IDENTIFICATION AND CHARACTERIZATION OF PEROXIDASES ACTIVITY IN LEMON THYME (THYMUS CITRIODORUS) ROOTS

Abstract

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 H 2 O 2. In this study, peroxidase activities were identified in an extract obtained from Thymus citriodorus roots by monitoring spectrophotometrically the H 2 O 2 -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 prof -1 and 0.5 min -1 .mg prot -1 for H 2 O 2 ; 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 H 2 O 2 ; 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 H 2 O 2 . The peroxidatic activities were sensitive to KCN, with IC 50 of 0.12 ± 0.02 μM for o-dianisidine, 0.75±0.05 μM for guaiacol and 0.6 ± 0.05 μM for ferulic acid. Results point out the predominance of lignin peroxidase activity over o-dianisidine and guaiacol peroxidases.