Home / Biology edcuation / Isolation, partial purification and stability studies of manganese peroxidase from white rot basodiomycetes, pleurotus tuber-regium

Isolation, partial purification and stability studies of manganese peroxidase from white rot basodiomycetes, pleurotus tuber-regium

 

Table Of Contents


Chapter ONE

1.1 Introduction
1.2 Background of Study
1.3 Problem Statement
1.4 Objective 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 Peroxidase
2.2 White Rot Basidiomycetes and Pleurotus Tuber-regium
2.3 Enzymatic Properties of Manganese Peroxidase
2.4 Biological Functions of Manganese Peroxidase
2.5 Production and Purification Methods
2.6 Structural Characterization of Manganese Peroxidase
2.7 Applications of Manganese Peroxidase
2.8 Studies on Stability of Manganese Peroxidase
2.9 Comparative Analysis with Other Peroxidases
2.10 Future Research Directions

Chapter THREE

3.1 Research Design and Methodology
3.2 Sampling Techniques
3.3 Data Collection Methods
3.4 Experimental Setup
3.5 Analytical Techniques Used
3.6 Statistical Analysis Procedures
3.7 Quality Control Measures
3.8 Ethical Considerations in Research

Chapter FOUR

4.1 Overview of Research Findings
4.2 Isolation of Manganese Peroxidase
4.3 Partial Purification Techniques
4.4 Stability Studies of Manganese Peroxidase
4.5 Enzyme Kinetics Analysis
4.6 Structural Analysis Results
4.7 Comparative Studies with Other Basidiomycetes
4.8 Implications and Interpretations of Results

Chapter FIVE

5.1 Summary of Research Findings
5.2 Conclusions Drawn from the Study
5.3 Contributions to Existing Knowledge
5.4 Recommendations for Future Research
5.5 Reflections on the Research Process

Thesis 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.

Thesis 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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