l‐Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l‐asparagine to l‐aspartic acid and ammonia. This property of l‐asparaginase inhibits the protein synthesis in cancer cells, making l‐asparaginase a mainstay of pediatric chemotherapy practices to treat acute lymphoblastic leukemia (ALL) patients. l‐Asparaginase is also recognized as one of the important food processing agent. The removal of asparagine by l‐asparaginase leads to the reduction of acrylamide formation in fried food items. l‐Asparaginase is produced by various organisms including animals, plants, and microorganisms, however, only microorganisms that produce a substantial amount of this enzyme are of commercial significance. The commercial l‐asparaginase for healthcare applications is chiefly derived from Escherichia coli and Erwinia chrysanthemi. A high rate of hypersensitivity and adverse reactions limits the long‐term clinical use of l‐asparaginase. Present review provides thorough information on microbial l‐asparaginase bioprocess optimization including submerged fermentation and solid‐state fermentation for l‐asparaginase production, downstream purification, its characterization, and issues related to the clinical application including toxicity and hypersensitivity. Here, we have highlighted the bioprocess techniques that can produce improved and economically viable yields of l‐asparaginase from promising microbial sources in the current scenario where there is an urgent need for alternate l‐asparaginase with less adverse effects.

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Background L‐Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L‐asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold‐ups during processing, and can result in product loss if stability is an issue, as is the case with L‐asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation. Methods and Results In this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L‐asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)‐based optimization was then performed for the refolding step. The net outcome was more than a three‐fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L‐asparaginase process intermediate was found to be stable for more than a week at room temperature and 2–8°C, while the unoptimized sample was stable at 2–8°C but did not show any activity at room temperature after 72 h. Conclusions The current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L‐asparaginase which are labile.