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PURIFICATION AND CHARACTERIZATION OF PAPAIN FROM CARICA PAPAYA LATEX: ITS APPLICATION IN THE HYDROLYSIS OF TIGERNUT PROTEIN HOMOGENATE

Topic Description

TABLE OF CONTENTS
Title page – – – – – – – – – – i
Certification – – – – – – – – – – ii
Dedication – – – – – – – – – – iii
Acknowledgement – – – – – – – – – iv
Abstract – – – – – – – – – – v
CHAPTER ONE: INTRODUCTION
1.1 Carica papaya – – – – – – – – – 1
1.1.1 Origin of C. papaya – – – – – – – – – 1
1.1.2 Taxonomy – – – – – – – – – – 2
1.1.3 Morphology- – – – – – – – – – 3
1.1.4 Importance of C. Papaya – – – – – – – – 3
1.2 Enzymes – – – – – – – – – 4
1.2.1 Enzyme structure – – – – – – – – – 5
1.2.2 Enzyme nomenclature – – – – – – – – – 6
1.7 Proteases (hydrolases) EC 3.4.- – – – – – – – 7
1.8 Cysteine proteases (3.4.22)- – – – – – – – 8
1.9 Papain (EC 3.4.22.2)- – – – – — – – – 9
1.10 Mechanism of action of papain – – – – – – – 12
1.11 Mechanism – – – – – – – – – – 14
1.12 Localisation and biosynthesis – – – – – – – – 15
1.2.5.3 Stability of papain – – – – – – – – 15
1.2.5.4 Papain specificity – – – – – – – – 16
1.2.5.5 Papain activators – – – – – – – – 16
1.2.5.6 Papain inhibitors- – – – – – – – – 16
1.2.5.7 Applications of papain – – – – – – – 17
1.3 Tiger nut (Cyperus esculentus var sativa) – – – – – 17
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1.3.1 Taxonomy of tiger nut – – – – – – – – 18
1.3.2 Origin of tiger nut Cyperus esculentus – – – – – – 19
1.3.3 Morphology – – – – – – – – – 19
1.3.4 Phytochemical composition of tiger nuts – – – – – 20
1.3.5 Economic importance of tiger nut – – – – – – 21
1.4 Protein – – – – – – – – – – 22
1.4.1 Classification of amino acids. – – – – – – – 22
1.4.2 Plant protein – – – – – – – – – 23
1.4.2.1 Importance of plant protein – – – – – – – 24
1.4.2.2 Enzymatic hydrolysis of protein- – – – – – – 24
1.5 Tiger nut milk – – – – – – – – – 25
1.6 Aim and objective of the study – – – – – – – 27
1.6.1 Aim of the study – – – – – – – – 27
1.6.2 Specific objectives of the study – – – – – – 27
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials – – – – – – – – – – 28
2.1.1 Carica papaya – – – – – – – – – 28
2.1.2 Equipment – – – – – – – – – 28
2.1.3 Chemicals – – – – – – – – – 29
2.2 Methods – – – – – – – – – – 30
2.2.1 Preparation of tiger nut sample – – – – – – 30
2.2.2 Preparation of tiger nut (C. esculentus) protein homogenate – – – 30
2.2.3 Extraction of papain from C. papaya latex – – – – – 30
2.2.4 Proximate analysis – – – – – – – – 30
2.2.4.1 Determination of moisture content – – – – – – 31
2.2.4.2 Determination of crude protein – – – – – 31
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2.2.4.3 Determination of crude fat – – – – – – – 33
2.2.4.5 Determination of ash content – – – – – – 33
2.2.4.5 Determination of crude fibre – – – – – – 33
2.2.4.6 Carbohydrate or nitrogen free extract (NFE) – – – – 34
2.2.5 Protein estimation – – – – – – – – 34
2.2.6 Purification of protein- – – – – – – – – 34
2.2.7 Assay of enzyme activity – – – – – – – 35
2.2.8 Purification of precipitated enzyme – – – – – – 36
2.2.8.1 Sephadex G50 gel purification- – – – – – – 36
2.2.8.2 Sephadex G200 gel purification- – – – – – – 36
2.2.9 Studies on the purified enzyme – – – – – – – 36
2.2.9.1 Effect of temperature change on papain activity – – – – 36
2.2.9.2 Effect of pH change on papain activity – – – – – 36
2.2.9.3 Effect of substrate concentration on papain activity – – – – 37
2.2.10 Hydrolysis of tiger nut protein by papain – – – – – 37
CHAPTER THREE: RESULTS
3.1 Proximate composition of tiger nut milk-like suspension – – – 38
3.2 Ammonium sulphate precipitation profile – – – – – 39
3.3 Studies on crude papain – – – – – – – 40
3.4 Purification table – – – – – – – – 41
3.5 Enzyme characterization – – – – – – – 42
3.5.1 Effect of pH change on papain activity.- – – – – – 42
3.5.2 Effect of temperature change on papain activity. – – – – – 43
3.5.3 Determination of kinetic parameters- – – – – – – 44
3.5.4 Effect of substrate on papain activity- – – – – – – 45
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CHAPTER FOUR: DISCUSSION
4.1 Discussion – – – – – – – – 60
4.2 Conclusion – – – – – – – – 64
REFERENCES – – – – – – – – 66
APPENDICES

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