The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion.
Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitidis, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and alpha-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an alpha-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce alpha-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.
Gilbert
M
M
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
Bayer
R
R
Cunningham
A M
AM
DeFrees
S
S
Gao
Y
Y
Watson
D C
DC
Young
N M
NM
Wakarchuk
W W
WW
D002384:Catalysis; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007474:Ion Exchange; D007785:Lactose / Q000378:metabolism; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D000216:N-Acylneuraminate Cytidylyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics; D009438:Neuraminic Acids / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012799:Sialyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D014462:Ultrafiltration; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D009097:Multienzyme Complexes; D009438:Neuraminic Acids; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D010728:Phosphoenolpyruvate; D012799:Sialyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase; D019158:N-Acetylneuraminic Acid; D007785:Lactose; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase