Purification of black rudish peroxidase (POD) enzyme and investigation of inhibition kinetic with benzohydrazides

In this study, it was aimed to establish a purification procedure in one step for black rudish Peroxidase (POD) enzymes by using 3-amino benzohydrazide and its derivatives. which are the inhibitors of the POD enzymes. In this aim, in order to contribute to the purification studies in the literature of plant peroxidase enzymes, 3-amino benzohydrazide and its derivatives which are 5-amino 2-methyl benzohydrazide, 3-amino 5-methyl benzohydrazide, 3-amino 4-nitro benzohydrazide and 3-amino 2-methyl benzohydrazide molecules were synthesized and firstly used at the study of purification of POD enzyme. The inhibition effects on POD enzyme were investigated by making hydrazine synthesis from the methyl benzoate forms of the 3-amino benzohydrazide derivative molecules. The purification of the single-stranded black rudish Peroxidase (POD) enzyme with affinity gels obtained by binding Sepharose-4B-L-Tyrosine gel material of molecules which have alternating inhibition effect was performed. The black rudish POD enzyme is purified using 3-amino benzohydrazide molecule with a yield of 11.9%, 246.6 fold, using 5-amino 2-methyl benzohydrazide molecule with a yield of 43%, 733.5 fold, using 3-amino 5-methyl benzohydrazide with a yield of 6.09%, 221.2 fold, using 3-amino 4-nitro benzohydrazide with a yield 14.6 fold 249.6 fold, using 3-amino 4-nitro benzohydrazide with a yield of 14.6%, 249.6 fold and using 3-amino 2-methyl benzohydrazide molecule with a yield of 21.4%, 172.5 fold. The molecular weight and enzyme purity of the POD enzymes purified from all the column materials were controlled by SDS-PAGE. Single band were observed at 43-47 kDa for the black rudish peroxidase enzyme. The purification results of all the column material were compared and the column holding capacity for Sepharose-4B-L-Tyrosine-5-amino-2-methyl benzohydrazide showing the result of being the best column, for POD were studied different pH, temperature and ionic strengths. Optimum parameter for radish POD were determined as 7.0 for pH, 15°C for temperature, 0.4 M for ionic intensity.