L-asparaginase

A clinical bacterial isolate from a patient urine sample in Kasr El-Aini was identified by biochemical and molecular means to be E. coli . This isolate was optimized for production of L-asparaginase (L-asparagine amidohydrolase), a relatively widespread enzyme found in bacteria, eukaryote and mammals but not man. This enzyme catalyzes the deamidation of L-asparagine to L-aspartic acid and ammonia. The production of L-asparaginase was achieved through optimization of fermentation parameters and it showed 6.05 IU of enzyme activity. The produced L-asparaginase was then purified by means of chromatography techniques and tested against three different cell lines for its anticancerous activity, human colon cancer CACO-2, Human breast Cancer MCF-7 and Human cancer prostate PC-3. The expression for the regulatory genes BAX, P53 and BCL2, was analyzed by RT-PCR and it was clear that L-Asparaginase enzyme shows anticancer activity against (MCF-7) and (PC-3), where it was non-effective to the cell line (CACO-2). It was also noticed that BAX and P53 genes were upregulated under the effect of Asparaginase enzyme and that BcL2 gene was down-regulated in Human Breast and prostate Cancer cell line while Human colon cell line was not.

L-asparaginase is an anticancer agent, especially for acute lymphoblastic leukemia. Nine E.coli strains were screened forits ability to produce an extracellular L-asparaginase enzyme. The optimum culture conditions for L-asparaginase production was found at pH 8.0, Incubation time 48 h, and 37oC temperature. Highest yields of L-asparaginase(140.5 and 96.4 IU/ml) by E.coli strains using glucose and beef extract as sole carbon and nitrogen sources respectively. 0.6 fold of higher L-asparaginase activity (168.4 IU/ml) was recorded using pUC18 UV60 than the parent strain. 0.54 fold increased L-asparaginase activity (220.6 IU/ml)was observed using pUC18 NTG90.The molecular mass of L-asparaginase was determined by SDS-PAGE and it was found to be 29KDa.   

A total of fifty six actinomycete isolates were isolated from the ten marine sediments of south India. Marine environment is a potential source of novel actinomycetes, which are a potent source of antibiotics and novel bioactive compounds. The isolates were identified as actinomycetes by morphological, biochemical and microscopic studies. The isolated actinomycetes were screened for L-asparaginase activity by rapid plate assay method. Based on screening, isolate 1, 2 and 18 were showed large clear pink zone and its diameter (dm) was measured as 9.0, 8.5 and 8.5 cm and the enzyme production has been determined by nesslerization method. The spectrophotometric assay of enzyme activity of the isolates 1, 2 and 18 were found to be 1.92, 1.48 and 1.46 U/ml.