• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave a fluorogenic substrate, 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid. The specific activity is >25,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Pro42-Gly403, with an N-terminal Met and 6-His tag Accession # BAA00852

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  40 kDa

• SDS-PAGE

  42 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Sodium Acetate, 150 mM NaCl, pH 4.5

• Recombinant M. viridifaciens Neuraminidase (rMvNA) (Catalog # 5084-NM)

• Substrate: 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid (Sigma, Catalog # M8639), 10 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rMvNA to 0.2 ng/µL in Assay Buffer.

2. Dilute Substrate to 400 µM with Assay Buffer.

3. Load into plate 50 µL of 0.2 ng/µL rMvNA and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing 50 µL of Substrate and 50 µL of Assay Buffer.

4. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).

• rMvNA: 0.010 µg

• Substrate: 200 µM

Background: Bacterial Neuraminidase

Neuraminidase is the common name for N-acetyl-neuraminyl hydrolase (Sialidase).   Micromonospora viridifaciens Neuraminidase catalyzes the hydrolysis of alpha 2-3 and alpha 2-6 and alpha 2-8 linked N-acetyl-neuraminic acid residues from glycoproteins and glycolipids (1).   The architecture of the full-length protein includes a canonical neuraminidase enzymatic domain, a linker domain and a C-terminal galactose binding domain (2). The full-length protein contains 647 amino acids and has a molecular weight of 68 kDa. It is easily degraded to 52 kDa and 41 kDa fragments (3). The expressed enzyme only contains the enzymatic domain.

• References:

1. Aisaka, K. & Uwajima, T. (1987) FEBS Microbiol. Lett. 44:289.

2. Gaskell, A. et al. (1995) Structure 3:1197.

3. Sakurada, K. et al. (1992) J. Bacteriol. 174:6896.

• Alternate Names:

  Bacterial Neuraminidase