SMB-1型金属β-内酰胺酶的原核表达及酶活性研究  

Prokaryotic expression and enzyme activity study of SMB-1 metallo-β-lactamase

作　　者：沈秉正[1] 宋金春[1] 彭燕[1] 李荣凌[1] 

机构地区：[1]武汉大学人民医院药学部,湖北武汉430060

出　　处：《广东药学院学报》2013年第4期439-442,共4页Academic Journal of Guangdong College of Pharmacy

摘　　要：目的原核表达SMB-1型金属β-内酰胺酶,并研究其水解β-内酰胺类抗生素的活性。方法利用化学合成方法合成blaSMB-1基因,经PCR扩增后克隆至pET28a载体,构建重组的原核表达质粒pET28a-SMB-1,转化至E.coli BL21(DE3),诱导表达。表达的重组蛋白经Ni2+-NTA纯化后,测定其水解底物时的酶促反应动力学参数及最适pH值和温度。结果重组质粒经测序证明其正确性;表达的重组蛋白相对分子质量约为33 000,纯化后蛋白的质量浓度为0.20 mg/mL,该酶对本实验中除氨曲南外的所有β-内酰胺类抗生素均具有较高水解活性,其最适pH值为8.0,最适温度为40℃。结论利用工程菌E.coliBL21(DE3)-pET28a-SMB-1成功实现了SMB-1型金属β-内酰胺酶的原核表达,为深入研究其生物学功能及作用机制提供了物质基础,同时也为研发该酶的抑制剂提供了靶标物质。Objective To express the SMB-1 metallo-β-lactamase（SMB-1） in prokaryotic cells and determine its hydrolytic activity of β-lactam antibiotics.Methods blaSMB-1 gene was obtained by chemical synthesis,amplified by PCR and cloned into vector pET28a.The constructed recombinant plasmid pET28aSMB-1 was transformed to E.coli BL21（DE3） for expression under induction.The expressed recombinant protein was purified by Ni2 ＋-NAT chromatography and determined for the kinetic parameters of the enzymic reaction,optimal pH and optimal temperature.Results Sequencing proved that the recombinant plasmid was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 33 000,reached a concentration of 0.20 mg /mL after purification,and showed high hydrolytic activity of β-lactam antibiotics except aztreonam.The optimal pH value of the recombinant protein was 8.0 and the optimal temperature was 40 ℃.Conclusion SMB-1 metallo-β-lactamase is successfully expressed in E.coli BL21（DE3）-pET28a-SMB-1,which provides a material for further research on its biological function and hydrolysis mechanism,and also a target material for development of inhibitors.

关 键 词：SMB-1金属β-内酰胺酶 原核表达 酶活性 

分 类 号：R978.1[医药卫生—药品]