2.6 Periplasmic extraction
After protein expression, culture media were centrifuged at 4000g for 12 min to harvest cells and the extraction of periplasmic fraction was done by osmotic shock method. Briefly, cells were resuspended in a hypertonic solution (Tris 30 mM, sucrose 20% w/v, EDTA 1 mM, pH 8) and incubated for 30 min at 4 °C. Cells were harvested by centrifugation (4000g for 12 min) and the supernatant collected. Cells were re-suspended in a hypotonic solution containing 5 mM MgSO4 that incubated 30 min at 4 °Cthen,the periplasmic fraction was obtained by centrifugation at 13000g for 20 min [20]. For improvement of periplas­mic extraction, the effect of sonication and lysozyme were investigated. In case of sonication, after resuspending the cells in hypotonc solution, it sonicated and incubated at at 4 °C for 30 min.