Identification and Characterization of Peroxidases Activity in Lemon Thyme (Thymus citriodorus) Roots

Enzymes are of critical importance in maintaining a species and protecting it against various forms of environmental stresses. In particular, the formation of reactive oxygen species and ensuing oxidative stress is a constant menace for any organism. Plants are equipped with a battery of antioxidative stress enzymes, including peroxidases that fulfill a variety of functions while scavenging H2O2. In this study, peroxidase activities were identified in an extract obtained from Thymus citriodorus roots by monitoring spectrophotometrically the H2O2-mediated oxidation of either o-dianisidine (at 460 nm), guaiacol (at 470 nm) or ferulic acid (at 310 nm) (lignin peroxidase) in the presence of extract aliquots. Assays were performed at pH 4 for o-dianisidine, 6 for guaiacol and 5 for ferulic acid oxidation, using extinction coefficients of, respectively, 11.3, 26.6 and 8.68 mM(-1).cm(-1). With o-dianisidine as the reducing substrate, apparent K-m, V-max and catalytic efficiency were, respectively, 0.65 +/- 0.06 mM, 0.17 +/- 0.007 mM.min(-1).mg prot(-1) and 0.27 +/- 0.01 min(-1).mg prot(-1) for o-dianisidine and 0.5 +/- 0.05 mM, 0.255 +/- 0.005 mM.min(-1).mg prot(-1) and 0.5 min(-1).mg prot(-1) for H2O2; with guaiacol, the kinetics parameters were, respectively, 4.5 +/- 3 mM, 0.6 +/- 0.06 mM.min(-1).mg prot(-1) and 0.13 +/- 0.002 min(-1).mg prot(-1) for guaiacol and 0.9 +/- 0.2 mM, 0.52 +/- 0.04 mM.min(-1).mg prot(-1) and 0.58 +/- 0.04 min(-1).mg prot(-1) for H2O2; with ferulic acid, kinetics parameters were, respectively, 0.07 +/- 0.01 mM, 0.9 +/- 0.09 mM.min(-1).mg prot(-1) and 13 +/- 1 min(-1).mg prot(-1) for ferulic acid and 0.025 +/- 0.005 mM, 0.7 +/- 0.01 mM.min(-1).mg prot(-1) and 28 +/- 3 min(-1).mg prot(-1) for H2O2. The peroxidatic activities were sensitive to KCN, with IC50 of 0.12 +/- 0.02 mu M for o-dianisidine, 0.75 +/- 0.05 mu M for guaiacol and 0.6 +/- 0.05 mu M for ferulic acid. Results point out the predominance of lignin peroxidase activity over o-dianisidine and guaiacol peroxidases.