Extraction and analysis of the enzyme

Crude l-asparaginase enzyme was extracted by centrifugation. The collected supernatant was then subjected to spectrophotometric analysis by measuring the ammonia liberated using Nessler’s reagent (Long et al. 2015). Enzyme assay mixture consisted of 900 µL of freshly prepared l-asparagine (10 mM) in Tris–HCl buffer (pH 8.6) (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) and 100 µL of crude extract of the enzyme was prepared. The reaction mixture was incubated at 37 °C for 30 min. The reaction was stopped by adding 200 µL of 1.5 M trichloroacetic acid (TCA, Sisco Research Laboratories Pvt. Ltd., Mumbai, India). The obtained mixture was centrifuged at 10,000 rpm for 15 min at 4 °C to remove the precipitates. The quantity of ammonia released in the supernatant was calculated using the colorimetric technique by adding 100 µL Nessler’s reagent into the sample containing 100 µL supernatant and 800 µL distilled water. The contents in the sample were centrifuged and then incubated at room temperature for 10 min, and the optical density (OD) was measured at 425 nm against the blanks to which TCA was added before crude enzyme. The ammonia released in the reaction was calculated based on the standard curve obtained with ammonium sulfate. One unit (U) of l-asparaginase activity was defined as the amount of the enzyme that liberates 1 mM of ammonia/min at 37 °C (Kumar et al. 2010).