Synthesis of the milk oligosaccharide 2′-fucosyllactose using recombinant bacterial enzymes

The enzymatic synthesis of GDP-β- l-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP- d-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy- d-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-α- d-mannose to GDP-β- l-fucose with 78% yield. For the transfer of the l-fucose to an acceptor, we cloned and overproduced the α-(1→2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2′-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP- l-fucose and lactose. The isolation of 2′-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield. The milk oligosaccharide 2′-frucosyllactose was synthesised using enzymatically synthesised GDP- l-fucose which was transferred to lactose using the recombinant fucosyltransferase FucT2 Helicobacter pylori.