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 β-galactosidase from Kluyveromyces lactis (KlβGal) 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(β-d-galactopyranosyl-(1→1)-d-fructose) with two main isomers in D(2)O was identified to be the major transgalactosylation product while lactulose(β-d-galactopyranosyl-(1→4)-d-fructose) turned out to be the minor one, indicating that KlβGal 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 15.4g/l, respectively, at 66.5% lactose conversion (200g/l initial lactose concentration).