Thesis Abstract

This study was carried out to screen, partially purify and characterize Manganese peroxidase from Rigidoporuslignosus. This study started with the optimization of enzymes production in the laboratory scale of submerged fermentation system.A pilot study was carried out for eight days to determine the day of highest Manganese peroxidase activity of which Day 7 wasthe highest. The optimal yield of Manganese peroxidase (0.888 U/ml) was found to be produced under the conditions of 20 mL of synthetic medium (containing (g/L) glucose, 10.0; NHâ‚„NO₃, 2.0; KHâ‚‚POâ‚„, 0.8; Naâ‚‚HPOâ‚„, 0.4; MgSOâ‚„ · 7Hâ‚‚O, 0.5; yeast extract, 2.0. pH 6.1) with 5% of glucose as the carbon sources, and with microelements (ZnSOâ‚„ · 7Hâ‚‚O, 0.001 g/L; FeSOâ‚„ · 7Hâ‚‚O, 0.005 g/L; CaClâ‚‚ · 2Hâ‚‚O, 0.06 g/L; CuSOâ‚„ · 7Hâ‚‚O, 0.05 g/L; MnSOâ‚„ · Hâ‚‚O, 0.05 g/L) with an initial pH of 4.5, and 4 cork borer of the pure culture of Rigidiporuslignosus, The specific activities for the crude enzyme was found to be 0.399 U/mg. Ammonium sulphate (80%) saturation was found suitable to precipitate protein with highest MnP activity. After ammonium sulphate precipitation and gel filtration, the specific activity was found to increase from 3.178U/mg protein to 1.707U/mg protein for fraction A with the purification of 4.28, while that for fraction B increased from 3.178 to 4.04U/mg protein with purification fold of 10.14. The optimum pH and temperature were found to be 5.0 and 50°C respectively. The Michealis-Menten constant, Kmand maximum velocity, Vmax obtained from Line-Weaver-Burk plot of initial velocity data at different substrate concentrations were found to be 1.102mg/ml and 11.561 U/ml using H2O2, 0.76mg/ml and 19.65U/ml using phenol red as substrate.Kinetics of MnP inactivation was studied over temperature range of 30- 70°C. The inactivation kinetics followed a biphasic pseudo first-order model with k values between 4.2×10-3 – 1.79×10-2 min-1. The decreasing trend of k values with increasing temperature indicates a faster inactivation of manganese peroxidase from Rigidiporuslignosusat higher temperatures. The activation energy (Ea) of 28.43kJ/mol was calculated from the slope of Arrhenius plot. Thermodynamic parameters (∆H, ∆G, ∆S) for inactivation of manganese peroxidase at different temperatures (30-70°C) were studied in detail.In conclusion, the results of this present study indicates that manganese peroxidase will be a good enzyme for delignification with a high capacity to remove xenobiotic substances and produce polymeric products which are useful in bioremediation.

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