摘要
Herein, we report the first characterization of a novel galactokinase from Meiothermus taiwanensis sp. nov. WR-220 (MtGalK), which is overexpressed in E. coli and exhibits a kcat/Km value of 168.47 mM−1 s−1 toward Gal at 75 °C. The thermophilic MtGalK shows specific substrate recognition toward the Gal configuration with limited tolerance for modifications at the C-2 position to form products such as GalN-1-P, GalN3-1-P, and GalNAc-1-P. Due to its unique thermostability toward elevated reaction temperatures, MtGalK served as an ideal biocatalyst in the synthesis of useful sugar-1-phosphates and was further combined with glucose-1-phosphate thymidylyltransferase (RmlA) and α-1,4-galactosyltransferase (LgtC) to achieve the efficient preparative-scale synthesis of globotriose analogs (Gb3) using a one-pot three-enzyme system. In combination with a chemical synthetic strategy, a carbohydrate antigen from breast cancer stem cells, stage specific embryonic antigen-3 (SSEA-3) pentasaccharide, was synthesized from Gb3 in ten steps with a 23% overall yield. This flexible chemoenzymatic strategy for the synthesis of SSEA-3 also allowed us to further expand the inventory of valuable natural and non-natural glycans.