β-D-半乳糖苷酶活性测定方法的研究

应用分光光度计建立一种简便检测β D 半乳糖苷酶(GAL)活性的方法。酶反应条件研究表明,在总体积为1mL的0.1mol/L柠檬酸反应缓冲体系(pH值为4.5)中含0.8μmol的底物邻硝基苯β D 吡喃半乳糖(ONPG),含相当于2mg左右蛋白质的酶提取液,37℃孵育30min后用0.5mol/L碳酸钠1mL终止反应,用紫外分光光度计于405nm波长测定的吸光度值最能反映该酶活性。结果显示,该方法的批内变异系数在3.92%～4.89%之间,批间变异系数为3.8%,均小于5%;在所检测该酶质量浓度范围(1～2.5mg/mL)内线性关系良好,当反应体系的蛋白含量低于0.5mg/mL时,酶活性不能被检出;每组的平均回收率均在95%～105%之间,说明在所检测浓度范围内回收良好。因此,该方法具有稳定性好、灵敏度高、简便易行等特点,适合一般实验室应用。

Study on Assay Method of β-D-Galactosidase Activity

To establish a simple method to measure Lactase activity, the activity is detected by using spectrophotometric method. The conditions for enzyme reaction are studied. The final concentrations of components in 1mL of reaction mixtures are as follows: 0.8 μmol o-nitrophenylβ-D-galactopyranoside (ONPG) in 0.1 mol/L citrate buffer (pH=4.5) and 2 mg protein of enzyme extract. After incubation for 30 min. at 37 ℃, the reactions are stopped by addition of 1mL 0.5 mol/L sodium carbonate. The absorbances at 405 nm are read. The results show that the variability within batchs is 3.92%～4.89% and the variability between batchs is 3.8%. These suggest that the method is so reliable that it could be used in Lactase assay. The method has the merits of high precision, good reproducibility and easy operation, so it could be used for detecting Lactase activity successfully.