O-Glycosidase

Specifictity
O-glycosidase cleaves only unsubstituted Gal-Beta(1-3)GalNAc-alpha disaccharides attached to the serine or threonine residues of glycoproteins or glycopeptides.

Substitutions such as sialic acid, galactose, fucose or N-acetylglucosamine must first be removed with the appropriate exoglycosidase prior to treatment with Endo-O-Glycosidase. At minimum, a sialadase such as Neuraminidase Au (alpha-2-3,6,8,9), part number E-S001, is almost always required to remove sialic acids

Source Recombinant Streptococcus pneumoniae in E.Coli

EC 3.2.1.97

CAS 9032-92-2

Contents
60 µl aliquot of enzyme (75 mU) in 50 mM sodium phosphate, pH 7.5.
Included with 20 µL and 60 µL pack sizes:
5x Reaction Buffer 5.0 (250 mM sodium phosphate, pH 5.0)

Specific Activity >12 U/mg

Activity >1.25 U/ml

Molecular weight 180,000 daltons

pH range 5-7, optimum 5.0

Suggested usage
1. Add up to 100 µg of glycoprotein to a tube.
2. Add de-ionized water to a total of 13 µl.
3. Add 4 µl of 5x Reaction Buffer 5.0.
4. Add 1 µl of Neuraminidase Au (alpha 2-,3,6,8,9), part number E-S001.
5. Add 2 µl of O-Glycosidase.
6. Incubate for 1 hour at 37˚C.

Specific Activity Defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pNP) in 1 minute at 37˚C, pH 5.0 from p-nitrophenyl- 2-acetamido-2-deoxy-3-O-(Beta-D-galactopyranosyl)-alpha- D-galactopyranoside.

Storage Store enzyme at 4˚C.

Stability Stable at least 24 months when stored properly. Several days exposure to ambient temperature will not reduce activity. Active for at lease 5 days under reaction conditions.

Purity O-Glycosidase is tested for contaminating protease as follows; 10 μg of denatured BSA is incubated for 24 hours at 37°C with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.

O-Glycosidase References

Bhavanandan, V.P., J. Umemoto and E.A. Davidson. Characterization of an endo-alpha-N-acetylgalactosaminidase from Diplococcus pneumoniae. Biochem Biophys 70: 738-74 5 (1976 ).

Fan, J.Q., K. Yamamoto, H. Kumagai and T. Tochikura. Induction and efficient purification of endo-alpha-N-acetyl- D-galactosaminidase from Alcaligenes sp. Agric Biol Chem 54: 233-23 4 (1990 ).

Glasgow, L R., J.C. Paulson and R.L. Hill. Systematic purification of five glycosidases from Streptococcus pneumoniae. J Biol Chem 252: 8615-8 623 (1977).

Iwase, H. and K . Hotta. Release of O-linked glycoprotein glycans by endo-alpha-N-acetyl-D-galactosaminidase. Meth Mol Biol 14: 151-159 (1993).

Unemoto, J., V.P. Bhavanandan and E.A. Davidson. Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J Biol Chem 252: 8609-8 614 (1977).