Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-β-N-acetylglucosaminidase A
Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-β-N-acetylglucosaminidase A
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DOI：
10.1006/bbrc.1999.1963
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发表时间：
2000-01-07
影响因子：
3.1
通讯作者：
Takegawa, K
中科院分区：
文献类型：
--
作者：
Fujita, K;Tanaka, N;Takegawa, K
描述了一种用于酶促合成新糖酶的方法。将原弗氏节杆菌（Arthrobacter proto-phormiae）的内切型β-N-乙酰氨基葡萄糖苷酶（Endo-A）基因在大肠杆菌中高效表达，并与谷胱甘肽S-转移酶（GST）融合。提取GST-Endo-A融合蛋白作为可溶性蛋白。融合蛋白用谷胱甘肽-Sepharose 4 B纯化至均一，并显示出对高甘露糖型糖肽的转糖基化活性，而不去除GST部分。谷胱甘肽-琼脂糖4 B固定化GST-Endo-A后，其转糖基活性仍保持不变。该固定化酶可将（Man）（6）GlcNAc整体转移到部分去糖基化的核糖核酸酶B上而不破坏其酶活性。固定化GST-Endo-A对于合成与均质N-连接低聚糖连接的活性新糖酶非常有用。(C)北京大学出版社.
A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter proto-phormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST-Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione-Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST-Endo-A immobilized on glutathione-Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST-Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides. (C) 2000 Academic Press.