Purification of peroxidase enzyme from cowpea (Vigna unguiculata) using affinity technique and, characterization study

In this thesis, a single step purification of cowpea (Vigna unguiculata) peroxidase enzyme was carried out for the first time by using amino benzohydrazide derivative molecules-based affinity chromatography technique and biochemical characterization of the purified enzyme was performed. 53,6% yield and 91,6 purification fold were achieved in a single chromatographic step using Sefaroz 4B-L-tyrosine-4-amino-3-bromo-2-methylbenzohydrazide affinity gel. A single band at 39 kDa was observed in SDS-PAGE performed to determine the purity and molecular weight of the obtained enzyme. To analyze the interactions of 4-amino-3-bromo-2-methylbenzohydrazide, 4-amino-3-bromo-5-fluorobenzohydrazide, 4-amino-3-bromo-5-chlorobenzohydrazide and 5-aminoisophthalohydrazide used as ligands in purification with the enzyme, the inhibition effect of each molecule on cowpea POD was assayedand the Ki value for 4-amino-3-bromo-2-methylbenzohydrazide, the most effective ligand in purification, was calculated as 970±130 µM. Within the scope of enzyme characterization studies, optimum conditions for enzyme; It was found that the pH was 6,0, the ionic strength was 0,3 M and the temperature was 40 ºC. Additionally, kinetic studies were carried out to determine the affinity of cowpea POD with guaiacol, H2O2, 4-methylcatechol and ABTS substrates, and the KM values of these substrates were calculated as 7,21 mM, 17,03 mM, 7.28 mM and 49,21 mM, respectively.