Isolation, purification and characterization of glucokinase from Staphylococcus aureus

Abstract 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 K m of 5.22 ± 0.17 mM and V max 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 .