Sialidase L
A NeuAca2,3Gal - linkage specific sialidase
Unique neuraminidase activity releasing 2,7-anhydro-NeuAc

Source - Recombinant Macrobdella leech sialidase

Specificities - Sialidase L (Mol. Wt. 84 KDa) is a unique sialidase which cleaves NeuAc from NeuAca2-3Gal linked glycoconjugates and releases 2,7-anhydro-NeuAc, not the free NeuAc, as the product.

Sialidase L hydrolyzes only the NeuAca2-3Gal linkage. Other NeuAc linkages, such as NeuAca2-6Gal, NeuAca2-6GalNAc, NeuAca2-6GlcNAc, NeuAc a2-8NeuAc, and NeuAca2-9NeuAc are not hydrolyzed. Sialidase L hydrolyzed the NeuAca2-3Gal linkage present in 3'-sialyllactose, bovine fetuin, keratan sulfate, a₁-acid glycoprotein and sialyl-Le^x glycosphingolipid,NeuAca2-3Galb1-3(Fuca1-4)GlcNAcb1-3Galb1-4 Glc-Cer.

Specific Activity - One unit of the enzyme releases 1 nmole of 4-methylumbelliferone (MU) per min at 37°C from 2'-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (4-MU-NeuAc). The specific activity of this preparation is 2,500 nmoles/mg/min.

Enzyme Assay - 2'-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid (4-MU-NeuAc) (100 microliter, 0.5mM) in 20 mM acetate buffer, pH 5.5 is incubated with an appropriate amount of sialidase L for a preset time. The reaction is terminated by the addition of 1.5 ml of 0.2 M sodium borate buffer, pH 9.8. The resulted fluorescence is measured by using a fluorometer with excitation at 360 nm and emission at 440 nm.

Optimal pH and stability - Sialidase L exhibits a broad pH optimum between pH 5.5 - 7.0 in sodium acetate and sodium phosphate buffers with MU-NeuAc as substrate. Sialidase L is not affected by the presence of 2mM EDTA, 20mM Ca⁺⁺, Mg⁺⁺, or Mn⁺⁺. The purified sialidase L lost 50% of its activity after one freeze and thaw cycle. As sold with 10% glycerol, the enzyme is stable for more than 4 months at -20°C and is able to withstand several freeze and thaw cycles.  If used frequently, store at 4°C.

Contaminants - This enzyme contains no detectable activities of ceramide glycanase, proteases, a- and b-galactosidases, a- and b-mannosidases, b-hexosaminidase, a-N-acetylglucosaminidase, a-N-acetylgalactosaminidase and a-fucosidase.

References
1. Li, Y.-T., Nakagawa, H., Ross, S., Hansson, G., and Li, S.-C. (1990), J. Biol. Chem. 265, 21629-21633.
2. Chou, M.-Y., Li, S.-C., Kiso M., Hasegawa, A., and Li, Y.-T. (1994), J. Biol. Chem. 269, 18821-18826.
3. Chou, M.-Y., Li, S.-C., and Li, Y.-T. (1996) J. Biol. Chem. 271, 19219-19224.

Specific Activity 2500 units/mg   pH containing 10% glycerol

This enzyme is in 20mM phosphate buffer
Protein = 420 µg/ml