Transglycosylation and Condensation of Disaccharide Units Using Endo-type Glycosidases

Two kinds of synthetic methods using transglycosylation or condensation with disaccharide units by endo-glycosidases have been developed. First, in a commercially available cellulase preparation of Trichoderma reesei, we found transglycosylation activity enabling it to transfer the entire lactose (Lac) and N-acetyllactosamine (LacNAc) from p-nitrophenyl β-lactoside (Lacβ-pNP) and p-nitrophenyl β-N-acetyllactosaminide (LacNAcβ-pNP), respectively. Using the enzyme activities, various β-lactosides such as alkyl β-lactosides were directly synthesized. The enzyme catalyzed not only transglycosylation but also condensation reaction between the disaccharides and aglycons. Although 1-alkanols, 2-alkanols, alkan-diols, allyl alcohol, glycerol, Man and Glc can be acceptors for transfer of the disaccharide units, the efficiency of transglycosylation and condensation decreased with the increase in length of the alkyl chain of alkanol acceptors from C2 to C12. The yield of condensation between lactose and glycerol reached up to 40% by elimination of coexistent β-galactosidases. Condensation using the enzyme enabled practical synthesis of various glycosides possessing Lac and LacNAc units and offered a novel method for enzymatic synthesis of neoglycolipid precursors. The results of purification of crude enzyme preparation followed by a substrate competition assay on LacNAcβ-pNP hydrolysis indicated that both transglycosylation and condensation are mediated by a single enzyme, which is thought to be one of the endo-β-(1-4)-glucanases. Next, endo-β-galactosidase from Escherichia freundii, which has been used for structural and functional analyses of glycans involved in glycoconjugates, was shown to catalyze regioselective transfer of the GlcNAcβ1-3Gal unit onto the OH-4 position of non-reducing end GlcNAc of acceptor substrates. As a result, the transglycosylation led to facile preparation of LacNAc-repeating oligosaccharides.