GalNAcEXO Enzyme

GalNAcEXO is an  exo-α-N-Acetylgalactosaminidase for efficient hydrolysis of α-N-acetylgalactosamine (GalNAc) linked to serine or threonine residues in glycoproteins (Tn antigen). The enzyme also displays activity on α1-3 linked terminal GalNAcs. GalNAcEXO hydrolyzes GalNAc on glycoproteins under native conditions and is highly active in the pH range 6.0 to 7.6. No cofactors or special buffers are required. The enzyme in GalNAcEXO is derived from  Akkermansia muciniphila, expressed in  E. coli with a His-tag, and has a molecular weight of 52 kDa.

Unit Definition

One unit of GalNAcEXO catalyzes the hydrolysis of α-linked GalNAc residues from ≥ 95% of 2 nmol 4-Nitrophenyl 2-acetamido-2-deoxy-D-galactopyranoside when incubated in 20 mM Tris, 1 % ethanol pH 6.8 at 37°C for 30 minutes, as monitored by reverse phase chromatography.

Content and Storage

GalNAcEXO enzyme is supplied as a lyophilized powder in Tris buffer saline (TBS) pH 7.6, with no preservatives added.

GalNAcEXO is shipped cold and should be stored at -20°C upon arrival.
After reconstitution GalNAcEXO is stable for at least 1 month at +4-8 °C.

Immobilized GalNAcEXO

Immobilized GalNAcEXO is a resin with the GalNAcEXO enzyme covalently coupled to agarose beads and is provided in microspin column format for efficient removal of GalNAc residues in glycoproteins. The enzyme in Immobilized GalNAcEXO is derived from  Akkermansia muciniphila  and expressed in  E. coli. GalNAc removal is achieved without the enzyme remaining in the final preparation. The glycoprotein sample is incubated in the immobilized GalNAcEXO column and the digested glycoprotein sample can then be easily recovered by a centrifugation step. No cofactors or special buffers are required. The recommended buffer is 20 mM Tris pH 6.8. The protocol may need optimization regarding buffer compatibility and incubation time depending on the individual glycoprotein. 

Immobilized Enzyme Activity

0.1 ml of settled Immobilized GalNAcEXO agarose resin catalyzes the hydrolysis of α-linked GalNAc residues from ≥ 95% of 5.5 µmol 4-Nitrophenyl 2-acetamido-2-deoxy-a-D-galactopyranoside when incubated in 20 mM Tris, 10 % ethanol pH 6.8 for 5 minutes at room temperature.  

Content and Storage

Immobilized GalNAcEXO microspin columns contain sufficient material to remove GalNAcs from 0.5 mg glycoprotein. The resin is supplied in 20% EtOH with no preservatives added. 

Immobilized GalNAcEXO is shipped cold and should be stored at +4-8°C upon arrival. 

Applications