Product Description 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 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.
Contents
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). Molecular Weight ~250,000 daltons
pH optimum 6.0, active over the range 5-7. The supplied buffer concentrate provides the optimal
pH for enzyme activity with the standard substrate.
If glycosidase treatment is performed at suboptimal
pH because of glycoprotein solubility or activity
requirements, expect some diminution in enzyme
activity.
Formulation
The enzyme is provided as a sterile-filtered solution in
20 mM Tris-HCl, 25 mM NaCl (pH 7.5).
Specific Activity One unit of ß-(1-4)-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.
Specificity
Non-reducing terminal ß(1-4)-Galactose. Number of
antennae does not affect cleavage rate. Fucose linked to
the penultimate N-acetylglucosamine will block cleavage
of the galactose.
Stability
Stable at least 12 months when stored properly. Several
days exposure to ambient temperatures will not reduce
activity.
Purity
ß(1-4)-Galactosidase is tested for contaminating protease
as follows: 10 µg of denatured BSA is incubated at 37°C
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.
Directions for use
1. Add up to 100 µg of asialoglycoprotein or 1 nmol of
oligosaccharide to tube.
2. Add deionized water to a total of 14 µl.
3. Add 4 µl of 5x Reaction Buffer 6.0.
4. Add 2 µl ß(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 pH for cleavage of oligosaccharides is ~6.
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