Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F19%3A00108192" target="_blank" >RIV/00216224:14310/19:00108192 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00159816:_____/19:00071091

Výsledek na webu

<a href="https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201800144" target="_blank" >https://onlinelibrary.wiley.com/doi/full/10.1002/biot.201800144</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/biot.201800144" target="_blank" >10.1002/biot.201800144</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

Popis výsledku v původním jazyce

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%.

Název v anglickém jazyce

Development of Fluorescent Assay for Monitoring of Dehalogenase Activity

Popis výsledku anglicky

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%.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Biotechnology Journal

ISSN

1860-6768

e-ISSN

—

Svazek periodika

14

Číslo periodika v rámci svazku

3

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

6

Strana od-do

1-6

Kód UT WoS článku

000460177400009

EID výsledku v databázi Scopus

2-s2.0-85053215310