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

An endo-beta-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 degrees C, and had the highest activity toward Man(6)GlcNAc(2)-PA at pH around 7.0, Oligomannose type sugar chains (Man(9-6)GlcNAc(2)-PA) and a hybrid type sugar chain (GlcNAc(1)Man(5)GlcNAc(2)-PA) were most favored substrates followed by Man(5)GlcNAc(2)-PA, Man(3)GlcNAc(2)-PA, and GlcNAc(2)Man(3)GlcNAc(2)-PA, but biantennary complex type sugar chain (Gal(2)GlcNAc(2)Man(3)GlcNAc(2)-PA) could not be hydrolyzed by the enzyme, The K-m values of the enzyme for Man(5)GlcNAc(2)-PA, Man(6)GlcNAc(2)-PA, and Man(9)GlcNAc(2)-PA were 0.40 mM, 0.25 mM, and 0.32 mM, respectively.