Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-beta-N-acetylglucosaminidase A

A procedure for the enzymatic synthesis of neoglycoenzymes is described. Th e gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter proto-p hormiae (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 g lutathione-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 act ivity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to part ially deglycosylated ribonuclease B without damaging its enzyme activity. T he immobilized GST-Endo-A should be very useful for synthesizing active neo glycoenzymes attached with homogeneous N-linked oligosaccharides. (C) 2000 Academic Press.