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FLORES-SANTOS, J. C. Culture of Escherichia coli BL21 (DE3) for the production of Lasparaginase II. 2017. 134f. Dissertação (Mestrado) Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, 2017. Widely used as a therapeutic agent in the treatment of acute lymphoblastic leukemia (ALL), L-Asparaginase II (ASNase) is an enzyme that works by reducing the concentration of free asparagine in plasma. Thus, it prevents the delivery of asparagine to the proliferation of malignant cells, which unlike healthy cell, cannot synthesize asparagine. ASNase currently used in Brazil is imported, which causes problems with cost and supply. Thus, the search for systems with high levels of asparaginase expression and the finding of ways to produce this enzyme for easy access and, if possible, with a lower allergic potential, are strikingly attractive. This encourages us to study the biotechnological production of ASNase in recombinant Escherichia coli BL21 (DE3) which super expresses this enzyme. The objective of this work was to establish, in shaker and batch bioreactor system, growth and induction parameters of the Escherichia coli BL21 aiming the production of ASNase, which will be useful for future studies in a fed-batch system. Our work evaluated factors that influenced the growth and induction phase of E. coli BL21 (DE3): culture medium based on elemental composition, pH control, use of glucose or glycerol as carbon source, formation of acetate, initial and final induction time, cellular permeabilization for ASNase secretion, inducer concentration, post-induction temperature. We performed a strategy for extracellular production of ASNase in E. coli BL21 (DE3) by growing in modified Luria Bertani (LB) medium for cell permeabilization. The volumetric productivity of extracellular ASNase was 484 IU L h on shaker, which reached 89% secretion at 24 h of post-induction with IPTG at 37°C. This represented an increase yield of 50 % regarding to the total ASNase formed and 15.5 times the ASNase secretion as compared to that attained with LB modified. While in batch 2L-bioreactor cultivation under the same conditions (except for the aeration employed: 500 rpm of stirring and 1 vvm of air flow, kLa = 88 h) it was obtained similar results in relation to shaker cultures. The volumetric productivity of extracellular ASNase was 525 IU L h at 20 h of post-induction. The biomass obtained for shaker and bioreactor were 3.26 and 2.63 g L, respectively. For this reason, we consider these promising results to increase productivity in future studies in bioreactor operated as fed-batch regimen.

Cultivo de <i>Escherichia coli</i> BL21 (DE3) para produção de L-asparaginase II