resumo
According to the World Health Organization (WHO), rising levels of bacterial resistance are leading to an increase in multidrug-resistant bacteria, limiting therapeutic options. There is therefore an urgent need for antibacterial alternatives capable of combating infections caused by pathogens classified as critical by the WHO, as well as diversifying therapeutic strategies. Pedopeptins are a mixture of three cationic, non-ribosomal antimicrobial peptides (PedA, PedB and PedC) produced by the strain Pedobacter lusitanus NL19, which inhibit the growth of important bacteria in both the clinical and food sectors. The main aim of this study was to develop more effective and sustainable methods for isolating and purifying active pedopeptins using Fast Protein Liquid Chromatography, enabling to obtain larger quantities of the peptides, facilitating future research. P. lusitanus NL19 was grown in TSB25% broth at 26 °C. The culture supernatants were concentrated using conventional n-butanol extraction, as well as alternative methods such as precipitation and ultrafiltration. In addition, various solvents to resuspend the n-butanol extracts were investigated to identify more (bio)compatible alternatives to acetonitrile, including with the downstream analysis by FPLC. The separation of n-butanol solubilized extracts as well as culture supernatants was tested using ion-exchange FPLC resins, resorting both to anion- and cation-exchangers. To evaluate the performance of the different methods, recurrent activity testing of fractions against Escherichia coli ATCC 25922 and Kocuria rhizophila ATCC 9341 to identify antibacterial activity were performed, which are indicative of the presence of pedopeptins. The results obtained within the scope of this thesis showed that 10% polypropylene glycol 400 g/mol allows for the effective dissolution of n-butanol extracts and that ultrafiltration and precipitation with ammonium sulphate can be alternatives for concentrating the supernatants of NL19 cultures. On the other hand, the application of a cation-exchange column and a mobile phase composed of MES-NaOH (pH 6.2)is a promising approach for obtaining single fractions with antibacterial activity. In addition, analysis using UPLC-ESI-Orbitrap MS confirmed the presence of the PedA and PedC peptides in these fractions. The minimum inhibitory concentrations of dissolved n-butanol extracts and supernatants were also determined to establish a procedure for consistent uniformity between assays. Overall, this study marks the first steps toward developing a procedure to purify pedopeptins on a preparative scale using alternative methods to existing ones, paving the way for future research, particularly in toxicity testing and pedopeptins encapsulation to formulate delivery vehicles.
autores
Ana Rita Correia das Neves
nossos autores
Rita Neves
Bolseiro de investigaçãoorientadores
Augusto Pedro and Tânia Caetano