Affinity chromatography of glycosidases. Preparation and properties of affinity column adsorbents.
A number of adsorbents useful for purifying glycosidases were synthesized and their adsorption and characteristics were examined using partially purified glycosidase mixtures from Takadiastase or soybean. These adsorbents were prepared by coupling di-epsilon-aminocaproyl-p-aminophenyl N-acetyl-1-thio-beta-D-glucosaminide, beta-D-glucoside, beta-D-galactoside or alpha-D-mannoside with CNBr-activated Sepharose 4B. Many glycosidases were adsorbed on the four adsorbents at low ionic strength, and increase of the ionic strength caused the enzymes to be eluted. However, the specificity of the adsorbents, contrary to our expection, was very low. All of these adsorbents adsorbed Taka N-acetyl-beta-D-glucosaminidase [EC 3.2.1.30], Taka beta-D-glucosidase [EC 3.2.1.21], and Taka beta-D-galactosidase [EC 3.2.1.23] at low ionic strength. The order of elution of these three enzymes by a linear gradient of ionic strength was the same in the four adsorbents, the order being beta-D-galactosidase, beta-D-glucosidase, and N-acetyl-beta-D-glucosaminidase. Soybean glycosidases also showed nearly the same elution pattern, though the ionic strength of the eluate was slightly lower than with Taka glycosidases. Soybean alpha-D-mannosidase [EC 3.2.1.24] was also adsorbed on these adsorbents, and was eluted between beta-D-glucosidase and N-acetyl-beta-D-glucosaminidase. These adsorption phenomena were not specific as regards the structure of the glycoside moiety, but they were useful for purifying glycosidases, possessing good reproducibility with easy activation and mild operating conditions.
D000681:Amylases / Q000302:isolation & purification; D002021:Buffers; D002846:Chromatography, Affinity; D010450:Endopeptidases; D005959:Glucosidases / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D006596:Hexosaminidases / Q000302:isolation & purification; D008024:Ligands; D008361:Mannosidases / Q000302:isolation & purification; D009994:Osmolar Concentration; D010944:Plants / Q000201:enzymology; D012685:Sepharose; D013025:Glycine max; D013865:Thioglycosides
D002021:Buffers; D008024:Ligands; D013865:Thioglycosides; D012685:Sepharose; D000681:Amylases; D005959:Glucosidases; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D008361:Mannosidases; D010450:Endopeptidases