Coloning Expression Production purification and characterization of novel glutaminase free recombinat l-asparaginase II of pectobacterium Carotovorum MTCC 1428 and Erwinia Carotovora Subsp. Atroseptica Scri 1043 in Escherichia Coli
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Bacterial L-asparaginase is used as a therapeutic agent for the treatment of acute lymphoblastic leukemia, and a processing aid in the manufacture of starchy food products such as potato chips and biscuits to reduce the formation of acrylamide. L-asparaginase is also used as a model enzyme for the development of new drug delivery system and L-asparagine biosensor to diagnose leukemia. The different side effects of this drug are attributed due to the presence of partial glutaminase activity of L-asparaginase. Therefore, glutaminase-free L-asparaginase is highly enviable for its successful clinical applications. Among the tested microorganisms, Pectobacterium carotovorum MTCC 1428 and Erwinia carotovora subsp. atroseptica SCRI 1043 have produced novel glutaminase-free periplasmic L-asparaginase. Hence, further studies were carried out with P. carotovorum MTCC 1428 and E. carotovora subsp. atroseptica SCRI 1043. The genes encoding L-asparaginase (L-asparaginase, L-asparaginase I and L-asparaginase II) in P. carotovaram MTCC were identified. These three genes of P. carotovorum MTCC 1428 and E. carotovora subsp. atroseptica SCRI 1043 were cloned and expressed in Escherichia coli BL21 (DE3), with and without 6x histidine at C-terminus. The histidine tag has proved to be facilitated the purification and also promoted expression level of the cytoplasmic L-asparaginase I and L-asparaginase II. But, in the case of L-asparaginase, lower expression of recombinant L-asparaginase was observed with histidine tag as compared to without tag. As L-asparaginase II is used as anticancer agent, the production of glutaminase-free recombinant L-asparaginase II of both the strains have been carried out both in shake flask and bioreactor (batch and fed-batch).Response surface methodology (RSM) was applied to optimize the production of recombinant L-asparaginase II of P. carotovorum MTCC 1428 and E. carotovora subsp. atroseptica SCRI 1043. Central composite design (CCD) was employed to optimize the medium components (tryptone, yeast extract and NaCl) and physical parameters (pH, agitation and inoculum size). For recombinant L-asparaginase II of P. carotovorum MTCC 1428, the optimal levels of tryptone, yeast extract and NaCl were found to be 13.30 g l-1, 6.38 g l-1 and 7.12 g l-1, respectively, and most favorable combination of pH, agitation and inoculum size were determined to be 7.1, 212 rpm and 2.50 %, respectively. At the optimal levels of medium components and physical parameters, the maximum production and productivity of recombinant L-asparaginase II of P. carotovorum MTCC 1428 was found to be 70.25 U mg-1 of protein (16.41 U ml-1) and 2735.00 U l-1 h-1, respectively. The production of recombinant L-asparaginase II of P. carotovorum MTCC 1428 was increased by 1.94 fold as compared to the un-optimized production conditions (36.15 U mg-1). For the production of recombinant L-asparaginase II of E. carotovora subsp. atroseptica SCRI 1043, the optimal levels of tryptone, yeast extract and NaCl were found to be 14.73 g l-1, 5.30 g l-1 and 4.03 g l-1, respectively, and the best levels of pH, agitation and inoculum levels were observed to be 7.4, 216 rpm and 2.34 %, respectively. After optimization of chemical and physical parameters, the production of rec.
Supervisor: V. V. Dasu
BIOSCIENCES AND BIOENGINEERING