PURIFICATION AND CHARACTERIZATION OF PAPAIN FROM CARICA PAPAYA LATEX: ITS APPLICATION IN THE HYDROLYSIS OF TIGERNUT PROTEIN HOMOGENATE BY OMEJE, KINGSLEY OZIOMA (PG/M.Sc/12/61361) DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA NSUKKA JULY, 2014 PURIFICATION AND CHARACTERIZATION OF PAPAIN FROM CARICA PAPAYA LATEX; ITS APPLICATION IN THE HYDROLYSIS OF TIGERNUT PROTEIN HOMOGENATE A RESEARCH PROJECT SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (M.Sc.) IN ENZYMOLOGY AND PROTEIN CHEMISTRY, DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA, NSUKKA BY OMEJE, KINGSLEY OZIOMA PG/M.Sc/12/61361 DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA, NSUKKA. SUPERVISOR.DR. S.O.O. EZE MAY, 2014. i CERTIFICATION Omeje, Kingsley Ozioma, a postgraduate student of the Department of Biochemistry with the Registration number PG/MSc/12/61361 has satisfactorily completed the requirement of research work for the Degree of Master of Science (M.Sc.) in Enzymology and Protein Chemistry. The work embodied in this project is original and has not been submitted in part or full for any other diploma or degree of this or any other University. _______________ _________________ Dr. S.O.O. Eze Prof. OFC Nwodo Supervisor Head of Department ___________________ External Examiner ii DEDICATION To my parents.Elder and Mrs P. E. Ugwudah. iii ACKNOWLEDGEMENT My unquantifiable gratitude goes to my supervisor Dr. S. O. O. Eze. Sir, you were not just a supervisor, you were a father, and a teacher. There is no volume of written work that is enough to show my appreciation for all your contributions throughout the period of this research work. Your fatherly advice, encouragement, openness and response to my challenges and useful suggestion are acknowledged, above all you taught me the principle of independence. Master, may God continue to bless, guide and protect you and your family. Also appreciated are all members of Biochemistry staff. I am thankful to the Head, Department of Biochemistry, Prof. O. F. C. Nwodo and to Prof. L. U. S. Ezeanyika, Prof. F. C. Chilaka, Prof, P. N. Uzoegwu, Prof. I. N.E, Onwurah, Prof. O. U. Njoku, Prof. H. A. Onwubiko, Dr V.N. Oguguo, Dr O.C. Enechi, Dr. C. S. Ubani, Dr. C. A. Anosike, Mrs. U. O. Njoku, Mr. P. A.C Egbuna, Dr. V.O.E. Ozougwu and Mr. O. E. Ikwuagwu, I thank the Chief Technologist, Mrs M. Nwachukwu, and the Technician in charge of the Departmental instrument Room, Mr Jude Chime. God bless you all. Greatly appreciated are Prof. E.O. Alumanah, Dr. B. C. Nwanguma and Dr. P. E. Joshua for their financial and moral support, May God replenishes your pockets a million times. I thank immensely the Dean, School of Postgraduate Studies, Prof. A.A. Ubachukwu, Sir may God bless you. My gratitude goes to the Dean of Biological Sciences, Prof. F. C. Chilaka for his support for the timely completion of the programme. I am ever grateful to members of the you 2012/2013 Postgraduate class for giving me the opportunity of being your chief servant, especially Ejembi Daniel, Odiba Solomon, Iroha Okechukwu, Vivian, Chidimma, Okuda Frank, Onons. Ebere, Ifeoma Ubachukwu, Som, chidiogo, GD, Obi, B. C. and Nnolim you guys are good. To my parents Elder and Mrs P.E. Ugwudah, and my siblings, Sunny, Joy, Queen, Emezie and Juliet may God bless you all. I thank Juliet Ugwu for typing this work. Finally, I remain grateful to God for giving me life. iv ABSTRACT In this study, the production of tiger nut suspension was carried out using a proteolytic enzyme (papain) isolated from the latex of C. papaya. The crude papain isolated was subjected to three steps purification system of 80% ammonium sulphate saturation using sephadex G50 and G200 filtrations at pH 7.2 and 37oC. The protein concentration of the crude enzyme obtained was 136 µg/ml, while its specific activity was 1.15U/mg. After 80% ammonium sulphate precipitation, the specific activity obtained was 1.31U/mg. Sephadex G50 and G200 filtrations gave specific activities of 1.48 and 1.28U/mg respectively. The optimal activity of papain was achieved at 90oC and pH 7.5 at 37oC and 1ml of 1% casein solution. The Vmax and Km were observed to be 1.133U/min/ml and 0.217µg/min/ml respectively. Pure papain obtained was used to hydrolyse tiger nut protein at 37oC and pH 7.5 compared with O-pthalaldehyde as a standard hydrolysing agent. The degree of hydrolysis was monitored with tiger nut protein concentrations ranging from 0.1-1.0g/ml and incubation times of 0, 10, 30, 60 and 120min at 340nm. The results obtained from this study suggest that the optimum incubation time for papain to hydrolyse tiger nut protein is 10min at pH 7.5 and 37oC and also, suggests that papain hydrolyses plant derive protein more than O- pthaldidehyde (OPA). All results obtained from this study suggest that it is highly promising to use papain extracted from unripe C. papaya as a proteloytic enzyme in the hydrolysis of tiger nut protein preparation to fortify and enrich the milk like beverage produced from tiger nut with amino acids at mild industrial conditions. v 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 vi 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 vii 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 viii CHAPTER FOUR: DISCUSSION 4.1 Discussion - - - - - - - - 60 4.2 Conclusion - - - - - - - - 64 REFERENCES - - - - - - - - 66 APPENDICES - - - - - - - - 79 ix LIST OF FIGURES Fig. 1: Amino acid composition of papain - - - - - - 9 Fig. 2: Three- Dimensional structure of papain mechanism - - - - 10 Fig. 3: L and R domain of papain - - - - - - - 12 Fig. 4: Papain mechanism of hydrolysis - - - - - - 13 Fig. 5: Ammonium sulphate precipitation profile for papain from C. papaya latex - 39 Fig. 6: Protein concentration at different purification steps - - - - 40 Fig. 7: Effect of pH change on papain activity - - - - - 42 Fig. 8: Effect of temperature changes on papain activity - - - - 43 Fig. 9: Lineweaver-Burk plot of papain from C. papaya latex - - - 44 Fig. 10: Effect of substrate concentration on papain activity - - - 45 Fig. 11: % yield of papain at different purification steps - - - - 46 Fig. 12: Sephadex G50 gel filtration graph - - - - - 47 Fig. 13: Sephadex G200 gel filtration graph - - - - - - 48 Fig. 14: Papain activity at different purification steps - - - - 49 Fig. 15: Degree of Hydrolysis against time at 0.1g/ml substrate concentration - 50 Fig. 16: Degree of hydrolysis against time at 0.2g/ml substrate concentration - 51 Fig.