Introduction of beta -galactosidase into a trans-sialidase reaction, i.e. sialic acid transfer reaction from a donor substrate (alpha2,3-sialyllactose) to an acceptor substrate (beta -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 alpha2,3-sialyllactose and Gal beta (1,3)GlcNAc, the yield of NeuAc alpha (2,3)Gal beta (1,3)GlcNAc increased from 45% to 75% by the coupling of Escherichia coli beta -galactosidase. Furthermore, by changing the substrate ratio in the coupled reaction, i.e. two-fold excess of alpha2,3-sialyllactose to Gal beta (1,3)GlcNAc, above 95% of yield was achieved based on the amount of Gal beta (1,3)GlcNAc. However, two-fold excess of Gal beta (1,3)GlcNAc to alpha2,3-sialyllactose in this reaction was more desirable for the purification of NeuAc alpha (2,3)Gal beta (1,3)GlcNAc, since complete consumption of alpha2,3-sialyllactose was achieved. Efficiency of the coupled reaction was affected by the specificity of P-galactosidase for acceptor substrate. When Gal beta (1.6)GlcNAc was used as the acceptor, E. coli beta -galactosidase hydrolyzed Gal beta (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 Gal beta (1,6)GlcNAc could be improved by employing Bacillus circulans beta -galactosidase. (C) 2001 Elsevier Science Inc. All rights reserved.