4 The G-6-P formation has essential role in the pathogen for energy generation in the catabolic Reverse Transcriptase inhibitor reactions to the synthesis of all the intermediates for the very survival of S. aureus. 5 The cytoplasmic glucokinase is detected in both Gram positive and Gram negative bacteria has 315–321 residues and a monomeric mass of 33–35 kDa, Km
values of glucokinase varied from 0.3 to 0.8 mM for glucose and 0.4–4 mM for ATP substrates in both Gram positive and Gram negative bacteria. 7 and 8 The bacterial glucokinases are found one ATP-dependent glucokinase and the other ATP-dependent glucokinase having ROK motifs. 9 In the occurrence of MDR and VRSA strains to understand the regulatory enzymes which are use full for biofilm formation and pathogen survival. 10 In the selleck present study we have focused on the isolation, purification and biochemical characterization of Glucokinase from S. aureus ATCC12600. In the present study
chemicals were obtained from Sisco Research Laboratories Pvt. Ltd., India, Hi-Media Laboratories Pvt. Ltd., India, Sigma–Aldrich, USA, New England Biolabs, USA and QIAGEN Inc., Valencia, CA. S. aureus ATCC12600 was grown on modified Baird Parkar media at 37 °C. After overnight incubation single black shiny coloured with distinct zone colony was picked and cultured in Brain heart infusion (BHI) broth then incubated at 37 °C. Thus, grown S. aureus ATCC12600 culture was used for the isolation, purification of Glucokinase enzyme. 11, 12 and 13 S. aureus ATCC12600 was grown in brain heart infusion broth (BHI) at 37 °C up to late log phase (OD540 = 0.9) from the culture the cytosolic fraction was isolated 11 and used for Glucokinase enzyme assay. In order to concentrate glucokinase, different concentrations of (NH4)2 SO4 were slowly added to the
cytosolic fraction. First it was concentrated to 0–10% (NH4)2 SO4, incubated overnight at 4 °C, centrifuged, pellet was dissolved in 0.1 M Tris–HCl pH 7.5 and upon assay activity was found to be very low. The pellet was discarded and the 0–10% saturated supernatant was recovered and concentrated to 10–20% crotamiton of (NH4)2 SO4, incubated overnight at 4 °C, the following day it was centrifuged at 10,000 rpm for 10 min at 4 °C and the obtained pellet was suspended in 2 mL of 0.1 M Tris–HCl pH 7.5, and dialysed against the same buffer the concentrate was used in glucokinase assay. From the assay results the protein was again fractionated using 20–40% (NH4)2 SO4 and the pellet thus obtained was 0.1 M Tris–HCl pH 7.5 and dialysed against the same buffer and the enzyme was used for glck assay. This fraction showed highest activity and was concentrated on lyophilization (Delvac).