ABSTRACT
Peroxidase activity from G. latifolium was done to see whether it could be used in industries. Crude peroxidase was extracted from G. latifolium with 0.05M sodium phosphate buffer of pH 6.0; 70% ammonium sulphate saturation to precipitated protein with the highest G. latifolium peroxidase activity. After gel filtration, two major peaks were seen and the active fractions were pooled differently together and characterized. The optimal pH for the enzyme peaks actually were found to be 6.5 and 6.0 and the optimum temperature was 30 and 40ºC for peak A and B respectively. The Michealis-Mentenconstant (Km) and maximum velocity (VmaxÂ) obtained from the Lineweaver-Burk plot of initial velocity of different substrate concentration were found to be 1.242 mM and 20.83 U/min for hydrogen peroxide concentration [H2O2] and 0.109 mM and 10.99 U/min for o-dianisidine concentrations. On the thermal stability assessment of the enzyme. Thermal inactivation profiles of these enzyme peaks follows first order kinetics with the time required varying with the product of the studies. The half-lives of the enzyme at the two peaks were obtained to be 770.16 mins at 30ºC for peak A and 330.07 mins at 40ºC for peak B, the activation energy for inactivation (Ea(inact)) calculated from the Arrhenius plot were found to be 67.55 KJmol-1 and 59.58 KJmol-1 forpeaks A and B, respectively. The Z-values were obtained to be 30.21 and 34.25 for the two enzyme peaks respectively. The thermodynamics parameters obtained for the two enzyme peaks were as follows: change in enthalpy of inactivation (ΔH(inact)) 65.026 KJmol-1K-1 for peak A at 30ºC and 56.982 KJmol-1K-1 at 40ºC for peak B; the change in free energy of inactivation, (ΔG(inact)) values for the two enzyme peaks were 102.229 KJmol-1K-1 at 30 ºC and 103.483 KJmol-1K-1 at 40ºC for peak A and B respectively. The entropy of inactivation (ΔS(inact)) values for the two enzyme peaks were calculated to be -0.1228 KJmol-1K-1 at 30ºC and -0.149 KJmol-1K-1 at 40ºC. Reactivation of the Gongronema latifolium peroxidase occurred rapidly, within first 30 minutes after the heated enzyme was cooled and incubated at room temperature. The extent of reactivation varied from 0 to 20% depending on the isoenzyme and heating conditions (temperature and time). The denaturation temperature allowing the maximum reactivation was 50°C and 40°C for peaks A and B respectively. In all cases, heat treatment at high temperatures for a long period prevented reactivation of the heated enzymes. The peak A peroxidase regained activity rapidly, within 30 minutes at 30 and 40°C and within 60 minutes at 50, 60, 70 and 80°C after the heated enzyme was cooled and incubated at room temperature. However, peak B peroxidase regained activity rapidly within 60 minutes at all the study temperature after the heated enzyme was cooled and incubated at room temperature.The kinetic and thermodynamic parameters and higher activation energies from this study suggest that this enzyme could be more suitable for several industrial applications.
