Single-step purification of soybean peroxidase using Aminobenzohydrazide-based affinity chromatography matrices

Soybean peroxidase (SBP, EC 1.11.1.7) is an industrially important enzyme due to its high catalytic activity, remarkable thermostability, and broad application potential in biotechnology, food processing, polymer synthesis, and biosensor development. In this study, novel benzohydrazide derivatives, namely 4-amino-3-bromo-5-fluorobenzohydrazide, 4-amino-3-bromo-5-chlorobenzohydrazide, 4-amino-3-bromo-2-methylbenzohydrazide, and 5-aminoisophytalohydrazide, were synthesized, and their usability as affinity chromatography ligands was investigated. For this purpose, the inhibition kinetics of the synthesized molecules were studied, and enzyme-ligand interactions were evaluated using in silico methods. The estimated free binding energies of the molecules were determined to range from -5.21 to -5.83 kcal/mol. Soybean peroxidase enzyme was purified in a single step from soybean sprout leaves using affinity chromatography columns prepared with synthesized ligands, and the optimization of purification conditions was investigated in detail. According to the results obtained, soybean peroxidase was obtained from the affinity gel containing the 4-amino-3-bromo-5-fluorobenzohydrazide ligand with a purification factor of 130.2-fold and a yield of 8.71%. In addition, a purification factor of 133.8-fold and a yield of 4.3% were obtained with the affinity column designed using the determined 4-amino-3-bromo-2-methylbenzohydrazide ligand, which has the smallest K_İ value of 80.55 ± 12.6 μM. The results show that benzohydrazide-based affinity matrices offer an effective and economical strategy for the purification of SBP.