Investigação da atividade de nuclease da albumina-2s (Mo-CBP3) de sementes de Moringa oleifera

Abstract

The plant Moringa oleifera (Moringaceae) is present in the semi-arid region. Its seeds have a high lipid content, and the flour obtained from its seeds is used to induce the flocculation of solid particles in turbid waters, allowing for removal and improving water quality for human consumption. At least three chitin-binding proteins, without chitinase activity, have been detected and studied from moringa seeds. However, a nuclease activity seems to coexist in one of the purified fractions of the chitin-binding proteins. This was the problem that motivated this research. Through chitin affinity chromatography, a protein fraction with affinity (PAC) is obtained, and this chromatographic peak was subjected to ion exchange, giving rise to 3 characteristic peaks, with peak 3 (CM-PIII) designated as Mo-CBP3. Preliminary studies with this sample demonstrated nucleolytic activities against plasmidial DNA in the amount of 1,25 ug of protein and 1 ug of nucleic acid. 15% polyacrylamide gel electrophoresis revealed 3 previously undescribed bands. Different purification approaches were used to separate Mo-CBP3 from its contaminants: reverse phase chromatography, ultra centrifugation filtration, and size exclusion chromatography (SEC). Size exclusion chromatography using PAC showed the best protein recovery and presented 4 peaks, which were subjected to electrophoresis analysis. Electrophoresis revealed the separation of the unknown proteins from the known proteins as Mo-CBPs (Moringa oleifera chitin-binding proteins). The SEC peaks were subjected to plasmidial DNA and RNA cleavage assays obtained from Klebsiella pneumoniae ATCC 10031, showing activity comparable to commercial nucleases in peak 3 (P3-SE). In summary, the refined purification by size exclusion chromatography (SEC) was crucial to isolate the biological activity. The results demonstrated that the P3-SE of the SEC, corresponding to the Mo-CBP3 protein, possesses remarkable nucleolytic activity against plasmidial DNA and RNA from Klebsiella pneumoniae. This discovery of a new activity for Mo-CBP3 expands the vast potential of Moringa oleifera seeds as a promising source for nuclease bioprospection.