Arthur Vieira Santos¹, Felipe Domingos de Sousa², Márcio Viana Ramos¹, Thalles Barbosa Grangeiro¹

Moringa oleifera is renowned for its coagulant properties in water purification, attributed to the cationic proteins in its seeds. Most studies focus on the group of lectins and coagulant proteins with specific biological activity unique to this species. Crude extracts of its seeds, when subjected to chitin-affinity chromatography, exhibit three characteristic peaks with a significant abundance of what was termed Moringa oleifera Chitin Binding Protein 3 (Mo-CBP3). Mo-CBP3 presents as a small ∼13 kDa homodimer, stable under acidic pH and thermostable (up to 60°C). It binds strongly to chitin and chitooligosaccharides via a chitin-binding domain (conserved in hevein-like lectins). Our recent findings revealed the presence of another protein, initially regarded as a contaminant, which exhibited nuclease activity after P2 fractionation. This chitin-retained fraction when subjected to size-exclusion chromatography, yielded two new fractions. Both were analyzed by SDS-PAGE and mass spectrometry to confirm purity. This new fraction (distinct from Mo-CBP3) was subjected to nucleolytic assays using concentrations ranging from 10 µg/mL to 1 mg/mL against 4 µL of genomic DNA (250 ng/µL) at 37°C for 30 minutes; commercial DNase served as the positive control and heat-denatured protein as the negative control. Agarose gel electrophoresis (1.5%, ethidium bromide staining) was subsequently performed.   SDS-PAGE revealed distinct bands in the MoCBP3 and contaminant fractions, corresponding to apparent molecular weights of 14 kDa and 66 kDa, respectively. The 14 kDa band matches literature data, confirming MoCBP3, while the 66 kDa band corresponds to an unknown protein. Mass spectrometry detected MoCBP3 peptides in the expected fraction but identified no known proteins in the contaminant fraction, supporting the hypothesis of a novel, previously undescribed protein. Additionally, agarose gel analysis demonstrated DNA degradation starting at 500 µg/mL, with a profile comparable to commercial DNase. Biological assays to evaluate antifungal and antibacterial activities are ongoing. The discovery of a novel nuclease from Moringa oleifera seeds is significant, as nucleases participate in defense mechanisms, cellular regulation, and frequently exhibit antimicrobial properties. This finding underscores the importance of bioprospecting in underexplored natural sources, which may yield innovative alternatives to conventional antibiotics.

Agradecimentos: FUNCAP, CNPq, CAPES e Fundação Edson Queiroz