Lipase Assays

Abstract

Lipase Assays Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) have broad applications in the food, oleochemical, pharmaceutical, and detergent industries, as well as in diagnostic settings (Hafkenscheid et al., 1983; Schmid and Verger, 1998). Over 100 lipases have been characterized to some extent, and more than 30 of these are commercially available. The identification of novel sources of lipases with unique patterns of reaction selectivity remains a strategic objective of lipase studies. Various levels of sophistication are required in the assessment of lipase activities, and they increase as one attains the differential goals of (1) screening for activity (qualitative and semi-quantitative); (2) quantifying activity; and (3) characterizing kinetic patterns of selectivity. The focus of this unit will be quantification of lipase activity, since methods developed for this purpose can often be adapted for the other two purposes as well. Lipases are unusual hydrolytic enzymes because they act on substrates providing an interface (with few exceptions). This feature has been historically used to distinguish lipases from esterases, the latter of which act on substrates in true solution (Jensen, 1983). The distinction of lipases as interfacial catalysts can make kinetic characterization a challenge, because relevant substrate concentrations are expressed in terms of area and not concentration. Even though esterases and lipases differ in their ability to act at interfaces, some assays can be used to measure both types of enzyme activities, since they share the general ability to hydrolyze carboxyl esters of various alcohols. In general, the use of water-soluble substrates (generally, shorter acyl chain length derivatives) is considered diagnostic for esterases, and the use of water-insoluble substrates (longer acyl chain length derivatives) is considered diagnostic for lipases. Of the three most commonly used assays, two exploit the ability to measure the free carboxylic acid or fatty acid residues liberated during lipase hydrolysis of native substrates, whether it be by titration of the released acid (Basic Protocol 1), or detection of the fatty acids by complexation with a colorimetric reagent of cupric acetate (Basic Protocol 2). Alternatively, the use of p-nitrophenyl acyl esters as chromogenic substrate analogs provides for a continuous, spectrophotometric assay (Basic Protocol 3). Throughout this unit, the term "fatty acid" will be used instead of the popular term "free fatty acid" since the former is preferred by IUPAC-IUB (1977), according to rules of nomenclature. BASIC PROTOCOL 1 TITRIMETRIC DETERMINATION OF LIPASE ACTIVITY In this procedure, native substrates (triacylglycerols) are hydrolyzed to yield fatty acids. Subsamples are withdrawn from reactive mixtures at predetermined intervals, and reactivity is quenched by the addition of ethanol. The amount of fatty acids released during the reaction is determined by direct titration with NaOH to a thymolphthalein end point. Materials 95% (v/v) ethanol 1% (w/...

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