Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose.
The enzymatic transgalactosylation from lactose to fructose leading to the prebiotic disaccharide lactulose was investigated using the beta-galactosidase from Aspergillus oryzae and the hyperthermostable beta-glycosidase from Pyrococcus furiosus (CelB). The conditions for highest lactulose yields relative to the initial lactose concentration were established on a 1 mL scale. Dependent on the initial molar ratio of lactose to fructose, more or fewer oligosaccharides other than lactulose were generated. Bioconversions on a 30 mL scale in a stirred glass reactor were performed, and lactulose yields of 46 mmol/L (44% relative to lactose) for CelB and 30 mmol/L (30% relative to lactose) for A. oryzae beta-galactosidase were achieved. Only <5% of other oligosaccharides were detectable. The corresponding productivities were 24 and 16 mmol/L/h, respectively. The molecular structure of lactulose was investigated in detail and confirmed after purification of the reaction solution by LC-MS and 1D and 2D NMR. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose) was unambiguously proved to be the major transglycosylation disaccharide.