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Quantitative turbidity assay for lipolytic enzymes in microtiter plates
Susann Barig1, Manja Schiemann, Vladimir M Mirsky
1Department of Biotechnology, Brandenburg University of Technology Cottbus - Senftenberg, 01968, Senftenberg, Germany.
Analytical and Bioanalytical Chemistry
|August 31, 2013
Summary
A novel microtiter plate assay quantifies lipase activity using tributyrin emulsion. This method accurately measures enzyme kinetics and has a low detection limit for various lipases.
Area of Science:
- Biochemistry
- Enzymology
- Analytical Chemistry
Background:
- Lipolytic enzymes play crucial roles in various biological and industrial processes.
- Accurate and sensitive assays are essential for characterizing lipase activity.
- Existing methods may have limitations in throughput or sensitivity.
Purpose of the Study:
- To develop and characterize a novel 96-well microtiter plate-based clearing assay for lipolytic enzymes.
- To optimize assay conditions for reliable lipase activity measurement.
- To determine the assay's sensitivity and applicability to different lipase sources.
Main Methods:
- A clearing assay utilizing emulsified tributyrin as a substrate in a gel matrix within 96-well plates.
- Detection of lipolysis by measuring the decrease in turbidity (extinction) using a microtiter plate reader.
- Analysis of signal kinetics, substrate concentration dependency, and enzyme diffusion through various gel-forming agents (agar, agarose, polyacrylamide).
- Confirmation of lipolysis via glycerol detection.
Main Results:
- The assay demonstrated a clear signal proportional to lipase activity, with reaction rates independent of substrate concentration at 0.5-1% tributyrin.
- Agar and agarose were identified as optimal gel-forming agents, allowing enzyme diffusion, unlike polyacrylamide.
- The optimized assay achieved a detection limit of 20-60 ng/well (30 μU/well for *T. lanuginosus* lipase).
- The assay was successfully validated with microbial and porcine pancreatic lipases.
Conclusions:
- A sensitive and efficient 96-well microtiter plate assay for quantifying lipase activity has been successfully developed.
- The assay's design, utilizing a tributyrin emulsion in agar gel, allows for accurate kinetic analysis and broad applicability.
- This method provides a valuable tool for high-throughput screening and characterization of lipolytic enzymes.
