The N-glycans in human immunoglobulin G were engineered through the bacterial glycosyltransferase-catalyzed galactosylation and sialylation with in situ regeneration of sugar-nucleotides. For the galactosylation and sialylation, beta 1,4-galactosyltransferase from H. pylori and alpha 2,3-sialyltransferase from N. gonorrhoeae were expressed in Escherichia coli. For the regeneration of UDP-galactose and CMP-NeuAc, the required enzymes were also expressed in E. coli and their extracts were employed as biocatalysts. Using the catalytic system, we carried out the galactosylation of terminal GlcNAc residues with UDP-Gal regeneration and sialylation of terminal Gal residues with CMP-NeuAc regeneration, respectively. When the oligosaccharides in human IgG were analyzed by HPLC (NP and WAX) and MALDI-TOF, the galactosylation reaction showed 86.1% increase in terminal galactosylation of N-linked oligosaccharide in IgG, and the sialylation reaction gave 69.6% increase in terminal sialylation. To achieve a maximum sialylation of IgG, simultaneous galactosylation and sialylation reaction was conducted by incubating all of the biocatalysts required for the galactosylation, sialylation and sugar-nucleotide regenerations in one reactor, resulting in 80.2% increase in terminal sialylation. (c) 2005 Elsevier Inc. All rights reserved.