Project Abstract

Peroxidase (EC 1.11.1.7) extracted from Sonalum lycopersicon was purified, on a two-step purification process of ammonium sulphate precipitation and gel filtration. The specific activity of the crude enzyme was 55.45μ/mg. The crude enzyme was purified to the level of gel filtration using Sephadex-G100 via ammonium sulphate precipitation. After ammonium sulphate precipitation and gel filtration, the enzyme was purified 3.3 fold and the specific activities were 35.93μ/mg and 119.61μ/mg respectively when o-dianisidine was used as substrate. The optimum pH and temperature was found to be 6.0 and 50°C respectively. Kinetics of peroxidase inactivation was studied over temperature range of 40-80°C. The enzyme obeyed Michealis-Menten kinetics and the Km and Vmax values were calculated and found to be 5.44mg/ml and 12.57μmol/min respectively. Biphasic inactivation curves were observed for the enzyme, where the initial heat inactivation is rapid followed by much slower inactivation periods. The inactivation kinetics followed a first-order model with k values between 3.5×10-2 – 8.14×10-2 min-1 and z value of 25.5°C. The decreasing trend of k values with increasing temperature indicates a faster inactivation of peroxidase at higher temperature. The study has shown that peroxidase from Sonalum lycopersicon is stable at temperature of 40 and 50°C as activity was maintained above 50% for 2 hours and less stable at a high temperature of about 60oC and stability dropped drastically at 70 and 80oC within 10min of heat treatment suggesting that high temperature short time treatment could easily inactivate the enzyme. The activation energy (Ea) of 127.34KJMol-1K- was calculated from the slope of Arrhenius plot. Thermodynamic parameters (^H, G ^, ^S) for inactivation of peroxidase at different temperatures (40-80°C) were studied. The Peroxidase activity was found to be pH-dependent and was stable at pH range of 6—8 …………. result from this research has shown that peroxidase from Sonalum lycopersicon has high pH and thermal stabilities and hence, could be a good source of peroxidase for industries where high temperature and pH stabilities are required for production processess.

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