Purification and substrate specificity of an endo-β-N-acetylglucosaminidase from pea (pisum sativum) seeds

An endo-β-N-acetyIglucosaminidase was purified to homogeneity from the seeds of pea (Pisum sativum). The molecular mass of the purified enzyme was estimated to be 41,669 Da by MALDI-TOF MS analysis and its isoelectric point to be 4.3 by isoelectric focusing. The enzyme was stable at pH 4–7 and at 25–50°C, and had the highest activity toward Man₆GlcNAc₂-PA at pH around 7.0. Oligomannose type sugar chains (Man_9–6GlcNAc₂-PA) and a hybrid type sugar chain (GlcNAc₁Man₅GlcNAc₂-PA) were most favored substrates followed by Man₅GlcNAc₂-PA, Man₃GlcNAc₂-PA, and GlcNAc₂Man₃GlcNAc₂-PA, but xylose-containing sugar chains (Man_4–3Xyl₁GlcNAc₂-PA and Man₃Fuc₁Xyl₁GlcNAc₂-PA) or a biantennary complex type sugar chain (Gal₂GlcNAc₂Man₃GlcNAc₂-PA) could not be hydrolyzed by the enzyme. The K_m values of the enzyme for Man₅GlcNAc₂-PA, Man₆GlcNAc₂-PA, and Man₉GlcNAc₂-PA were 0.40 mm, 0.25 mm and 0.32 mm, respectively.