Caracterização estrutural e bioquímica da l-asparaginase ans-z de bacillus subtilis e avaliação de sua atividade antiproliferativa em linhagens de células tumorais hematológicas

Abstract

L-asparaginase catalyzes the hydrolysis of L-asparagine into aspartic acid and ammonia. It is found in animals, plants, and microorganisms and is part of a therapeutic strategy based on the restriction of essential amino acids, as are other enzymes that exploit the dependence of cancer cells on these nutrients to survive. Therefore, microbial sources are preferred for large-scale production due to their efficiency and ease of cultivation. In this study, we recombinantly produced and characterized an L-asparaginase from Bacillus subtilis (Asp- Z). Asp-Z was heterologously expressed in Escherichia coli and purified by affinity chromatography, resulting in a soluble protein with optimal activity at 55 °C and pH 7.5. The kinetic parameters under these conditions were a Km of 0.47 mM and a Vmax of 52.13 U/mg. Asp-Z proved specific for L-asparagine, showing no detectable glutaminase activity, and exhibited antiproliferative effects against hematologic cancer cell lines, particularly RAJI and JURKAT, with IC₅₀ values in the micromolar range. In silico analyses revealed distinct immunogenic epitopes between Asp-Z and commercial E. coli L-asparaginase, suggesting divergent antigenic profiles, while crystallographic data revealed a conserved tetrameric structure with a highly flexible active site loop. Taken together, these findings highlight Asp-Z as a thermostable enzyme, free of glutaminase activity and with low immunogenicity, representing a promising structure for optimization through protein engineering, targeting therapeutic and biotechnological applications.