Production, purification, Characterization and cytotoxic effect of L-asparginase from locally isolated Pseudomonas aeruginosa

 In this study, a total of 46 clinical samples were collected for the isolation of P .aeruginosa, 14 samples taken from wounds, 20 samples from burns and 12 samples from ear infections. All samples were collected from Al-yarmouk hospital. Among these samples, a total of 29 bacterial isolates were successfully cultured .Six of them were subjected to the identification according to their morphological,
cultural, biochemical characteristics and VITEK2 system since they were identified as P. aeruginosa. The ability of these isolates for L-asparginase production was examined. Results showed that all six isolates of P. aeruginosa were L-asparginase producers with variable degrees. Among them, the isolate P. aeruginosa P4 was the most efficient in L-asparginase production. Its specific activity of L-asparginase in crude filtrate was 0.15U/mg protein. Therefore, P. aeruginosa P4 was selected for enzyme characterization and purification. The optimum conditions for L-asparginase production were studied. Results showed that maximum L-asparginase production was achieved after supplementation of the production medium (pH7) with 0.1% glycerol and 0.1% tryptone , inoculated with a 10  cells/ml of fresh bacterial culture and incubation at 37 C in shaker incubator (150 rpm) for 24h. Under these conditions, the specific activity of L-asparginase produced in culture medium was sharply increased to 0.6
U/mg protein. have high potential for cytotoxicity on cancer cell. L-asparginase produced under the optimum conditions was purified in three purification steps, first by precipitation with 80% saturation of ammonium sulfate, second by ion exchange chromatography using DEAE-Cellulose column, while the gel filtration chromatography throughout Sephadex G-200 column was the third step. Specific activity of the purified enzyme was increased up to15 U/mg with 25 folds of purification and 60% enzyme recovery. Some biochemical characteristics of the purified enzyme were studied. It was found that the molecular weight of L-asparginase produced by P. aeruginosa P4 was about 120000 Dalton, the highest enzyme activity at pH 7, while that for stability was 8. Also 37 C gave the higher activity, hence the enzyme was stable with full activity at a range of temperatures between 20-37 C. Enzyme activity was inhibited in the presence of HgCl mercaptoethanol, and cysteine that were added individually at different ncentrations. Also the enzyme is not affected by EDTA indicating it was not a metalloenzyme. In this study, crude and purified asparginase were tested for in-vitro cytotoxicity activity using MTT assay against MCF7 cancer cell line and the purified asparginase was found to inhibit the growth of cell line with an IC50 of 185.7 Ϻg\ml in comparison to an IC50 of 355.6 Ϻg\ml for crude enzyme. On the other hand, the enzyme didn’t show significant effect on normal cell that it may  , CaCl