Abstract
A number of adsorbents useful for purifying glycosidases were synthesized and their adsorption characteristics were examined using partially purified glycosidase mixtures from Takadiastase or soybean. These adsorbents were prepared by coupling di-ε-aminocaproyl-p-aminophenyl N-acetyl-l-thio-β-D-glucosaminide, β-D-glucoside, β-D-galactoside or α-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-β-D-glucosaminidase [EC 3.2.1.30], Taka β-D-glucosidase [EC 3.2.1.21], and Taka β-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 β-D-galactosidase, β-D-glucosidase, and N-acetyl-β-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 α-D-mannosidase [EC 3.2.1.24] was also adsorbed on these adsorbents, and was eluted between β-D-glucosidase and N-acetyl-β-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.
