TEM-116型ESBL天然酶与重组酶的动力学特性研究

目的 研究TEM-116型ESBL天然酶与重组酶的动力学特性，并比较它们的差异。方法 采用紫外分光光度法检测酶促抗生素水解反应，以Lee-Wilson改良双倒数方程数据处理法进行数据处理，测定天然酶与重组酶的Km、Vmax及kcat，观察温度和pH对酶促反应的影响。结果 以Lee-Wilson改良双倒数方程数据处理法进行数据处理方便而准确地测定了天然酶与重组酶的Km与Vmax并计算出kcat,温度和pH对天然酶与重组酶酶促反应效应相似。天然酶与重组酶最优先的底物均为头孢哌酮，其次是头孢氨苄；对氨苄西林、阿莫西林、青霉素和哌拉西林有最高的催化效率。结论 天然酶与重组酶的动力学参数无显著差异。

Kinetic characteristics of native and recombinant TEM-116 beta-lactamases

Objective To investigate and compare the kinetic characteristics of native and recombinant TEM-116 beta-lactamases.Methods Hydrolysis reactions of beta-lactam antibiotics were detected by ultraviolet photometry.Data was processed according to Lee and Wilson's improved double-reciprocal equation.Kinetic parameters of native and recombinant TEM-116 beta-lactamases,including Km,Vmax and kcat,were determined.Effects of temperature and pH on enzymatic reaction were observed.Results Michaeles constant(Km)and maximal velocity(Vmax)were obtained by means of Lee and Wilson's improved double-reciprocal equation,and the catalytic constant(kcat)was calculated.There were similar pH or temperature effects on either native or recombinant beta-lactamases.The most preferential substrate for both native and recombinant TEM-116 beta-lactamases is cefoperazone and followed by the next cephalexin.Among the highest catalytic efficiency are ampicillin,amoxicillin,penicillin and piperacillin.Conclusion There is no difference of kinetic characteristics between native and recombinant TEM-116 beta-lactamases.