Properties
Description
Recombinant Enterococcus faecalis O-glycosidase expressed in E. coli catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins.
Unit Definition
One unit will remove 0.68 nmol of O-linked disaccharide from 5 mg of neuraminidase digested, non-denatured fetuin in 1 hour at 37°C in a total reaction volume of 100 µl. One unit of both O-glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively.
Expression System
E. coli
Source
Enterococcus faecalis
Components
O-Glycosidase (40,000,000 units/ml); 10 x GlycoBuffer (50 mM sodium phosphate, pH 7.5, 25°C); 10 x Glycoprotein denaturing buffer (0.5% SDS, 40 mM DTT); NP-40 (10%)
Reaction Conditions
1X GlycoBuffer (50 mM sodium phosphate, pH 7.5, 25°C), Incubate at 37°C
Heat Inactivation
10 minutes at 65°C
Production Methods
Fermentation
Detection Method
A two-fold gradient dilution of O-glycosidase is added to a reaction mixture of 5 mg of neuraminidase-digested fetuin and 1X GlycoBuffer (50 mM sodium phosphate, pH 7.5, 25°C). The reaction mixture is incubated at 37°C for 1 h. O-linked disaccharide carbohydrates are analyzed by the Morgan and Elson assay.
Concentration
40,000,000 units/ml
Purity
≥95%, determined by SDS-PAGE.
Buffer
20 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.5 at 25°C
Applications
Recombinant Enterococcus faecalis O-glycosidase expressed in E. coli can be used to remove more complex O-glycan recombinases.
