L- Asparaginase is capable of hydrolyzing L-asparagine into L-aspartic acid and ammonia. This catalytic function is used for the treatment of children with acute lymphoblastic leukemia resulting in the depletion of L- asparagine in the leukemia cells. Bacterial asparaginase used in current leukemia therapies has side effects and short serum half-life. This study presents the purification and characterization of nitrile hydratase (NHase) with asparaginase (ASNase) and glutaminase (GLNase) from Rhodococcus rhodochrous DAP 96253and 96622.The influence of pH, lysis buffer composition, dialysis process, anion exchange chromatography’s flow rates and pH were incorporated into the approach to purify NHase exhibiting ASNase activity.A NHase purified at pH 7.6, using 50 mM phosphate buffer, 5mM 2-mercaptoethanol as a lysis buffer, then dialyzed, followed by anion exchange chromatography and subsequent size exclusion chromatography showed a higher ASNase and GLNase activity and lower NHase activity. This enzyme shows potential as leukemia treatment.