The enzyme releases non-reducing terminal α(1-3,4,6)-linked galactose from oligosaccharides.
Green coffee bean.
NOTE: Highly purified; β-galactosidase contaminating activity is not detectable, as determined by extended incubation with 2-AB NA2 (G2).
Coffee bean α-galactosidase cleaves non-reducing terminal galactose residues linked α1-3, α1-4 and α1-6 to polysaccharides, glycoproteins and glycans. The precise specificity of the enzyme is somewhat dependent on the nature of the glycoconjugate.
Figure 1: Cleavage specificity for GKX-5007 α(1-3,4,6)-Galactosidase.
Terminal α-linked galactose residues have been reported to occur on several glycoproteins and are of increasing interest. The galactose-α-1-3Gal group is widely found in glycoconjugates from non-primate mammals and New World monkeys although it is absent from humans and Old World monkeys. The potential presence of this modification and its associated antigenicity is an important consideration in the expression of recombinant glycoproteins by mammalian cell cultures. Both α(1-3)-linked galactose and the less abundant α(1-6)-linked galactose are cleaved by this enzyme. Other workers have used the enzyme for the modification of blood group specificity. The enzyme also has diagnostic applications for the determination of raffinose in food.
Suggestions for Use:
Procedure For De-galactosylation
1. Add up to 100 μg of glycoprotein or 3 nmol of oligosaccharide to tube.
2. Add de-ionized water to a total of 14 μl.
3. Add 4 μl of 5x Reaction Buffer.
4. Add 2 µl containing 5 mU α(1-3,4,6)-Galactosidase.
5. Incubate at 37 °C for 18 hours.
Note: The enzyme is stable. At 37 °C in pH 6.0 buffer, full activity is recovered after 19 hours and 70% after 48 hours.
Note: The enzyme has a slow turnover. After 18 hours at 37 °C with 5 U/ml enzyme, a 40 µM solution of asialo, biantennary, fucosylated oligosaccharide with outer arm α(1-3)-galactose is 67% de-α-galactosylated.
WS0061 5x Reaction Buffer [500 mM sodium citrate/phosphate (pH 6.0)]
Reaction Buffer: 5X concentrated buffer which when diluted gives 100 mM sodium citrate-phosphate pH 6.0.
Lyophilized from 100 mM sodium phosphate pH 6.5, containing 0.25 mg/ml bovine serum albumin. See note above on BSA amount.
MW: ~26 kD
One unit is defined as the amount of enzyme required to hydrolyze 1 μmole of pNP-α-D-galactopyranoside per minute at pH 6.5 and 37oC.
Size: 5 U lyophilized
Product Code: GKX-5007
Enzyme Commission (EC) number: 22.214.171.124
CAS Number: 9025-35-8
Q. Can I use this enzyme in a digestion with multiple glycosidases?
A. We use an ammonium acetate buffer when we run multi-enzyme exoglycosidase digests that include GKX-5007 α(1-3,4,6) galactosidase. We suggest a 10X reaction buffer of 500mM ammonium acetate pH 5.5 w/ 0.05% azide. The reaction buffer can also be used diluted to 20X (25mM ammonium acetate) and lower with purified glycans. This buffer works with most of our exoglycosidases in an overnight digestion: GK80040, GK80021, GKX-5014, GKX-5013, GKX-5023, GKX-5007, GKX-5010. Generally, 2 μl of each exoglycosidase is used in a 20μl reaction with an overnight incubation (16 hours) at 37°C. Please Contact Us for more details.
If the glycans need to purified from the reaction prior to analysis by e.g. LC-MS, samples can be spun through Nanosep® 10K Omega spin filters to remove protein. For data using these methods, please see our poster, Characterizing Low-Abundance Glycans on Therapeutic Proteins.
Also, acetate buffers work better than phosphate for injecting onto LC (phosphate phase separates in acetonitrile), and acetate is volatile so samples can be dried down if necessary.
Product Safety Documentation for GKX-5007:
|GKX-5007||Glyko® α(1-3,4,6)-Galactosidase (green coffee bean)|||