Analytical and Bioanalytical Chemistry

, Volume 405, Issue 26, pp 8539–8547 | Cite as

Quantitative turbidity assay for lipolytic enzymes in microtiter plates

  • Susann Barig
  • Manja Schiemann
  • Vladimir M. MirskyEmail author
  • K. Peter Stahmann
Research Paper


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.


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 are 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


Lipase assay Lipolytic enzymes Microtiter plates Turbidity Tributyrin 



The work was supported by Brandenburg ministry for science and culture. The authors are grateful to Professor Dr-Ing. K. Schnitzlein, Dr R. Schmid and Ph.D. students S. Schröter and M. Muth for fruitful discussions.

Supplementary material

216_2013_7283_MOESM1_ESM.pdf (205 kb)
ESM 1 (PDF 205 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Susann Barig
    • 1
  • Manja Schiemann
    • 1
  • Vladimir M. Mirsky
    • 1
    Email author
  • K. Peter Stahmann
    • 1
  1. 1.Department of BiotechnologyBrandenburg University of Technology Cottbus - SenftenbergSenftenbergGermany

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