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Assaying Fucosidase Activity

  • Zoë Anne Megson
  • Paul Messner
  • Christina SchäfferEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)

Abstract

The characterization of a recombinant glycosidase can be done with commercially available substrates, which enable testing of enzyme functionality and determination of linkage specificity. Colorimetric assays with p-nitrophenyl substrates provide a relatively simple and fast way of screening conditions which could affect enzyme activity (buffer, pH, ion dependence, temperature). These substrates are useful for the determination of activity optima and the characterization of basic activity parameters. However, testing for linkage specificity should be performed on more complex sugars presenting a range of different glycosidic bonds and might need more sophisticated methods of analysis. This protocol provides comprehensive instructions on how to perform an initial characterization of your glycosidase using a recombinant α-l-fucosidase as an example.

Key words

Fucosidase Enzymatic activity Substrate specificity pH optimum Cation dependence HPAEC-PAD Reaction rate 

Notes

Acknowledgments

This work was supported by the Austrian Science Fund FWF, projects P24317-B22 (to C.S.) and the Doctoral Programme W1224 Biomolecular Technology of Proteins.

References

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Zoë Anne Megson
    • 1
  • Paul Messner
    • 2
  • Christina Schäffer
    • 2
    Email author
  1. 1.Sandoz GmbHLangkampfenAustria
  2. 2.Department of NanoBiotechnology, NanoGlycobiology UnitUniversität für Bodenkultur WienViennaAustria

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