Characterization of α-galacto-oligosaccharides formed via heterologous expression of α-galactosidases from Lactobacillus reuteri in Lactococcus lactis.
α-Galacto-oligosaccharides (α-GOS) are produced by transgalactosylation reactions of α-galactosidase (α-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to β-GOS, α-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of α-Gal remain scarce. The α-Gal gene sequence from Lactobacillus reuteri was cloned into an α-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of α-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. α-Gal of Lactobacillus reuteri formed (1 → 3)-, (1 → 4)- or (1 → 6)-linked α-GOS but exhibited a preference for formation of (1 → 6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for α-Gal of L. reuteri, resulting in formation of (1 → 3)-, (1 → 4)- or (1 → 6)-linked hetero-oligosaccharides. By determining the structural specificity of α-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess α-GOS pathogen adhesion prevention in mammalian hosts.
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada.
Yvonne
Y
Wang
Brenna A
BA
Black
Jonathan M
JM
Curtis
Michael G
MG
Gänzle
D000117:Acetylglucosamine / Q000378:metabolism; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D005643:Fucose / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D007785:Lactose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D063214:N-Terminal Acetyltransferase F; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D011887:Raffinose / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D000519:alpha-Galactosidase / Q000235:genetics* / Q000378:metabolism*
D009844:Oligosaccharides; D011994:Recombinant Proteins; D005643:Fucose; D008553:Melibiose; D063214:N-Terminal Acetyltransferase F; D000519:alpha-Galactosidase; D007785:Lactose; D011887:Raffinose; D000117:Acetylglucosamine