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 Km value of this peroxidase for guaiacol was 60 °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 °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, β-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.

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► A novel heme peroxidase from Jatropha curcas leaves was isolated and purified to homogeneity.
► The purified enzyme showed high thermal stability, wide pH resistance, high H2O2 tolerance and broad substrate specificity.
► This new peroxidase showed high activity under high temperature ranging from 55 °C to 75 °C.
► The activity of this peroxidase was significantly enhanced by 2.5 M NaCl.
► The purified enzyme had long shelf life with 180 days at 4 °C and 14 days at room temperature.