Endo H cleaves asparagine-linked hybrid or high mannose oligosaccharides but not complex oligosaccharides

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Part Number – Amount of Enzyme
E-EH02     – 60 µLs¹
E-EH02-20   – 20 µls¹
E-EH02-200 – 200 µls²
¹ includes buffer and denaturant
² includes enzyme only

Product Description

Endo H, endo-beta-N-acetylglucosaminidase H, Endoglycosidase H

Endo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.

Source recombinant from Streptomyces plicatus in E.Coli


Specific Activity One unit of Endo H activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 µmole of denatured Ribonuclease B in one minute at 37˚C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved Ribonuclease B migrates faster).

60 µl aliquot of enzyme (300 mU) in 20 mM Tris-HCl, 25 mM NaCl, 1 mM EDTA (pH 7.5).
Included with 20 µL and 60 µL pack sizes:
200 µl 5x Reaction Buffer 5.5 (250 mM sodium phosphate, pH 5.5)
Denaturation Solution – 2% SDS, 1 M Beta-mercaptoethanol

Specific Activity >40 U/mg

Activity >5 U/ml

Molecular weight 29,000 daltons

pH range 5-6, optimum 5.5

Suggested usage
1. Add up to 200 µg of glycoprotein to Eppendorf tube. Adjust to 37.5 µl final volume with deionized water.
2. Add 10 µl 5X Endo H Buffer and 2.5 µl of Denaturation Solution (SDS/-ME). Heat at 100˚C for 5 minutes.
3. Cool, and then add 2.0 µl of Endo H to the reaction. Incubate 3 hours at 37˚C.

Storage Store enzyme at 4˚C.

Stability Stable at least 12 months when stored properly. Several days exposure to ambient tempertures will not reduce activity.

Purity Endoglycosidase H is tested for contaminating protease as follows; 10 μg of denatured BSA is incubated for 24 hours at 37oC with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.

The production host strain has been extensively tested and does not produce any detectable glycosidases.

Endo H References

Robbins P. W., D. F. Wirth and C. J. Hering. Expression of the Streptomyces enzyme endoglycosidase H in Escherichia coli. Biol Chem 256:10640-10644 (1981).

Robbins P. W., R. B. Trimble, D. F. Wirth, C. Hering, F. Maley , G. F. Maley, R. Das, B. W. Gibson, N. Royal and K. Biemann. Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H. J Biol Chem 259:7577-7583 (1984).

Trimble R. B., A. L. Tarentino , G. E Aumick and F. Maley. Endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus. Methods Enzymol 83:603-610 (1982).

Trimble R. B. and F. Maley. Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H. Anal Biochem 141:515-522 (1984).

Trimble R. B., R. J. Trumbly and F. Maley. Endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus. Methods Enzymol 138:763-770 (1987).

Trumbly R. J., P. W. Robbins, M. Belfort, F. D. Ziegler, F. Maley and R. B. Trimble. Amplified expression of Streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product. J Biol Chem 260:5683-5690 (1985).