Purification and partial characterization of a thermostable peroxidase isoenzyme from Bambara groundnut (Vigna subterranea L. Verdc) seedlings

Abstract Background: Some applications of peroxidases imply reactions proceeding at high temperatures. In our previous studies, thermostable peroxidase isoenzymes were detected in the seedlings of Vigna sp. One of these isoperoxidases (named peroxidase A6) especially had a great activity in these seedlings. Its isolation and characterization is thus necessary for a thorough study of its biotechnological potential. Results: Peroxidase A6 was purified from Bambara groundnut seedling roots by a combination of gel filtration on Sephadex G-100, heat treatment, CM-cellulose chromatography and DEAE-cellulose chromatography. It has a molecular weight of about 41 kDa and exhibits a great activity toward the oxidation of O-dianisidine, ABTS, TMB, DAB and OPD at acid optimum pH (pH 3 for ABTS, pH 4 for OPD and pH 6 for the others) and toward the reduction of H2O2. Apparent Km values for these substrates were respectively 3.50 mM, 0.12 mM, 1.81 mM, 0.05 mM, 17.22 mM and 2.53 mM; catalytic efficiencies were 5.12×104 mM-1.min-1, 2.22×106 mM-1.min-1, 1.59×105 mM-1.min-1, 1.82×105 mM-1.min-1, 3.17×105 mM-1.min-1and 1.79×106 mM-1.min-1. It has an optimum temperature of activity around 60°C, and its heat inactivation fit to the first-order kinetics, with half-lives of 3.06 weeks, 13.5 hours, 15 min and 3.5 min at 50°C, 70°C, 80°C and 90°C respectively. The calculated activation energy (E) for its thermal inactivation was found to be 221.5 KJ/mol at pH 8. It loose only 5% of its initial activity over a period of 4 months. Mg2+ inhibits the activity of the enzyme. The Ca2+ions greatly increase the stability of this peroxidase at 80 °C, while Mn2+and Zn2+ reduce it. The enzyme is inhibited by sodium azide at concentrations above 1 µM with an IC50value around 10 µM. This inhibition, in addition to the RZ value (A403nm/A280nm) evaluated at 2.4 confirms the presence at its active site of a heme group common to class III peroxidases. Conclusion: The unusual catalytic and thermal characteristics of peroxidase A6 could make it a potent tool in several biotechnological applications, especially as part of kit for enzyme immunoassays and clinical diagnosis.