Quantitative turbidity assay for lipolytic enzymes in microtiter plates

  • 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 suitableA 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.show moreshow less

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Metadaten
Author: Susann BarigORCiD, Manja Schiemann, Vladimir M. MirskyORCiDGND, Klaus-Peter Stahmann
DOI:https://doi.org/10.1007/s00216-013-7283-5
ISSN:1618-2650
Title of the source (English):Analytical and Bioanalytical Chemistry
Document Type:Scientific journal article peer-reviewed
Language:English
Year of publication:2013
Tag:Lipase assay; Lipolytic enzymes; Microtiter plates; Tributyrin; Turbidity
Volume/Year:405
Issue number:26
First Page:8539
Last Page:8547
Faculty/Chair:Fakultät 2 Umwelt und Naturwissenschaften / FG Nanobiotechnologie
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