Issue 40, 2022

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

Abstract

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 LacOX (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.

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

Supplementary files

Article information

Article type
Paper
Submitted
20 jul 2022
Accepted
06 sep 2022
First published
13 sep 2022
This article is Open Access
Creative Commons BY-NC license

RSC Adv., 2022,12, 26042-26050

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

E. Kaddouch, M. E. Cleveland, D. Navarro, S. Grisel, M. Haon, H. Brumer, M. Lafond, J. Berrin and B. Bissaro, RSC Adv., 2022, 12, 26042 DOI: 10.1039/D2RA04485D

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