Synthesis of potentially-bioactive lactosyl-oligofructosides by a novel bi-enzymatic system using bacterial fructansucrases.

Efficient enzymatic synthesis of lactosyl-oligofructosides (LFOS) with a degree of polymerization from 4 to 8 was achieved in the presence of sucrose:lactosucrose and sucrose:lactose mixtures by transfructosylation reaction. The main synthesized LFOS which consist of β-2,1-linked fructose to lactosucrose: β-d-galactopyranosyl-(1→4)-α-d-glucopyranosyl-[(1→2)-β-d-fructofuranosyl]n-(1→2)-β-d-fructofuranoside (where n refers to the number of transferred fructose moieties) was structurally characterized by nuclear magnetic resonance (NMR). The maximum formation of LFOS was 81% (in weight with respect to the initial amount of lactosucrose) and was obtained after 24h of transfructosylation reaction based on sucrose:lactosucrose (250gL each) catalyzed by an inulosucrase from Lactobacillus gasseri DSM 20604 (IS). The production of LFOS in the presence of sucrose:lactose mixtures required a previous high-yield lactosucrose synthesis step catalyzed by using a levansucrase from Bacillus subtilis CECT 39 (LS) before the inulosucrase-catalyzed reaction. This novel one-pot bi-enzymatic system led to the synthesis of about 22% LFOS in weight, with respect to the initial amount of lactose (250gL). The results revealed a high specificity for the substrate involved in the inulosucrase-catalyzed reaction given that, although lactosucrose (O-β-d-galactopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside) acted as a strong acceptor of β-2,1-linked fructose, lactose (β-d-galactopyranosyl-(1→4)-α-d-glucose) was found to be an extremely weak acceptor.