N-Acetylglucosaminidase

ß-N-acetylglucosaminidase, N-acetyl-β-d-glycosaminide N-acetylglucosaminohydrolase, glucosaminidase, hexosaminidase

N-acetylglucosaminidase cleaves all non-reducing terminal β-linked N-acetylglucosamine residues from complex carbohydrates and glycoproteins.

The cleavage rates of different linkages of GlcNAc on bi-, tri- and tetraantennary oligosaccharides is greatly dependent on the steric hindrance by neighboring residues. The β(1-2)GlcNAc residue linked to the β(1-3)-linked mannose is cleaved at the highest rate and the β(1-2) GlcNAc residue linked to the β(1-6)-linked mannose at the lowest rate for all three oligosaccharides. The β(1-6) GlcNAc residue, when present, is removed at the second highest rate and the β(1-4) GlcNAc, third. On a triantennary structure, this residue is removed at the second highest rate. A bisecting β(1-4) GlcNAc linked to the β-linked mannose severely hinders cleavage of other GlcNAc residues–high concentrations of enzymes and prolonged incubation times are required for cleavage.

Source recombinant from Streptococcus pneumoniae in E. Coli.

EC 3.2.1.52

Contents
N-acetylglucosaminidase in 20 mM Tris-HCl, 25 mM NaCl, pH 7.5

Included with 20 µL and 60 µL pack sizes:
5x Reaction Buffer 250 mM sodium phosphate, pH 5.0

Specific Activity >80 U/mg
Activity >50 U/ml

Molecular weight ~140,000 daltons

pH range 5-7, optimum 5.0

Suggested usage
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).
4. Add 2 µl of N-acetylglucosaminidase.
5. Incubate 3 hours at 37˚C.

NOTE: If bisecting GlcNAc or beta(1-2)GlcNAc-alpha(1-6)Man are present increase incubation nation time to 18 hours.

Specifictity Cleaves all non-reducing terminal Beta-linked N-acetylglucosamine. Bisecting GlcNAc slows the reaction.

Specific Activity Assay 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.

Purity Each lot of N-acetylglucosaminidase is tested for contaminating protease as follows; 10 μg of denatured BSA is incubated for 24 hours with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.

The production host strain has been extensively tested and does not produce any detectable glycosidases.

Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity.

N-acetylglucosaminidase 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).

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).