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

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• 作者：Nazaret Hidalgo-Cuadrado ; Patricia P茅rez-Galende ; Teresa Manzano ; C谩ndido Garcia De Maria ; Valery L. Shnyrov ; Manuel G. Roig
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2012
• 出版时间：May 16, 2012
• 年：2012
• 卷：60
• 期：19
• 页码：4765-4772
• 全文大小：343K
• 年卷期：v.60,no.19(May 16, 2012)
• ISSN：1520-5118

文摘

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^鈥?) did not vary for at least 15 months when stored at 4 掳C (k_inact = 0.146 year^鈥?, t_1/2聽inact = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL^鈥?) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k_inact = 2.2 脳 10^鈥? day^鈥?, t_1/2聽inact = 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 semipurified 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_max^appH₂O₂ = 6.4 mM min^鈥?; K_m^{app聽guaicol} = 32 mM; V_max^{app聽guaicol} = 9.1 mM min^鈥?), 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 渭M; V_max^appGD = 23 渭M min^鈥?). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.

Keywords:

agricultural wastes; screening; crude extracts; lentil stubble peroxidase; kinetics; guaiacol; Green Domalan textile dye