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Quantitative turbidity assay for lipolytic enzymes in microtiter plates

Barig, Susann, Schiemann, Manja, Mirsky, Vladimir M., Stahmann, K. Peter
Analytical and bioanalytical chemistry 2013 v.405 no.26 pp. 8539-8547
agar, agarose, detection limit, emulsions, enzyme substrates, gels, glycerol, lipolysis, polyacrylamide, swine, triacylglycerol lipase, tributyrin, turbidity
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.