| NEVOLOVÁ, Šárka, Elisabet MAŇÁSKOVÁ, Stanislav MAZURENKO, Jiří DAMBORSKÝ a Zbyněk PROKOP. Development of Fluorescent Assay for Monitoring of Dehalogenase Activity. Biotechnology Journal. WEINHEIM: WILEY-V C H VERLAG GMBH, 2019, roč. 14, č. 3, s. 1-6. ISSN 1860-6768. Dostupné z: https://dx.doi.org/10.1002/biot.201800144. |
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@article{1638600,
author = {Nevolová, Šárka and Maňásková, Elisabet and Mazurenko, Stanislav and Damborský, Jiří and Prokop, Zbyněk},
article_location = {WEINHEIM},
article_number = {3},
doi = {http://dx.doi.org/10.1002/biot.201800144},
keywords = {activity screening; enzyme assay; fluorescence; haloalkane dehalogenase; sulfur mustard},
language = {eng},
issn = {1860-6768},
journal = {Biotechnology Journal},
title = {Development of Fluorescent Assay for Monitoring of Dehalogenase Activity},
url = {https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201800144},
volume = {14},
year = {2019}
}
TY - JOUR
ID - 1638600
AU - Nevolová, Šárka - Maňásková, Elisabet - Mazurenko, Stanislav - Damborský, Jiří - Prokop, Zbyněk
PY - 2019
TI - Development of Fluorescent Assay for Monitoring of Dehalogenase Activity
JF - Biotechnology Journal
VL - 14
IS - 3
SP - 1-6
EP - 1-6
PB - WILEY-V C H VERLAG GMBH
SN - 18606768
KW - activity screening
KW - enzyme assay
KW - fluorescence
KW - haloalkane dehalogenase
KW - sulfur mustard
UR - https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201800144
L2 - https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201800144
N2 - 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%.
ER -
NEVOLOVÁ, Šárka, Elisabet MAŇÁSKOVÁ, Stanislav MAZURENKO, Jiří DAMBORSKÝ a Zbyněk PROKOP. Development of Fluorescent Assay for Monitoring of Dehalogenase Activity. \textit{Biotechnology Journal}. WEINHEIM: WILEY-V C H VERLAG GMBH, 2019, roč.~14, č.~3, s.~1-6. ISSN~1860-6768. Dostupné z: https://dx.doi.org/10.1002/biot.201800144.
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