We have developed efficient procedure for isolation of horseradish peroxidase (HRP) using aminobenzohydrazide‐based affinity chromatography. Sepharose 4B‐bounded aminobenzohydrazides are suitable for long‐term use and large‐scale purification. In this study, 26 aminobenzohydrazide derivatives were synthesized, characterized and defined as new HRP inhibitors. In addition, detailed inhibition effects of these molecules on HRP enzyme were investigated. Affinity matrix was formed by bonding aminobenzohydrazides, which exhibited inhibitory activity to sepharose‐4B‐l‐tyrosine. HRP was isolated from crude homogenate in single step and purification factors were recorded as 1,151‐fold (recovery of 8.5%) with 4‐amino 3‐bromo benzohydrazide and as 166.16‐fold (recovery of 16.67 %) with 3‐amino 4‐chloro benzohydrazide.

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In this study, a new affinity process was developed for the purification of Lactoperoxidase with synthesized sixteen aminobenzohydrazide derivatives. For this purpose, ligands were covalently bound to CNBr-activated Sepharose-4BÀ L-tyrosine matrix and affinity columns were prepared, and LPO was purified in one step with high yield and purity. Among all synthesized molecules, the 4-amino-3-bromo-2-methylbenzohydrazide molecule had a high usable potential in the purification of Lactoperoxidase from mammalian milk. Lactoperoxidase was purified 411.8 times with a yield of 17.38 % from goat milk, 187.25 times with a yield of 9.72 % buffalo milk, 2772.4 times with a yield of 18.98 % from bovine milk, and 1246.65 times with a yield of 4.43 % from sheep milk. It was demonstrated for the first time that aminobenzohydrazide molecules could be used as ligands in the purification of Lactoperoxidase enzyme.

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Lactoperoxidase enzyme (LPO) is secreted from salivary, mammary, and other mucosal glands including the bronchi, lungs, and nose, which had functions as a natural and the first line of defense towards viruses and bacteria. In this study, methyl benzoates were examined in LPO enzyme activity. Methyl benzoates are used as precursors in the synthesis of aminobenzohydrazides used as LPO inhibitors. For this purpose, LPO was purified in a single step using sepharose‐4B‐l‐tyrosine‐sulfanilamide affinity gel chromatography with a yield of 9.91 % from cow milk. Also, some inhibition parameters including the half maximal inhibitory concentration (IC50) value and an inhibition constant (Ki) values of methyl benzoates were determined. These compounds inhibited LPO with Ki values ranging from 0.033±0.004 to 1540.011±460.020 μM. Compound 1 a (methyl 2‐amino‐3‐bromobenzoate) showed the best inhibition (Ki=0.033±0.004 μM). The most potent inhibitor (1 a) showed with a docking score of −3.36 kcal/mol and an MM‐GBSA value of −25.05 kcal/mol, of these methyl benzoate derivatives (1 a–16 a) series are established H‐bond within the binding cavity with residues Asp108 (distance of 1.79 Å), Ala114 (distance of 2.64 Å), and His351 (distance of 2.12 Å).