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Non-reducing terminal β(1-4)-Galactose. Number of antennae does not affect cleavage rate. Fucose linked to the penultimate N-acetylglucosamine will block cleavage of the galactose.
β 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, β(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 µg of asialofetuin can be completely β(1-4)-degalactosylated in less than 1 hour using 3 mU of enzyme.
Source: Recombinant Streptococcus pneumoniae in E. coli
Alternate Names: β-D-galactoside galactohydrolase, Exo-(1-4)-β-D-galactanase, Lactase
β-(1-4) Galactosidase in 20 mM Tris-HCl, 25 mM NaCl (pH 7.5).
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.
Specificity: Non-reducing terminal β(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 β 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-β-D-galactopyranoside.
Storage: Store enzyme at 4°C.
Purity: Each lot of β 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.