Development of Fluorescent Assay for Monitoring of Dehalogenase Activity
Nevolova, S., Manaskova, E., Mazurenko, S., Damborsky, J., Prokop, Z.
BIOTECHNOLOGY JOURNAL XX: XXX-XXX (2018)
The rapid accumulation of sequence data and powerful protein engineering techniques providing a large mutant libraries have greatly heightened interest in efficient methods for biochemical characterization of proteins. Here we report a continuous assay for screening of enzymatic activity. The assay was developed and tested with the model enzymes haloalkane dehalogenases and relies upon a fluorescent change of 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 has been successfully validated with gas chromatography and then applied for screening of twelve 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%.