title: Quantitative turbidity assay for lipolytic enzymes in microtiter plates.
abstract: A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5-1% tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1% tributyrin emulsion in 2% agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20-60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.
authors.affiliation: Department of Biotechnology, Brandenburg University of Technology Cottbus - Senftenberg, 01968, Senftenberg, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Barig
authors.forename: Manja
authors.initials: M
authors.lastname: Schiemann
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Mirsky
authors.forename: K Peter
authors.initials: KP
authors.lastname: Stahmann
mesh_terms: D000818:Animals; D001419:Bacteria / Q000201:enzymology; D004655:Emulsions / Q000737:chemistry / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005658:Fungi / Q000201:enzymology; D007700:Kinetics; D057230:Limit of Detection; D008049:Lipase / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004655:Emulsions; D014280:Triglycerides; D008049:Lipase; C005830:tributyrin