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

• Published in: Bioscience, biotechnology, and biochemistry Vol. 60; no. 2; pp. 228 - 232
• Main Authors: Kimura, Yoshinobu, Iwata, Koushi, Sumi, Yoshiko, Takagi, Shigeaki
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 01.01.1996
• Subjects: Base Sequence; Cations; Detergents - pharmacology; endo-β-N-acetylglucosaminidase; Enzyme Stability; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; MALDI-TOF; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - chemistry; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - isolation & purification; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - metabolism; Molecular Sequence Data; Molecular Weight; N-linked oligosaccharide; Pisum sativum; Pisum sativum - enzymology; plant glycoprotein; Seeds - enzymology; Substrate Specificity; Temperature
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.60.228

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 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 xylose-containing sugar chains (Man 4-3 Xyl 1 GlcNAc 2 -PA and Man 3 Fuc 1 Xyl 1 GlcNAc 2 -PA) or a 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.