A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-D-galactopyranosyl azide to alpha-galacto-oligosaccharides.

The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while beta-glycosynthase can be produced with a rather well-established technology, examples of alpha-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. alpha-l-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other alpha-glycosynthases is not trivial and remains to be confirmed. Here, beta-d-galactopyranosyl azide was converted to alpha-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel alpha-galactosynthase based on the GH36 alpha-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting alpha-galacto-oligosaccharides and demonstrate more widespread use of beta-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.