TY - GEN A1 - Barig, Susann A1 - Schiemann, Manja A1 - Mirsky, Vladimir M. A1 - Stahmann, Klaus-Peter T1 - Quantitative turbidity assay for lipolytic enzymes in microtiter plates T2 - Analytical and Bioanalytical Chemistry N2 - 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. KW - Lipase assay KW - Lipolytic enzymes KW - Microtiter plates KW - Turbidity KW - Tributyrin Y1 - 2013 U6 - https://doi.org/10.1007/s00216-013-7283-5 SN - 1618-2650 VL - 405 IS - 26 SP - 8539 EP - 8547 ER -