Purificação e caracterização de uma nova peroxidase oriunda de raízes de feijão-de-corda [Vigna unguiculata (L.) Walp.] e prospecção de atividade antifúngica e removedora de fenóis

Abstract

Peroxidases (POXs) are enzymes that catalyze the oxidation of a variety of substrates and play important functions in plant defense against pathogens, acting on H2O2 scavenging and lignin biosynthesis. Because of their broad applicability in medicine, agriculture and biotechnology as biosensor systems, POXs became significantly relevant. In this work, a peroxidase was purified from cowpea (Vigna unguiculata) roots of genotype TE97-411-1F. After total soluble proteins extraction with 0.05 M sodium acetate buffer pH 5.2, containing 0,15 M NaCl, followed by ammonium sulfate precipitation (0-90% saturation), the obtained fraction (F0/90) was inserted in a chromatography column containing chitin as stationary phase, by which the peroxidase activity was concentrated on the non-retained peak (PNRQ). The PNRQ was loaded in an ion exchange column, and the peroxidase of interest, denominated POX100, was purified after been eluted with 0,1 M NaCl, as shown by SDS-PAGE. Analysis by mass spectrometry (ESI-QUAD-TOF) indicated a high similarity between the POX100 and others leguminous peroxidases. An analysis of a pure protein showed that POX100 is a glycoprotein composed by a single 58 kDa polipeptidic chain, with IEP = 3,79. It presented an excellent thermal stability up to 60°C, during 120 minutes of treatment. Its high enzymatic activity, using guaiacol as substrate, was achieved at pH 6.0. In vitro phenolic compounds removal assays, the degradation of 48% of guaiacol was observed within 1 h of reaction. On the assay with presence of ions and additives, significant enhancement of the activity in the presence of EDTA and significant inhibition by DTT, Sodium Azide and Sodium Bisulfite were noticed. Furthermore, the analysis of antifungal potential exhibited a mild inhibitory activity of the vegetative growth of phytopathogenic fungi Colletotrichum gloeosporioides and Fusarium oxysporum.