The use of mannan-Sepharose 4B affinity chromatography for the purification of endo-beta-N-acetylglucosaminidase from Bacillus alvei.
In order to facilitate the isolation of endo-beta-N-acetylglucosaminidase for the structural analysis of glycoconjugates, we have isolated a strain of Bacillus alvei which produces a high level of endo-beta-N-acetylglucosaminidase. We have also devised a simple procedure for the purification of endo-beta-N-acetylglucosaminidase from B. alvei using mannan-Sepharose affinity chromatography. By using this method, endo-beta-N-acetylglucosaminidase was purified 3300-fold with 85% yield from the crude enzyme obtained by ammonium sulfate precipitation of the culture medium. The molecular weight of this enzyme was estimated to be about 66 000 by gel filtration. When using (Man)6(GlcNAc)2-Asn-Dns as substrate, the optimal activity occurs at pH 6.5 with Km of 1.9 mM. The action of endo-beta-N-acetylglucosaminidase toward several glycopeptides was also studied.
D000118:Acetylglucosaminidase / Q000302:isolation & purification*; D001407:Bacillus / Q000201:enzymology*; D002846:Chromatography, Affinity / Q000379:methods; D006596:Hexosaminidases / Q000302:isolation & purification*; D008351:Mannans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
D008351:Mannans; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase