Expression and Purification of Glycerol Kinase and Its Application for Ketose Synthesis

Abstract:

In this paper，the expression of glycerol kinase（GK） from Escherichia coli MG1655 and its application for ketose synthesis were investigated. The gene glpK encoding GK was amplified and cloned into the expression plasmid pET-28a. The recombinant plasmid was transformed into the E.coli Rosetta（DE3） to express the protein induced by IPTG. GK enzyme was purified by Ni2+ chelating affinity column chromatography. The GK enzyme can catalyze cheap substrate glycerol to produce L-glycerol 3-phosphate which is converted into DHAP by glycerol phosphate oxidase（GPO）.The generated DHAP can be coupled with the acceptor D-glyceraldehyde catalyzed by different DHAP-dependent aldolases to produce ketose-1-phosphate，which can be dephosphorylated with acid phosphatase（AP） to get a series of ketoses. The results demonstrated that a series of ketoses including rare sugars were successfully synthesized using one-pot multienzymatic reaction with the cheap substrate glycerol as the precursor. The product yields and ratios were detected by TLC and HPLC. This approach could provide the theory basis for the synthesis of more other rare sugars and their derivatives.