Releases α(2,3)-, α(2,6)-, α(2,8)-, and α(2,9)-linked N-acetylneuraminic acid from oligosaccharides and glycoproteins. Also capable of releasing N-glycolylneuraminic acid.
Recombinant gene from Arthrobacter ureafaciens, expressed in E. coli.
The enzyme releases α(2,3)-, α(2,6)-, α(2,8)-, and α(2,9)-linked N-acetylneuraminic acid from complex carbohydrates. The initial rate of hydrolysis of α(2,6) linkages is reported to be approximately twice that of α(2,3)-linked sialic acid however, in practice, this kinetic selectivity is of little consequence during extended incubations. Effective digestion of glycolipid substrates is facilitated by addition of a detergent, such as sodium taurodeoxycholate to the incubation.
Sialidase A is capable of releasing N-glycolylneuraminic acid (Neu5Gc, NGNA) in addition to N-acetylneuraminic acid (Neu5Ac, NANA) , although similarly to other sialidases  the activity is lower toward Neu5Gc than Neu5Ac.
The enzyme can also be used to remove sialic acid from gangliosides , glycosphingolipds (ceramide & oligosaccharide) with sialic acid.
May be used for exoglycosidase sequencing in conjunction with Sialidase S (GK80021), which is specific for α(2,3)-linked sialic acids.
WS0049 5x Reaction Buffer B [250 mM sodium phosphate pH 6.0]
5X concentrated buffer which when diluted gives 50 mM sodium phosphate pH 6.0.
20 mM Tris HCl pH 7.5, containing 25 mM NaCl.
One unit is defined as the amount of enzyme required to catalyze the release of 1 μmole of p-nitrophenol from p-nitrophenyl-α-D-N-acetylneuraminic acid per minute at 37° C, pH 5.5.
Size: 1 U (200 µl)
Concentration: ≥ 5 U/ml
Product Code: GK80040
Q. Does GK80040 contain a purification tag, such as 6xHis?
A. No. Although GK80040 Sialidase A is expressed recombinantly, it does not contain a purification tag.
Q. I do not want to use the sodium phosphate reaction buffer supplied with GK80040. Can I use an acetate buffer? Is this good for digestion with multiple exoglycosidases?
A. We use an ammonium acetate buffer when we run multi-enzyme exoglycosidases digests that include GK80040 Sialidase A. 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. 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.
Q. Can GK80040 be used with detergents and reductants?
A. The enzyme can be used with sodium dodecyl sulfate (SDS) and β-mercaptoethanol (βME) at certain levels.
The 5x Denaturation Solution used in the GK80110 kit is 2% sodium dodecylsulfate (SDS) and 1 M βME, and 2.5μl is used in a reaction volume of around 50μl giving a final reaction concentration of 0.1% SDS, 50mM bME. Denaturation Solution and 5x Incubation Buffer are added to the glycoprotein substrate and the mixture is heated to 100°C for 5 minutes. After cooling, 2.5μl of a Detergent Solution (15% NP-40, final reaction concentration 0.75%) is added to counteract the inhibitory effect of SDS on PNGase F. This is followed by the addition of N-Glycanase, Sialidase A (GK80040) and O-Glycanase (GK80090), with incubation for 3 hours at 37°C.
Product Safety Documentation for GK80040:
|GK80040||Glyko® Sialidase A™|||
|WS0049||5x Reaction Buffer B|