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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

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.

Figure

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

Keywords

Lipase assay Lipolytic enzymes Microtiter plates Turbidity Tributyrin 

Notes

Acknowledgement

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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