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 and Vojtěch SEDLÁČEK

Basic information

Original name

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

Authors

KUČERA, Igor (203 Czech Republic, guarantor, belonging to the institution) and Vojtěch SEDLÁČEK (203 Czech Republic, belonging to the institution)

Edition

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

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 2.245

RIV identification code

RIV/00216224:14310/17:00094834

Organization unit

Faculty of Science

DOI

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

UT WoS

000399014600017

Keywords in English

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

Abstract

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.

Links

GAP503/12/0369, research and development project

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

Investor: Czech Science Foundation

Citovat

KUČERA, Igor and Vojtěch SEDLÁČEK. An Enzymatic Method for Methanol Quantification in Methanol/Ethanol Mixtures with a Microtiter Plate Fluorometer. Food Analytical Methods. New York: Springer, 2017, vol. 10, No 5, p. 1301-1307. ISSN 1936-9751. Available from: 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 and 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, vol.~10, No~5, p.~1301-1307. ISSN~1936-9751. Available from: https://dx.doi.org/10.1007/s12161-016-0692-2.

Detailed Information on Publication Record