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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2022
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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 |
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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 |
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