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.