Purification and characterization of a novel peroxidase from pepper (Piper nigrum L.)

Peroxidase (POD) isolated from Piper nigrum L. was tested against various substrates including pyrogallol, catechol, and guaiacol. It was determined that POD in its monomeric form has a molecular weight and theoretical isoelectric point calculated to be 37.43 kDa and 7.62, respectively, based on native PAGE and the amino acid sequence. Kinetic parameters, such as K m and V max , were determined for pyrogallol, catechol, and guaiacol, with pyrogallol being the most effective substrate. The enzyme exhibited peak activity at pH 5.0 to 7.0 and temperatures between 20°C and 60°C. However, activity significantly declined after exposure to 80°C for 15 min. The presence of K + , Ba 2+ , citric acid, malic acid, benzoic acid, and EDTA•Na 2 enhanced POD activity, while Zn 2+ , Cu 2+ , Fe 3+ , glutathione, cysteine, and ascorbic acid inhibited it. The thermal inactivation of POD at temperatures from 60 to 80°C over 30 min was accurately described using the Weibull model. A three-dimensional homology model of POD was constructed using SWISS-MODEL and evaluated with the SAVES system. Molecular docking was subsequently qualified using AutoDock. The binding affinities of three substrates including pyrogallol, catechol, and guaiacol were evaluated using binding free energies, and key residues identified, which acquired a deep insight into the workings of enzyme-substrate interactions. • A novel of peroxidase (POD) was isolated from Piper nigrum L. • The molecular weight of POD was 37.43 kDa. • POD exhibited the highest affinity for pyrogallol. • Docking results revealed the dynamics between POD and its substrates.