Publication details

Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

Authors

NEVOLOVÁ Šárka MAŇÁSKOVÁ Elisabet MAZURENKO Stanislav DAMBORSKÝ Jiří PROKOP Zbyněk

Year of publication 2019
Type Article in Periodical
Magazine / Source Biotechnology Journal
MU Faculty or unit

Faculty of Science

Citation
Web https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201800144
Doi http://dx.doi.org/10.1002/biot.201800144
Keywords activity screening; enzyme assay; fluorescence; haloalkane dehalogenase; sulfur mustard
Description The rapid accumulation of sequence data and powerful protein engineering techniques providing large mutant libraries have greatly heightened interest in efficient methods for biochemical characterization of proteins. Herein is reported a continuous assay for screening of enzymatic activity. The assay is developed and tested with the model enzymes haloalkane dehalogenases and relies upon a fluorescent change of a derivative of 8-hydroxypyrene-1,3,6-trisulphonic acid due to the pH drop associated with the dehalogenation reactions. The assay is performed in a microplate format using a purified enzyme, cell-free extract or intact cells, making the analysis quick and simple. The method exhibits high sensitivity with a limit of detection of 0.06 mM. The assay is successfully validated with gas chromatography and then applied for screening of 12 haloalkane dehalogenases with the environmental pollutant bis(2-chloroethyl) ether and chemical warfare agent sulfur mustard. Six enzymes exhibited detectable activity with both substrates. The within-day variability of the assay for five replicates (n = 5) was 21%.
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