The present investigation focused on L- asparaginase activity by Bacillus sp. isolated from soil. L-asparaginase activity was analyzed using phenol red indicator growth medium showed significant zone of hydrolysis. Five different carbon sources such as glucose, maltose, soluble starch, rhamnose and fructose were analyzed for the enzyme production. Glucose was found to be the best carbon source. The partial purification of L-asparaginase was done by two way ammonium sulfate precipitation (45% initial & 85% final saturation respectively). After purification dialysis was carried out by using dialysis membrane-60 to remove excess salt results into the total protein decreased from 400 mg to 220 mg & specific activity increased from 1.18 IU/ml/min to 2.15 IU/ml/min. The characterized enzyme exhibited maximal enzyme activity at pH 7 and 37°C after 24 hrs of incubation. The L- asparaginase activity was increased by 0.97 IU/mg.
