A new approach for affinity‐based purification of horseradish peroxidase

Abstract

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.

A New Affinity Matrixe Synthesized from Aminobenzohydrazide Derivatives for Purification of Lactoperoxidase Enzyme

“…[16] 3-Amino benzohydrazide derivatives (10 a-12 a) and 2-amino benzohydrazide derivatives (13 a-16 a) were synthesized from methyl aminobenzoate forms according to described procedure. [17] Synthesis mechanisms were presented in Figure 1.…”

A novel peroxidase from runner bean ( Phaseolus coccineus L.): Enhanced affinity purification, characterization, and dye decolorization activity

“…Purification of plant PODs with aminobenzohydrazides is one of the current approaches that have attracted considerable attention from researchers (Somtürk, Kalın, & Özdemir, 2014). Plant‐origin PODs were purified 613‐fold with a yield of 33.8% from black radish, 781‐fold with a yield of 34.4% from sweet radish (Oztekin et al., 2019), and 166.2‐fold with a yield of 16.7% from horseradish roots (Almaz et al., 2020) by the monosubstituted 4‐aminobenzohydrazides‐based affinity chromatography. In our current report, disubstituted 4‐aminobenzohydrazides have been designed and synthesized for the first time to purify the RBP with high yield and purity.…”

“…In recent studies, PODs were separated from radish species and horseradish roots using monosubstituted 4‐aminobenzohydrazides as an affinity agent (Almaz et al., 2020; Oztekin et al., 2019). In this study, new disubstituted 4‐aminobenzohydrazide compounds [4‐amino‐3‐bromo‐2‐methylbenzohydrazide ( a ), 4‐amino‐3‐bromo‐5‐fluorobenzohydrazide ( b ) and 4‐amino‐3‐bromo‐5‐chlorobenzohydrazide ( c )] were designed and synthesized for the single‐step affinity separation of RBP.…”

“…In recent studies, PODs were separated from radish species and horseradish roots using monosubstituted 4-aminobenzohydrazides as an affinity agent (Almaz et al, 2020;Oztekin et al, 2019).…”

Evaluation of Peroxidase in Herbal Medicines Based on an Electrochemical Sensor

“…Non-denaturing discontinuous polyacrylamide gel electrophoresis (native PAGE) does not easily denature proteins. It essentially does not disrupt the natural conformation of proteins and the subunit interactions, so it maintains protein biological activity ( Rajhans et al, 2020 ; Almaz et al, 2021 ). This method is most widely used in the detection of plant peroxidases because it does not denature proteins.…”