Partial purification and characterization of a novel endo-beta-mannosidaseacting on N-linked sugar chains from Lilium longflorum thumb

An enzyme catalyzing the hydrolysis of the Man beta 1-4GlcNAc linkage of N- Iinked sugar chains was partially purified and characterized. Endo-p-mannos idase activity was detected using pyridylaminated (PA-) Man alpha 1-6Man be ta 1-4GlcNAc beta 1-4GlcNAc as the substrate in a homogenate of lily flower s (Lilium longflorum Thumb). The enzyme was partially purified by ammonium sulfate precipitation, and Q-Sepharose, Superdex 200, hydroxyapatite, Poros PE/M, Mono Q, and Superdex 200 column chromatographies. The optimum pH was 5.0 and the estimated molecular weight of the enzyme was 78,000, as determ ined by gel filtration. The K-m value found for Man alpha 1-6Man beta 1-4Gl cNAc beta 1-4GlcNAc-PA was 1.4 mM. The enzymatic activity was not influence d by the addition of 10 mM EDTA or 2 mM Ca2+. Experiments on the hydrolysis of several PA-N-linked sugar chains revealed that the enzyme hydrolyzed Ma n(n)Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc-PA (n = 0-2) into a mixt ure of Man(n)Man alpha 1-6Man and GlcNAc beta 1-4GlcNAc-PA, indicating that it is an endoglycosidase in nature. However, the enzyme did not hydrolyze beta 1-4mannohexaose or p-nitrophenyl beta-mannopyranoside.