ImiS的表达、纯化及催化3类β-内酰胺类抗生素水解的动力学研究  

Expression,purification of ImiS and kinetic studies on hydrolysis of three types of β-lactam antibiotics catalyzed by ImiS

作　　者：冯吉利[1] 杨霞[1] 闫小艳[1] 高会洲[1] 冯雷[1] 程序[1] 贾超[1] 吴迪[1] 杨科武[1] 

机构地区：[1]合成与天然功能分子化学教育部重点实验室西北大学化学与材料科学学院,西安710069

出　　处：《中国抗生素杂志》2011年第3期197-200,216,共5页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(20972127);教育部博士点基金(200806970005);人力资源部基金(SLZ2008013);陕西省自然科学基金(2009JM2002);陕西省教育厅基金(09JK786)

摘　　要：目的研究金属β-内酰胺酶(MBL)ImiS催化β-内酰胺类抗生素水解的动力学特征,为研发MBLs的通用型抑制剂提供实验数据。方法在大肠埃希菌中表达ImiS,以SP-Sepharose离子交换层析柱纯化,通过MALDI-TOF MS测定其分子量,用UV-VIS测定ImiS催化法罗培南等3类6种β-内酰胺类抗生素水解的酶动力学参数,并考察不同pH、温度及底物浓度对酶活性的影响。结果表征获得ImiS的分子量为25209Da,ImiS对法罗培南、青霉素G、氨苄西林及羧苄青霉素水解的催化常数Kcat分别为(2.15±0.03),(0.85±0.03),(0.71±0.02)和(0.58±0.02)s-1,对头孢三嗪和头孢噻肟无活性。底物为法罗培南时,ImiS最适活性的pH为7.5±0.1,在酸性环境中酶活性降低较为明显,最适活性的温度为(44.8±0.8)℃。结论治疗携带ImiS的病菌引起的感染,使用法罗培南不妥,而头孢三嗪或头孢噻肟为合理。实验结果为ImiS抑制剂的设计与合成提供了很有价值的信息。Objective Kinetic studies on the hydrolysis of β-lactam-containing antibiotics catalyzed by metallo-β- lactamases （MBLs） ImiS and providing experimental data for research and development of universal inhibitors of MBLs. Methods ImiS was expressed in BL21 （DE3） E. coli. and purified SP-Sepharose chromatography. The molecular weight of ImiS was determined by MALDI-TOF MS, the kinetic parameters for hydrolysis of 3 groups （6 kinds） of β-lactam antibiotics catalyzed by ImiS were determined by UV-Vis, and the effects of pH, temperature and substrate concentration on enzymatic activity were investigated. Results The molecular weight of ImiS is 25209Da. Kat values of ImiS catalyzing the hydrolysis of faropenem, penicillin G, ampicillin and carbenicillin are （2.15±0.03）, （0.85±0.03）, （0.71±0.02） and （0.58±0.0）s-1, respectively, and ImiS is inactive to ceftriaxone and cefotaxime. The optimum pH value for ImiS hydrolyzing faropenem is 7.5±0.1 and the optimum temperature is （44.8±0.8）℃. Conclusion For treatment of the bacteria with ImiS, choosing ceftriaxone or cefotaxime is rational, but faropenem is irrational. The experimental results provide very valuable information for rationally design and synthesis of the universal inhibitors of ImiS.

关 键 词：细菌耐药 金属β-内酰胺酶ImiS Β-内酰胺类抗生素 酶催化动力学 

分 类 号：R978.11[医药卫生—药品]