Development of Fluorescent Assay for Monitoring of Dehalogenase Activity
| Autoři | |
|---|---|
| Rok publikování | 2019 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Biotechnology Journal |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201800144 |
| Doi | http://dx.doi.org/10.1002/biot.201800144 |
| Klíčová slova | activity screening; enzyme assay; fluorescence; haloalkane dehalogenase; sulfur mustard |
| Popis | 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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