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A total of 84 soil samples were collected from different locations in Baghdad. Only six of them were L-asparaginase producers with variable degrees. Among them, the isolate Bacillus sp. B5 was the most efficient in L-asparaginase production as the specific activity of enzyme in crude filtrate was 2.92 U/mg. Therefore, Bacillus sp. B5 was chosen to determine the optimum conditions for L-asparaginase production. L-asparaginase purification steps: first by ammonium sulfate precipitation with 80% saturation, second by ion exchange chromatography using DEAE-Cellulose and finally by gel filtration chromatography uses Sephadex G-200. The specific activity of purified enzyme was increased up to 85.7 U/mg with 20.1 folds of purification and 91.7% enzyme recovery. Characterization of the purified enzyme showed that the molecular weight of L-asparaginase from Bacillus spB5 was about 47000 Dalton, optimum pH …