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

Part of the Methods in Molecular Biology book series (MIMB, volume 1588)


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.

Key words

Carbohydrate-active enzyme Kinetics Hydrolysis Transglycosylation HPLC HPAEC-PAD Oligosaccharide 



Funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) via the Strategic Partnership Grants for Networks (for the Industrial Biocatalysis Network) and Discovery Grant programs is gratefully acknowledged. Equipment infrastructure was funded by the Canada Foundation for Innovation and the British Columbia Knowledge Development Fund. We thank Kazune Tamura (Brumer group, UBC) for comments on an early version of this chapter.


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Nicholas McGregor
    • 1
    • 2
  • Gregory Arnal
    • 1
  • Harry Brumer
    • 1
    • 2
    • 3
  1. 1.Michael Smith LaboratoriesUniversity of British ColumbiaVancouverCanada
  2. 2.Department of ChemistryUniversity of British ColumbiaVancouverCanada
  3. 3.Department of Biochemistry and Molecular BiologyUniversity of British ColumbiaVancouverCanada

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