Muller-Santos_2006_Anal.Biochem_351_305

The ability of lipases (triacylglycerol hydrolases, E.C. 3.1.1.3) to catalyze the hydrolysis of triacylglycerols has been known for many decades. However, since the early 1990s their importance in biotechnology has expanded significantly, due to their ability to catalyze reactions in nonconventional media such as organic solvents and to recognize various different esters as substrates. For example, lipases have been used to catalyze the transesterification of triacylglycerols and in the racemic resolution of various chiral esters. These special uses have driven a search for lipases able to catalyze novel reactions. Considering that typically such reactions are carried out under aggressive reactions conditions, such as the presence of organic solvents, it is necessary to find lipases that have high stability and activity under these conditions.The discovery of new enzymes through bioprospection, screening of metagenomic libraries, or molecular evolutionary approaches demands a practical and sensitive screening method capable of manipulating a large number of samples of either whole cells or crude enzyme extracts. The development of such methods for screening lipases is hampered by the physicochemical properties of lipids. Methods that rely on titrimetric or radiometric measurements have been used, but methods in the first group have low sensibility while those in the second group require expensive radiolabeled substrates. When a large number of samples are assayed, chromogenic or fluorogenic substrates are used. These substrates are esters from which the enzymatic hydrolysis liberates either a chromogenic or a fluorogenic agent that can be accompanied by UV/visible or fluorescence spectrophotometry, respectively. Note that fluorogenic substrates such as 4-methylumbelliferone (MUF)1 esters are more sensitive than 4-nitrophenyl esters. The use of MUF esters with quantification by fluorescence spectrophotometry to determine lipase and esterase activity has been described [6]. Here, we describe an image-based screening approach to quantify the activity of lipases using MUF-butyrate as the substrate. Images of the reactions in microtiter plates were captured with a charge-coupled device (CCD) camera.