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

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 77; pp. 59 - 66
• Main Authors: Cai, Feng, OuYang, Chao, Duan, Peipei, Gao, Shun, Xu, Ying, Chen, Fang
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2012; Elsevier
• Subjects: ambient temperature; ammonium sulfate; beta-mercaptoethanol; Bioconversions. Hemisynthesis; biodiesel; Biological and medical sciences; Biotechnology; catalytic activity; cetyltrimethylammonium bromide; dimethyl sulfoxide; dithiothreitol; EDTA (chelating agent); Enzyme purification; enzyme stability; fractionation; Fundamental and applied biological sciences. Psychology; gel chromatography; guaiacol; guanidines; hydrogen peroxide; ion exchange chromatography; Jatropha curcas; leaves; Methods. Procedures. Technologies; molecular weight; Peroxidase; polyacrylamide gel electrophoresis; Salt activation; shelf life; sodium azide; sodium chloride; solvents; substrate specificity; Thermal stability; toluene; urea; Peroxidase; Salt activation; Enzyme purification; Jatropha curcas; Thermal stability; Purification; Enzyme; Activation; Plant leaf; Jatropha; Characterization; Peroxidases; Dicotyledones; Angiospermae; Euphorbiaceae; Spermatophyta; Oxidoreductases
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2011.12.002

[Display omitted] ► 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 2.5M NaCl. ► The purified enzyme had long shelf life with 180 days at 4°C and 14 days at room temperature. 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 48kDa 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 0.17mM, respectively. In addition, NaCl (2.5M) significantly enhanced the activity of this peroxidase. The purified enzyme was stable under high temperature (70% activity retained after 1h incubation at 70°C), extreme pH environment (93% or more activity retained after 2h incubation under pH 3–12), high NaCl concentration (88% or more activity retained after 2h incubation with 1–4M NaCl) and organic solvents (95% or more activity retained after 54h incubation with various organic solvents). Moreover, this peroxidase was resistant against 20mM hydrogen peroxide, 8M urea, 3M guanidine hydrochloride and 20mM 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.