Glucose 1-Phosphate Thymidylyltransferase in the Synthesis of Uridine 5'-Diphosphate Galactose and its Application in the Synthesis of N-Acetyllactosamine

摘要

In this study, we describe the direct synthesis of uridine 5'-diphosphate galactose (UDP-Gal) by a wild-type bacterial thymidylyltransferase (RmlA), which is used to synthesize thymidine 5'-diphosphate glucose (TDP-glucose) in nature. By using magnesium (Mg~(2+)) as a cofactor and a reaction temperature of 55 ℃, a one hundred milligram-scale synthesis of UDP-Gal was achieved by RmlA. In addition, RmlA was site-specifically and covalently immobilized on magnetic nanoparticles (MNPa) The resulting RmlA-MNP complex retained almost 95% of its activity after reuse in ten consecutive enzyme assays. Furthermore, β-1,4-galactosyltransferase (GalT) from Neisseria meningitides was successfully overex-pressed and purified by using an intein-mediated protein expression system. GalT was relatively stable at 25 ℃, and its activity was enhanced in the presence of DTT and BSA. Thus, it was feasible to synthesize N-acetyllactosamine (LacNAc) using RmlA and GalT in a sequential addition of enzyme and adjustment of thereaction temperature. These results demonstrate the potential applications of bacterial RmlA in carbohydrate synthesis.