Cargando…

A simple and direct ionic chromatography method to monitor galactose oxidase activity

Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases (CROs). The reaction catalyzed by GalOx leads to the oxidation of the C-6 hydroxyl group of galactose and galactosides (including galactosylated polysaccharides and glycoproteins) to the correspondin...

Descripción completa

Detalles Bibliográficos
Autores principales: Kaddouch, Eden, Cleveland, Maria E., Navarro, David, Grisel, Sacha, Haon, Mireille, Brumer, Harry, Lafond, Mickaël, Berrin, Jean-Guy, Bissaro, Bastien
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469488/
https://www.ncbi.nlm.nih.gov/pubmed/36199594
http://dx.doi.org/10.1039/d2ra04485d
_version_ 1784788657250500608
author Kaddouch, Eden
Cleveland, Maria E.
Navarro, David
Grisel, Sacha
Haon, Mireille
Brumer, Harry
Lafond, Mickaël
Berrin, Jean-Guy
Bissaro, Bastien
author_facet Kaddouch, Eden
Cleveland, Maria E.
Navarro, David
Grisel, Sacha
Haon, Mireille
Brumer, Harry
Lafond, Mickaël
Berrin, Jean-Guy
Bissaro, Bastien
author_sort Kaddouch, Eden
collection PubMed
description Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases (CROs). The reaction catalyzed by GalOx leads to the oxidation of the C-6 hydroxyl group of galactose and galactosides (including galactosylated polysaccharides and glycoproteins) to the corresponding aldehydes, coupled to the reduction of dioxygen to hydrogen peroxide. Despite more than 60 years of research including mechanistic studies, enzyme engineering and application development, GalOx activity remains primarily monitored by indirect measurement of the co-product hydrogen peroxide. Here, we describe a simple direct method to measure GalOx activity through the identification of galactosylated oxidized products using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). Using galactose and lactose as representative substrates, we were able to separate and detect the C-6 oxidized products, which were confirmed by LC-MS and NMR analyses to exist in their hydrated (geminal-diol) forms. We show that the HPAEC-PAD method is superior to other methods in terms of sensitivity as we could detect down to 0.08 μM of Lac(OX) (eq. 30 μg L(−1)). We believe the method will prove useful for qualitative detection of galactose oxidase activity in biological samples or for quantitative purposes to analyze enzyme kinetics or to compare enzyme variants in directed evolution programs.
format Online
Article
Text
id pubmed-9469488
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher The Royal Society of Chemistry
record_format MEDLINE/PubMed
spelling pubmed-94694882022-10-04 A simple and direct ionic chromatography method to monitor galactose oxidase activity Kaddouch, Eden Cleveland, Maria E. Navarro, David Grisel, Sacha Haon, Mireille Brumer, Harry Lafond, Mickaël Berrin, Jean-Guy Bissaro, Bastien RSC Adv Chemistry Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases (CROs). The reaction catalyzed by GalOx leads to the oxidation of the C-6 hydroxyl group of galactose and galactosides (including galactosylated polysaccharides and glycoproteins) to the corresponding aldehydes, coupled to the reduction of dioxygen to hydrogen peroxide. Despite more than 60 years of research including mechanistic studies, enzyme engineering and application development, GalOx activity remains primarily monitored by indirect measurement of the co-product hydrogen peroxide. Here, we describe a simple direct method to measure GalOx activity through the identification of galactosylated oxidized products using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). Using galactose and lactose as representative substrates, we were able to separate and detect the C-6 oxidized products, which were confirmed by LC-MS and NMR analyses to exist in their hydrated (geminal-diol) forms. We show that the HPAEC-PAD method is superior to other methods in terms of sensitivity as we could detect down to 0.08 μM of Lac(OX) (eq. 30 μg L(−1)). We believe the method will prove useful for qualitative detection of galactose oxidase activity in biological samples or for quantitative purposes to analyze enzyme kinetics or to compare enzyme variants in directed evolution programs. The Royal Society of Chemistry 2022-09-13 /pmc/articles/PMC9469488/ /pubmed/36199594 http://dx.doi.org/10.1039/d2ra04485d Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Kaddouch, Eden
Cleveland, Maria E.
Navarro, David
Grisel, Sacha
Haon, Mireille
Brumer, Harry
Lafond, Mickaël
Berrin, Jean-Guy
Bissaro, Bastien
A simple and direct ionic chromatography method to monitor galactose oxidase activity
title A simple and direct ionic chromatography method to monitor galactose oxidase activity
title_full A simple and direct ionic chromatography method to monitor galactose oxidase activity
title_fullStr A simple and direct ionic chromatography method to monitor galactose oxidase activity
title_full_unstemmed A simple and direct ionic chromatography method to monitor galactose oxidase activity
title_short A simple and direct ionic chromatography method to monitor galactose oxidase activity
title_sort simple and direct ionic chromatography method to monitor galactose oxidase activity
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9469488/
https://www.ncbi.nlm.nih.gov/pubmed/36199594
http://dx.doi.org/10.1039/d2ra04485d
work_keys_str_mv AT kaddoucheden asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT clevelandmariae asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT navarrodavid asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT griselsacha asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT haonmireille asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT brumerharry asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT lafondmickael asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT berrinjeanguy asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT bissarobastien asimpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT kaddoucheden simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT clevelandmariae simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT navarrodavid simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT griselsacha simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT haonmireille simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT brumerharry simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT lafondmickael simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT berrinjeanguy simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity
AT bissarobastien simpleanddirectionicchromatographymethodtomonitorgalactoseoxidaseactivity