Otimização do processo produtivo de L-asparaginase II recombinante de Erwinia carotovora em biorreator e análise da sua ação antileucêmica

Abstract

The L-asparaginase II enzyme is one of the drugs chosen for the multidrug treatment of acute lymphoblastic leukemia (ALL) in children and adults. Leukemia cells use plasma L-asparagine for protein synthesis. Through the hydrolysis mechanism of L-asparaginase, there is depletion of asparagine in the plasma, resulting in interruption of protein synthesis and induction of apoptosis. The L-asparaginase II used in clinical practice is derived from Escherichia coli, however, approximately 30% of patients develop immune reactions against asparaginase, limiting its therapeutic action and requiring replacement with different preparations. Alternative enzymes derived from Erwinia subspecies have a shorter half-life, although they are associated with a reduced risk of hypersensitivity reactions due to their lack of cross-reactivity and reduced glutaminase activity. The aim of the work was to optimize the production process of recombinant Erwinia carotovora L-asparaginase II in bioreactor and analyze its antileukemic potential. Different bioreactor cultivation strategies were adopted for the production of the enzyme in E. coli C43 (DE3) cells, with the DO-stat and linear feeding strategies. Cultivation kinetic parameters were evaluated for yield, productivity, enzymatic activity and microbial growth rate. After obtaining the recombinant enzyme and its subsequent purification, in vitro and in vivo tests were carried out, verifying its antileukemic potential. The highest values of enzymatic activity, yield per biomass and productivity were obtained using the DO-stat strategy with induction in 18 h of cultivation. In the in vitro tests, the enzyme showed cytotoxic potential against different strains of ALL, reducing cell viability and inducing apoptosis in the cells. In the in vivo tests, the enzyme showed greater bioavailability when administered intramuscularly and was effective in increasing the survival of C57Bl6/J mice transplanted with primary murine ALL. Based on the results obtained, the antileukemic potential of the L-asparaginase II enzyme from E. carotovora against ALL cells was verified