BALB/c小鼠小肠乳糖酶活性双糖酶法测定条件的优化

A study on the method of lactase activity determination in small intestine of BALB/c mouse

目的:建立一种方便、准确测定小鼠小肠乳糖酶活性的方法。方法:采用不同pH的底物溶液与肠黏膜提取物反应,计算相应的酶活性;取不同反应时间(45min,60min,75min),不同显色时间(45min,60min,75min)和不同显色剂用量(2.0ml,2.5ml,3.0ml)按L9(34)正交实验设计方法,测定不同条件下的底物溶液与肠黏膜反应的A值作为正交实验的评分标准。结果与结论:由正交实验得出,该方法的最佳反应条件为:pH4.6,水浴75min,显色时间45min,显色剂用量3.0ml;批内酶活性(0.029±0.003)U/ml,CV=2.49%,批间酶活性(0.032±0.004)U/ml,CV=4.19%。该方法具有很好的重复性和可靠性。

Aim: To optimize the conditions of determining the activity of lactase in the small intestine of mice.Methods: The mucosa extract of small intestine were mixed with substrate solutions of different pH, then the lactase activities of samples were calculated;different reaction time(45 min,60 min,75 min),developing time(45 min,60 min,75 min),and dosage of developer(2.0 ml,2.5 ml,3.0 ml) were applied in orthogonal experiment,and absorbency at 420 nm was determined. Results and conclusion: The best conditions of this method were: pH 4.6, water bath for 75 min, color developing for 45 min, and 3.0 ml of developer. The method is precise and convenient with better replication and liability.