Recombinant F. graminearum Galactose Oxidase His Protein, CF

Recombinant F. graminearum Galactose Oxidase His-tag Protein (Catalog # 11206-GX) oxidizes free and terminal D-Galactose to 6-Aldehyde-D-Galactose with the concomitant production of H2O2 that allows convenient detection through horseradish peroxidase.

2 μg/lane of Recombinant F. graminearum Galactose Oxidase His-tag Protein (Catalog # 11206-GX) was resolved with SDS-PAGE under reducing (R) conditions and visualized by Coomassie® Blue staining, showing a band at 65 kDa.

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

Recombinant F. graminearum Galactose Oxidase His Protein, CF Summary

• Details of Functionality: Measured by its ability to oxidize galactose. The specific activity is >75,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived f. graminearum Galactose Oxidase protein
  Ala42-Gln680, with a C-terminal 6-His tag
• Accession #: I1S2N3.1
• N-terminal Sequence: Ala42
• 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: 60-68 kDa, under reducing conditions.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -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 7.0
  • Recombinant F. graminearum Galactose Oxidase His-tag (rF. GalOx) (Catalog # 11206-GX)
  • D-(+)-Galactose, 750 mM stock in deionized water
  • Horseradish Perosidase (HRP), 250 U/mL stock in 0.1 M Sodium Phosphate, pH 8.0
  • 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 50 mM stock in deionized water
  • Hydrogen Peroxide, 30% in water
  • Copper (II) Sulfate, 1 M stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader
  1. Dilute rF. GalOx to 0.3 ng/µL in Assay Buffer.
  2. Activate rF. GalOx by adding 1 mM Copper (II) Sulfate and incubate on ice for 30 minutes.
  3. Dilute 30% Hydrogen Peroxide 1/4080 using deionized water. Then, dilute the Hydrogen Peroxide 1/10 using Assay Buffer. This is the first point of the standard curve.
  4. Complete the standard curve by performing six one-half serial dilutions using Assay Buffer. The standard curve has a range of 0.625 to 40 nmol per well.
  5. Prepare a Substrate Mixture containing 30 U/mL HRP, 8 mM ABTS, and 16 mM D-(+)-Galactose in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 50 µL of activated rF. GalOx into empty wells of the same plate as the curve. Include a Control containing 50 μL of Assay Buffer.
  8. Start the reactions by adding 50 µL of substrate mixture to all wells, including standard curve wells and sample wells.
  9. Seal and incubate plate at room temperature for 5 minutes.
  10. Immediately read plate at 420 nm (absorbance) in endpoint mode.
  11. Calculate specific activity:

  Specific Activity (pmol/min/µg) = ([Adjusted Sample OD420 x Conversion Factor]* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the Hydrogen Peroxide standard curve using linear fitting and adjusted for Control
  Per Well:
  • rF. GalOx: 0.015 µg
  • HRP: 15 U/mL
  • ABTS: 4 mM
  • D-(+)-Galactose: 8 mM

Notes

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

Alternate Names for Recombinant F. graminearum Galactose Oxidase His Protein, CF

• Galactose Oxidase

Background

Fusarium graminearum galactose oxidase (GalOx) is an extracellular copper-containing oxidoreductase. Its catalytic site contains a free radical ligand formed by covalently cross-linked cysteine and tyrosine side chains coordinating to a copper center (1, 2, 3). GalOx catalyzes the two-electron oxidation of the C6-hydroxyl group of D-galactose as well as a range of primary alcohols to the corresponding aldehydes with concomitant reduction of oxygen to hydrogen peroxide. GalOx is strictly regioselective on primary alcohols and displays remarkable stereospecificity in its reaction with sugars, showing activity only with galactose but not glucose. Because of this specificity, GalOx is used to determine lactose levels in biological fluids, human milk and dairy products and examine histochemistry of mucus-secreting cells (4, 5). Furthermore, GalOx has been used to modify cell surface carbohydrates for cell labeling and histochemical staining and induce interferon in human lymphocyte culture (2, 6). GalOx is viewed as a competitive and cost-effective catalyst compared to chemical conversion for manufacturing fine chemicals in pharmaceutical and food industry. For example, GalOx is used for conversion of D-galactose to food-grade cross-linking agents (7).

1. Baron, AJ. et al. (1994) J. Biol. Chem. 269:25095. 
2. Whittaker, J.W. (2002) Adv. Protein Chem. 60:1.  
3. Parikka K, et al.  (2015) J. Mol. Catal., B Enzym. 120:47. 
4. Roberts, G.P. and Gupta, S.K. (1965) Nature 207:425. 
5. Kanyong, P. et al. (2013) Anal. Biochem. 435:114. 
6. Dianzani, F. et al.  Infect. Immun. (1979) 26:879. 
7. Schoevaart, R. and Kieboom, T.  (2001) Carbohydr. Res. 334:1.

Bioinformatics

• Uniprot:
  • I1S2N3.1()