α1-2,3,6 Mannosidase is a broad specificity exoglycosidase that catalyzes the hydrolysis of terminal, non-reducing α1-2, α1-3 and α1-6 linked mannose residues from oligosaccharides.
• Recombinant enzyme with no detectable endoglycosidase or other exoglycosidase contaminating activities
• Glycerol -free for optimal performance in HPLC and mass spectrometry analysis
• ≥95% purity, as determined by SDS-PAGE and intact ESI-MS
• Optimal activity and stability for up to 12 months
Product Information
α1-2,3,6 Mannosidase, cloned from Jack Bean, and also known as JBM, is a broad specificity exoglycosidase that catalyzes the hydrolysis of terminal α1-2, α1-3 and α1-6 linked mannose residues from oligosaccharides. α1-2,3,6 Mannosidase has a slight preference for α1-2 mannose residues over α1-3 and α1-6 mannose residues.
Substrate Specificity
Product Source
Cloned from Canavalia ensiformis (Jack Bean) and expressed in Pichia pastoris.
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
P0768S
4
α1-2,3,6 Mannosidase
P0768SVIAL
4
1 x 0.04 ml
2,000 units/ml
GlycoBuffer 4
B1703SVIAL
-20
1 x 1 ml
10 X
Zinc
B0768SVIAL
4
1 x 1 ml
10 X
Properties & Usage
Unit Definition
One unit is defined as the amount of enzyme required to cleave > 95% of the terminal mannose from 1 nmol of Man(α1,3)-Man(β1,4)-GlcNAc-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37°C in a total reaction volume of 10 µl.
Reaction Conditions
1X GlycoBuffer 4
Supplement with 1X Zinc
Incubate at 37°C
Two fold dilutions of α1-2,3,6 Mannosidase are incubated with 1 nmol AMC-labeled substrate, 1X GlycoBuffer 4 and 1X Zinc in a 10 µl reaction. The reaction mix is incubated at 37°C for 1 hour. Separation of reaction products are visualized via thin layer chromatography (1).
α1-2,3,6 Mannosidase is a glycosylated protein. The theoretical molecular weight is 110 kDa, with two subunits at 66 kDa and 44 kDa (2).
The optimal pH for α1-2,3,6 Mannosidase is 4.5. Enzyme activity is enhanced by the presence of 2 mM Zn2+ in the reaction.
In order to achieve complete removal of all non-reducing terminalα-linked mannose residues, it may be necessary to use an increasedenzyme concentration as well as an overnight incubation time (18 hours).
To achieve complete removal of α1-6 mannose residues from a complexsubstrate, a sequential digest using α1-2,3,6 Mannosidase followed byα1-6 Mannosidase (NEB #P0727) may be necessary.
Reactions may be scaled-up linearly to accommodate larger reaction volumes.
The amount of exoglycosidase enzyme required varies when differentsubstrates are used. Start with 1-2 μl for 1 μg of glycoprotein or 100nM of oligosaccharide for one hour in a 10-25 μl reaction. If there isstill undigested material, let the reaction go overnight.
References
Wong-Madden, S.T. and Landry, D. (1995). Glycobiology. 5, 19-28.
Kumar, S.N. et al (2014). Glycobiology. 24, 252-261.
Degani, G., Barbiroli, A., Magnelli, P., Digiovanni, S., Altomare, A., Aldini, G., & Popolo, L. (2019) Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris Glycoconjugate Journal; 36-1, 27-38. PubMedID: 30612271, DOI: 10.1007/s10719-018-09855-x
Publications
Degani, G., Barbiroli, A., Magnelli, P., Digiovanni, S., Altomare, A., Aldini, G., & Popolo, L. (2019) Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris Glycoconjugate Journal; 36-1, 27-38. PubMedID: 30612271, DOI: 10.1007/s10719-018-09855-x
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