BIOTRANSFORMATION USING RECOMBINANT CMP SIALIC ACID SYNTHETASE AND α-2, 6-SIALYLTRAN SFERASE: ENZYMATIC SYNTHESIS OF SIALOSIDES

In this research, we successfully expressed recombinant CMP-sialic Acid Synthetase (CSS) from Neisseria meningitides and 2,6-Sialyltransferase (SAT) from Photobacterium damsela in E. coli BL21(DE3) fermented at a scale of up to 8 litres using individual plasmids pIRL-1 and pIRL-4b, respectively. After cell lysis with BugBuster, enzyme levels of 2U and 22U per litre were produced for CSS and SAT, respectively. The enzyme solutions were either used directly as crude preparations or further purified by affinity chromatography. Characterization of the CSS and SAT confirmed that both enzymes had comparable properties to those described in the literature. The production of cytidine 5’-monophosphate N-acetylneuraminic acid (CMP-NeuAc) and CMP-9-azido-NeuAc using crude CSS was successful with >90% conversion at scales from 100 mg to 5 g. Activated sugar purification by ethanol precipitation was optimized. Finally, the CSS and SAT enzymes were applied to a large-scale synthesis of a sialylated lactosamine glycoside via a two-step biotransformation. The initial step employed crude CSS to convert Cytidine Triphosphate (CTP) and 9-azido-NeuAc to CMP-9-azido-NeuAc at a conversion efficiency of 98%. This reaction mixture, after ultrafiltration to remove β-galactosidase activity co-expressed by E. coli BL21, was used as the donor substrate for the second step involving SAT. The sialoside 9-azido-sialyl-α-2,6’-lactosamine glycoside was produced with 86% conversion of the starting glycoside. Purification of the product was achieved by chromatography on Diaion HP-20 (a hydrophobic styrenic resin).