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Related Experiment Video

Updated: Jan 27, 2026

Assaying Protein Kinase Activity with Radiolabeled ATP

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

Assaying Fucosidase Activity.

Zoë Anne Megson¹, Paul Messner², Christina Schäffer³

¹Sandoz GmbH, Langkampfen, Austria.

Methods in Molecular Biology (Clifton, N.J.)

|March 14, 2019

Summary

This summary is machine-generated.

Characterize recombinant glycosidases using simple colorimetric assays and complex substrates to determine enzyme functionality and linkage specificity. This protocol guides initial enzyme characterization for research applications.

Keywords:

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

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Last Updated: Jan 27, 2026

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Published on: May 26, 2017

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Area of Science:

Biochemistry
Enzymology

Background:

Recombinant glycosidases require thorough characterization to understand their functionality and specificity.
Commercially available substrates are crucial tools for assessing enzyme activity and specificity.

Purpose of the Study:

To provide a protocol for the initial characterization of recombinant glycosidases.
To demonstrate the use of colorimetric assays and complex substrates for enzyme analysis.
To use a recombinant α-L-fucosidase as an example for glycosidase characterization.

Main Methods:

Utilizing colorimetric assays with p-nitrophenyl substrates for screening enzyme activity under various conditions (buffer, pH, ion dependence, temperature).
Employing more complex sugars with diverse glycosidic bonds to determine linkage specificity.
Applying sophisticated analytical methods for detailed characterization.

Main Results:

Colorimetric assays efficiently screen optimal conditions and basic activity parameters for glycosidases.
Complex substrates are necessary for accurate determination of glycosidase linkage specificity.
The protocol provides a framework for comprehensive initial characterization.

Conclusions:

Initial characterization of recombinant glycosidases involves a combination of simple and complex substrate assays.
Colorimetric assays are valuable for preliminary screening and determining activity optima.
Accurate linkage specificity determination requires advanced methods and diverse substrates.