Part number E-GL01-200
Source recombinant from Streptococcus pneumoniae in E. Coli.
200 µl aliquot of enzyme (8 U) in 20 mM Tris-HCl, 175 mM NaCl, pH 7.5
5x Reaction Buffer 250 mM sodium phosphate, pH 5.0
Specific Activity >80 U/mg
Activity 40 U/ml
Molecular weight ~140,000 daltons
pH range 5-7, optimum 5.0
1. Add up to 100 µg of glycoprotein or 1 nmole of oligosaccharide to a tube.
2. Adjust to 14 µl final volume with de-ionized water.
3. Add 4 µl 5x reaction buffer (pH 5.0)
3. Add 2 µl of Glucosaminidase
4. Incubate 18 hours at 37¡C.
NOTE: Shorter incubation times are possible if bisecting GlcNAc or beta(1-2)GlcNAc-alpha(1-6)Man are present.
Specifictity Cleaves all non-reducing terminal Beta-linked N-acetylglucosamine. Bisecting GlcNAc slows the reaction.
Specific Activity Defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol in 1 minute at 37¡C, pH 5.0 from <->p-nitrophenyl-Beta-D-N-acetyl-glucosaminide
Storage Store enzyme at 4¡C.
References Clarke, V. A., N. Platt and T.D. Betters. Cloning and expression of the beta-N-acetylglucosaminidase gene from Steptococcus pneumoniae. Generation of truncated enzymes with modified aglyconn specificity. J Biol Chem 270:8805-8814 (1995).
Dwek, R. A., C. J. Edge, D. J. Harvey, M. R. Wormald and R. B. Parekh. Analysis of glycoprotein-associated oligosaccharides. Ann Rev Biochem 62: 65-100 (1993).
Glasgow, L.R., J.C. Paulson and R.L. Hill. Systematic purification of five glycosidases from Streptococcus pneumoniae J Biol Chem 252:8615-8623(1977).
Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100: 1-14 (1979).
J Biochem (Tokyo) 106: 1086- 1089 (1989).
Prime, S., J. Dearnley , A. M. Venton, R. B. Parekh and C. J. Edge. Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. J Chromatogr A 720: 263-274 (1996).