New England Biolabs, P0749S, α1-2,4,6 Fucosidase O

α1-2,4,6 Fucosidase O is a broad specificity exoglycosidase that catalyzes the hydrolysis of terminal α1-2, α1-4 and α1-6 linked fucose residues from oligosaccharides. α1-2,4,6 Fucosidase O cleaves α1-6 fucose residues more efficiently than other linkages. Related Categories Exoglycosidases,, Proteome Analysis Applications Glycan Sequencing,, Proteomics,, Recombinant Glycoprotein Expression, Specification Unit Definition One unit is defined as the amount of enzyme required to cleave > 95% of the fucose from 1 nmol of GlcNAc(Fucα1-6)β1-4GlcNAc-AMC in 1 hour at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 1 Incubate at 37°C 1X GlycoBuffer 1 5 mM CaCl2 50 mM sodium acetate (pH 5.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 1 mM EDTA pH 7.5 @ 20°C Heat Inactivation 65°C for 10 minutes Molecular Weight Apparent: 49 kDa Unit Assay Conditions Two fold dilutions of α1-2,4,6 Fucosidase O are incubated with 1 nmol AMC-labelled substrate and 1X GlycoBuffer 1 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). FAQ Q: What is the difference between α1-2,3,4,6 Fucosidase and α1-2,4,6 Fucosidase O? A: α1-2,4,6 Fucosidase O, cloned from Omnitrophica bacterium, is a broad specificity exoglycosidase that catalyzes the hydrolysis of terminal α1-6 fucose residues more efficiently than α1-2 and α1-4 linked fucose residues. In addition, α1-2,4,6 Fucosidase O can cleave reductively aminated glycans as well as glycans with instant labels. Whereas, α1-2,3,4,6 Fucosidase (NEB# P0748) is cloned from bovine kidney and expressed in E. coli and is a broad specificity enzyme that catalyzes the hydrolysis of α1-2, and α1-6 linked fucose residues more efficiently than α1-4 and α1-3 linked fucose residues. Q: Can α1-2,4,6 Fucosidase O cleave fucose residues in the presence of instant labelled glycans? A: Yes, α1-2,4,6 Fucosidase O can cleave fucose residue from oligosaccharides or released glycans that have been labelled with instant labels such as Instant 2AB, Instant Procainamide or RapiFluor. Instant labelled samples may require longer incubation times (18 hours) for complete digestion. Q: Can I use α1-2,4,6 Fucosidase O in a double digest with other exoglycosidases? A: Yes. We recommend simultaneous digestion with other exoglycosidases using 1X GlycoBuffer 1 (50mM NaAcetate, pH 5.5, 5mM CaCl2). For example, α1-2,4,6 Fucosidase O can be combined in a simultaneous digest with other exoglycosidases such as α2-3,6,8,9 Neuraminidase A (NEB# P0722), α1-3,4,6 Galactosidase (NEB# P0747), β1-4 Galactosidase S (NEB# P0745) and β-N-Acetylglucosaminidase S (NEB# P0744) Q: Can α1-2,4,6 Fucosidase O cleave α1-3 fucose residues? A: No, α1-2,4,6 Fucosidase O will not cleave α1-3 fucose residues. Q: Can α1-2,4,6 Fucosidase O cleave branched and complex substrates? A: α1-2,4,6 Fucosidase O will cleave branched α1-6 fucose residues and has minimal activity on branched α1-4 fucose residues. The enzyme will not cleave branched α1-2 fucose residues. Longer incubation times (4 hours to overnight) may be needed for digestion of complex oligosaccharide substrates with multiple branches. Q: Can α1-2,4,6 Fucosidase O cleave fucose residues from an intact antibody? A: α1-2,4,6 Fucosidase O is only active on an intact antibody if it is trimmed to the trimannosyl core. Q: What is the optimal incubation temperature for α1-2,4,6 Fucosidase O? A: The optimal incubation temperature when using the enzyme alone is 50°C. The enzymatic unit definition is calculated using a 37°C reaction temperature, to accommodate for simultaneous glycosidase digestions and N-glycan sequencing protocols. A 37°C reaction temperature results in ~35% lower activity. Q: What is a good method to re-purify a glycan or glycopeptide after exoglycosidase treatment? A: Filtration with micro-spin filters, or solid-phase extraction (for example: graphitized carbon cartridges).