Caracterização bioquímica da L-asparaginase II ansZ recombinante do Bacillus subtilis 168 expressa em Escherichia coli e avaliação de seu potencial citotóxico

Abstract

L-asparaginase type II is a therapeutic enzyme used to treat acute lymphoid leukemia. Leukemic lymphoblasts have a deficiency in the asparagine synthetase gene, requiring free Lasparagine in plasma to proliferate. L-asparaginase acts on the hydrolysis of L-asparagine, depleting the serum levels of this amino acid and, consequently, inhibiting its proliferative potential. The formulations currently available on the market use Escherichia coli and Erwinia chrysanthemi L-asparaginases. There is a high rate relative to side effects and immunogenicity associated with treatment with these enzymes in patients. Therefore, the search for new sources that present an effective treatment with reduction of side effects is important. The objective of this work was to characterize the recombinant L-asparaginase II of Bacillus subtilis, which had been expressed heterologously in Escherichia coli, determining, from microscale tests, the influence of pH and temperature on enzymatic activity and its kinetic parameters, in addition to the evaluation of cytotoxicity in leukemic lymphoblastic lineage. The colorimetric method based on the use of the Nessler reagent was used for the assays that involved the evaluation of enzymatic activity, while the alamarBlue® cytotoxicity assay was performed. The optimum pH was 7.0 and the optimum temperature was 50 ° C. For cytotoxicity, it was possible to observe that the treatment of Raji cells with B. subtilis L-asparaginase only showed a higher efficacy at the highest concentrations tested (70 and 80 μg/mL). The adaptation to the microscale was successful, providing benefits regarding the use of the enzyme and use of the Nessler reagent.