Purification of peroxidase enzyme from cauli̇flower by affi̇ni̇ty chromatography techni̇que and investigation of inhibition kinetics

Within the scope of this thesis, the purification of Cauliflower (Brassica oleracea var. botrytis L.) peroxidase enzyme using 4-amino 3-bromo 2-methyl benzohydrazide molecule was carried out for the first time and in a single step by affinity chromatography technique. The inhibition type for the Cauliflower peroxidase (POD) enzyme of the 4-amino 3-bromo 2-methyl benzohydrazide molecule we used was investigated. Purification of this enzyme was carried out with the affinity gel obtained by binding this molecule to the Sepharose-4B-L-Tyrosine matrix, which showed a reversible inhibition effect. The purity of the Cauliflower POD enzyme purified from this affinity gel was checked by the SDS-PAGE technique and its molecular weight was determined. A single band at 44 kDa was observed for the cauliflower POD enzyme. Cauliflower POD enzyme was purified 562 fold with a yield of 15.4% with 4-amino 3-bromo 2-methyl benzohydrazide molecule used as a ligand in purification by affinity technique. The 4-amino 3-bromo 2-methyl benzohydrazide inhibitor exhibited a competitive type of inhibition for Cauliflower peroxidase. In this study, which was carried out to determine the Ki and IC50 values for the guaiacol substrate, the Ki value for the Cauliflower POD enzyme was calculated as 0.113±0.012 mM and the IC50 value as 0.196 mM. In addition, in this study, the usage area of peroxidase enzyme, its importance in terms of industry, purification methods and the products from which it is obtained were mentioned. The peroxidase working methods in the literature and the sources from which it was obtained were examined in detail.