Migliolo, LudovicoNunes, Ellynes Amancio Correia2024-12-262024-12-262024-10-15NUNES, Ellynes Amancio Correia. Lectina manganês-dependente da peçonha de bothrops moojeni com atividade bacteriostática: isolamento e caracterização. Orientador: Dr. Ludovico Migliolo. 2024. 96f. Tese (Doutorado em Bioquímica e Biologia Molecular) - Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 2024.https://repositorio.ufrn.br/handle/123456789/60986The rich diversity of bioactive molecules present in animal venoms offers vast potential for the development of new drugs. Snake venoms are a complex of molecular proteins, whose diversity of toxins and their mechanisms of action explain the variety of clinical manifestations observed in snake envenomations. The study of snake venom proteins allows us to explore their biological activities. In this context, the aim of this study was to investigate the biotechnological potential of lectins present in Bothrops moojeni venom. Based on a literature review on snake lectins, the hypothesis was formulated that these proteins may have antibiotic activities. To test these hypotheses, the isolation, biochemical characterization and evaluation of the biological activities of a purified lectin from the venom were performed. Lectin isolation and identification were performed by size exclusion chromatography on a Sephacryl S-100 column, equilibrated with 50 mM ammonium bicarbonate and pH 7.8, with a flow rate of 680 µL.min-1. The fractions with hemagglutinating activity were subjected to reversed-phase high-performance liquid chromatography on a C18 column, using a linear gradient of acetonitrile and water in 0.1% trifluoroacetic acid as the mobile phase. Protein purity was assessed by electrophoresis on a 12.5% SDS-PAGE polyacrylamide gel under reducing conditions. The presence of lectins in different fractions was monitored by hemagglutinating activity using 2% (v/v) human erythrocytes in PBS. The determination of specificity and ion dependence was investigated by hemagglutination in the presence of different sugars and ions, respectively. The influence of pH and temperature on hemagglutinating activity was investigated over a pH range of 2 to 11 and temperatures from -4 to 100°C. Hemolytic activity was assessed by the release of hemoglobin from human type A erythrocytes at a concentration of 2% v:v using the spectrophotometric method at 425 nm. Antibacterial activity against Acinetobacter baumannii and Staphylococcus aureus was determined by modified broth microdilution growth inhibition assays. Biofilm formation was quantified by the crystal violet method. Size exclusion chromatography investigated a profile with six distinct protein fractions. Analysis by hemagglutination assays revealed that fractions 5 and 6 exhibited hemagglutinating activity. Fraction 5, with the highest activity, was designated BmoojL. The biochemical characterization of the BmoojL lectin revealed that its activity is manganese-dependent and inhibited by several monosaccharides, such as glucose, fructose and mannose. Furthermore, the protein was stable over a wide range of pH (5-9) and temperature (up to 80°C). BmoojL was not hemolytic at the concentrations evaluated (31.25-500 µg. mL-1 ), with a hemolysis percentage below 10%. The antibacterial assays demonstrated that, despite not reaching the minimum inhibitory concentration, BmoojL impairs the bacterial growth of A. baumannii from the concentration of 64 µg. mL-1, being, therefore, bacteriostatic. The antibiofilm assay showed that for S. aureus, BmoojL was effective, considerably reducing biofilm growth at all concentrations tested (16 - 512 µg. mL-1) by approximately 50%. Thus, the study allowed the isolation of a molecule with manganese-dependent lectin activity with the potential to exert bacteriostatic and antibiofilm activity, contributing to the research of bioactive molecules derived from venomous snakes. Although there are some studies involving such molecules and their heterologous activity, when compared to other molecular groups, the study involving snake lectins is an area that is still little explored, and these findings show what there is still to know about these molecules and directions and propose new activities that may add to the biotechnological research related to these animal groups.Acesso AbertoAtividade biológicaPeçonhas ofídicasBioprospecçãodoctoralThesisCNPQ::CIENCIAS BIOLOGICAS