Purification and partial characterization of a thermostable peroxidase isoenzyme from Vigna sp seedlings

Abstract

A peroxidase isoenzyme (named A6 in a previous study) was purified from radicles of a Vigna species by a combination of gel filtration on Sephadex G-100, heat treatment, CM-cellulose chromatography and DEAE-cellulose chromatography. It has been successfully separated from other anionic isoperoxidases expressed in the same tissue. 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 optimum pH (pH 3 for ABTS, pH 4 for OPD and pH 6 for the others) and toward the reduction of H2O2. Its very acid optimum pH for the oxidation of ABTS is not a characteristic of other peroxidases except African oil palm tree peroxidase. 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 pH8. This peroxidase isoenzyme is stable for 4 months at room temperature, loosing only 5% of its initial activity over this period. 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 IC50 value around 10 µM. This inhibition, in addition to the RZ value (A403nm/A280nm) evaluated at 2.4 confirms the presence at the active site of the enzyme of a heme group common to class III peroxidases. The unusual catalytic and thermal characteristics of A6 could make it a potent tool in several biotechnological applications, especially as part of kit for enzyme immunoassays and clinical diagnosis.