紫外分光光度法检测金属β-内酰胺酶活性的研究

Study on determination of metallo-β-lactamase used in sterility test by UV spectrophotometry

目的建立以紫外分光光度法检测金属β-内酰胺酶活性的方法。方法采用以美罗培南作反应底物，中性羟胺与硫酸铁胺为显色体系，通过紫外分光光度计检测反应液中美罗培南剩余量计算金属β-内酰胺酶的分解量，从而计算出其酶活性。结果美罗培南的浓度在1～5mg／mL范围内，线性良好，相关系数大于0．999。该方法检测限为0．05U，回收率在90％～119％范围内，重复性相对标准偏差为6．1％～8．7％．检测出的酶活性与酶蛋白含量成正相关。结论该方法能有效区分金属β-内酰胺酶与非金属β-内酰胺酶，并准确反应金属p．内酰胺酶分解碳青霉烯类抗生素的能力。

Objective To eatablish a method for determination of metallo-β-lactamase used in sterility test by UV spectrophotometry. Methods Neutral hydroxylamine and ammonium iron （III） sulfate were used as chromogenic system, and the meropenem was reaction substrates. Then the enzymatic activity can be calculated by the residue of meropenem. Results The correlation coefficients of linear calibration curves were over 0.999 within the meropenem concentration range of 1-Smg/mL. The detection limit was 0.05U, the recovery were 90%-119%, the RSDs of repeatability were 6.1%-8.7%. There was a positive correlation between the content of metallo-β-1actamase and the activity determinated by this method. Conclusion This method can effectively distinguish metallo-β-1actamase from penicillinase and ceohalosporinase, and it also can accurately reflect the ability of decomposin~ carbapenem antibiotic.