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L-Asparaginase appears to be highly effective especially in children with newly diagnosed Acute lymphoblastic leukemia. Prolonged L-Asparaginase intensification improved the outcome significantly. Nowadays L-Asparaginase is an essential drug that is used to treat children with Acute lymphoblastic leukemia all over the world. Its potential is now well established, as it has remarkably induced remission in most of the patients suffering with this disease. This therapy has brought a major breakthrough in modern oncology and with the development of its new functions, a great demand for L-Asparaginase is expected in the coming years. Keeping the potential of this particular enzyme in cancer treatment, the present work is planned with an intention of optimizing the production of L-Asparaginase both by conventional as well as by genetic engineering techniques. Pectobacterium carotovorum is selected for the present work based on its potentiality in cancer treatment and due to less glutaminase activity compared to the enzyme sourced from E. coli. Microbial culture was procured from MTCC, Chandigarh and screened for L-Asparaginase production in plate method and identified the activity based on the formation of pink color zone after overnight incubation.