Purification of jack bean meal beta-D-galactosidase by a new affinity adsorbent.
A simple procedure has been developed for the purification of jack bean beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) by affinity chromatography employing a new affinity adsorbent. The ligand 6-N-beta-(4-aminophenyl)-ethylamino-3-O-beta-D-galactopyranosyl-6-deoxy-L-gulitol was prepared by the reaction between lactose and beta-(4-aminophenyl)-ethylamine and was coupled to cyanogen bromide activated Sepharose 4B via the amino groups of the 4-aminophenyl moiety. This affinity gel resulted in a 111-fold purification of beta-D-galactosidase with a 64% recovery of the enzyme. With p-nitrophenyl-beta-D-galactopyranoside as the substrate the apparent Km and V values were 0.59 mM and 1.87 mumol/min per mg, respectively. The method for purification of beta-D-galactosidase may be applicable to other glycosidases depending upon the choice of specific di- or oligosaccharides of known structures to be used in the preparation of ligands.
D002846:Chromatography, Affinity; D007887:Fabaceae / Q000201:enzymology; D005696:Galactosidases / Q000302:isolation & purification*; D007700:Kinetics; D008024:Ligands; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D008024:Ligands; D005696:Galactosidases; D001616:beta-Galactosidase