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

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Protein-Carbohydrate Interactions

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

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.

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Acknowledgments

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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Authors and Affiliations

  1. Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4

    Nicholas McGregor, Gregory Arnal & Harry Brumer

  2. Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4

    Nicholas McGregor & Harry Brumer

  3. Department of Biochemistry and Molecular Biology, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada, V6T 1Z4

    Harry Brumer

Authors
  1. Nicholas McGregor
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  2. Gregory Arnal
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  3. Harry Brumer
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Correspondence to Harry Brumer .

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Editors and Affiliations

  1. Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada

    D. Wade Abbott

  2. Biotechnology Institute, University of Groningen, Groningen, The Netherlands

    Alicia Lammerts van Bueren

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McGregor, N., Arnal, G., Brumer, H. (2017). Quantitative Kinetic Characterization of Glycoside Hydrolases Using High-Performance Anion-Exchange Chromatography (HPAEC). In: Abbott, D., Lammerts van Bueren, A. (eds) Protein-Carbohydrate Interactions. Methods in Molecular Biology, vol 1588. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6899-2_2

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  • DOI: https://doi.org/10.1007/978-1-4939-6899-2_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6898-5

  • Online ISBN: 978-1-4939-6899-2

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