硫酸铵反抽提法纯化细菌β-内酰胺酶的实验研究

Purification of Bacterial β-lactamase with Ammonium Sulfate Backfractionation

目的比较硫酸铵反抽提和硫酸铵抽提两种方法对临床耐药菌β-内酰胺酶的纯化效果,为研究β-内酰胺酶提供简便易行的纯化方法。方法采用超声破碎法提取耐药菌β-内酰胺酶;分别用硫酸铵反抽提法和硫酸铵抽提法对酶初提物进行初步纯化;紫外分光光度法测定酶活性;蛋白质定量检测试剂盒测定蛋白质含量;计算β-内酰胺酶的得率和纯化倍数,用配对t检验分析两者之间的差异。结果两法之间在酶的得率上没有显著性差异(p=0.84),在纯化倍数上有显著性差异(p<0.0001)结论硫酸铵反抽提法对β-内酰胺酶的纯化效果优于硫酸铵抽提法。

Objective To analyze the usefulness of ammonium sulfate backfractionation in bacterial β-lactamase purification. Methods Bacterial β-lactamases were prepared by using sonication and centrifugation. The crude β-lactamascs were purified by ammonium sulfate fraetionation and ammonium sulfate backfractionation, respectively. The enzyme activities of the purified products were determined by ultraviolet speetrophotometer. Bradford method was used to measure protein concentration. The yield and purification fold were calculated and compared. Results No significant difference was found in the yield of β-laetamase between the two methods （p = 0.84）, but the purification fold of ammonium sulfate backfractionation was significantly higher than that of ammonium sulfate fractionation （p 〈 0. 0001 ）. Conclusion Ammonium sulfate backfraetionation was better than ammonium sulfate fraetionation in bacterial β-lactamasc purification.