Purification et caractérisation biochimique d’une peroxydase thermostable du pois Bambara (Vigna subterranea (L.) Verdc) en vue de son application en biotechnologie

Abstract

Peroxidases are of a great biotechnological interest because of their utility as catalysts in clinical biochemistry and in diverse industries. In our study, we obtained a crude peroxidase extract with very great thermostability from a Bambara groundnut (Vigna subterranea (L.) Verdc) phenotype, cultivated in the far-north region of Cameroon (very hot region, with monthly average temperatures of approximately 29°C). We purified and characterized a thermostable peroxidase isoenzyme from radicles of that landrace. That peroxidase has been successfully separated from several other less active isoperoxidases expressed in the same tissue. It is a single polypeptide and has a molecular weight of around 40,1 kDa and exhibit 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 for African oil palm tree peroxidase. Apparent Km values for the reducing substrates are respectively 3.50 mM, 0.12 mM, 1.81 mM, 0.05 mM and 17.22 mM for a saturating concentration of H2O2 (30 mM) and 2.53 mM for H2O2, at a saturating concentration of ABTS (9 mM) ; catalytic efficiencies are 5.12×104mM-1 .min-1, 2.22×106mM 1.min-1, 1.59×105mM-1.min-1, 1.82×105mM-1.min-1, 3.17×105mM-1.min-1and 1.79×106mM 1.min-1 respectively. 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. This peroxidase isoenzyme is stable for 4 months at room temperature, loosing only 5% of its initial activity over this period. The Ca2+ ions greatly increase the stability of this peroxidase at 80°C. However, Mn2+and Zn2+ reduce that thermostability. The enzyme is inhibited by sodium azide at concentrations above 1 µM with an IC50 value around 10 µM, for an enzyme concentration of 1 unit/ml. 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 this peroxidase could make it a potent tool in several biotechnological applications, especially as part of kit for enzyme immunoassays and clinical diagnosis.