ABSTRACT
An antifungal class III peroxidase was purified from Marsdenia megalantha latex (named Mo-POX) using DEAE-cellulose and gel filtration chromatography on a Superose 12 HR 10/30 column. Mm-POX has an apparent molecular mass of 67.0kDa and a pI of 5.2, shares identity with other peroxidases, and follows Michaelis-Menten kinetics. It has a high affinity for guaiacol and hydrogen peroxide. The pH and temperature optima for Mm-POX were 5.0-7.0 and 60°C, respectively. The catalytic activity of Mm-POX was decreased in the presence of classic peroxidase inhibitors including azide, dithiothreitol, ethylenediamine tetraacetic acid, and sodium metabisulfite and high concentrations of Na+, Mn+, and salicylic acid. In contrast, Ca+ and Mg+, even at low concentrations, enhanced the Mm-POX enzymatic activity. This protein inhibited the germination of the conidia of the phytopathogenic fungi Fusarium oxysporum and Fusarium solani by acting through a membrane permeabilization mechanism. Mm-POX also induced oxidative stress in F. solani. Mm-POX is the first enzyme to be isolated from the M. megalantha species and it has potential use in the control of plant disease caused by important phytopathogenic fungi. This adds biotechnological value to this enzyme.
Subject(s)
Cell Membrane Permeability/drug effects , Fusarium/drug effects , Latex/chemistry , Marsdenia/chemistry , Peroxidase/isolation & purification , Peroxidase/pharmacology , Plants/microbiology , Amino Acid Sequence , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Stability , Fusarium/cytology , Fusarium/metabolism , Fusarium/physiology , Hydrogen-Ion Concentration , Kinetics , Metals/pharmacology , Microbial Viability/drug effects , Molecular Weight , Peroxidase/antagonists & inhibitors , Peroxidase/chemistry , Reactive Oxygen Species/metabolism , Salicylic Acid/pharmacology , Spores, Fungal/drug effects , Spores, Fungal/growth & development , Substrate Specificity , TemperatureABSTRACT
Marsdenia tenacissima extract (MTE) is a new plate-derived biotechnology product that is frequently used, but occasionally reported, in the field of chemotherapy. In this study, we assessed the antitumor activity and related mechanisms of MTE by various biotechnological methods. The survival rates of MG63 osteosarcoma cells treated with MTE and doxorubicin were measured, individually or jointly, and the changes in cellular shape, apoptotic rates, and Fas expression were observed. The results indicated that combination of MTE and doxorubicin up-regulated Fas expression and induced apoptosis. The survival rate of combined application of 50 mg/mL MTE and 1 µg/mL doxorubicin was significantly lower than that of the individual application (P < 0.01). Other biotechnology methods also showed an apoptosis-inducing effect of combined application that was much stronger than individual application. All of these results suggested that MTE may promote the effects of doxorubicin chemotherapy, perhaps related to the up-regulation of Fas expression in tumor cells.