Abstract
In this study, the recombinant galactokinases (GalK) from Meiothermus taiwanensis ATCC BAA-400 was cloned, over-expressed, and purified by IMPACTTM-CN system with a yield of 16 mg/L cell cultures. The optimal reaction conditions for recombinant GalK are at 75 °C, and pH at 9.0. The kcat/Km value of recombinant GalK toward galactose is 168.47 s−1 mM−1 and specific unit at 55 °C and 75 °C are 240 U/mg and 388 U/mg, respectively. The GalK was combined with glucose-1- phosphate thymidylyltransferase (RmlA) to synthesize uridine 5’-diphosphate galactose (UDP-Gal) in one-pot reaction. In addition, the genes of α-1,4-galactosyltransferases (GalTs) from Neisseria meningitides (19 residues at C-terminal deleted; LgtC-19) and Haemophilus influenzae Rd KW2 (38 residues of N-terminal was deleted;N-cut-38-Hi0258) were cloned and the corresponding proteins were over-expressed. Both α-1,4-GalTs were respectively combined with GalK and RmlA to synthesize Pk antigen derivatives (α-D-Gal-(1-4)-β-D -Gal-(1-4)-β-D-Glc-OC6H12N3) in sequential addition one-pot reaction manner.