Glucose 1‐Phosphate Thymidylyltransferase in the Synthesis of Uridine 5′‐Diphosphate Galactose and its Application in the Synthesis ofN‐Acetyllactosamine

Abstract

AbstractIn 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 (Mg2+) as a cofactor and a reaction temperature of 55 °C, 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) fromNeisseria meningitideswas successfully overexpressed and purified by using an intein‐mediated protein expression system. GalT was relatively stable at 25 °C, and its activity was enhanced in the presence of DTT and BSA. Thus, it was feasible to synthesizeN‐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.