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A Quantitative Fluorescence-Based Lipase Assay

Giovanna Lomolino, Giovanna Di Pierro and Anna Lante*


Department of Agronomy, Food Natural Resources, Animals and Environment, Agripolis, University of Padova, Viale dell’Università 16, IT-35020 Legnaro, PD, Italy


Article history:

Received January 13, 2011

Accepted February 9, 2012

Key words:

agar plate assay, hydrolytic activity, lipase, milk

Summary:

An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed 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-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.


*Corresponding author:           anna.lante@unipd.it
                                               ++39 049 827 2920
                                               ++39 049 827 2929

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