Transglycosylation activity of endoglycosidases and its application

Many exoglycosidases have been shown to have transglycosylation activity in addition to hydrolytic activity. The transglycosylation activities of endoglycosidases acting on complex carbohydrates have not been studied in detail, the first report being on endo-β-N-acetylglucosaminidase from Flavobacterium meningosepticum (Endo-F). This enzyme transfers high-mannose oligosaccharide (Man₆GlcNAc) to glycerol during cleavage of the chitobiose linkage in GlcNAc₂Man₆Asn, as judged from the observation that the Endo-F released oligosaccharide had no reducing end in the presence of glycerol. Endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was also found to have transglycosylation activity. Digestion of Man₆GlcNAc₂Asn with Endo-A in the presence of 4-L-aspartylglycosylamine (GlcNAc-Asn) gave a mixture of hydrolytic (Man₆GlcNAc) and transglycosylic (Man₆GlcNAc₂Asn) products. By means of translycosylation, Man₆GlcNAc was transferred en bloc to partially deglycosylated ribonuclease B having a GlcNAc-Asn residue, concomitant with the hydrolysis of Man₆GlcNAcAsn. Thus, N-linked oligosaccharides in the native ribonuclease were converted into Man₆GlcNAc, and the neoribonuclease containing homogeneous oligosaccharide was synthesized through the transglycosylation activity of Endo-A. This enzyme exhibited exclusive transglycosylation activity in the presence of acetone. The novel endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), which can cleave not only the high-mannose type of N-linked oligosaccharide but also the complex type of oligosaccharide, has transglycosylation activity. This enzyme transfers the sialo complex type oligosaccharide from the human transferrin glycopeptide to an appropriate acceptor having a GlcNAcAsn residue. Using the transglycosylation reaction of Endo-M, a sialo complex type oligosaccharide was attached to a synthetic peptide containing GlcNAc, such as peptide T-GlcNAc. The transglycosylation product was ascertained to have more resistance against proteolysis. Endo-α-N- acetylgalactosaminidase from Diplococcus pneumoniae exhibits transglycosylation and transfer reaction (reversed hydrolysis) activities. Treatment of asialoglycoproteins having Galβ1→3GalNAcα1→Ser/Thr linkages with the enzyme in the presence of glycerol resulted in the formation of Galβ1→3GalNAcβ1→1(3)-glycerol. Moreover, D-glucose, D-galactose,p-nitophenol, threonine, etc. were good substrates (acceptors) for the transfer reaction (reverse hydrolysis). The endoglycoceramidases (ceramide glycanase) from leech and Corynebacterium sp. exhibit transglycosylation activity that transfers the oligosaccharides en bloc from various glycosphingolipids to suitable acceptors. They transfers the intact oligosaccharide from GM1 to various 1-alkanols having a long carbon chain. The transglycosylation activity of these enzymes will be useful for synthesizing neoglycolipids and novel alkylglycosides.