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L-asparaginase is a cardinal biotherapeutic drug for treating acute lymphoblastic leukemia, which is highly prevalent in children worldwide. In the current investigation, L-asparaginase producing marine bacterial isolate, Bacillus australimaris NJB19 (MG734654), was observed to be producing extracellular glutaminase free L-asparaginase (13.27 ± 0.4 IU mL-1). Production of L-asparaginase was enhanced by the Box-Behnken design approach that enumerated the significant variables affecting the enzyme production. The optimum levels of the de