Purification and Characterization of Endo-β-N-acetylglucosaminidase from aFlavobacteriumsp.

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Abstract
A gram negative bacterium isolated from soil was found to produce a high level of endo-β-N-acetylglucosaminidase in the culture medium. The organism was identified as a Flavobacterium sp. from various bacteriological characteristics. The enzyme from the Flavobacterium sp. was purified to homogeneity from culture broth by fractionation with ammonium sulfate and column chromatographies on DEAE-cellulose, hydroxylapatite, and Sephadex G-150 and G-100. The molecular weight of the enzyme was estimated to be 27,000 and 30,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, and it appeared to consist of a single polypeptide chain. The optimal pH for activity was 5.0 to 6.0 and the stable pH range was 5~7. The Michaelis constant was 0.30 mm with dansyl-Asn-(GlcNAc)2(Man)6 as the substrate. The enzyme hydrolyzed oligosaccharides of native ovalbumin, bovine pancreatic ribonuclease B and a yeast invertase.