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Forty two isolates were isolated from different sources (water and soil). Vitek 2 system were used for identification the isolates as P. aeruginosa.. Twelve biosurfactant producer isolates were selected due to primary screening assay of hemolysis, CTAB, oil dispersion, drop collapse, emulsification index(E24%) methods. Twelve biosurfactant producer isolates were confirmed by 16SrDNA gene identification. Biosurfactants were purified by column chromatography method to obtain honey sticky product. TLC, FTIR, GC-MS, and HPLC were used to characterize purified biosurfactant as rhamnolipid. Rhamnolipid showed anticancer activity against MCF7 and SKG cell lines causing inhibition of viability at IC50 concentration. Acridine orange ethidium bromide staining revealed that rhamnolipid causing necrosis of cancer cells for 72 hrs. treatment with IC50 concentration.