ABSTRACT
A cationic soluble peroxidase isoenzyme (CysPrx) has been purified and characterized from artichoke (Cynara cardunculus subsp. scolymus (L.) Hegi) leaves by combination of aqueous two phase extraction, ion exchange chromatography, and gel filtration. The purification fold was 149 and the activity recovery 5.5%. CysPrx was stable from 5 to 45 °C with a pH optimum around 5.5; the pI was 8.3 and the MW of 37.7 ± 1.5 kDa. MALDI-TOF MS analysis provided partial peptide sequences and resolved CysPrx isoenzyme into two putative isoforms. The presence of these isoforms was confirmed by the isolation of full-length cDNA encoding CysPrx that generate two slightly different sequences coding for two putative CysPrx: CysPrx1 and CysPrx2. The obtained MS peptides showed a 35% coverage with 100% identity with the two CysPrx deduced protein sequences. A molecular modeling analysis was carried out to predict in silico the protein structure and compare it with other plant Prx structures. Considering that CysPrx is quite stable, the study carried out in this paper will offer new insights for the production of the recombinant protein for utilization of CysPrx as an alternative Prx for food technology, biomedical analysis and bioremediation.
Subject(s)
Cynara scolymus/enzymology , Peptides/analysis , Peroxidases/isolation & purification , Plant Leaves/enzymology , Plant Proteins/isolation & purification , Amino Acid Sequence , Cloning, Molecular , Cynara scolymus/chemistry , Cynara scolymus/genetics , DNA, Complementary/isolation & purification , Hydrogen-Ion Concentration , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/isolation & purification , Models, Molecular , Molecular Sequence Data , Molecular Structure , Peroxidases/chemistry , Peroxidases/genetics , Phylogeny , Plant Proteins/chemistry , Plant Proteins/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Olive oil production generates large amounts of recalcitrant compounds, the olive oil mill wastewater (OMWW), which represent one of the most contaminating effluents among those produced by the agrofood industries. Nowadays, this view has changed to one that recognizes the waste as a low-cost starting material rich in bioactive compounds, particularly biophenols, that can be extracted and applied as natural antioxidants for the food and pharmaceutical industries. The data reported in this paper indicate that the OMWW extracts, besides low molecular weight antioxidant phenolics such as tyrosol and hydroxytyrosol, also contain phenolics with a molecular weight in the range of 600-5000 Da, which exhibit efficient scavenging activities against hydroxyl and peroxyl radicals. This group of phenolics includes, besides verbascoside, isoverbascoside, and an oxidized form of verbascoside, a number of higher molecular weight phenolics arising from oxidative polymerization of hydroxytyrosol and caffeic acid. Overall, these higher molecular weight phenolics prove to be, in some in vitro tests, more efficient scavengers of hydrophilic hydroxyl radicals than hydroxytyrosol, which could be used for industrial applications as natural nontoxic antioxidants.
