Purification and Substrate Specificity of an Endo-β-N-acetylglucosaminidase from Pea (Pisum sativum) Seeds

Abstract

An endo-β-N-acetylglucosaminidase 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.