Releases non-reducing terminal α(1-2,3,6)-linked mannose from oligosaccharides.
The enzyme has broad specificity, cleaving α(1-2,3,6)-linked mannose, although some kinetic preference has been observed (α1-2, 6 > 3). The enzyme will not cleave a single α(1-6) linked mannose residue from core β-mannose, but will, however, remove a single α1-3 linked mannose from the core β-mannose. By using enzyme concentrations of around 50 U/ml and extended incubation times (up to 18 hours) at 37°C, complete removal of all non-reducing terminal α linked mannose residues may be achieved.
To expedite glycan sequencing studies, the sluggish activity of GKX-5010 Jack Bean α mannosidase toward α(1-6)-linked mannose residues can be overcome by using the enzyme in combination with the α mannosidase from Xanthomonas mannihotis which rapidly cleaves α(1-6) linkages. The X. mannihotis α-mannosidase may inhibit the action of other mannosidases if a branched (α1-6) mannose is present in the substrate, so is typically added after incubation of the substrate with Jack bean α-mannosidase (GKX-5010).
Figure 1: Cleavage specificity for GKX-5010 α(1-2,3,6)-Mannosidase.
WS0095 GKX-5010 Incubation Buffer (500 mM sodium acetate, 10 mM zinc chloride (pH 5.0).
5X concentrated buffer which when diluted gives 100 mM sodium acetate pH 5.0 containing 2 mM Zn2+.
20 mM Tris-HCl, 20 mM NaCl (pH 7.5)
MW: ~190 kD
One unit is defined as the amount of enzyme that will hydrolyze 1 μmole of pNP-α-mannopyranoside per minute at pH 4.5 and 37°C.
Size: 10 U (70 μl)
Concentration: ≥ 150 U/ml
Product Code: GKX-5010
Q. Can this enzyme cleave O-Linked mannose?
A. Yes, α(1-2,3,6)-Mannosidase from jack bean has been shown to be capable of digesting O-linked oligomannose chains on O-mannosylated glycoproteins, and can also cleave the Man-α-Ser/Thr glycosidic bond .
1. Gomathinayagam S, Hamilton SR. In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins. Appl Microbiol Biotechnol. 2014 Mar;98(6):2545-54.
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-5010 α(1-2,3,6) mannosidase. 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-5010:
|GKX-5010||Glyko® α(1-2,3,6)-Mannosidase (jack bean)|
|WS0095||5X Reaction Buffer|