Recombinant S. pneumoniae beta(1-4)-Galactosidase, CF

• Reactivity: Spneu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant S. pneumoniae beta(1-4)-Galactosidase, CF Summary

• Details of Functionality: 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.
• Source: E. coli-derived s. pneumoniae beta (1-4)-Galactosidase protein
  Thr2-Leu595, with an N-terminal Met and a 6-His tag
• Accession #: YP_815516
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 70 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 62 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant S. pneumoniae beta(1-4)-Galactosidase, CF

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

Background

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).

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.

Bioinformatics

• Uniprot:
  • YP_815516()