Isolation, partial purification and stability studies of manganese peroxidase from white rot basodiomycetes, pleurotus tuber-regium
Table Of Contents
Chapter ONE
1.1 Introduction1.2 Background of Study
1.3 Problem Statement
1.4 Objectives of Study
1.5 Limitation of Study
1.6 Scope of Study
1.7 Significance of Study
1.8 Structure of the Research
1.9 Definition of Terms
Chapter TWO
2.1 Overview of Manganese Peroxidase2.2 Biological Functions of Manganese Peroxidase
2.3 Enzymatic Properties of Manganese Peroxidase
2.4 Production of Manganese Peroxidase
2.5 Applications of Manganese Peroxidase
2.6 Factors Affecting Manganese Peroxidase Activity
2.7 Comparative Studies on Manganese Peroxidase
2.8 Stability Studies of Manganese Peroxidase
2.9 Role of Manganese Peroxidase in White Rot Basidiomycetes
2.10 Future Directions in Manganese Peroxidase Research
Chapter THREE
3.1 Research Methodology Overview3.2 Selection of Basidiomycetes Samples
3.3 Isolation and Partial Purification Techniques
3.4 Characterization of Manganese Peroxidase
3.5 Stability Testing Methods
3.6 Data Collection and Analysis Procedures
3.7 Experimental Design and Setup
3.8 Statistical Analysis Methods
Chapter FOUR
4.1 Analysis of Isolated Manganese Peroxidase4.2 Purification Efficiency Evaluation
4.3 Enzyme Activity Assays
4.4 Stability Studies Results
4.5 Comparison with Other Peroxidases
4.6 Discussion on Enzyme Characteristics
4.7 Implications of Findings
4.8 Future Research Directions
Chapter FIVE
5.1 Summary of Research Findings5.2 Conclusion on Manganese Peroxidase Study
5.3 Contributions to the Field
5.4 Practical Applications and Recommendations
5.5 Limitations of the Study
5.6 Suggestions for Future Research
Project Abstract
Manganese peroxidase (MnP) enzyme is the most common lignin-modifying peroxidase produced by almost all wood-colonizing basidiomycetes causing white-rot and various soil-colonizing litter-decomposing fungi. Multiple forms of this glycosylated haeme protein with molecular weight normally at 40 to 50 kDa are secreted by ligninolytic fungi into their micro-environment. In the present study, a ligninolytic fungus was able to produce manganese peroxidase. The organism was identified as Pleurotus tuber-regium. Parameter such as pH, temperature, carbon source, nitrogen source wereassayed and characterized for better production of manganese peroxidase. The mass production of the enzyme was carried out using optimized conditions and the activity and specific activity was found to be 1.50U/ml and 0.76U/mg, respectively. Seventy percent ammonium sulphate precipitation was found suitable to precipitate protein with higher peroxidase activity. After ammonium sulphate precipitation, the specific activity was found to be 0.77U/mg. After gel filtration, two peaks were obtained with specific activities 2.75U/mg and 2.09U/mg specifically for peak A and B. The optimum pH for the two peaks were 4.5 and 5.0, respectively with optimum temperature of 40oC. The pH and temperature stability studies showed that the purified enzyme had a residual activity within the range of 80 to 70% after pre-incubation for 120 min for pH of 4.5 and temperature of 40oC.The kinetic parameters, maximum velocity (Vmax) and Michaelis Menten constant(Km)obtained from Lineweaver-Burk plot of initial velocity data and V0 at different substrate concentrations [S] were found to be 0.08mg/ml and 0.69µmol/min, respectively using H2O2 as substrate for peak A. After using phenol red, Km and Vmax were 0.08mg/ml and 1.49µml/min. More so, for peak B, Km and Vmax were 0.18mg/ml and 0.96µmol/min using H2O2 as substrate while 0.08mg/ml and 1.46µmol/min were obtained using phenol red as substrate, respectively.
Project Overview
Manganese peroxidase (MnP) enzyme is the most common lignin-modifying peroxidase produced by almost all wood-colonizing basidiomycetes causing white-rot and various soil-colonizing litter-decomposing fungi. Multiple forms of this glycosylated haeme protein with molecular weight normally at 40 to 50 kDa are secreted by ligninolytic fungi into their micro-environment. In the present study, a ligninolytic fungus was able to produce manganese peroxidase. The organism was identified as Pleurotus tuber-regium. Parameter such as pH, temperature, carbon source, nitrogen source wereassayed and characterized for better production of manganese peroxidase. The mass production of the enzyme was carried out using optimized conditions and the activity and specific activity was found to be 1.50U/ml and 0.76U/mg, respectively. Seventy percent ammonium sulphate precipitation was found suitable to precipitate protein with higher peroxidase activity. After ammonium sulphate precipitation, the specific activity was found to be 0.77U/mg. After gel filtration, two peaks were obtained with specific activities 2.75U/mg and 2.09U/mg specifically for peak A and B. The optimum pH for the two peaks were 4.5 and 5.0, respectively with optimum temperature of 40oC. The pH and temperature stability studies showed that the purified enzyme had a residual activity within the range of 80 to 70% after pre-incubation for 120 min for pH of 4.5 and temperature of 40oC.The kinetic parameters, maximum velocity (Vmax) and Michaelis Menten constant(Km)obtained from Lineweaver-Burk plot of initial velocity data and V0 at different substrate concentrations [S] were found to be 0.08mg/ml and 0.69µmol/min, respectively using H2O2 as substrate for peak A. After using phenol red, Km and Vmax were 0.08mg/ml and 1.49µml/min. More so, for peak B, Km and Vmax were 0.18mg/ml and 0.96µmol/min using H2O2 as substrate while 0.08mg/ml and 1.46µmol/min were obtained using phenol red as substrate, respectively.
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