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Affinity chromatography of glycosidases
Acta Universitatis Carolinae. Medica. Monographia
|January 1, 1977
Summary
Researchers purified beta-galactosidases from rat intestinal mucosa using affinity chromatography. A p-aminophenyl-beta-D-thioglucoside Sepharose 4B material effectively isolated lactase, achieving high purity.
Area of Science:
- Biochemistry
- Enzymology
- Chromatography
Background:
- Intestinal mucosa contains various glycosidases, including glucoamylase and beta-galactosidases.
- Efficient separation of these enzymes is crucial for biochemical studies and potential applications.
Purpose of the Study:
- To investigate the efficacy of different affinity chromatographic materials for separating glucoamylase and beta-galactosidases.
- To purify beta-galactosidase (lactase) from rat intestinal mucosa.
Main Methods:
- Affinity chromatography using Sepharose 4B functionalized with different ligands: p-aminophenyl-beta-D-thioglucoside, gluconate, galactonate, and phlorizin.
- Optimization of adsorption and elution conditions for glycosidases.
- Analysis of enzyme purity using polyacrylamide gel electrophoresis.
Main Results:
- The p-aminophenyl-beta-D-thioglucoside bound to Sepharose 4B demonstrated effectiveness in adsorbing target glycosidases.
- Optimal conditions for enzyme adsorption and elution were determined for the tested materials.
- Purification of a lactase preparation to a single electrophoretic zone using the beta-thioglucoside affinant was achieved.
Conclusions:
- Affinity chromatography, particularly with p-aminophenyl-beta-D-thioglucoside Sepharose 4B, is a viable method for purifying beta-galactosidases from rat intestinal mucosa.
- The developed method yields highly purified lactase, suitable for further biochemical characterization.