Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis

The enzymatic transgalactosylation of lactose in the presence of fructose using beta-galactosidase from Kluyveromyces lactis (KI beta Gal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(beta-D-galactopyranosyl-(1 -> 1)-D-fructose) with two main isomers in D2O was identified to be the major transgalactosylation product while lactulose(beta-o-galactopyranosyl-(1 -> 4)-D-fructose) turned out to be the minor one, indicating that KI beta Gal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4 g/l, respectively, at 66.5% lactose conversion (200 g/l initial lactose concentration). (C) 2012 Elsevier Ltd. All rights reserved.