Development of Fluorescent Assay for Monitoring of Dehalogenase
Activity

J 2019

Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

NEVOLOVÁ, Šárka, Elisabet MAŇÁSKOVÁ, Stanislav MAZURENKO, Jiří DAMBORSKÝ, Zbyněk PROKOP et. al.

Basic information

Original name

Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

Authors

NEVOLOVÁ, Šárka (203 Czech Republic, belonging to the institution), Elisabet MAŇÁSKOVÁ (203 Czech Republic, belonging to the institution), Stanislav MAZURENKO (643 Russian Federation, belonging to the institution), Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution) and Zbyněk PROKOP (203 Czech Republic, belonging to the institution)

Edition

Biotechnology Journal, WEINHEIM, WILEY-V C H VERLAG GMBH, 2019, 1860-6768

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

Germany

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 3.912

RIV identification code

RIV/00216224:14310/19:00108192

Organization unit

Faculty of Science

DOI

http://dx.doi.org/10.1002/biot.201800144

UT WoS

000460177400009

Keywords in English

activity screening; enzyme assay; fluorescence; haloalkane dehalogenase; sulfur mustard

Abstract

V originále

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%.

Links

GA16-07965S, research and development project

Name: Řízená evoluce dynamických elementů v enzymech s využitím mikrofluidních čipů

Investor: Czech Science Foundation

LM2015051, research and development project

Name: Centrum pro výzkum toxických látek v prostředí (Acronym: RECETOX RI)

Investor: Ministry of Education, Youth and Sports of the CR

LM2015055, research and development project

Name: Centrum pro systémovou biologii (Acronym: C4SYS)

Investor: Ministry of Education, Youth and Sports of the CR

LO1214, research and development project

Name: Centrum pro výzkum toxických látek v prostředí (Acronym: RECETOX)

Investor: Ministry of Education, Youth and Sports of the CR

Citovat

NEVOLOVÁ, Šárka, Elisabet MAŇÁSKOVÁ, Stanislav MAZURENKO, Jiří DAMBORSKÝ and Zbyněk PROKOP. Development of Fluorescent Assay for Monitoring of Dehalogenase Activity. Biotechnology Journal. WEINHEIM: WILEY-V C H VERLAG GMBH, 2019, vol. 14, No 3, p. 1-6. ISSN 1860-6768. Available from: https://dx.doi.org/10.1002/biot.201800144.

@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 and Zbyněk PROKOP. Development of Fluorescent Assay for Monitoring of Dehalogenase Activity. \textit{Biotechnology Journal}. WEINHEIM: WILEY-V C H VERLAG GMBH, 2019, vol.~14, No~3, p.~1-6. ISSN~1860-6768. Available from: https://dx.doi.org/10.1002/biot.201800144.

Detailed Information on Publication Record