·mg) and catalytic efficiency kcat/Kmvalue of 0.691 s·mmol/L were estimated,respectively.
引文
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