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

• Published in: Carbohydrate research Vol. 334; no. 2; pp. 97 - 103
• Main Authors: Albermann, Christoph, Piepersberg, Wolfgang, Wehmeier, Udo F
• Format: Journal Article
• Language: English
• Published: OXFORD Elsevier Ltd 23.08.2001; Elsevier
• Subjects: 2′-Fucosyllactose; Animals; Bacterial α-(1→2)-fucosyltransferase; Biochemistry & Molecular Biology; Chemistry; Chemistry, Applied; Chemistry, Organic; Enzymatic synthesis; Escherichia coli - enzymology; Fucosyltransferases - metabolism; GDP- l-fucose; Guanosine Diphosphate Fucose - biosynthesis; Guanosine Diphosphate Fucose - metabolism; Helicobacter pylori - genetics; Helicobacter pylori - metabolism; Helicobacter pylori, Escherichia coli; Hydro-Lyases - metabolism; Lactose - metabolism; Life Sciences & Biomedicine; Milk - chemistry; Physical Sciences; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Science & Technology; Trisaccharides - biosynthesis; Trisaccharides - chemistry; Trisaccharides - isolation & purification; Enzymatic synthesis; Bacterial α-(1→2)-fucosyltransferase; Helicobacter pylori, Escherichia coli; 2′-Fucosyllactose; GDP- l-fucose; COLANIC ACID; 2 '-fucosyllactose; bacterial alpha-(1 -> 2)-fucosyltransferase; GDP-FUCOSE; Escherichia coli; enzymatic synthesis; Helicobacter pylori; GENE-CLUSTER; CLONING; GDP-L-fucose; BIOSYNTHESIS; ESCHERICHIA-COLI K-12; MANNOSE PYROPHOSPHORYLASE; HELICOBACTER-PYLORI; EXPRESSION; TRANSFERASE
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/S0008-6215(01)00177-X

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.