β-D-galactoside galactohydrolase, Exo-(1-4)-beta-D-galactanase, Lactase, beta-(1-3,4,6) galactosidase
Beta-(1-3,4,6) Galactosidase cleaves all β(1-3) and β(1-4) linked non-reducing, terminal galactose. β(1-6) linked galactose is released at a slower rate. The enzyme is a glycoprotein.
β(1-4) galactose is by far the most common linkage found in N-linked oligosaccharides. For cleavage of this linkage we recommend β(1-4) Galactasidase (E-BG07).
Source Bovine Testes
200 µls of Beta-(1-3,4,6) Galactosidase in 20 mM Tris-HCl, 50 mM NaCl, 0.5mg/ml BSA,pH 7.5
5x Reaction Buffer- 500 mM sodium citrate/phosphate pH 4
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.
Specific Activity >10.2 U/mg
Activity >2.5 U/ml
Molecular weight 68,000 daltons
Rxn pH 4
1. Add up to 100 μg of asialoglycoprotein or 1 nmol of oligosaccharide to tube.
2. Add deionized water to 14 µl.
3. Add 4 µl 5x Reaction Buffer 4.0.
4. Add 2 µL of enzyme
5. Incubate one hour at 37˚C.
For glycoproteins, cleavage may be monitored by SDS-PAGE if the size differential between native and de-galactosylated protein is sufficient for detection.
Specifictity Cleaves all ß(1-3) and ß(1-4) linked non-reducing, terminal galactose.
Specific Activity One unit of ß(1-3,4,6)Galactosidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pNP) in 1 minute at 37˚C, pH 4.0 from p-nitrophenyl-Beta-D-galactopyranoside.
Storage Store enzyme at -20˚C.
Purity Each lot of β(1-3,4,6) Galactosidase is tested for contaminating protease as follows: 10 μg of denatured BSA is incubated for 24 hours at 37°C with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.
Each lot is also tested for contaminating activities by incubating the enzymes with the appropriate substrates for 24 hours; the detection limit is 5 μU/mL (IUB). A passing lot will have no detectable activity.
Stability Stable at least 12 months when stored frozen.
Beta-(1-3,4,6) Galactosidase References
Guile GR, Rudd PM, Wing DR, Prime SB, Dwek RA. A rapid high resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal Biochem. 1996; 240(2):210-26.
Jacob GS, Scudder P. Glycosidases in structural analysis. Methods Enzymol. 1994;230:280-99.
Distler JJ, Jourdian GW. The purification and properties of beta-galactosidase from bovine testes. J Biol Chem. 1973; 248(19):6772-80.