Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Abstract

Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.

Naked-eye sensitive ELISA-like assay based on gold-enhanced peroxidase-like immunogold activity

“…AuNPs were synthesized according to the reported method with slight modifications [7]. All glassware used in the preparation procedure were bathed in freshly prepared aqua regia (1:3 (v/v) HNO 3 -HCl), thoroughly rinsed with doubledeionized water, and dried in an oven prior to use.…”

“…For example, horseradish peroxidase (HRP) is one of most common enzymes used in the ELISA since it serves as a catalyst to facilitate a series of biological processes [4,5]. However, natural enzyme-labeled systems have some inevitable shortcomings, such as easy denaturation and digestion by proteases, and complicated preparation, purification, and storage processes, which severely limit their routine use in diagnosis [6,7]. Hence, it is necessary to synthesize new non-natural enzymes with high catalytic activity to overcome the restrictions of natural enzymes.…”

A Defense Associated Peroxidase from Lemon Having Dye Decolorizing Ability and Offering Resistance to Heat, Heavy Metals and Organic Solvents

“…The class III plant peroxidases have been reported to play crucial roles in the plant life cycle [13] such as cell wall metabolism [9], lignification [10], suberization [11], auxins metabolism [12], wound healing, reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism [13,14], fruit growth and ripening [15], defense against pathogens [16] etc.…”

Hydrophobic Interaction Chromatography: Fundamentals and Applications in Biomedical Engineering

“…Lavery et al (2010) reported the purification of a peroxidase from horseradish roots (Armoracia rusticana) by means of a three-step strategy, consisting of ultrasonication, ammonium sulfate precipitation, and HIC (using Phenyl Sepharose). In this strategy, the only high-resolution purification step corresponded to HIC.…”