Characterization of a Galactosynthase Derived from Bacillus circulans beta-Galactosidase: Facile Synthesis of D-Lacto- and D-Galacto-N-bioside

Abstract

Glycosynthasesretaining glycosidases mutated at their catalytic nucleophilecatalyze the formation of glycosidic bonds from glycosyl fluorides as donor sugars and various glycosides as acceptor sugars. Here the first glycosynthase derived from a family 35 -galactosidase is described. The GluGly mutant of BgaC from Bacillus circulans (BgaC-E233G) catalyzed regioselective galactosylation at the 3-position of the sugar acceptors with -galactosyl fluoride as the donor. Transfer to 4-nitophenyl -D-N-acetyl-glucosaminide and -D-N-acetylgalactosaminide yielded 4-nitophenyl -lacto-N-biose and -galacto-N-biose, respectively, in high yields (up to 98%). Kinetic analysis revealed that the high affinity of the acceptors contributed mostly to the BgaC-E233G-catalyzed transglycosylation. BgaC-E233G showed no activity with -(1,3)-linked disaccharides as acceptors, thus suggesting that this enzyme can be used in one-pot synthesis of LNB- or GNB-containing glycans.