THE SYNTHESIS OF SIALYLATED OLIGOSACCHARIDES USING A CMP-NEU5AC SYNTHETASE SIALYLTRANSFERASE FUSION/

Large-scale enzymatic synthesis of oligosaccharides, which contain ter minal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, w hich 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-si alyltransferase activities. The fusion protein was produced in high yi elds (over 1200 U/L, measured using an alpha-2,3-sialyltransferase ass ay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusi on protein could sialylate various oligosaccharide accepters (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion prote in was also used to produce alpha-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, siali c acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.