Screening of Postharvest Agricultural Wastes as Alternative Sources of Peroxidases: Characterization and Kinetics of a Novel Peroxidase from Lentil (Lens culinaris L.) Stubble

摘要

Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL~(-1)) did not vary for at least 15 months when stored at 4 °C (k_(inact) = 0.146 year~(-1), t_(1/2inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL~(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k_(inact) = 2.2 X 10~(-3) day~(-1), t_(1/2inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurined by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H2O2-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K_m~(appH2O2) = 1.87 mM; V_m~(appH2O2) = 6.4 mM min~(-1); K_m~(app guaicol) = 32 mM; V_(max)~(app guaicol) = 9.1 mM min~(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases, Finally, after the effectiveness of the crude extracts of LSP in oxidizing and removing from solution a series of last-generation dyes present in effluents from textile industries (1) had been checked, a steady-state kinetic study of the H2O2-mediated oxidation and decolorization of Green Domalan BL by the catalytic action of the lentil stubble extract was carried out, with the observation of the same apparent Michaelian kinetic behavior (K_m~(appGD) = 471 μM; K_(max)~(appGD) = 23 μM min~(-1)). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.