Direct Fluorescence-Based Lipase Activity Assay

Lipases and esterases (glycerol ester hydrolases, E.C. 3.1.1.-) are enzymes acting on the carboxyl ester bonds present in acylglycerols. They have been recognized as useful biocatalysts because of their wide-ranging versatility in industrial applications (2,5). However, the physical properties of lipids have caused a great difficulty in studying lipolytic enzymes (4). Traditionally, lipases have been assayed by radiometric or titrimetric techniques, but these methods require the use of radiolabeled and expensive substrates or they suffer from low sensitivity (8). For experiments involving a large number of assays or non-purified samples, it is convenient to use chromogenic or fluorogenic substrates, which can be tested simply and rapidly. Unfortunately, many chromogenic substrates show low specificity and sensitivity, or they become spontaneously hydrolyzed (4). On the contrary, the use of fluorogenic substrates like 4-methylumbelliferone (MUF) derivatives provides a sensitive method for determination of enzyme activity (3,10). Here, we describe a fast, simple assay to detect lipolytic activity from purified and non-purified samples, using MUF-derivative substrates. Identification of lipase producing microbial strains was performed after growth on a solid medium by spreading the plate surface with a 100 μM solution of MUF-butyrate (Sigma, St. Louis, MO, USA) in 50 mM phosphate buffer, pH 7.0, prepared from a 25 mM stock solution in ethyleneglycol monomethylether (Merck, Germany). Under UV illumination, only strains bearing lipolytic activity showed fluorescence emission due to substrate hydrolysis and MUF release (Figure 1A). Lipolytic activity detection can also be performed from cell suspensions or cell fractions, by transferring a small aliquot (5 μL) of each sample onto filter paper and adding 5 μL of 25 mM MUF-butyrate stock solution. UV illumination of the paper allows identification of the samples bearing lipolytic activity (not shown). This simple procedure was also used to select the most convenient working concentration of MUF-substrate for a given sample (Figure 1B) or to determine the amount of enzyme required for hydrolyzing a defined MUF-substrate. For most of our samples, detection of lipolytic activity against MUF-butyrate was achieved in less than 30 s, while hydrolysis of MUF-oleate usually required a 15-min incubation at room temperature. Electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gels has widely been used to study lipases and esterases. Activity detection often requires zymographic analysis, usually performed by overlay techniques (11). We standarized a new fluorescence-based assay for lipase activity detection in SDS-polyacrylamide gels (Figure 2). After protein separation, SDS was removed from the gels by soaking them for 30 min in 2.5% Triton X-100 at room temperature (1). The gels were then briefly washed (1 min) in 50 mM phosphate buffer, pH 7.0 and covered by a solution of 100 μM MUF-butyrate or 200 μM MUFoleate in the same buffer. After UV illumination, fluorescent activity bands became visible in 30 s for MUF-butyrate and in about 15 min for MUF-oleate. In contrast to other zymographic systems (11), the sensitivity of the method described is extremely high, and allows detection of 1.5 × 10-7 U of Pancrealipase (USP NF-190; Inquiaroma, LIB, France) using MUF-butyrate as a substrate (Figure 2A, left). Nevertheless, the degree of sensitivity of the system is a function of the properties of the enzyme analyzed and the substrate used. Accordingly, higher amounts of enzyme were needed (2.5 × 10-6 Pancrealipase U) when MUF-oleate was used Benchmarks