Regioselectivity of β-d-galactosyl-disaccharide formation using the β-d-galactosidase from Bacillus circulans

β-d-Galactosidase from Bacillus circulans catalyzed the transfer of galactose from lactose predominantly to the OH-4 position of, respectively, GlcNAc and GalNAc to afford β-d-Gal-(1 → 4)-d-GlcNAc and β-d-Gal-(1 → 4)-d-GalNAc. Thus, preponderant formation of (1 → 4)-linkages occurs and (1 → 6)-linkages are formed to a lesser extent, but no (1 → 3)- or (1 → 1)-linkages are formed. When 3-acetamido-3-deoxy-d-glucose (Glc3NAc, N-acetylkanosamine) was used as an acceptor, the enzyme catalyzed the β-d-galactosyl transfer to, respectively, the β-anomeric position (OH-1) and OH-6 of this sugar to afford β,β-d-Gal-(1 → 1)-d-Glc3NAc and β-d-Gal-(1 → 6)-d-Glc3NAc. In contrast, with methyl β-d-glucoside and methyl β-d-galactoside as acceptors, the enzyme induced the formation of (1 → 3)-linked disaccharide glycoside other than (1 → 4)- and (1 → 6)-linked ones. This demonstrates that the regioselectivity of β-d-galactosyl transfer onto GlcNAc, GalNAc, and Glc3NAc acceptors as catalyzed by the enzyme is strongly determined by the presence of the N-acetyl group.