trans-Sialidase catalyzed sialylation of β-galactosyldisaccharide with an introduction of β-galactosidase

Introduction of β-galactosidase into a trans-sialidase reaction, i.e. sialic acid transfer reaction from a donor substrate (α2,3-sialyllactose) to an acceptor substrate (β-galactosyldisaccharide), could improve the yield of desired sialylated trisaccharide by hydrolyzing lactose, a byproduct from the donor. When trans-sialidase reaction was performed with stoichiometric amounts (2 mM) of α2,3-sialyllactose and Galβ(1,3)GlcNAc, the yield of NeuAcα(2,3)Galβ(1,3)GlcNAc increased from 45% to 75% by the coupling of Escherichia coli β-galactosidase. Furthermore, by changing the substrate ratio in the coupled reaction, i.e. two-fold excess of α2,3-sialyllactose to Galβ(1,3)GlcNAc, above 95% of yield was achieved based on the amount of Galβ(1,3)GlcNAc. However, two-fold excess of Galβ(1,3)GlcNAc to α2,3-sialyllactose in this reaction was more desirable for the purification of NeuAcα(2,3)Galβ(1,3)GlcNAc, since complete consumption of α2,3-sialyllactose was achieved. Efficiency of the coupled reaction was affected by the specificity of β-galactosidase for acceptor substrate. When Galβ(1,6)GlcNAc was used as the acceptor, E. coli β-galactosidase hydrolyzed Galβ(1,6)GlcNAc as well as lactose in the coupled reaction, resulting in a significant decrease in the yield of desired sialylated trisaccharide. The conversion yield of the sialylation of Galβ(1,6)GlcNAc could be improved by employing Bacillus circulans β-galactosidase.