Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017;1588:15-25.
doi: 10.1007/978-1-4939-6899-2_2.

Quantitative Kinetic Characterization of Glycoside Hydrolases Using High-Performance Anion-Exchange Chromatography (HPAEC)

Affiliations

Quantitative Kinetic Characterization of Glycoside Hydrolases Using High-Performance Anion-Exchange Chromatography (HPAEC)

Nicholas McGregor et al. Methods Mol Biol. .

Abstract

High-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) is a powerful analytical technique enabling the high-resolution separation and sensitive quantification of oligosaccharides. Here, we describe a general method for the determination of glycoside hydrolase kinetics that harnesses the intrinsic power of HPAEC-PAD to simultaneously monitor the release of multiple products under conditions of low substrate conversion. Thus, the ability to track product release under initial-rate conditions with substrate concentrations as low as 5 μM enables the determination of Michaelis-Menten kinetics for glycosidase activities, including hydrolysis and transglycosylation. This technique may also be readily extended to other carbohydrate-active enzymes (CAZymes), including polysaccharide lyases, and glycosyl transferases.

Keywords: Carbohydrate-active enzyme; HPAEC-PAD; HPLC; Hydrolysis; Kinetics; Oligosaccharide; Transglycosylation.

Similar articles

See all similar articles

Cited by 2 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback