The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the beta-galactosidases from Kluyveromyces lactis (Kl beta Gal) and Bacillus circulans (Bc beta Gal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcbGal. Bc beta Gal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L-1, corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using Kl beta Gal, whereas the regioselectivity of Bc beta Galproved to be subject to variations, the 1-4 and 1-6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N-acetyl-allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by Bc beta Galwas a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 386-394, 2011