豌豆（Pisum sativum）种子中一种内切-β-N-乙酰氨基葡萄糖苷酶的纯化及底物特异性

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

作者信息

Kimura Y, Iwata K, Sumi Y, Takagi S

机构信息

Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Japan.

出版信息

Biosci Biotechnol Biochem. 1996 Feb;60(2):228-32. doi: 10.1271/bbb.60.228.

DOI:10.1271/bbb.60.228

PMID:9063968

Abstract

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 Man6GlcNAc2-PA at pH around 7.0. Oligomannose type sugar chains (Man9-6GlcNAc2-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 (Man4-3Xyl1GlcNAc2-PA and Man3Fuc1Xyl1GlcNAc2-PA) or a biantennary complex type sugar chain (Gal2GlcNAc2Man3GlcNAc2-PA) could not be hydrolyzed by the enzyme. The Km values of the enzyme for Man5GlcNAc2-PA, Man6GlcNAc2-PA, and Man9GlcNAc2-PA were 0.40 mM, 0.25 mM, and 0.32 mM, respectively.

摘要

从豌豆（Pisum sativum）种子中纯化出一种内切β-N-乙酰氨基葡萄糖苷酶，使其达到同质。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析，纯化酶的分子量估计为41,669道尔顿，通过等电聚焦测定其等电点为4.3。该酶在pH 4-7和25-50摄氏度下稳定，在pH约7.0时对Man6GlcNAc2-PA具有最高活性。寡甘露糖型糖链（Man9-6GlcNAc2-PA）和杂合型糖链（GlcNAc1Man5GlcNAc2-PA）是最适宜的底物，其次是Man5GlcNAc2-PA、Man3GlcNAc2-PA和GlcNAc2Man3GlcNAc2-PA，但含木糖的糖链（Man4-3Xyl1GlcNAc2-PA和Man3Fuc1Xyl1GlcNAc2-PA）或双天线复合型糖链（Gal2GlcNAc2Man3GlcNAc2-PA）不能被该酶水解。该酶对Man5GlcNAc2-PA、Man6GlcNAc2-PA和Man9GlcNAc2-PA的米氏常数（Km值）分别为0.40 mM、0.25 mM和0.32 mM。