Purificação, caracterização bioquímica e atividade contra Candida spp. de uma nova proteína ligante à quitina de sementes de Moringa oleifera LAM.

Abstract

Candida species encompass a group of yeast that normally lives on the skin and mucous surfaces of human beings, in which the infectious disease candidiasis can occur, with severe consequences particularly for immunocompromised patients. The available antifungal drugs used for the candidiasis treatment are usually toxic and can lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. Moringa oleifera is a plant with valuable nutritional and medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. Mo-CBP2 (Mo: Moringa oleifera; CBP: Chitin Binding Protein) was purified from the albumin fraction of M. oleifera seeds through chitin affinity chromatography followed by cation exchange chromatography. Mo-CBP2 represented about 0.14% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,160 Da. However, in contrast, after native gel filtration chromatography (pH 7.5) two protein peaks with molecular masses of 33.0 and 66.0 kDa emerged from Mo-CBP2. By SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 25.0 kDa. Nevertheless, Tricine-SDS-PAGE of Mo-CBP2 under non-reduced conditions revealed two protein bands in the range of 13.0 and 17.0 kDa. After DTT and β-mercaptoethanol treatments, Mo-CBP2 appeared as been composed of masses of 7.0 and 4.0 kDa. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9) with 4.1% sugar, with did not display hemagglutinating or hemolytical activity upon rabbit erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei and C. tropicalis, with MIC ranging from 9.45 to 37.9 µM. In addition, Mo-CBP2 (18.9 µM) increased the cell membrane permeabilization and oxigen reactive species production in C. albicans and promoted degradation of circular plasmid DNA (pUC18) from Escherichia coli. The results presented in this work point out the potential of Mo-CBP2 as a new antifungal protein active against Candida spp