β-D-galactoside galactohydrolase, Exo-(1-4)-beta-D-galactanase,
Beta Galactosidase from Streptococcus pneumoniae releases only β(1-4)- linked, non-reducing terminal galactose from complex carbohydrates and glycoproteins. β(1-4) galactose is by far the most common linkage found in N-linked oligosaccharides. For other galactosidase linkages, ß(1-3,4,6)-Galactosidase (E-BG02) from Bovine testes is recommended. The enzyme is as active on tetraantennary oligosaccharides as on disaccharides containing β(1-4)-linked galactose. Fucose linked to the penultimate N-acetylglucosamine will block cleavage of the galactose. Up to 100 υg of asialofetuin can be completely β(1-4)-degalactosylated in less than 1 hour using 3 mU of enzyme.
Source recombinant Streptococcus pneumoniae
ß-(1-4) Galactosidase in 20 mM Tris-HCl, 25 mM NaCl (pH 7.5).
Included with 20 µL and 60 µL pack sizes:
5x Reaction Buffer 6.0 (250 mM sodium phosphate, pH 6.0).
Specific Activity >6 U/mg
Activity >3 U/ml
Molecular weight ~250,000 daltons
1. Add up to 100 μg of asialoglycoprotein or 1 nmol of oligosaccharide to tube.
2. Add water to 14 μL
3. Add 4 μL 5X Reaction Buffer
4. Add 2 μL β(1-4) Galactosidase
5. Incubate at 37°C for 1 hour.
For glycoproteins, cleavage may be monitored by SDS-PAGE if the size differential between native and de-galactosylated protein is sufficient for detection.
Note: The optimum for cleavage of oligosaccharides is ~ pH 6.0.
Specifictity Non-reducing terminal Beta-(1-4)-galactose. Number of antennae does not affect cleavage rate. Fucose linked to the penultimate N-acetylglucosamine will block cleavage of the galactose.
Specific Activity Assay One unit of Beta Galactosidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pNP) in 1 minute at 37˚C pH 5 from p-nitrophenyl-beta-D-galactopyranoside.
Storage Store enzyme at 4˚C.
Purity Each lot of Beta Galactosidase 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.
Beta Galactosidase References
1. Glasgow, LR., J.C. Paulson and R.L. Hill. Systematic purification of five glycosidases from Streptococcus pneumonia. J. Biol Chem 252: 8615- 8623(1977).
2. Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100: 1- 14 (1979).
3. 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)
4. 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.