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Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples
Yurong Chen, Zachary Armstrong1, Marta Artola
1Department of Chemistry, York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.
Journal of the American Chemical Society
|January 26, 2021
Summary
New probes enable selective detection of amylase activity in various biological samples. This aids in discovering novel amylase inhibitors and microbial amylases for industrial biotechnology and therapeutic applications.
Area of Science:
- Biochemistry
- Enzymology
- Chemical Biology
Background:
- Amylases are crucial enzymes for starch processing in industry and human digestion.
- Developing selective assays for amylase activity is vital for discovering inhibitors and biotechnological enzymes.
- Activity-based protein profiling (ABPP) offers a suitable method for assay development.
Purpose of the Study:
- To design and synthesize novel pseudodisaccharide probes for activity-based protein profiling of retaining amylases.
- To utilize these probes for identifying and characterizing amylase activity in diverse biological sources.
- To validate the selectivity of these probes for amylases against related enzymes.
Main Methods:
- Synthesis of 1,6-epi-cyclophellitol-based pseudodisaccharides with reporter tags.
- Application of these probes in activity-based protein profiling (ABPP).
- Biochemical analysis to determine probe activity, efficiency, and selectivity; structural studies for validation.
Main Results:
- Successful design and synthesis of pseudodisaccharide probes.
- Demonstrated utility of probes in ABPP for detecting retaining amylase activity in human saliva, murine tissue, and fungal secretomes.
- Biochemical and structural data confirming probe efficacy and selectivity for amylases.
Conclusions:
- The developed pseudodisaccharide probes are effective tools for activity-based protein profiling of retaining amylases.
- These probes facilitate the discovery of new amylase inhibitors and biotechnologically relevant microbial amylases.
- The study validates a powerful approach for selective enzyme activity assessment in complex biological systems.
