Production purification and characterization of glutaminase-free L-asparaginase from pectobacterium carotovorum MTCC 1428

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Bacterial L-asparaginase has been widely used as a therapeutic agent in the treatment of certain kinds of cancer, mainly in treatment of acute lymphoblastic leukemia. Moreover, L-asparaginase is used in food industry for the production of acrylamide free food, as model enzyme for development of new drug delivery system and L-asparagine biosensor for diagnosis of leukemia. The various side effects of this drug are mainly due to the presence of partial glutaminase activity of L-asparaginase. Hence, glutaminase-free L-asparaginase is highly desirable for its successful application. Among the tested microorganism, Pectobacterium carotovorum MTCC 1428 has the ability to produce novel glutaminase-free L-asparaginase. Hence, further studies were carried out using P. carotovorum MTCC 1428. Localization, production, purification and characterization of glutaminase-free L-asparaginase have been performed by P. carotovorum MTCC 1428. Production of glutaminase-free L-asparaginase was performed both in the shake flask and bioreactor (batch and fed-batch). Furthermore, Kinetic models were developed for dual substrate growth, substrates utilization and L-asparaginase production by P. carotovorum MTCC 1428 in batch bioreactor. The localization of L-asparaginase was carried out using cell fractionation techniques. The specific activity of L-asparaginase was found to be 85 and 77% in the cytoplasm of Pectobacterium carotovorum MTCC 1428 that was grown on medium containing L-asparagine, and a combination of both L-asparagine and glucose, respectively. Among the carbon sources tested, L-asparagine and a combination of both L-asparagine and glucose were found to be the most suitable carbon sources for maximum production of L-asparaginase. Yeast extract, L-asparagine and peptone showed significant effect on the production of L-asparaginase. L-asparagine was used as an essential carbon source for the maximum production of L-asparaginase by P. carotovorum MTCC 1428.........
Supervisor: V Venkata Dasu and K Pakshirajan