An Enzymatic Method for Methanol Quantification in
Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer

J 2017

An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer

KUČERA, Igor a Vojtěch SEDLÁČEK

Základní údaje

Originální název

An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer

Autoři

KUČERA, Igor (203 Česká republika, garant, domácí) a Vojtěch SEDLÁČEK (203 Česká republika, domácí)

Vydání

Food Analytical Methods, New York, Springer, 2017, 1936-9751

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 2.245

Kód RIV

RIV/00216224:14310/17:00094834

Organizační jednotka

Přírodovědecká fakulta

DOI

http://dx.doi.org/10.1007/s12161-016-0692-2

UT WoS

000399014600017

Klíčová slova anglicky

Methanol; Alcohol oxidase; Formaldehyde dehydrogenase; Diaphorase; Resazurin; Fluorescence

Štítky

NZ, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 28. 3. 2018 14:25, Ing. Nicole Zrilić

Anotace

V originále

In response to the need for a rapid, high-throughput screening of methanol contamination in spirits, a new microplate-based assay was developed. In this assay, alcohol oxidase first oxidizes methanol to formaldehyde, which is further oxidized to formate by formaldehyde dehydrogenase while reducing NAD+ to NADH. The latter product then reacts with resazurin under catalysis by FerB, a diaphorase-type enzyme, to give the highly fluorescent resorufin. These reactions are run simultaneously in 200 uL final volume in a 96-well plate and quantified using a plate reader and fluorescence detector. It is shown that the rate of fluorescence change is related to methanol and ethanol concentrations according to the rate law for two competing substrates. Quantification of methanol in real samples is carried out by applying the standard additions technique with four different spiking concentrations of the methanol standard; methanol content in the sample is calculated from the x-intercept of the fitted line. The high activity of FerB with resazurin and low rate of further conversion of resorufin to non-fluorescent dihydroresorufin indicate that FerB may be advantageous over commercially available diaphorases for use in fluorescence enzyme assays.

Návaznosti

GAP503/12/0369, projekt VaV

Název: Nové flavin-dependentní enzymy Paracoccus denitrificans: reakční mechanismy, metabolické funkce a úloha v buněčném oxidačním stresu

Investor: Grantová agentura ČR, Nové flavin-dependentní enzymy Paracoccus denitrificans: reakční mechanismy, metabolické funkce a úloha v buněčném oxidačním stresu

Citovat

KUČERA, Igor a Vojtěch SEDLÁČEK. An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer. Food Analytical Methods. New York: Springer, 2017, roč. 10, č. 5, s. 1301-1307. ISSN 1936-9751. Dostupné z: https://dx.doi.org/10.1007/s12161-016-0692-2.

@article{1385132,
   author = {Kučera, Igor and Sedláček, Vojtěch},
   article_location = {New York},
   article_number = {5},
   doi = {http://dx.doi.org/10.1007/s12161-016-0692-2},
   keywords = {Methanol; Alcohol oxidase; Formaldehyde dehydrogenase; Diaphorase; Resazurin; Fluorescence},
   language = {eng},
   issn = {1936-9751},
   journal = {Food Analytical Methods},
   title = {An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer},
   url = {https://link.springer.com/article/10.1007%2Fs12161-016-0692-2},
   volume = {10},
   year = {2017}
}

TY  - JOUR
ID  - 1385132
AU  - Kučera, Igor - Sedláček, Vojtěch
PY  - 2017
TI  - An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer
JF  - Food Analytical Methods
VL  - 10
IS  - 5
SP  - 1301-1307
EP  - 1301-1307
PB  - Springer
SN  - 19369751
KW  - Methanol
KW  - Alcohol oxidase
KW  - Formaldehyde dehydrogenase
KW  - Diaphorase
KW  - Resazurin
KW  - Fluorescence
UR  - https://link.springer.com/article/10.1007%2Fs12161-016-0692-2
L2  - https://link.springer.com/article/10.1007%2Fs12161-016-0692-2
N2  - In response to the need for a rapid, high-throughput screening of methanol contamination in spirits, a new microplate-based assay was developed. In this assay, alcohol oxidase first oxidizes methanol to formaldehyde, which is further oxidized to formate by formaldehyde dehydrogenase while reducing NAD+ to NADH. The latter product then reacts with resazurin under catalysis by FerB, a diaphorase-type enzyme, to give the highly fluorescent resorufin. These reactions are run simultaneously in 200 uL final volume in a 96-well plate and quantified using a plate reader and fluorescence detector. It is shown that the rate of fluorescence change is related to methanol and ethanol concentrations according to the rate law for two competing substrates. Quantification of methanol in real samples is carried out by applying the standard additions technique with four different spiking concentrations of the methanol standard; methanol content in the sample is calculated from the x-intercept of the fitted line. The high activity of FerB with resazurin and low rate of further conversion of resorufin to non-fluorescent dihydroresorufin indicate that FerB may be advantageous over commercially available diaphorases for use in fluorescence enzyme assays.
ER  -

KUČERA, Igor a Vojtěch SEDLÁČEK. An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer. \textit{Food Analytical Methods}. New York: Springer, 2017, roč.~10, č.~5, s.~1301-1307. ISSN~1936-9751. Dostupné z: https://dx.doi.org/10.1007/s12161-016-0692-2.

Podrobný výpis o publikaci