Cultivos em meios complexos para produção de L-asparaginase em Escherichia coli BL21(DE3)

Abstract

Acute Lymphoblastic Leukemia (ALL) is a type of neoplasia that has L-asparaginase as a highly effective treatment. This biopharmaceutical cleaves asparagine and releases ammonium and aspartic acid; this neoplastic cells do not produce asparagine due to their still immature cellular machinery and end up dying in the absence of that amino acid. In Brazil, patients have difficulty in accessing this medicine because it is imported, has a high cost, in addition to problems in supply. Therefore, a group of researchers seeks to develop a national drug, with less allergic potential, therefrom the country will have autonomy, enabling access to the population through SUS. Escherichia coli BL21 (DE3) has been widely used for the expression of heterologous proteins and to reach a high cell density. This work consists of evaluating different complex media, micronutrients and appropriate inducer for the production of recombinant L-asparaginase. For this study was used E.coli BL21 (DE3) containing the expression vector pET28a, with T7 promoter, selection marker for kanamycin and carrying the KY305877 gene sequence. Experiments in stirred flask were performed at 300 rpm, 37 ° C in: Luria Bertani (LB), Semi Defined (SD), Super Broth (SB), Terrific Broth (TB), TB mod., TB mod. + nutrients, TB mod. in different carbon sources, TB mod. in lactose as an inducer. Bioreactor tests were also carried out with the modified TB medium. Samples were collected every hour for analysis of biomass, enzymatic activity and electrophoresis. The TB medium showed the highest DO600nm and enzymatic activity. The tryptone of the TB medium was replaced by soytone, a component of plant origin, and adjusted in its concentration (17 g / L) to achieve similar values in growth and asparaginase activity obtained with the tryptone originating the TB mod medium. In this composition there was no need for micronutrient supplementation. The increment of the TB medium mod. with glucose, glycerol and lactose, with lactose also acting as an inducer, showed promising enzymatic activity in stirred flasks of 5 IU / ml of culture through 6UI / ml of culture with IPTG. The use of IPTG is inappropriate due to the high cost, toxicity and limited monitoring during cultivation, on the other hand, lactose may be introduced in the formulation of the medium together with other components and will act as an inducer, when the glucose runs out, and as a carbon source. This type of medium is known as a self-inducing medium. In a bioreactor the DO600nm reached 39.9 AU with enzymatic activity of 29 IU / Cultivation.