Endo-Beta-Galactosidase

Part number E-XBG01
Source Recombinant from Bacteroides fragilis
EC 3.2.1.103

Contents
60 µl aliquot of enzyme (0.9 U) in 20 mM tris-HCl, pH 7.5
1 vial reaction buffer- 250mM Sodium phosphate, pH 5.8

Specific Activity >150 U/mg
Activity 15 U/ml

Molecular weight ~32,000 daltons

Suggested usage For glycoproteins:
1. Add up to 100 µg of glycoprotein to a tube.
2. Add 4 ul 5X buffer and water to 19 µl.
3. Add 1 µl enzyme.
4. Incubate at 37C for 2 hrs.

Specificity Cleaves internal b(1-4) galactose linkages in unbranched, repeating poly-N-acetyllactosamine [GlcNAcb(1-3)Galb(1-4)]n structures are the preferred substrate. Sulfated structures such as keratan sulfate are also cleaved. Branching and/or fucosylation of the substrate may decrease or eliminate cleavage. Sulfation of C-6 on galactose will block cleavage. Oligosaccharidesof the neo-lacto group are cleaved at greatly educed rates depending on the deviation from the preferred substrate.

For example, Galb(1-3)GlcNAcb(1-3)Galb(1-4)Glc is cleaved at 5x10-5 the rate of keratan sulfate(see ref.4). Specificity is similar to the Escherichia freundii enzyme.pecificity is similar to the Escherichia freundii enzyme except that it is limited to cleaving N-acetyllactosamine extensions on tetraantennary structures of erythropoietin see ref 5).

Specific Activity One unit of endo-b-Galactoctosidase is defined as the amount that will liberate one µmole of reducing sugar per minute at 37oC and pH 5.8 from bovine corneal keratan sulfate.

References

1. Scudder,P., Uemura, K., Doby, J., Fukuda, M.N. & Feizi, T.(1983) Isolation and characterization of an endo-b-galactosidase from Bacteroides fragilis Biochem. J. 213 , 485-494.

2. Scudder,P., Hanfland, Pl, Uemura, K. & Feizi, T. (1984) Endo-b-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series. J. Biol. Chem . 259, 6586-6592.

3. Scudder, P. Tang, P.W., Hounsell, E.F., Lawson, A.M., Mehmet, H. & Feizi, T. (1986) Isolation and characterization of sulfated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-b-galactosidase. Eur. J. Biochem. 157, 365-373.

4. Murata, T., Hattori, T. Amarume, S. Koicki, A. & Usui, T. (2003) Kinetic studies on endo-b-galactosidase by a novel colorimetric assay and sythesis of N-acetyllactosamine-repeating oligosaccharide b-glycosides using its transglycosylation activity. Eur. J. Biochem 270, 3709-3719.

5. Hokke, C.H., Bergwerff, A.A., Van Dedem, D.W., Kamerling, J.P, and Vliegenthart, J.F. (1995) Structural analysis of the N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster overay cells. Sialylation patters and branch location of dimeric N-acetyllactosamine units. Eur. J. Biochem. 228, 981-1008.