A quantitative fluorescence-based lipase assay

An easy and fast gel diffusion assay for detecting and monitoring lipase activity through a quantification of fluorescein is described. Measuring the intensity of fluorescence it is possible to obtain a calibration curve with a regression coefficient better than that one built up attained using the radius of fluorescent haloes. Since some synthetic compounds contain fluorescein, a fluorescent molecule which is released after enzyme catalysis, this method allowed measurement of the hydrolytic activity of a commercial preparation of lipase and of fresh skim milk. Through the quantification of fluorescence intensity of fluorescein released after hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plate, commercial and skim milk lipase activity were studied. Moreover, with this method lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectofotometer and fluorimeter. In this experiment boiled skim milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as buffer of reaction of milk lipase to reproduce natural conditions. The development of such an assay has potential for applications in industries ranging from pharmaceuticals to food production and monitoring.