CHARACTERISTICS OF PEROXIDASE FROM CASSAVA LEAVES Manihot esculenta Crantz cv. KU50 AND ITS APPLICATION IN POLYCHLORINATED BIPHENYL DEGRADATION AND THIOL DETECTION BY VOLTAMMETRY

Abstract

Cassava peroxidase (CSP) isolated from cassava leaves (Manihot esculenta Crantz of cv. KU50) was purified by column chromatography. It was a haem-containing cationic glycoprotein with the molecular weight of 38 and 44 kDa determined by MALDI-TOF-MS. Its characteristics and kinetic catalysis were investigated and compared to those of horseradish peroxidase (HRP). It was found that CPS showed higher stability at 70°C in 0.1 M phosphate buffer pH 6 and more tolerant in the buffer containing 50% of acetonitrile than those of HRP. In preliminary study, the inhibitory effect of thiourea on peroxidases was investigated and then the possible use of peroxidases for the detection of thiol compounds such as thiourea and thiram by voltammetry was studied. It was based on enzyme inhibition assay and square-wave voltammetric measurement of the peak height at the potential of -0.24 V versus Ag/AgCl electrode was performed in 0.04 M Britton-Robinson buffer, pH 6 containing urea hydrogen peroxide and 3, 3ʹ-diaminobenzidine. It was found that the peak height was inversely proportional to the concentration of thiol and the relationship could be used for the detection of thiourea and thiramin in the range of 5-100 µM. In addition, the catalytic potency of CSP for the enhancement of polychlorinated biphenyl (PCB) such as 2, 4, 4ʹ-trichlorobiphenyl (PCB-28) degradation was carried out. PCB-28 concentration of 2.2 µM was clearly decreased to 50% of its initial concentration with the higher rate than HRP catalysis in 0.04 M Britton-Robinson buffer, pH 6 during 6 hours at room temperature. From these results, it can be concluded that CSP is a novel peroxidase with thermal and acetonitrile tolerant properties which can potentially be applied as a valuable tool in bioremediation and other biotechnological applications.