Isolation, purification and characterization of glucokinase from Staphylococcus aureus

In Staphylococcus aureus 85% of glucose is catabolised through EMP pathway and the very first step of the formation of glucose-6-phosphate (G-6-P) in the cytoplasm is catalysed by glucokinase (glck) which was isolated and purified from S. aureus ATCC12600. This G-6-P formation has essential role in the pathogen from energy generation in the catabolic reactions to the synthesis of all the intermediates for the very survival of S. aureus. This enzyme was purified by 20-40% ammonium sulphate fractionation and Diethylaminoethyl (DEAE) cellulose ion exchange chromatography followed by reverse phase high performance liquid chromatography (RP-HPLC) the glck was eluted at a retention time of 15 min. The purified glck in 10% SDS-PAGE showed single band with a molecular weight of 33 kDa confirming the monomeric in nature. The pure glck obtained from DEAE cellulose ion exchange chromatography followed by RP-HPLC exhibited Km of 5.22 ± 0.17 mM and Vmax 2.24 ± 0.06 with Hill coefficient of 1.71 ± 0.025 mM. The enzyme kinetics showed very high degree of cooperativity towards glucose which means this enzyme is highly involved in the phosphorylation glucose in S. aureus.