2.5.1. Preparation of cell free extracts.
Frozen cell paste (25-35 g) was thawed and suspended in 50 mM sodium phosphate buffer (pH 7.0). The cell suspension was disrupted by sonication (12 W, 6 x 15 pulses with 15 sec intervals) in 2 mL Eppendorf tubes containing 1.5 mL of cell suspension, supplemented with lysozyme (250 µg/mL,) in a sonicator at 4 °C. Unbroken cells and cell debris were removed by centrifugation at 30000 x g for 30 min at 4 °C. The pellets were re-suspended in 50 mM sodium phosphate buffer (pH 7.0) and made up to appropriate volume and subjected to ammonium sulphate precipitation. Solid ammonium sulphate was added and the proteins precipitated at 40-60 % saturation was collected by centrifugation, dialyzed and used for assay.