RESUMO
This study aimed to partially characterize the three main serine carboxypeptidases (SCP3, SCP20, and SCP47) from Nepenthes mirabilis. Furthermore, one peptidase (SCP3) was chosen for further heterologous expression in Escherichia coli Shuffle®T7. SCP3 also was characterized in terms of its allergenic potential using bioinformatics tools. SCP3, SCP20, and SCP47 showed very similar 3D structures and mechanistic features to other plant serine peptidases belonging to clan SC and family S10. Although SCP3 was obtained in its soluble form, using 1% ethanol during induction with 0.5 mM IPTG at 16 °C for 18 h, it did not show proteolytic activity by zymography or in vitro analysis. SCP3 presented a few allergenic peptides and several cleavage sites for digestive enzymes. This work describes additional features of these enzymes, opening new perspectives for further studies for characterization and analysis of heterologous expression, as well as their potential biotechnological applications.
Assuntos
CarboxipeptidasesRESUMO
MAIN CONCLUSION: Seeds of native species from the rain forest (Amazon) are source of chitinases and their protein extracts exhibited strong and broad antifungal activity. Numerous plant species native to the Amazon have not yet been chemically studied. Studies of seeds are scarcer, since adversities in accessing study areas and seasonality pose constant hurdles to systematic research. In this study, proteins were extracted from seeds belonging to endemic Amazon species and were investigated for the first time. Proteolytic activity, peptidase inhibitors, and chitinases were identified, but chitinolytic activity predominated. Four proteins were purified through chromatography and identified as lectin and chitinases by MS/MS analyses. The proteins were examined for inhibition of a phytopathogen (Fusarium oxysporum). Analyses by fluorescence microscopy suggested binding of propidium iodide to DNA of fungal spores, revealing that spore integrity was lost when accessed by the proteins. Further structural and functional analyses of defensive proteins belonging to species facing highly complex ecosystems such as Amazonia should be conducted, since these could provide new insights into specificity and synergism involving defense proteins of plants submitted to a very complex ecosystem.