Deglycosylation of Hen Ovalbumin in Native Form by Endo-β-N-Acetylglucosaminidase

Abstract

The properties of endo-β-N-acetylglucosaminidase isolated from a Flavobacterium sp. were examined with native hen ovalbumin as a substrate for the first step in preparing a large amount of the deglycosylated glycoprotein with the native structure. This enzyme cleaved the oligosaccharide chain in native ovalbumin not only for the A₁ ovalbumin containing two phosphoryl residues per molecule, but also for the A₂ and A₃ ovalbumins, containing one and no phosphoryl residues, respectively. With polyacrylamide gel electrophoresis, glycosylated ovalbumin of the native form was separated from the deglycosylated variety, which was then transferred to a vinylidene fluoride membrane and stained with concanavalin A and peroxidase to confirm the deglycosylation. The apparent Km value was about 2.67mM at pH 6.0 when native ovalbumin was used as the substrate, the optimum pH for the enzyme reaction with native ovalbumin being around 5.0. This enzyme was not inhibited by its end product.