Cloning and characterization of D-xylulose kinase (XK) from the marine bacterium, Kocuria gwangalliensis

Abstract

D-Xylulose is phosphorylated to D-xylulose-5-phosphate by D-xylulokinase before it enters glycolysis through the nonoxidative pentose phosphate pathway. A gene encoding a novel D-xylulose kinase (XK) from K. gwangalliensis has been sequenced and expressed in E. coli. The nucleotide sequence of XK consisted of 1419 bp encoding 463 amino acids residues and turned out to be well conserved during evolution. XK gene was subcloned into the pET-44a(+) expression vector which allows the expression of the recombinant pET44a(+)-XK plasmid. The resulting plasmid was transformed into the E. coli strain BL21(DE3) cell and the expression of the recombinant XK protein was induced by the addition of IPTG at a final concentration of 0.1 mM. The predicted size of recombinant XK protein is approximately 48 kDa including and expressed proteins was purified to homogeneity by affinity chromatography and showed enzymatic activity corresponding to D-xylulose kinase. The results obtained from this study will provide a wider base of knowledge on the primary structure and characterization of the D-xylulose kinase at the molecular level.