(469g) Enzymatic Synthesis of Prebiotic Human Milk Oligosaccharide Analogs Using Five ?-Galactosidases From Bifidobacterium Longum Subsp. Infantis ATCC 15697

Human milk is established as the ideal nutrition for term infants. Specifically, it contains a large variety of prebiotic human milk oligosaccharides (HMO) with the potential to provide selection for beneficial probiotic organisms in the gut. Synthesis of complex oligosaccharides resembling those found in human milk would therefore be desirable as a nutritional additive to infant formula. We investigated the production of galactooligosaccharides (GOS) using five β-galactosidases cloned from Bifidobacterium longum subsp. infantis ATCC 15697. One ml reactions containing 30% w/v initial lactose concentration were followed over 46 hours and samples analyzed by HPLC. A Central Composite Design was used to establish optimum pH and temperature for transgalactosylation activity (i.e., maximum initial rate of reaction). Kinetic parameters (i.e., V_max and K_m) were subsequently calculated using lactose as the sole reactant. All enzymes showed transgalactosylation activity, with two of them producing GOS with a degree of polymerization higher than 3. Optimum pH varied from 5.9 to 7.4 and optimum temperature from 46 to 64 °C. V_max varied more than 10x fold. While formation of GOS is important, HMO are typically decorated with fucose and sialic acid residues that improve the prebiotic efficacy. Therefore, production of decorated GOS was also investigated by using different combinations of lactose, sialyllactose and fucosyllactose as building block donors. Products were analyzed with LCMS.