Purification and characterization of a novel thermal stable peroxidase from Jatropha curcas leaves

A novel heme peroxidase from Jatropha curcas, an important source of bio-diesel, was purified to homogeneity using ammonium sulfate fractionation, desalting chromatography and ion exchange chromatography. Molecular mass of this purified enzyme was around 48 kDa as detected by SDS-PAGE. Gel filtration analysis revealed that the enzyme was a monomer under native conditions. The purified enzyme had broad substrate specificity with the ideal substrates of guaiacol and o-phenylenediamine. The optimum temperature, pH and K-m value of this peroxidase for guaiacol was 60 degrees C, 5.0 and 0.17 mM, respectively. In addition, NaCl (2.5 M) significantly enhanced the activity of this peroxidase. The purified enzyme was stable under high temperature (70% activity retained after 1 h incubation at 70 degrees C), extreme pH environment (93% or more activity retained after 2 h incubation under pH 3-12), high NaCl concentration (88% or more activity retained after 2 h incubation with 1-4 M NaCl) and organic solvents (95% or more activity retained after 54 h incubation with various organic solvents). Moreover, this peroxidase was resistant against 20 mM hydrogen peroxide, 8 M urea, 3 M guanidine hydrochloride and 20 mM EDTA. However, the peroxidase activity was significantly inhibited by sodium azide, dithiothreitol. CTAB, beta-mercaptoethanol, DMSO, toluene and ferrous ion. The enzyme had long shelf life with 180 days at 4 C and 14 days at room temperature. This new robust peroxidase may bring a better understanding for the high anti-adversity property of J. curcas. Meanwhile, the broad substrate specificity, wide stability against high temperature, extreme pH. organic solvent and hydrogen peroxide suggested that the enzyme could be a potential candidate peroxidase source for industrial and biomedical applications. (C) 2011 Elsevier B.V. All rights reserved.