• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• 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, 4-Methylumbelliferyl-beta -D-galactopyranoside. The specific activity is >600 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Thr2-Leu595, with an N-terminal Met and a 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  70 kDa

• SDS-PAGE

  62 kDa, reducing conditions    
   

Assay Procedure

Materials

• Assay Buffer: 50 mM Sodium Phosphate, pH 6.0

• Recombinant S. pneumoniae beta (1?4)-Galactosidase (rSp beta (1-4)-Galactosidase) (Catalog # 5549-GH)

• Substrate: 4-methylumbelliferyl-beta -D-galactopyranoside (Sigma, Catalog # M1633), 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 rSp beta (1-4)-Galactosidase to 4 ng/μL in Assay Buffer.

2. Dilute Substrate to 500 μM in Assay Buffer.

3. Load into plate 50 μL of 4 ng/μL rSp beta (1-4)-Galactosidase and start the reaction by adding 50 μL of 500 μM Substrate. Include a Substrate Control containing 50 μL Assay Buffer, 50 μL of 500 μM Substrate.

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:

   Specific Activity (pmol/min/μg) =	Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
   	amount of enzyme (μg)

        *Adjusted for Substrate Blank
        **Derived using calibration standard 4-methylumbelliferone (Sigma, Catalog # M1381).

Per Well:

• rSp beta (1-4)-Galactosidase: 0.20 μg

• Substrate: 250 μM

Background: beta (1-4)-Galactosidase

Majority of secreted and membrane proteins are glycosylated (1, 2). Proper glycosylation could be critical for protein folding and biological functions (3, 4). Galactosides are commonly found on various glycan conjugates and galactosidases are tools for removing these sugar residues (5). beta 1-4 Galactosidase from   Streptococcus pneumoniae is especially efficient in removing beta 1-4 linked non-reducing galactosides from Gal-beta 1-4-GlcNAc or Gal-beta 1-4-GalNAc linkage (6).

• References:

1. Lis, H. and Sharon, N. (1993) Eur. J. Biochem. 218:1.

2. Hart, G.W. (1992) Curr. Opin. Cell Biol., 4:1017.3.

3. Dwek, R.A. (1995) Biochem. Soc. Trans. 23:1.

4. Wormald, M.R. and Dwek, R.A. (1999) Structure 7:R155.

5. Hood, J.M. et al. (1977) Proc. Natl. Acad. Sci. USA 75:113.

6. Glasgow, L. et al. (1977) J. Biol. Chem. 252:8615.

• Alternate Names:

  beta (14)Galactosidase; beta (1-4)-Galactosidase; beta-Galactosidase-3