| Autor: |
Nicholas, McGregor, Gregory, Arnal, Harry, Brumer |
| Rok vydání: |
2017 |
| Předmět: |
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| Zdroj: |
Methods in molecular biology (Clifton, N.J.). 1588 |
| ISSN: |
1940-6029 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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