EXTRACTION, PARTIAL PURIFICATION AND PRELIMINARY CHARACTERIZATION OF PERIOXIDASE USING OCIMUM GRATISSIMUM USING TRIS BUFFER

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TABLE OF CONTENTS
CERTIFICATION i
DEDICATION iii
ACKNOWLEDGEMENTS iv
TABLE OF CONTENTS v
LIST OF FIGURES viii
ABSTRACT ix
CHAPTER ONE 1
1.0 INTRODUCTION 1
1.1 Background of the Study 1
1.2 Statement of the problem 2
1.3 Justification 2
1.4 Aims and Objectives 3
CHAPTER TWO 4
2.0 LITERATURE REVIEW 4
2.1 Peroxidase 4
2.2 Classification of Peroxidases 5
2.3 Physiological Roles of Plant Peroxidases 10
2.3.1 Lignification and Suberization 11
2.3.2 Plant defense against pathogen infection 12
2.3.3 Wound healing 13
2.3.4 Reactive Oxygen Species (ROS) metabolism 13
2.3.5 Auxin catabolism 14
2.3.6 Seed germination 14
2.3.7 Fruit ripening 15
2.4 Applications of peroxidases 15
2.4.1 Application as biosensor 16
2.4.2 Application in analytical and diagnostic kits 17
2.4.3 Application in de-colorization of industrial dyes 18
2.5 Botany of Ocimum gratissimum 18
2.6 Morphology 19
2.7 Classification of O. gratissimum 20
2.8 Chemical Composition 21
2.9 Pharmacological Studies 22
2.9.1 Antimicrobial and Antifungal Activity 22
2.9.2 Ovicidal Activity 23
2.9.4. Antidiarrhoeal Effect 24
2.9.5 Gastro Intestinal Tract 24
2.9.6 Wound Healing 25
2.9.7 Anti-Inflammatory 25
2.9.8 Analgesic Activity 26
2.9.9 Antimutagenic 26
2.9.10 Cytotoxic Activity 26
2.9.11 Antidiabetic Effect 27
2.9.12 Hair Loss 27
2.9.13 Antioxidant Capacity 28
2.9.14 Suspending Activity 29
2.10 Clinical Studies 29
2.11 Toxicology 30
2.12 Nutritional and Flavouring Properties 31
2.13 Traditional Uses 32
CHAPTER THREE 34
3.0 MATERIALS AND METHODS 34
3.1 Materials 34
3.1.1 Equipment 34
3.1.2 Chemicals and Reagents 35
3.1.3 Sample Collection 35
3.2 Methods 35
3.2.1 Preparation of Buffer Solution 35
3.2.1.1 Tris buffer (stock solution) 35
3.2.1.2 Preparation of different pH using Acetate buffer 35
3.2.1.3 Extraction of Enzyme (Peroxidase) 36
3.2.2 Preparation of Enzyme Extract 36
3.2.3 Preparation of Substrate Solution 36
3.2.4 Peroxidase Assay using guaiacol as Substrate 37
3.2.5 Purification of Peroxidase from O. gratissimum Leaves 37
3.2.5.1 Ammonium Sulphate Precipitation Profile 37
3.2.6 Studies on the Purified Enzyme 38
3.2.6.1 Characterization of Enzyme 38
3.2.6.1.1Effect of pH on Peroxidase Activity 38
3.2.6.1.2 Effect of Temperature on Peroxidase Activity 38
3.2.6.2 Kinetic Study on the Partially Purified Enzyme 38
3.2.6.2.1 Effects of H2O2 on Peroxidase Activity 38
3.2.6.2.2 Effects of Different Concentrations of Guaiacol on Peroxidase Activity 38
3.2.6.4 Determination of Protein Concentration and Peroxidase Activity 40
3.2.7 Thermal Inactivation and Regeneration Studies 40
3.2.7.1 Determination of Percentage Residual Activity 41
CHAPTER FOUR 42
4.0 RESULTS AND DISCUSSION 42
4.1 Results 42
4.2 Partial Purification Characteristics 42
4.3 Preliminary Characterization Studies 42
4.3.1 Study of pH on peroxidase activity 42
4.3.2 Study of Wavelength on peroxidase activity of Ocimum gratissimum 43
4.3.3 Effect of temperature 44
4.4 Discussion 44
CHAPTER FIVE 46
DISCUSSION, CONCLUSION AND RECOMMENDATION 46
5.1 Conclusion 46
5.2 Recommendation 46
References 47
Appendices 54

LIST OF FIGURES
Figure 2.1 Schematic representation of classification of peroxidase 5
Figure 2.2 Schematic representation of various roles of plant perioxidases 10
Figure 2.3: Ocimum gratissimum plant 20
Figure 4.1: Plot of pH against Peroxidase Enzyme Activity 43
Figure 4.2: Plot of Wavelength against Peroxidase Enzyme Activity 43
Figure 4.3: Plot of Temperature against Peroxidase Enzyme Activity 44

CHAPTER ONE
1.0 INTRODUCTION

1.1 Background of the Study
Peroxidase, an antioxidative enzyme, is widely distributed in microbes, plants, and animal tissues and represents a heme-containing enzymes family (Huystee and Cairns 1982). This oxidoreductase catalyzes a reaction in which hydrogen peroxide acts as the acceptor and another compound acts as the donor of hydrogen atoms (Rodrigo et al. 1996). In the presence of peroxide, peroxidase from plant tissues are able to oxidize a wide range of phenolic compounds, such as guaiacol, catechol, pyrogallol, chlorogenic acid, and catechin (Onsa et al. 2004). This enzyme can provide value for multiple industrial applications, of which the most important ones include decolorization of waste (Jadhav et al. 2009), treatment of waste water containing phenolic compounds (Lai and Lin 2005; Dalal and Gupta 2007), and synthesis of various aromatic chemicals and removal of peroxides from food stuffs and industrial wastes (Kim and Yoo 1996; Saitou et al. 1991). In the biological field, e.g. as diagnostic kits for enzyme immunoassays and as an important component of ELISA system, this enzyme has also been widely used (Castillo et al., 2002; Deepa and Arumughan 2002).
Ocimum gratissimum is a herbaceous plant which belongs to the Labiatae family. The plant is indigenous to tropical areas especially India and it is also in West Africa. In Nigeria, it is found in the Savannah and coastal areas. It is cultivated in Ceylon, South Sea Islands, and also within Nepal, Bengal, Chittagong and Deccan. It is known by various names in different parts of the world. In India it is known by its several vernacular names, the most commonly used ones being Vriddhutulsi (Sanskrit), Ram tulsi (Hindi), Nimma tulasi (Kannada). In the southern part of Nigeria, the plant is called “effinrin-nla” by the Yoruba speaking tribe. It is called “Ahuji” by the Igbos, while in the Northern part of Nigeria, the Hausas call it “Daidoya”. The present of peroxidases have been reported to play diverse functions in this plant life cycle such as in cell wall metabolism, lignification, suberization, ROS metabolism, wound healing, fruit growth and ripening, seed germination etc. (Passardi et al., 2005).

1.2 Statement of the problem
Development of environmentally sustainable processes is a challengeable task for the current bioeconomy. In this direction, the use of biocatalysts, enzymes, in various processes is considered as an ecofriendly approach. The stability, activity and specificity of enzymes are the fundamental parameters that are required to develop enzymes for their optimal applications in various industrial processes. Therefore, identification of newer sources for such novel enzymes with desired properties is important. Furthermore, modern approaches such as characterization and purification to develop such novel enzymes with improved attributes are also in great demand.

1.3 Justification
Peroxidases are important from the point of view of their industrial applications by virtue of their ability to catalyze the oxidation-reduction reaction of a wide range of phenolic as well as non-phenolic substrates in the presence of hydrogen peroxide. A number of industrial applications of peroxidases have been reported in the area of agriculture, analytical, environmental, medical sectors etc. Thus, peroxidases have been used in bioremediation of contaminating environmental pollutants such as phenols, delignification in paper and pulp industry, diagnosis kit development, immunoassay, organic and polymer synthesis as well as in and biosensor technology. They are also used for developing convenient and quick methods for the determination and quantification of hydrogen peroxide in both the biological and industrial sample.
Therefore, this work will help the general public:
To know the constituents of Ocimum gratissimum
To know the optimum time for the enzyme activity
The findings of this study are expected to find application in drug producers (pharmaceuticals) would hopefully benefit from the findings of this study by fitting in for the purpose of drug production.

1.4 Aims and Objectives
This research work was aimed at the extraction, partial purification and characterization of perioxidase from low cost Ocimum gratissimum
In order to achieve the stated aim, the following objectives included to:

EXTRACTION, PARTIAL PURIFICATION AND PRELIMINARY CHARACTERIZATION OF PERIOXIDASE USING OCIMUM GRATISSIMUM USING TRIS BUFFER