• 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, 4-Methylumbelliferyl-beta -D-galactopyranoside. The specific activity is >14,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Ser25-Glu613 with an N-terminal Met and 6-His tag Accession # NP_638243

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  67 kDa

• SDS-PAGE

  63 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 0.1 M MES, pH 5.5

• Recombinant X. campestris beta (1?3)-Galactosidase (rXc beta -Galactosidase) (Catalog # 5704-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 rXc beta -Galactosidase to 1 ng/μL in Assay Buffer.

2. Dilute Substrate to 400 μM in Assay Buffer.

3. Load into plate 50 μL of 1 ng/μL rXc beta -Galactosidase, and start the reaction by adding 50 μL of 400 μM Substrate. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL 400 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:

     *Adjusted for Substrate Blank

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

Per Well:

• rXc beta -Galactosidase: 0.050 μg

• Substrate: 200 μM

Background: beta (1-3)-Galactosidase

The majority of secreted and membrane proteins are glycosylated (1, 2). Proper glycosylation might be critical for protein folding and biological functions (3, 4). Galactoside is an essential sugar commonly found on various glycan conjugates and galactosidases are among the earliest enzymes to be studied (5). beta 1?3 Galactosidase from   Xanthomanas   capestris is a useful tool for removing beta 1-3 linked galactosides from the non-reducing terminus of glycoconjugates (6, 7).

• 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. 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. Taron, C. et al. (1995) Glycobiology 5:603.

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

• Alternate Names:

  beta (13)Galactosidase; beta (1-3)-Galactosidase