甲硫氨酸氨肽酶高通量微型化检测方法的建立

Establishment of a High Throughput Method for Detecting Methionine Aminopeptidase Activity

采用甲硫氨酸对硝基苯胺底物检测甲硫氨酸氨肽酶酶活,通过优化甲硫氨酸氨肽酶检测条件提高检测方法的准确性和灵敏性,并在此基础上建立了微型化的检测方法———酶标板法,适应微型化高通量发酵培养对高通量酶活检测的要求,以实现氨肽酶产生菌真正意义上的高通量筛选。优化后的甲硫氨酸氨肽酶酶活微型化检测条件为:氨-氯化铵缓冲液40 mmol/L、pH 8.0、甲硫氨酸对硝基苯胺浓度6 mmol/L、温度60℃、酶反应时间10 min,于380nm波长比色测定。

The substrate methionine p-nitroaniline in this paper had been used to measure methionine aminopeptidase activity. The accuracy and sensitivity of the detection were improved and the enzyme panel method was established baseed on the optimization of determination conditions, to adapt to the detection for miniaturization fermentation and the high throughput screening for high aminopeptidase-produeing strains. The optimal determination conditions were as follows： 40 mmol/L ammonium chloride/ammonia buffer, pH 8.0, 6 mmol/L methionine p-nitroaniline, reaction temperature 60℃ , reaction time 10 min, measured colormetrically at 380 nm.