Assessment of Kinetic Parameters of Peroxidase Isolated from Maturing Solanum lycopersicum Fruits for Analytical and Biotechnological Applications

Abstract

The wide application of peroxidase in biotechnology, food industries, environmental remediation and medical diagnosis has necessitated the interest for further research on the enzyme. This study investigated the kinetic parameters of maturing Solanum lycopersicum (tomato) fruit peroxidase with the prospect to ascertain its potentials and viability for analytical and biotechnological applications. Ammonium sulphate precipitation and gel filtration with sephadex G-100 were used to purify Sonalum lycopersicum peroxidase to homogeneity in two phases. Using o-dianisidine as a substrate, the optimal pH and temperature were found, while the Michaelis constant (Km) and maximum velocity (Vmax) were obtained using the Lineweaver Burk graph. The purification factor and specific activity of the crude enzyme were 2.16 and 55.5 μ/mg respectively. Maturing Solanum lycopersicum fruit peroxidase was purified to homogeneity via a dual-step purification phases of gel filtration preceded by ammonium sulphate precipitation with specific activities of 34.11 μ/mg and 117.20 μ/mg in that order. The substrate used for the reaction was o-dianisidine. The enzyme adhered to Michaelis-Menten kinetics with Michaelis constant and maximum velocity of 5.23 mg/mL and 12.27 μmol/min, respectively. Maturing Solanum lycopersicum fruit peroxidase showed sensitivity under a range of pH (6-8) and temperature (40-90oC) in its activity with 50°C and 5.9 as the temperature and pH optima, respectively. The result of this research has revealed that peroxidase from Sonalum lycopersicum exhibited physiochemical properties that are similar to what is obtainable in vivo which makes it suitable for analytical and biotechnological applications that in most cases mimics physiological conditions.