Assaying Fucosidase Activity
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 wordsFucosidase Enzymatic activity Substrate specificity pH optimum Cation dependence HPAEC-PAD Reaction rate
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.
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