ANTI - CANCER ENZYME(L - ASPARAGINASE) PRODUCTION, PURIFICATION AND CHARACTERIZATION FROM A SOIL ISOLATE OF PSEUDOMONAS SP
Journal: International Journal of Advanced Research (Vol.7, No. 12)Publication Date: 2019-12-17
Authors : Narendra Kumar S Mohammed Haseeb Nawaz Shyam Shankar Mishra Satya Suman Lingayya Hiremath Praveen Kumar Gupta Ajeet Kumar Shrivastava; Mahesh M;
Page : 753-761
Keywords : L-Asparaginase Pseudomonas Sp Optimization Purification Lowrys Method Characterization Sds-Page;
Abstract
L-Asparaginase or L-asparagine amido hydrolases are enzymes that catalyze the substrate hydrolysis of L-asparagine. It results in the formation of L-aspartate and ammonia. It has immense application in the treatment of lymphoblastic leukemia as an antineoplastic agent and also finds its use in food technology. Its vast application in the pharmaceutical industry has led to the need for more sources of production of L-asparaginase. The current work is focused on production, purification, and characterization of L-asparaginase. The enzyme is produced using the batch mode of cultivation with critical media components disodium phosphate (0.042M), sodium chloride (0.0854M) and Asparagine (0.060M) respectively. The extracellular L-asparaginase was later purified by the following techniques: salt dialysis, ion-exchange chromatography, and gel filtration chromatography. Meanwhile, protein estimation was done using lowrys method. The molecular weight of the enzyme was found to be at 55KDa as revealed through SDS-PAGE. The biochemical analysis revealed that the species producing the enzyme belonged to Pseudomonas sp. The culture condition favoring the production of the enzyme L-asparaginase was found to be at a temperature of 400C, pH-9, and incubation time of 24hr. Optimization with critical carbon and nitrogen sources with varying concentrations disclosed sucrose and ammonium sulfate at 1.5%(w/w) to maximize the enzyme production. The purified enzyme was characterized by the above parameters (400C, pH-9) and incubation period of 40 minutes was found to be having an enzyme activity 434.10(U/ml). Additionally, to overproduce the enzyme, strain development was performed with the treatment of UV-B rays exposed at different heights and X-rays to yield more amount of enzyme.
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