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Subtilisin producing Bacilluswas isolated from Soil sample, collected from the different agricultural area of Coimbatore, Tamil nadu, India, followed by bacterial isolates were screened and identified. After identification of bacteria using molecular biology techniques subtilisin was extractedfrom Bacillus subtilis(MK156386) and Bacillus tequilensis (MK156389). Gibbon’s media was used for the production of subtilisinwhich was precipitated and purified with ammonium sulphate and column chromatography techniques. Characterization of the compound wasdone byusing TLC,UV-Visible and FTIR study. The antibacterial activity of the subtilisin against the food borne pathogens ofEscherichia coli (OL601965), Klebsiellapneumoniae (OL601967) and Citrobacterwerkmanii (OL602111) was tested using well diffusion method.