Purification and Characterization of Papain from Carica Papaya Latex: Its Application in the Hydrolysis of Tigernut Protein Homogenate

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

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

OMEJE, KINGSLEY OZIOMA

PG/M.Sc/12/61361

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA, NSUKKA.

SUPERVISOR.DR. S.O.O. EZE

MAY, 2014.

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

DEDICATION

To my parents.Elder and Mrs P. E. Ugwudah.

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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.

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.

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

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 ------79

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. 17: Degree of hydrolysis against time at 0.3g/ml substrate concentration - 52 Fig. 18: Degree of hydrolysis against time at 0.4g/ml substrate concentration - 53 Fig. 19: Degree hydrolysis against time at 0.5g/ml substrate concentration - - 54 Fig. 20: Degree hydrolysis against time at 0.6g/ml substrate concentration - - 55 Fig. 21: Degree hydrolysis against time at 0.7g/ml substrate concentration - - 56 Fig. 22: Degree of hydrolysis against time at 0.8g/ml substrate concentration - 57 Fig. 23: Degree of hydrolysis against time at 0.9g/ml substrate concentration - 58 Fig. 24: Degree of hydrolysis against time at 1.0g/ml substrate concentration - 59

LIST OF TABLES

Table 1: Enzyme nomenclatute ------7 Table 2: Proximate analysis of C. esculentus milk-like suspension - - - 38 Table 3: The purification profile for papain ------41

CHAPTER ONE

INTRODUCTION

Carica papaya, the sole species in the genus Carica of the plant family Caricaceae cultivated in most countries with tropical climate like Nigeria (Akinloye and Morayo, 2010), is commonly and erroneously referred to as a “tree”. The plant is properly a large herb growing at a rate of 6-10 feet in the first year and reaching 20-30 feet in height, with a hollow green or deep purple stem between 30-40cm or more thick at the base and roughened by leaf scars. It is a herbaceous soft wooded, typically unbranched, cultivated worldwide in tropical and subtropical climates, mainly for its melon- like fruits Organisation for Economic Cooperation and Development (2005). Europeans encountered papaya first in the western Hemisphere tropics and various interests disseminated it widely (Sauer, 1966; Ferrao, 1992).

Carica papaya Linn is more commonly called pawpaw in Nigeria. The generic name is derived from the Latin “Carica”, meaning edible fig’, on account of the similarity of their leaves (Orwa et al., 2009). It has many local names, such as (Fafay, babaya), Arabic, (Bi- sexual paw paw, tree, melon tree, papaya) English, French (Papailler, papaya, papaye), German (Melonbraum), Spanish (Figuera del monte, fruta bomba, papaya) (Orwa et al, 2009). It is known as okwulu bekee by the Igbos, ibepe by the Yoruba and Kawuse by the Hausa tribes of Nigeria (Abo et al, 2008, Udeh and Nwaehujor, 2013).

1.1 C. Papaya 1.1.1 Origin of C. Papaya C. papaya, originally is from south Mexico (Udeh and Nwaehujor, 2013). Though opinions differ on the origin of C. papaya, it is native in northern- tropical western hemisphere Organisation for Economic Cooperation and Development (2005). It is likely that C. Papaya originated from the low-lands of East Central America, from Mexico to the Panama (Nakasone and Paull, 1998). Its seeds were distributed to the Carribean and South-east Asia during Spanish exploration in the 16th century, from where it spread rapidly to India, the pacific and Africa (Villegas, 1997). The genus vasconcellea (formerly in carica) is found in South America along the Andes, especially in Ecuador (Badillo, 1993; Morales Astudillo, et al., 2004), with outlying species reaching as far as Chile, Mexico, Argentina and Uruguay (Aradhya et al., 1999; Van Droogenbroeck et al., 2004). This led some to propose South America as the origin for C. papaya (Prance, 1984). Evidence to the contrary is provided by finding only domesticated – type feral C. papaya in South America (Manshardt and Zee, 2

1994; Morshidi, 1996), but finding wild plants in Mexico and Honduras (Moreno, 1980; Manshardl and Zee 1994: Manshardt, 1998; Paz and Vazguez–yanes, 1998). Papaya was probably domesticated in northern tropical America.

1.1.2 Taxonomy of C. Papaya Taxonomy is defined as the analysis of an organism’s characteristics for the purpose of classification. C. papaya is classified as follows:- Kingdom Plantae

Subkingdom Angiosperms

Division Magnoliophyta

Class Rosids

Order Brassicales

Family Caricaceae

Genus Carica

Species Carica papaya.

(Wikipedia, 2013)

Caricaceae family was thought to comprise 31 species in three genera, namely Carica, Jacaritia and Jarilla (Nakasone and Paull, 1998). A recent taxonomic revision proposed that some species formerly assigned to Carica were more appropriately classified in the genus vasconcellea (Badillo, 2002). However, concensus has been developed that the genus Carica L. has only one species Caricapapaya, and that Caricacae may contain six genera (Aradhya et al., 1999; Badillo, 2000; Van Droogenbreeck et al, 2002, 2004; Kubitzki, 2003). Most of the genera are Neotropical forest plants, occurring in South America and Mesoamerica andVasconcelleae, the largest genus with 21 species had usually been considered as a section with Carica. The other members of the genera include Jacaritia (7 Spp). Jarilla (3 Spp), Horovitaia (1 Spp) (Badillo, 1993), and Cylicomorpha (2 Spp) which occur mainly in montane forests in equatorial Africa (Badillo, 1971), with Carica papaya the only species within the genus carica (Badillo, 2001).The highland papaya, vasconcelleae is the closest relatives to Carica papaya (Badillo, 1993; Aradhya et al, 1999; Van Droogenbroeck et al, 2002, 2004). 3

1.1.3 Morphology of C. Papaya Carica papaya is an evergreen, fast-growing, tree-like herb, usually unbranched, although sometimes branched due to injury. It is a tufted tree of about 2-10m in height (OCED, 2005), that contains white latex in all its parts (Orwa et al., 2009). It is a soft wooded perennial plant that lives for about 5-10years (Chay-prove et al., 2000). It has large palmately lobed leaves with long stout leaf- stalks attached densely round the terminus of the straight trunk forming a loose open crown. The leaf stalks end in a leaf blade 20-60cm across (Campostrini and Yamanishi, 2001a) with each blade usually 5-7 lobes and each lobe cut pinnately. The trunk patterned conspicuously with large leaf – scars, it is thin barked and often hollow between nodes with ageing (Elias, 1980), that has 15-30 mature leaves with a leaf persisting 3-8 months and new leaves arising at the rate of 2-4 per week (Sippel et al., 1989; Allan et al., 1997; Mabberhey, 1998; Nakasone and Paulll, 1998; Fourier et al., 2003). Leaf’s positon within the plant canopy rather than simply increasing age (Ackerly, 1999). Papaya flowers are born on inflorescences which appear in the axils of the leaves. Female flowers are held close against the stem as single flowers or in clusters of 2-3 (Chay-Prove et al., 2000). Male flowers are smaller and more numerous and are born on 60-90cm long pendulous inflorescences (Nakasone and Paull, 1998).

C. papaya fruits are ready to harvest five to six months after flowering, which occurs five to eight months after seed germination (Chay-Prove et al, 2000). The fruits range in size from 7- 30cm long and vary in mass from about 250 to 300g (OCED, 2003). Fruits from female trees are spherical whereas the shape of fruits from bisexual trees are affected by environmental factors especially temperature, that modify floral morphology during early development of the inflorescence (Nakasone and Paull, 1998). Ripe C. papaya fruits have smooth, thin yellow-orange coloured skin depending on the cultivar, flesh thickness varies from 1.5 to 4cm (Nakasone and Paull, 1998) and flesh colour may be pale yellow to red (Villegas, 1997; Nakasone and Paull, 1998). Mature fruits contain numerous grey-black spherical seeds of about 5mm in diameter (Villegas, 1997). The life span of floral trees is about 15-20 years (Anon, 2006). All parts of the plant contain a thin acrid milky latex including the unripe fruits, which exerts protection on the plant against herbivores and other pests (OGTR, 2003)

1.1.4 Importance of C. Papaya C. papaya is the most economically important member of Caricacea family Office of the Gene Technolgy Regulator (2003a). It is cultivated widely for its consumption as a fresh fruit and used in making fruits salad, refreshing drinks, and jams candies and as dried crystallized 4 fruit (Villegas, 1997; OGTR, 2003a; Orwa et al. 2009). Nutritionally, it is a good source of the minerals such as potassium, magnesium, vitamins A and C (Nakasone and Paull, 1998; Hardisson et al, 2001). The vitamin A and C contents exceed the Dietary Reference intake established by the US food and Nutrition Board for adult minimum daily requirement (USDA, 2001).

C. papaya organs such as the leaves, fruits, flowers and extracts contain many biologically active compounds and phytochemicals such as proteins, alkaloids, carapine which makes it possess important medical, pharmaceutical and industrial applications (El- Moussaoui et al., 2001; Orwa et al., 2009). Carapine present in papaya can be used as a heart depressant, amoebicide and diuretic (Orwa et al., 2009), as a haemostat and antidote against venoms and rabies and for treatment ofDiabetes due to its diuretic activity (Burkill, 1985). C. papaya latex has been shown to have activity against Candida albicans (Giordani et al., 1996), Heligmosomoides polygyrus, antihelminthic, Ascaris Suum and Ascardia galli (Satrija et al, 1994, 1995), Enterobacteria, antimicrobial (Osato et al, 1993). Extracts of papaya leaves have shown potential activity in the management of dengue fever (Ahmed et al., 2011), anti- tumour and immunomodulatory activities (Otsuki et al., 2010).

There has been evidence of isolation of two important proteases, (chymopapain and papain) which aid in digestion of proteins (Brocklehurst et al., 1985).The plant parts have various industrial applications such as meat tenderization, protein hydrolysis and juice clarification due to the presence of these proteolytic enzymes in the sap and milky latex (Burkill, 1985). Papain may be associated with protection from frugivorous predators and herbivores (El- Moussaoui et al, 2001). Papain purified from the extract is used in food beverages and pharmaceutical industries, tenderizing of meat, brewing and manufacturing of baby food (Mabberley, 1998; Wiersema and Leon, 1999; Orwa et al., 2009). Papain has also been used in textiles industry, for degumming of silk and for softening wools (Villegas, 1997) and in cosmetics industry, in soaps and shampoo Office of the Gene Technology Regulator (2003). Also, the latex from papaya has been used in manufacturing of chewing gum (de Wit, 1966), oil from the seeds and other components from the fruits and leaves have been used in cosmetics and soap (Quenum, 2001).

1.2 Enzymes Enzymes are produced by all living cells as catalysts for specific chemical reaction. Life depends on series of chemical reactions, most of which proceed too slowly on their own to 5 sustain life, hence, nature has designed catalysts which we refer to as enzymes to greatly accelerate the rates of these chemical reactions (Copeland, 2000). The process of cheese making, leaven bread, vinegar production, and other food processes such as wine and beer clarification which are as old as man are all catalysed by enzymes (Dewdney, 1973; Nwachukwu and Chilaka, 2002), hence, enzymes are organic biological catalyst, secreted by living cells of organisms which accelerate the rate of biological reaction, without being consumed in the process.

Biological catalysis was first recognized and described in the late 1700s, in studies on the digestion of meat by the secretion of stomach and continued in the 1800s with examinations of the conversion of starch to sugar by saliva and various plant extracts (Nelson and Cox, 2005). In 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalysed by “ferments”. Edward Buchner, in 1897 discovered that yeast extracts could ferment sugar to alcohol and carbon (iv) oxide, indicating that yeast juice contains complex mixture of enzymes required for effective fermentation, showing that enzymes can act intra- and extra-cellularly (Nwachukwu and Chilaka, 2002).

Furthermore, in 1897-1900, W. Kuhen, studying catalysis in yeast extracts, first coined the term “enzyme” which was derived from the Medieval Greek word “enzyme” which relates to the process of leavening bread (Copeland, 2000). Summer (1926) isolated urease as crystalline protein. Subsequently, Northrop and Kunitz isolated and crystallized pepsin, trypsin and chymotypsin and these led to further purification and proving that all enzymes are protein (Nwachukwu and Chilaka, 2002). All living systems synthesize their own specific enzymes needed for the various metabolic reactions upon which their life depends (Dewdney, 1973).

1.2.1 Enzyme Structure All enzymes are globular proteins and so are soluble in water and have definite functional role to play in living systems (Nwachukwu and Chilaka, 2002). Their function is determined by their complex structure that contains a small part of the enzyme called the active site, while the rest of the protein acts as scaffolding (Nwachukwu and Chilaka, 2002).

The general nature of enzymes as enumerated by Nwachukwu and Chilaka (2002) are listed below:-

i. All known enzymes have been shown to be biologically active proteins, which may require a non-protein component to be catalytically active.

ii. They increase the rate of chemical reactions within the living cells without being consumed or changed in the overall reaction.

iii. They are specific in reaction. A particular enzyme acts on the specific substrate or substrates to produce a given product or products. This is attributed to the complex conformation of the protein, uniqueness of the active site and the structural conformation of the substrate molecule. Specificity could be group absolute group and relative group specificity.

iv. There is formation of intermediate complex between the enzyme and its substrate in course of catalysis.

v. Enzymes have active sites. The conformation of an enzyme is such that certain R- groups in the polypeptide backbone are brought into close proximity with a highly specific manner to form the active site, which may be a deep cleft (e.g.) carbonic anhydrase or small crevice (e.g.) papain.

vi. Enzymes as proteins are easily denatured at high temperature, pH and ionic concentration.

vii. They are point of regulation for the rate of reactions in living systems.

1.2.2 Enzyme Nomenclature As thousands of enzymes were being discovered and characterized, problems emanated. Such problems as ambiguous name, (eg) Chymosin also known as Rennin.Some names of enzymes do not specify their substrate nor type of reaction they catalyse (Nwachukwu and Chilaka, 2002). To avoid such confusion, inconsistencies and clumsiness in enzyme nomenclature, the International Union of Pure and Applied Chemistry (IUPAC) formed the Enzyme Commission (EC) in 1973 to develop a systematic numerical nomenclature for enzymes (Copeland, 2000). The commission classified enzymes into six (6) general categories according to the reaction they catalyse. Within each of these broad categories, the enzymes are further differentiated by a second number that more specifically defines the substrates on which they act, third digits also describes the type of reaction catalysed since enzymes 7 catalyse very similar but different reactions and fourth digit specifies the actual substrate being catalysed (Copeland, 2000; Nwachukwu & Chilaka, 2002).

Table 1: Enzyme nomenclature FIRST (EC) ENZYME CLASS REACTION NUMBER 1 Oxidoreductases Oxidation-Reduction Reaction 2 Transferases Chemical group transfer 3 Hydrolases Hydrolytic bond cleavages 4 Lyases Non-hydrolytic bond cleavages 5 Isomerases Change in arrangement of atom in molecules 6 Ligases Joining together of two or more molecules (Copeland, 2000)

1.2.3 Proteases (Hydrolases) EC. 3.4 Proteases are involved in numerous physiological processes that include food digestion, cell maintenance, cell signaling, wound healing, cell differentiation for approximately 2% of the genes in most organism, second in number only to transcription factor (Hedstrom, 2002). Proteases are present in all living beings and play an important role in normal and abnormal physiological conditions catalyzing various metabolic reactions (Sandhya et al., 2004). They are significant in that,they do not only govern proteolytic reactions, but also regulate various enzymatic cascades, which ultimately lead to all metabolic reactions involving the breaking down of fats, proteins and carbohydrates; hence, proteases are enzymes that catalyse hydrolytic reactions in which protein molecules are degraded into peptides and amino acids (Sumantha et al., 2006). They constitute a very large and complex group of enzymes, which differ in properties such as substrate specificity, active site, catalytic mechanism, temperature and pH optima and stability profile, its specificity, is governed by the nature of amino acids and other functional groups close to the bond being hydrolysed (Sumantha et al., 2006).

According to Enzyme Commission (EC) classification, proteases belong to group 3 (hydrolases) and subgroup four, (4), which hydrolyse peptide bonds (Copeland, 2000). Also, proteases can be classified into two major groups based on their abilities to cleave N-or C- terminal peptide bonds (exopeptidases) or internal peptide bonds (endopeptidases), while aminopeptidases cleave the N-terminal peptide linkage, carboxyl peptidases cleave the C- terminal peptide bonds (Sumantha et al., 2006). Ward (1985) classified proteases based on the presence and absence of charged groups in position relative to the susceptible bond and are classified on a number of basis; their pH optima into, acidic, alkaline and neutral; 8 substrate specificity. Collagenase, keratinase, elastase or their homology to well-studied proteins such as trypsin, pepsin; trypsin-like, pepsin-like.Endopeptidases are classified into four groups on the basis of their active sites and sensitivity to various inhibitors into aspartic or carboxyl proteases, cysteine or thiol proteases, serine proteases and metal proteases (Hartley, 1960; Barrett, 1994).

1.2.4 Cysteine Proteases (3.4.22) Cysteine proteases (EC 3.4.22) are endopeptidyl hydrolases with a cysteine residue in their active site and are identified base on the effect of their active site inhibitors and activation of the enzymes by thiol compounds (Grudkowska and Zagdanska, 2004). Cysteine proteases are widely distributed throughout nature, having been found in viruses, bacteria, protozoa, plants, mammals and fungi, with 21 families discovered (Otto and Schirmeister, 1997; Rawling and Barrett, 1999). They are made up of three structurally distinct clans, which include the papain family, the caspases and picornaviridae family. Majority of cysteine proteases belong to the papain family (Leung, et al., 2000). They are responsible for many biochemical processes occurring in living organisms and they have also been implicated in the development and progression of several diseases that involves abnormal protein turnover, with the main physiological role being metabolic degradation of peptides and proteins (Grzonka et al., 2001).

Cysteine proteases are proteins with molecular mass of about 21-30KDa, which show highest hydrolytic activity at pH 4 - 6.5 (Grzonka et al., 2001). They are involved in protein maturation, degradation and protein rebuilt in response to different external stimuli and also play a house- keeping function to remove abnormal, misfolded proteins, in each case, the proteolysis by cysteine proteases is a highly regulated process (Grzonka et al., 2001). Their enzymatic activity is related to the cysteine and histidine residues which in the pH interval of 3.5-8.0, exists as an ion- pair (Bert and Storer, 1995; Turk et al., 1997). This activity is regulated by proper gene transcription and the rate of protease synthesis and depredation, as well as by their specific inhibitors (Grzonka et al. 2001). The active site of cysteine proteases contain three amino acids, cysteine, Histidine and Asparagine that facilitate the hydrolysis or cleavage of the peptide bond and the substrate orientation for catalysis occur by interaction between subsites of the enzyme and amino acids residues of the substrate.

1.2.5 Papain (EC 3.4.22.2) Papain (EC 3.4.22.2) is an endolytic plant cysteine protease which is isolated from C.papaya latex. The greener the fruit the more active is papain (Amri and Mamboya, 2012; Otto and Schirmeister, 1997). It belongs to papain super family, and of great importance in many biological processes (Tsuge et al., 1999). The latex of C. papaya is a rich source of cysteine endopetidases including Glycyl endopeptidases, chymopapain, papain and carican, which consist more than 80% of the whole enzyme fraction (Azarkan et al., 2003).

Papain is a single chain polypeptide with molecular weight between 23,000-23,406 daltons, consisting of 212 amino acid residues with three disulfide bridges Cys22- Cys63, Cys 56- Cys95 and Cys153- Cys200 and catalytically important residues at Gln19, Cys 25, His 158 and His 159 (Dreuth et al, 1968; Mitchel et al., 1970; Robert et al., 1974; Tsuge et al. 1999). It is isolated in an inactive form (Garret and Grisham, 1999) in which the active site is blocked by a disulpide bondbetween the active site Cys-25 and Cys-22. This replaces the disulfide bond between Cys-22 and Cys-63 residues in the active papain. The zymogen form of papain is important forming the correct quaternary structure of the enzyme before it is activated (Otto and Schirmeister, 1997). Activation of papain occurs either by disulfide exchange with thiol reagents or reducing agent (Otto and Schirmeister, 1997; Arnon, 1970).

Fig. 1: Amino acid composition of papain (Dreuth et al., 1968) 10

Papain is stable and active under a wide range of conditions and stable even at elevated conditions of temperature and pH (Cohen et al., 1986). The pH and temperature optima range between 3.0-9.0 and 65-80oC respectively (Harton et al., 2002; Ding et al, 2003) which varies with different substrate (Edwin and Jagannadham, 2000; Ghosh, 2005). It preferentially cleaves peptide bonds involving basic amino acids such as arginine, lysine and phenylalanine (Menard et al., 1990).

The protein is stabilized by three disulfide bridges in which the molecule is folded along these bridges creating a strong interaction among the side chains which contributes to the stability of the enzyme (Tsuge et al, 1999; Edwin and Jagannadham, 2000).

Fig. 2: Three-dimensional structure of papain (Dreuth et al., 1969)

The three dimensional structure consists of two distinct structural domains, with a cleft that contains the active site and catalytic diad that has been likened to the catalytic triad of chymotypsin with amino acids Cys-25 and His-159 present at the catalytic diad (Amri and Mamboya, 2012). The two domains of the polypeptide chain is folded into the L-domain consisting of residues 10-11 and 207-212 while the R-domain is made up of the remaining 11 residues (Kim et al, 1992). The L and R domains are separated by a cleft which contains the active site thiol Cys-25 positioned at the entrance of the alpha-helix residues 24-42 on the L- domain, and His-159 residue is on the opposite side of the R-domain but still in close proximity (Madej et al., 2012).

The molecule has an all alpha domain and an anti-parallel beta-sheet domain (Kamphuis et al, 1984, Madej et al., 2012). The conformational behavior of papain in aqueous solution has been reported to show high alpha- helical content (Huet et al., 2006). It shows evidence of substantial secondary structure as beta-sheet and exhibits a great tendency to aggregate at lower concentration of salt (Edwin and Jagannadham, 2000). The hydrophobicity and hydrophilicity values of the amino acids in a protein sequence influence the protein conformation and interaction within its environment, since hydrophobic residues tend to be more buried in the interior of the molecule and hydrophilic residues are more exposed to solvent; hence, determining its overall folding pattern. Such hydrophobic amino acid side chains of papain include alanine, valine, leucine, methionine and isoleucine which vary in degrees of hydrophobicity (Amri and Mamboya, 2012). The hydrophobic-hydrophilic interaction of papain amino acids in the side chain seem to be the major thermodynamic force which drive protein folding and play an important role in inducing the two different intermediates along the two different thermodynamic pathways (Chamani et al., 2009). The native protein possesses hydrophobic core and a charged and polar group on the surface that helps to stabilize the tertiary structure of the protein by hydrophobic interaction and the outer polar surface preferentially interact with the exterior aqueous medium (Wang et al., 2006).

1.2.5.1 Mechanism of Action of Papain

Papain is characterized by a two- domain structure.

Fig. 3: L and R Domain of papain (Dreuth, 1968)

The active site where substrate binds is located between the domains, with catalytic residues of Cys-25 and His-159 (Grzonka et al, 2001). The His-159 residue is positioned to act as the base catalyst for activation of the thiol group during peptide hydrolysis. Asn-175 orientateHis-159 for interaction with Cys-25, which results in the activation of both the His- 159 and cys-25 (Leung et al. 2000, Beers et al, 2004). The backbone amino group of cys-25 and the side chain NH2 group of Gln-19 form the oxyanion hole, for binding substrate and promoting reactivity for nucleophilic attack (Lin and Welsh, 1996). The active site of papain binds at least seven amino acid residues in appropriate subsites but only five subsites are important for substrate binding. S2, S1 and S11 subsites are important for both back bone and side chain binding and S3 and S2 and important for amino acid side chain binding (Schechter and Berger, 1967; Turk et al., 1998). Smith et al., (1958) proposed two step mechanisms for the hydrolysis of N-acyl-amino acid derivatives.

The catalytic activity of papain involves hydrolysis of protein with broad specificity for peptide bonds. It has been reported that papain preferentially cleaves peptide bonds with Arg 13

in P1 position or phe at P2 position or amino acid bearing a large hydrophobic side chain at the P2 position but does not accept val in P1 (Kamphius et al., 1985; Menard and Storer, 1998; Fischer et al., 2000).

The reaction pathway of papain-catalysed hydrolysis is represented by three steps according to Lowes (1970) as follows;

E+S K 1 ES ES`+ P1 E +P2 K2 K2 K-1

E----- Enzyme (papain)

S----- Substrate

ES---- Enzyme- Substrate complex

P1---- Hydrolysed product

P2--- final product hydrolysed

Fig. 4: Mechanism of hydrolysis of papain (Lowes, 1964)

In the above equation, an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, and by deacylation of the acyl-enzyme, where ES is the Michaelis complex, ES1 is the acyl-enzyme formed through the thiol group of Cys-

25, P2 is the N-terminal peptide and P1 hydrolysed substrate.The first step involves the acylation of the enzyme through an essential thiol group, whereas, in the second step, deacylation of the acyl- enzyme occurs. When the substrate binds to the active site, an intermediate, S-acyl-enzyme moiety is formed through nucleophilic attack of the thiolate group of the Cys-25 residue portion of the triad in the active site that attacks the carbonyl carbon in the backbone of the peptide chain of the hydrolysed peptide bond with a release of the C-terminal fragment of the cleaved product freeing the amino terminal portion, thus, leading to the breaking of the protein molecule (Amri and Mamboya, 2012).

The peptide bond breakage involves the deprotonation of Cys-25 by His-159. The Cys-25 and His-159 present at the catalytic triad form a thiolate-imidazolium ion pair which confers high nucleoplilicity upon the cysteine thiol, which acts as a nucleophile to hydrolyse the scissile bond of the substrate. Asn-175 allows this deprotonation to take place. Cys-25 then performs a nucleophilic attack on the carbonyl carbon of the peptide backbone (Menard et al, 1990; Tsuge et al., 1999). The initial tetrahedral intermediate is stabilized by hydrogen bond formed between Cys-25 and Gln-19 which makes up the oxyanion hole. This is followed by acylation of the enzyme and release of the C-terminal substrate fragment. Hydrolysis of the acyl- enzyme forms a second tetrahedral intermediate, which reacts with water molecules to cleave the peptide terminal to regenerate the free enzyme and release the N-terminal substrate fragment (Storer and Menard, 1994; Leung et al, 2000).

1.2.5.2 Localisation and Biosynthesis of Papain Cysteine proteinases (Papain) are synthesized at membrane bound polysomes in the cytoplasm as large precursors with short N-terminal and much longer C-terminal propeptides, and the inactive proenzymes enter the lumen of the endoplasmic reticulum (ER) and transported to the vacuole or cell wall (Grudkowska and Zagdariska, 2004). Most soluble plant proteins have a C-terminal KDEL/HDEL tetrapeptide sequence recognized by the ERD2-KDEL receptor or the Golgi apparatus and are transported to the trans-Golgi network (Okamoto et al., 2003). Papain, containing an endoplasmic reticulum retention signal is transported into large KDEL vesicle that bud off from the endoplasmic reticulum and by pass the Golgi to fuse directly with protain storage vacuole (Toyooka et al., 2000; Okamoto et al., 2003). A KDEL /HDEL 15 sequence in plants function as protein retention signal in the endoplasmic reticulum and also regulate the delivery of proteins to other compartment (Grudkowska and Zagdanska, 2004). Sequential removal of the N- and C-terminal propeptides in vacuoles is required to produce the mature form (Yamada et al., 2001). Papain has the endoplasmic reticulum retention signal KDEL at the C-terminal of their cDNA –deduced amino acid sequences removed post- translationally (Okamoto and Minamikawa, 1999; Fischer et al., 2000). KDEL has been detected in papain, a sulfhydryl endopeptidase (Okamoto and Minamikawa, 1998). Papain is synthesized as an inactive zymogen, with a carboxy – terminal KDEL motif (Okamoto and Minamikawa, 1998). The post - translational processing of sulfhydryl endopeptidase includes removal of the KDEL motif prior to translocation and activation of mature protein (Okamoto et al., 2003). The KDEL – ion containing proteinases appear to be a distinct plant specific enzyme subset linked to the endoplasmic reticulum- derived precursor protease vesicles and have been identified mainly in higher plants (Chrispeels and Herman, 2000; Toyooka et al., 2000; Okamoto et al.,2003) . The C-terminal extension sequences are composed of two domains; a pro-rich domain and a domain of high homology to animal proteins of the epithelin/ granulin family (Bhandari et al., 1992). At the N-terminal propeptides, the papain proteinase contains the ERFNIN (EX3 RX3 FX3 NX3I/VX3N) motif about 110 residues propeptide which is strongly conserved and seerns to function as an autoinhibitory domain (Beers et al., 2004).

1.2.5.3 Stability of Papain Papain stability depends on the nature of the enzyme, whether crystalline or native. The crystalline enzyme shows a high degree of stability in sodium chloride solution. It can be kept at 4oC for months without detectable less in activity, whereas, the active enzyme losses 1-2% of its activity per day due to autolysis or oxidation (Arnon, 1970). Also, papain has broad stability at high environmental conditional of temperature and organic solvents and reagents that could cause denaturation to other enzymes (Harton et al., 2002). It is unstable under strongly acidic conditions but has wide range of pH values3.8-9.0 (Ding et al., 2003). Though, the enzyme has good temperature stability, it is strongly pH dependent. Papain is stable to several denaturing agents such as dimethylsulfoxide with organic solvent, 8m urea solution and does not undergo any conformational changes (Shapira and Arnon, 1969). Also in the presence of stronger denaturing agents such as 10% trichloro acetic acid or 6.0M guanidine hydrochloride, causes irreversible changes in papain which are manifest in drastic changes in loss of activity, and under strongly acidic pH values below 2.8, the enzyme activity decreases significantly (Shapira and Arnon, 1969). 16

1.2.5.4 Specificity of Papain Papain possesses wide range of activities including endopeptidase, amidase and esterase activities (Arnon, 1970). The specificity of papain for peptides containing a phenylalanine residue largely occurs during the acylation step (Lowe and Yuthavong, 1971a). Schechter and Berger (1967) concluded that the active site of papain comprises seven subsites, each capable of accommodating a single amino acid residue of peptide substrate, four of the subsites (S1-

S4) are located on the N-terminal side of the point of cleavage of the peptide and other three subsites (S1-S3) on the C-terminal side one being numbered away from the site of cleavage. The Si subsite of papain is stereospecific L-amino acid residues (Berger & Schechter, 1970), but the cleavage pattern of certain polypeptides shows largely S2 subsite domination. The Si subsite has a preference for hydrophobic residues such as L-leucine and L-tryptophan (Robert et al., 1974; Lowe and Williams, 1965). The S2 subsite was reported by Schechter and Berger (1968) to be selective for phenylalanine, the hydrolysis of peptides containing this residue, almost take place at the next peptide bond towards the C-terminus. The S2 subsite of papain is most important primary substrate recognition site of the protease and contains mainly Try-69, Try-67, Phe-207, Pro-68 Ala-160, Val-133 and Val-157, with others not showing any strict amino acid preference (Kim, 1999); it prefers those amino acids with bulky non polar side chain such as phe.

1.2.5.5 Activators of Papain Papain is being a sulfhydryl enzyme and hence, requires a free sulfhydryl group for its catalytic activity (Arnon, 1970). Activation improves in the presence of reducing agents such as cysteine, sulfite, sulfide as well as cyanide (Arnon, 1970). Kimmel and Smith (1954) also observed that optimum activation occur upon simultaneous application of cysteine, a heavy metal binding agent such as Ethylenediaminetetraacetic acid (EDTA) or a compound 2,3- dimercaptopropanol, which combines the function of a thiol group and a metal-binder.

1.2.5.6 Papain Inhibitors Heavy metal ions such as Cd2+, Zn2+, Fe2+, Cu2+, and Hg2+ inhibit activity of papain (Shyterman, 1967). Similarly, iodoacetic acid reacts with the free sulfhydryl group of papain causing an irreversible inactivation (Kimmel et al., 1965). The reaction of papain with the chloromethyl ketones of phenylalanine and lysine was observed to cause total loss of activity (Whitaker and Perez-Villaserior, 1968) acting specifically on the active sulfhydryl group of the enzyme. Other papain inhibitors include phenylhydrazine, hydroxylamine and other 17 aldehyde reagent (Masuda, 1959), and carbobenzoxy –L-glutamic acid referred to as specific structural inhibitor for papain in the region of pH 3.9-4.5 (Kimmel et al, 1965).

1.2.5.7 Applications of Papain Papain has potential application in food and biotechnological industries due to its broad substrate specificity, high stability in extreme conditions, good solubility and activity over a wide range of pH and temperature. It has a wide range of applications in different industries, including personal care products such as soap, shower gel and food industries (Neidlema, 1991; Uhlig, 1998; Orwa et al., 2009), beer clarification (Caygill, 1979; Chaplin, 2002). It is also being used to tenderize meat and meat products, batting of hides, degumming silk and softening of wool (Orwa et al, 2009; Chaplin, 2002). It is also used in the protein hydrolysis and preparation of protein hydrolysates (Dupaigne, 1973) in confectionary industry to prepare chewing gum and in dairy industries for cheese – making (Chaplin, 2002). It is applied in pharmaceutical preparations, drug preparation for digestive ailments and treatment of gangrenous wounds (Orwa et al., 1999; Amri and Mamboya, 2012). Uhlig (1998) reported its uses in textile industries and tanning industry.

1.3 Tiger nut (Cyperus esculentus) Tiger nut is an underutilized crop which produces rhizomes from the base and tubers that are somewhat spherical (Bamishaiye and Bamishaiye, 2011), hence, has attracted very little scientific and technological interest, except for the production and studies on its oil (Elena- Sanchez-Zapata et al., 2012). Tiger nut is known by various names, such as Chufa (Spannish), earthnut, yellow nut sedge, ground nut, rush nut, edible galingale (Oderinde and Tairu, 1988). In Nigeria, the Hausas call it “Aya”, Yoruba “imumu,” Igbos “Ofio,” “aki awusa” (Omode et al., 1995). It is an edible perennial grass-like plant native to the old world and has been considered a food stuff since ancient times (Pascual et al, 2000; Elena Sanchez– Zapata et al, 2012).

Fig. 5 : Tiger nut (Cyperus esculentus)

(Bamishaiye and Bamishaiye, 2011)

Tiger nut is erroneously called nut because of its small size, like that of a peanut growing at the rhizome of the plant and it most inhabits wet marshes and edges of streams and ponds where it grows in coarse tufts (Temple et al., 1989; Mason, 2005). It is one of the earliest domesticated crops and was used to embalm bodies of the Egyptian pharoahs.

1.3.1 Taxonomy of Tiger nut Tiger nut (C. esculentus) commonly called “aki-hausa” in South-East Nigeria, is classified as follows;

Kingdom _____ Plantae

Subkingdom _____ Angiosperms

Division ______Magnoliophyta

Class ______Liliopsia

Order ______Cyperales

Family ______Cyporaceae 19

Genus ______Cyperus

Species ______C. esculentus

(Udeozor, 2012)

1.3.2 Origin of Tiger nut (C. esculentus) Tigernut was first discovered some 4000 years in ancient Egypt and is cultivated today in China, Spain and West-Africa (Childers, 1982). It was found to be a cosmopolitan, perennial crop of the same genus as the papyrus plant (Belewu and Belewu, 2007). It is a crop of early domestication and was added to other crops of the Nile valley. Its dry tubers have been found in tombs from predynastic times about 6000 years ago (Zohary, 1986). It has been cultivated since early times for its small tuberous rhizomes, which are eaten raw or roasted, used as log feed or pressed for cooking oil, baking flour, fish baits, milk in lieu of cow’s milk (Bamishaiye and Bamishaiye, 2011). In Southern Europe, tigernut has been cultivated for several centuries. It is believed to have been introduced in Europe during the middle Ages by the Arabs after their expansion across the north of Africa (Elena- Sanchez-Zapataet al., 2012), furthermore, there are written records from the 13th century which mention the consumption of a drink made from tigernut in some Mediterranean areas, mainly the Valencia Region. This beverage could be considered an ancestor of the modem “horchata” (Pascual et al., 2000). This plant was originally native to the Mediterranean region but its cultivation has now spread to many warm countries (Mohammed et al., 2005).

Today, tigernut is cultivated in Northern Nigeria, Niger, Mali, Senegal, Ghana and Togo where they are used primarily uncooked as a side dish (Omode et al., 1995). It is considered as weed in some areas (De Vries, 1991).

1.3.3 Morphology of Cyperus esculentus Cyperus esculentus is a tough erect, fibrous-rooted perennial plant. It grows between 1-3 feet high reproducing by seed-slender rhizomes, which form weak runners above the ground and small nuts at the tips of underground stem (Consejo, 2006). The planting period lapses between April – November, with mainly three varieties; namely black, brown and yellow. But only brown and yellow are available in the Nigerian markets. The yellow variety is preffered because of its inherent properties such as bigger size, attractive colour and fleshier body (Bamishaiye and Bamishaiye, 2011). The yellow variety also yields more milk, contains lower fat and higher protein and less anti-nutritional factors especially polyphenols (Okafor et al., 2003). Its leaf blades are up to 1.5 inches long and 0.33 inches wide; they are light green, 20 spreading outward from the stem (Bamishaiye and Bamishaiye, 2011). The leaf sheaths are whitish green, closed and hairless, they become pale and red towards the base of the plants (Kelley, 1990). The central stem terminates in an umbel with varying size and shape. With the central stalk flattened and narrowly winged. The overlapping scales are slightly spreading along the length of each spikelet, with each scale having 2-3 mm in length. Each floret has a white tripartite style and yellowish brown anthers; the tips of the styles are curly. The blooming period occurs from mid-summer to early fall (Bamishaiye and Bamishaiye, 2011).

Pollination is by wind (James et al., 1991). The shallow root system is fibrous, rhizomatous and tuberous. The rhizomes are connected to small globoid tubers up to 0.5 inch across. Young tubers are white, but gradually covered by a yellow membrane as it grows older (Bamishaiye and Bamishaiye, 2011).

The nutlets are almost smooth at maturity and unevenly globe shaped (James et al., 1991). High temperatures and low nitrogen levels increase tuber production and an increased day length will reduce tuber formation (Bamishaiye and Bamishaiye, 2011). It grows best in moist sandy-loam soils but will grow in the hardest clay (James et al., 1991; Oderinde and Tairu, 1988).

1.3.4 Phytochemical Composition of Tiger nuts Tigernut, an under-utilized crop, has been reported by many researchers to contain varieties of phytochemicals and nutrients. Kordyias (1990) and Umerie and Enebeli (1997) reported that tigernuts are valued for their highly nutritional starch content, dietary fibre, carbohydrates, sodium, calcium, potassium, magnesium, zinc and traces of copper (Omode et al., 1995). Its moisture content and carbohydrate contents are much higher and the lipid and protein contents are lower than in free nuts (Venkatachalam and Sathe, 2006; Carcel et al., 2011; Panahia and Khezri, 2011). The nutritional value of lipids depends in part on the presence of essential fatty acids such as linolenic and linoleic acid of which plant is the major source. The organs of rats fed with tiger nut- based diet showed the presence of lauric, myristic, palmitic, stearic, oleic and Linoleic acids as reported by Bamishaiye et al., (2010). Other phytoesterols present in tigernut as reported by Yeboah et al (2011) include vitamin E, β-sitosterol, stigmasterol and comypesterol and F- Omega-avenasterol. This moderately high content of phytoesterols further enriches the quality and value of tigernut.

1.3.5 Economic Importance of Tiger nut Tiger nut has been cultivated for both human and livestock consumption (Sanful, 2009). Mason (2008) reported that tiger nut has long been recognized for its health benefits as they are high in fibre, natural sugars and proteins. Also, they have high content of soluble glucose and oleic acid, high energy content and rich in minerals such as phosphorus, potassium, vitamin E and vitamin C. The presence of these phytochemicals in tigernut could bestow many health and pharmaceutical benefits on it. C. esculentus had been reported to be a “health” food since its consumption can help prevent heart diseases and thrombosis which is attributed to the presence of high proportion of monosaturated acid such as oleic acid and is said to activate blood circulation (Chukwuma et al., 2010), reduce the risk of colon cancer (Adejuyitan et al., 2009), obesity and gastrointestinal disease (Anderson et al., 1994). Similarly, its rich content of starch, fat, sugar and protein, minerals and vitamins E and C makes this tuber suitable for diabetic patients and those who wish to lose weight (Belewu and Belewu, 2007; Berge et al, 2008).

Muhammed et al. (2011) and Bamishaiye et al. (2010) reported the presence of essential fatty acids such as linolenic acid, linoleic acid, oleic acid, and arachidonic, lauric and myristic acid. The presence of these essential fatty acids gives C. esculentus its nutritional value (Bamishaiye et al 2010). It is believed the acids help those seeking to reduce cholesterol or lost weight, hasten the inception of menstruation and considered a stimulant sedative and tonic in China (Maton et al., 1993; Osagie and Eka, 1998; Adejuyitan, 2010). Chukwuma et al. (2010) observed that C. esculentus contains essential phytochemical compounds that can serve as potential source of drugs because they are biologically active compounds. They have been reported to exhibit diverse pharmacological and biochemical actions when ingested by animals (Amadi et al., 2006).

C. esculentus has wide range of industrial applications. In Spain, it is used mainly in making a milk-like, non-alcoholic beverage called “horchata de chufa” usually consumed in summer time (Mosquera et al., 1996) and several other European and Latin-American countries (Cortes et al, 2005). Similarly, tiger nut milk is commonly called “Kunnu aya” in northern Nigeria (Bamishaiye and Bamishaiye. 2011). It has been included in the production of yorghurt (Sanful, 2009). Tigernut tuber has been used in compounding rat’s feeds (Bamishaiye and Bamishaiye, 2011).

Its high content of essential fatty acids indicates it could be a good source of edible oil and for industrial use such as in salad-making (Bamishaiye and Bamishaiye, 2011). In the textile 22 industry, the oil is used to water-proof textile fibers (Bamishaiye and Bamishaiye 2010). It has been successfully used as a flavouring agent in ice cream, added to biscuits and other bakery products (Coskuner et al, 2002; Elena-Sanchez-Zapata et al, 2012). Also, its oil is used in making soap (Adejuyitan, 2011). In Nigeria, it is used in preparing kunnu, a local non-alcoholic beverage which does not contain lactose, casein sugar, proteins of the milk, cholesterol making it ideal milk (Belewu and Abodunrin, 2006) .

1.4 Protein Proteins are macromolecules found in all biological systems, from lower prokaryotes to higher eukaryotes that occupy prominent position in living cells both quantitatively. Protein is derived from the Greek word Protos, meaning ‘first rank of importance (Alain, 2002).Quantitatively, it represents over 50% of dry weight of cells and are involved in virtually all biological processes (Alain, 2002).

Protein components are amino acids, with each amino acid residue joined to the other neighbor by a specific type of covalent bond (Nelson and Cox, 2005). Each amino acid comprises of an amino group, a carboxyl group, a hydrogen atom and a specific R group bonded to a carbon atom called the alpha-carbon. Of the 20 naturally re-occuring amino acids usually present in proteins, 19 amino acids have the general structure shown below, but proline has its side – chain bonded to nitrogen atom to give an imino acid.

H2N C COOH

General structure of amino acid (Copeland, 2000)

1.4.1 Classification of Amino Acids Generally, the ionization properties of the constituent amino acids and their side chains influence their solubility, stability and structural organization of proteins.

Classification of the 20 amino acids as proposed by Alain (2002) 23

Aliphatic (5 amino acids) which include amino acids containing non- cycling hydrophobic non polar chains, poorly insoluble in water they are Glycine, alanine, valine, leucine and isoleucine.

Hydroxylic (2 amino acids). They contain primary and secondary alcohol group, polar and are very soluble in water, they are serine and threonine.

Acidic and corresponding amides: They possess a second carboxyl group in their side chain that is ionized, negatively charged and very polar under physiological conditions. They include glutamic acid, and aspartic acid and their corresponding derivatives Asparagine and glutamine.

Basic (3 amino acids) lysine and arginine, which are ionized, positively charged and very polar , histidine, with a imidazole group which is poorly protonated at pH 7.

Cyclic amino acids, contain aromatic cycle (e.g.) phenylalanine, tyrosine and typtophan; proline, the imino acid with an aliphatic heterocycle.

Sulphur-containing; methionine, with a hydrophobic non polar chain and cysteine, which is polar because of its sufhydryl group.

1.4.2 Plant Protein Plant protein (Phyto-protein) is derived from plant sources such as legumes, nuts and grains (Hermann, 2002). They play significant roles in human nutrition, particularly in developing countries where average protein intake is less than that required (Yufei and MaO, 2012). They have a reduced content of essential amino acids in comparison to animal proteins (Krajcovicova et al, 2005). Plant proteins are richer in non-essential amino acids, which sum is between 111-129% compared with egg (Krajcovicova et al, 2005). Content of alanine and serine is higher in pulses, grains, legumes hazel nuts, some seedoil such as puppy, flax sunflower compared with meat (Holcikova et al, 1994; Krajcovicova et al, 2005).

The high content of amino acids was found in whole grains or dark grain products in comparison to white grain product (Souci et al, 1986; Holcikova et al., 1994).

1.4.2.1 Importance of Plant Protein 24

Proteins are essential nutrients for human body, growth and maintenance (Hermann, 2002). It is well known from experimental studies that cholesterol – free diets containing milk casein or other animal proteins include an elevation of plasma total and LDL cholesterol concentrations that can be prevented by using a vegetable protein such as soy protein (Carroll and Kurowska, 1995). It was shown that the hypercholesterolemia was primarily due to essential amino acids, higher intake of arginine and pyrivugenic amino acids in vegetarians provides the protective effects of plant proteins against cardiovascular diseases and cancer.

1.4.2.2 Enzymatic Hydrolysis of Protein Proteins are broken down into smaller peptides at optimal hydrolysis conditions. Several methods have been employed by researchers in hydrolyzing proteins; and such methods include pH-stat, Osmometry, trintrobenzene-sulfonic acid and strong acid hydrolysis (Nielsen et al., 2001). These methods do not go without their attendant disadvantages. The strong acid (6M HCl) used for complete hydrolysis of proteins destroy some amino acids, such as tryptophan, asparagine and glutamine, other amino acids are destroyed to a lesser extent or in certain cases are released incompletely from peptide linkage (Hill and Schmidt, 1962). These difficulties could be overcome by employing proteolytic enzymes as the sole agents for hydrolysis.

Enzymatic hydrolysis is commonly used in the modification of protein structure in order to enhance the functional properties of proteins (Corredig, 1997). Enzymatically hydrolysed proteins possess functional properties such as low viscosity, increased whipping ability and high solubility which make them advantageous for use in many food products (Adler-Nissen, 1979). Properties possessed by enzymatically hydrolyzed protein make them attractive as a protein source in human nutrition such as diets for the elderly and patients with impaired gastrointestinal absorption, hypoallergenic infants’ formulas, sport nutrition and weight- control diets, as well as in consumer products for general uses (Frokjaer, 1994; Schmidl et al, 1994). Peptide solubility mainly under acidic conditions and during heat treatment is an essential property of protein hydro lysate (Frokjaer, 1994). Others include improved texture, and water binding capacity (Lin et al, 1997).

Ziegler et al. (1998) concluded that protein hydrolysates are physiologically better than intact proteins because they possess more intestinal absorption than intact proteins. Modified hydrolysates have a potential for use in soluble high nutritional products (Calderon de La Barca et al., 2000). Specifically, the nature of these released peptides can be affected by 25 many factors, such as the origin of enzymes, enzyme activity and selectivity, enzyme to substrate ratio and hydrolysis conditions such as pH, temperature and time. The released peptides lead to enhanced functional properties depending on their physicochemical properties (Mietsch et al, 1989; Jung et al., 2005). Another benefit of hydrolysis is the induction of changes in the balance of net charges that lead to enhanced solubility (Zorin and Baiarzhargal, 2009). Similarly, food protein derived bioactive peptides can be released upon limited enzymatic hydrolysis, which may exert regulatory and physiological effects on the human body beyond normal and adequate nutrition (Shahidi and Zhong, 2008), released peptides upon hydrolysis play an important role in protein functionality and bioactivity (Wang et al, 2013).

Enzyme assisted hydrolysis has replaced acid hydrolysis, because it possesses more advantages over other means of hydrolysis. These advantages include, enzyme hydrolysis occurring under milder conditions, which reduces industrial accidents, reduced and safe costs, products are produced under more control.

1.5 Tigernut Milk-Like Beverage Milk is an excellent source of nutrients except iron and ascorbate (Ukwuru et al, 2011). The origin of the use of this tuber for making milk is exclusive to the Spaniards to which it may have been introduced by the Arabs (Bamishaiye and Bamishaiye, 2011). Milk is an excellent source of all nutrients and has been recognized as an important food for infants and growing children (Obizoba and Anyika, 1995; Ukwuru et al, 2011), yet the cost of dairy milk and their products are prohibitive in Nigeria and other developing countries which decreases their consumption (Kerven, 1986). Similarly, some individuals suffer intolerance of lactose and other dairy milk components such as casein and gluten.

Development of milk substitutes extracted from legumes, grains and nuts serve as an alternative source of producing acceptable nutritious milk (Harkins, and Sarret, 1967). Tiger nut milk, a phyto milk is less expensive and possesses no intolerance, hence, serves as a substitute for dairy milk(Iwuoha and Umunnakwe, 1997). In view of the scarcity of dairy milk supply in Nigeria and the ever increasing gap between its requirement and population, efforts are being made to develop alternative milk-like beverage with enriched amino acids from vegetable sources using a plant protease (Papain) extracted from C. Papaya latex. C. papaya, a common plant that grows as weed at different dump sites, could be an important source of cysteine protease (papain) which occur naturally in papaya latex found in the 26 leaves, trunk and unripe fruits (Chu Chi Ming et al, 2002). The greener the fruit the more active is the papain (Chu Chi Ming et al, 2002).

Papain is one of the sulfhydyl proteases isolated from the latex of the green fruits of C. papaya (Arnon, 1970). The latex of C. papaya contains other types of cysteine endopeptidases, such as chymopapain carican, glycyl- endopeptidase (Sarote et al, 2008). Papain obtained by traditional methods is usually contaminated with other proteins present in the latex thereby reducing the activity of the enzyme (Sarote et al, 2008).

Tiger nut milk-like beverage, commonly called ‘Kunnu aya’ in northern Nigeria, is a healthy drink with many nutrients (Bamishaiye and Bamishaiye, 2011). It is a good source of nutrients such as vitamin E and C, minerals such as phosphorus, Magnesium, potassium, Calcium, iron and carbohydrates, unsaturated fatty acids, proteins and some enzymes which help in digestion and contains more Fe, Mg and carbohydrates than cow milk (Elena Sanchez–Zapata et al, 2012). It does not contain lactose, casein, sugar, proteins of the milk, cholesterol making it ideal milk for people that do not tolerate gluten or cow’s milk (Belewu and Abodunrin, 2006). It is an important food for infants and growing children (Obizoba and Anyika, 1995). In developing countries, the cost of dairy milk and their products are ex- orbital and prohibitive, leading to decrease in the consumption of milk and milk products (Udeozor, 2012). This led in part, the processing of milk from different seeds and nuts (Belewu and Belewu, 2007). Efforts have been made over the years to develop alternative milk-like products from vegetable sources in view of bridging the gap created by milk scarcity and the ever increasing population (Singh and Bains, 1988).

Among the source of vegetable milk, soy bean has received very high research attention and more researches are still being designed to improve the quality of soy milk (Sun-Young et al., 2000). But little or no attention has been given to tiger nut milk.

Development of milk substitutes extracted from legumes serves as an alternative source of producing an acceptable nutritious drink (Harkins and Sarret, 1967), prior to the development of such phyto milk like tigernut milk which serves as a less expensive substitute for dairy milk, direct milk consumption as a beverage was not common in Nigeria (Iwuoha and Umunnakwe, 1997; Onweluzo and Owo, 2005).

1.6 Aim and Objectives of the Study 27

1.6.1 Aim of the Study This work was aimed at investigating the qualities of papain extracted from the latex of Carica papaya and fortification and enrichment of milk–like beverage produced from tiger nut with amino acids hydrolysed from the protein of tiger nut by papain.

1.6.2 Specific Objectives of the Study The work was designed to achieve the following objectives. • To extract crude papain from the latex of C. papaya. • To purify the enzyme • To assay for the activity at different purification step. • To characterize the enzyme using pH, temperature and substrate concentration. • To prepare tiger nut protein. • To determine the optimum incubation time for papain to hydrolyse tiger nut (C. esculentus) protein. • To compare the degree of hydrolysis of tiger nut protein by the purified papain and O- phthaldialdehyde 28

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials 2.1.1 Carica papaya Carica papaya used for this study was obtained from Edem-Ani Community in Nsukka Local Government Area of Enugu state, Nigeria. The tigernuts used were purchased from Ogige modern market, Nsukka. The tigernuts were carefully selected to remove stones and other unwanted components before washing with distilled water and sun dried to remove dirts from it.

Fig. 6: Papaya fruit

2.1.2 Equipment Spectrophotometer 6405 UV/Visible, Jenway, England. Refrigerator Haier Thermocool, Nigeria. Stop watch Labtech, Germany. Thermometer Pyrex, England. Water Bath Galenkamp, England. Adjustable Micropipette Perfect (USA) Certrifuge Wischerfuge Model Blender Kelvinator, Korea 29

pH Meter Fischer Lab, England Weighing Balance Gallenkamp England Magnetic Stirrer Sward, England.

2.1.3 Chemicals The chemicals used for this study were of analytical grade and purchased from Lavan’s and Altran Scientific stores, Nsukka and RMRDC labouratory, Univeristy of Nigeria Nsukka.

Chemicals Product

Hydrochloric Acid BDH chemicals, England

(NH4)2SO4 BDH chemicals, England

Sodium Hydroxide Pellet Kermel chemicals, China

Tris salt BDH chemicals, England

Acetic Acid BDH chemicals, England

Phosphoric Acid BDH chemicals, England

Na2HPO4 Fluka chemicals, Germany

EDTA Pharmacia fine chemicals, Sweden

Coomassie Blue J.T. Baker chemicals co. philipsburg.

Ethanol BDH chemicals, England

Ovalbumin Bio-Rad, lab. India

Casein May and Baker, England

Sephadex G50 AB Uppsala, Sweden.

Sephadex G200 AB Uppsala, Sweden.

Sodium Dodecyl sulfate Riedel-Dettaen Hannaves (Germany)

Di sodium tetraborate decahydrate Sigma-Aldrich, USA.

Dithio threitol (DTT) Sigma-Aldrich, USA. 30

2.2 Methods 2.2.1 Preparation of tiger nut flour Tigernut was purchased, destoned and 500g was ground to produce tiger nut flour

2.2.2 Preparation of Tiger nut (C. esculentus) Protein Sedimentation fractionation of ground C. esculentus sample was carried out as described by Bautista et al. (1990). A quantity, 200g of ground sample was suspended in 500ml of distilled water and allowed to stand for 20mins to achieve uniform dispersion of the flour. The suspension was allowed to sediment for 15mins to enable the suspension resolve into three phases: a protein fraction that formed sediment at the bottom of the beaker; a soluble fraction in middle, and a lignocellulosic fraction that floated at the top. The lignocellulosic fraction was removed manually, while the soluble fraction and protein fraction was separate using separation funnel.

Tigernut sample solutions were prepared as follows:

A quantity, 0.1g-1.0g of tigernut flour sample containing 1.485% protein was dissolved in 10ml of distilled water respectively. They were allowed to standard for 120minutes with continuous stirring to produce tiger nut suspension.

2.2.3 Extraction of Papain from C. papaya Latex The procedure for extracting papain was carried out according to the method described by Chu chi Ming et al. (2002). The latex was obtained by making incisions on the surface of green fruits in the morning (9:00am), on the same tree for two weeks. A plastic bowl was held under the fruit to collect the latex. The collected latex (373.08 g) was dissolved in 1120 ml of 20 mM EDTA (3:1) ratio. The solution was stirred for 15min using a magnetic stirrer to produce a homogenous solution. Then the latex solution was centrifuged at 4000 rpm for 30min and filtered with whatman’s No1 filter paper, hence crude papain. Total volume of crude papain produced was 1,021ml.

2.2.4 Proximate Analysis The percentage concentrations of moisture, fats, ash, fibre and crude protein were determined for Cyperus esculentus using the AOAC (1990).

2.2.4.1 Determination of Moisture Content Tiger nut suspension, 50ml was measured and dried in an oven at 110oC to a constant weight. The dishes and samples were cooled and reweighed and percentage moisture content was calculated using.

Where W1 = Weight of sample

W2 = Initial weight of sample and dish

W3 = Final weight of dry sample.

2.2.4.2 Determination of Crude Protein The crude protein content was determined using the method of Micro-Kjeldahl (1883). This method is generally used to determine nitrogen (N) in substances that contain nitrogen (N) as ammonium salts, nitrates or organic nitrogen compounds. It measures the total amount of nitrogen in a compound, hence, only a rough indication of the total protein content can be obtained and is termed crude protein.

The quantity of nitrogen measured is then multiplied by a factor of 6.25 to calculate the protein content of the sample; this multiplication factor can vary with some materials

(AOAC, 1990). The nitrogen of protein and other compounds are converted in (NH4)2SO4 by acid digestion by boiling with H2SO4. The acid digest is cooled, diluted with water and made strongly basic with NaOH. Ammonium is released and distilled into a 2% boric acid solution.

The amount of ammonium borate formed is determined with standardized H2SO4 or HCl.

The indicator used, bromocresol green, gives a pink coloured end point at a hydrogen ion concentration corresponding to a solution of NH4Cl. Boric acid is a weak acid that has no appreciable influence on the pH. The reactions are represented as follows;

- NH3+HBO3 NH4 +BO2

+ H +BO2 HBO2 (AOAC, 1990)

The process has three stepsnamely;

a. Digestion of sample 32

b. Distillation of the ammonia into a 2% boric acid solution

c. Quantification of ammonia by titration

Digestion: A small quantity of sample (0.1g) was weighed into a kjeldahl flask with 2.0g sodium sulfate copper sulphate as catalyst. Concentrated tetraoxosulphate (vi) acid (20ml) was poured into the flask and the content until the content of the flask was completely digested giving a clear solution.

Distillation: The content of the flask was washed with 220ml distilled water into a distillation flask and cooled under ice blocks. 2% Boric acid (100ml) was poured into the flask and three (3) drops of the indicator were added.

Back Titration:

Cooled 40% NaOH (50ml) was added and the distillate was titrated against 0.5N Na2SO4 solution.

% Nitrogen =

Where T = Titre volume

N = Normality of acid

Df = Dilution factor

MWN = Molecular weight of nitrogen

% protein = % Nitrogen 6.25

Where 6.25 = conversion factor of nitrogen to protein.

2.2.4.3 Determination of Crude Fat The sample was continuously extracted with ether using a quick –fit flask. After extraction, the ether extract was evapourated to dryness. The ether extract contains fat portion, organic acids, oils, pigments, alcohol and fat-soluble vitamin and is hence termed crude fat. 33

A clean, dried and cooled quick-fit flask was weighed. Fresh sample of the suspension was weighed into extraction thimble and placed in the quick fit Soxhlet apparatus with a solvent flask containing 250ml of diethyl ether connected to a condenser. The set-up was heated for 16hours for complete extraction. The extract was evapourated at 70oC to remove any remaining solvent. The apparatus was reweighed and percentage crude fat calculated

%crude fat=

2.2.4.4 Determination of Ash Content Ash is defined as the mineral matter of a food, since it includes for the most part, the inorganic or mineral components of the food (Ensiminger and Odentine, 1978; Cullison, 1982). The sample was ashed in a muffle furnance at 600oC to burn off all organic materials. The inorganic material which did not volatilize at this temperature was designated ash.

Into weighed porcelain dishes, 5g of sample was added and reweighed. The crucible and samples were placed in a muffle furnance at 600oC for 3hr. The ash and crucible were cooled in a desicator and reweighed and percentage ash content was calculated using

% Ash =

Where W1- weight of crucible

W2- weight of crucible and sample

W3 – weight of crucible and ash

2.2.4.5 Determination of Crude Fibre Crude fibre includes those materials in food which are of low digestibility, such as cellulose, some hemi cellulose and lignin. A moisture – free, ether extract is digested first with weak acid solution (1.25% H2SO4), then with a weak basic solution (1.25% NaOH). The organic residue left after digestion was collected. The loss of weight on ignition was termed crude fibre.

The sample milk – like beverage, (2 g) was weighed into 500 ml beakers containing pre- heated dilute H2SO4 of about 40 ml. The content was boiled for 30 minutes and filtered. The 34 residue was washed three times with hot water, then, 150ml of pre-heated KOH and drops of antiform (Lactanol) were added to sample in the beaker and heated to boiling.

The mixture was boiled slowly for 30min more, filtered and washed three times with hot water. Acetone was used in washing it three times in cold extraction unit and the content dried at 130oC for 1 hr.

The content was ashed at 500oC and the ash weighed and percentage fibre calculated

% crude fibre =

2.2.4.6 Carbohydrate or Nitrogen Free Extract (NFE) These include mostly sugars, starches and more soluble hemicelluloses and lignins (Cullinson, 1982). Since it was designed to include the more soluble carbohydrate, it is sometimes referred to as the carbohydrate portion of the material being analyzed. Total carbohydrate was calculated by the difference method as reported by Onyeike et al., 2003. That is, summing the values of moisture, ash, crude protein, fibre, and crude fat and substracting the sum from 100.

100 – (%Moisture + % Crude protein + %Fat + %Fibre + %ash) = % NFE

2.2.5 Protein Estimation The protein content was estimated using Bradford method (1976). This method relies on the binding of coomassie Blue G250 to protein molecules. The amount of dye bound to protein can be estimated by monitoring absorbance at 595 nm. The dye appears to bind arginine residues in protein and this can lead to variation in protein-dye binding activity.

The commonly used standard bovine serum albumin (BSA) gives a very high absorbance value with this method. Therefore, a more suitable standard protein for this method is ovalbumin, because its’ response to dye binding is intermediary compared to several proteins.

2.2.6 Purification of Protein Determination of Ammonium sulphate percentage saturation suitable for papain production. Ammonium sulphate precipitation profile was carried out to determine the percentage at which papain production will be maximum. Nine test tubes were used; papain was precipitated at 20-90% saturation of ammonium sulphate salt at each test tube. The 35 ammonium sulphate-crude enzyme solutions were allowed to stand at 4oC for 12hr. The test tubes were centrifuged at 3500rpm for 30min.

Precipitates from the tubes were dissolved in equal volume of 0.05M Tris-HCl buffer pH 7.2. The precipitates were assayed for papain activity to ascertain the percentage with maximum activity. Crude enzyme filtrate (10 ml) was used in this process. From the result of ammonium sulphate profile, 80% ammonium sulphate saturation was suitable for precipitation of the enzyme.

Eighty percent ammonium sulphate precipitation was carried out by dissolving 154.8g of the salt in 300ml of crude enzyme and stirred gently until complete dissolution was achieved; the mixture was incubated for 12hr at 4oC. The precipitate obtained was redissolved in 50ml of 0.05M Tris buffer pH 7.2 after centrifugation and stored in a thermocool refridgerator at 4oC for further studies.

2.2.7 Assay of Enzyme Activity Papain activity was assayed using the method of Arnon (1970) by measuring the rate of digestion of proteins or by following the rate of hydrolysis of small molecular weight synthetic substrates such peptides, esters or amide derivatives of amino acids (Arnon, 1970). Papain exhibits very low activity at its native state; hence, all assays were carried out in the presence of an activator 0.002M EDTA at 275 nm.

This method is based on the estimation of the amount of the small molecular weight digestion products formed from proteins in the presence of the enzyme. Papain activity was assayed using a modified method described by Arnon (1970).The casein solution was brought to 37oC prior to the assay. Papain solution in amounts of 0.1 to 0.8ml was pipetted into test tube. The activating agent 0.2ml (0.002M EDTA) was added to each tube and the volume adjusted to 1ml with Tris-HCl buffer (pH 7.2). 1ml aliquot of the casein solution was added to each tube at intervals of 1 minute.

After mixing and incubation at 37oC for 10 minutes the reaction was stopped by adding 3 ml of trichloro-acetic acid which precipitates the residual large molecular weight material. The precipitate formed was filtered after standing for 60 minutes at 25oC. Absorbance was read at 275 nm against blank. The blank contained all the assay reagents except the enzyme.

2.2.8 Purification of Precipitated Enzyme 2.2.8.1 Sephadex G50 Gel Filtration A quantity, 10g of sephadex G-50 was dissolved in 700ml of warm distilled water, and allowed to stand of for 24hour. The gel was packed in a chromatography column 70.50cm and diameter 3.20cm. The length of gel was 52.43cm and equilibrated with 300ml of Tris buffer pH 7.2. Gel filtration was carried out with a view to removing ions that may interfere with enzyme activity. After the fractions were collected, they were stored at 4oC for further purification step.

2.2.8.2 Sephadex G200 Gel Filtration A quantity, 10g of sephadex G-200 was dissolved in 700ml of warm distilled water and allowed to stand for 24hour. The gel was packed in a chromatography column length 70.50cm and diameter 3.20cm. The length of gel was 53.44cm and equilibrated with 330ml of Tris buffer pH 7.2.

2.2.9 Studies on the Purified Papain

2.2.9.1 Effect of Temperature on the activity of papain The optimum temperature of papain was determined by incubating the enzyme with 1% casein solution at 40–110oC interval of 5oC for 60 minutes and pH 7.2. The activity was then assayed using the method described by Arnon (1970). Papain activity was assayed using a modified method described by Arnon (1970).The casein solution was brought to 37oC prior to the assay. Papain solution in amounts of 0.1 to 0.8ml was pipetted into test tube. The activating agent 0.2ml (0.002M EDTA) was added to each tube and the volume adjusted to 1ml with Tris-HCl buffer (pH 7.20). 1ml aliquot of the casein solution was added to each tube at intervals of 1minute.

After mixing and incubation at 37oC for 10minutes the reaction was stopped by adding 3ml of trichloro-acetic acid which precipitates the residual large molecular weight material. The precipitate formed was filtered after standing for 60 minutes at 25oC. Absorbance was read at 275 nm against blank. The blank contained all the assay reagents except the enzyme

2.2.9.2 Effect of pH on the activity of Papain The optimum pH for enzyme activity was determined using 0.05M sodium acetate buffer pH 3.5-5.5, phosphate buffer pH 6.0-7.5 and Tris buffer pH 8.0-9.0 at intervals of pH 0.5. The purified enzyme was incubated with the reaction medium containing 0.3ml of the respective 37 buffers for 60minute. Papain activity was assayed using the method described by Arnon (1970).

2.2.9.3 Effect of Substrate Concentration on the Activity of Papain The effect of substrate concentration on the activity of papain was determined by incubating the enzyme with 0.1, 0.2 0.3 0.4, 0.5, 0.6 0.7, 0.8, 0.9, and 1.0g/ml of 1% casein solution at pH 7.2 and 37oC.

The Vmax and Km values of enzyme were determined using the Lineweaver- Burk double reciprocal plot.

2.2.10 Hydrolysis of Tiger nut protein by Papain Protein hydrolysis was carried out as described by Nielsen et al. (2001); Calderon de la Barca et al. (2000). Degree of hydrolysis (DH) is defined as the percentage of cleaved peptide bonds.

DH = h/htot X 100. h is the number of hydrolysed bonds htot is the total number of peptide bond per protein equivalent. Protein hydrolysis was monitored by the degree of hydrolysis of the peptide bond present in the protein by papain and OPA at 340 nm.

The degree of protein hydrolysis was determined by incubating varying concentrations of tigernut sample (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0g) with 0.2 ml of papain at pH 7.5 and 37oC. The hydrolysate was read at 340 nm. Degree of hydrolysis was calculated according to the method of Nielsen et al. (2001).

Method: A quantity 0.1 -1.0g of the flour was dissolved in 100 ml of distilled water to produce a homogenous solution. Casein, which weighed 0.1-1.0 g, was also dissolved in 100 ml of distilled water respectively. Tris-HCl buffer pH 7.5 (2 ml) was first added to the tubes. 0.2 ml of OPA was added to tubes containing casein, and 0.2ml of papain was added to tubes containing the tigernut sample. The mixture was thoroughly mixed and incubated at 37oC for 30min before measuring absorbance in a spectrophotometer at 340 nm. 38

CHAPTER THREE

RESULTS

3.1 Proximate Composition of C. esculentus Milk-like Suspension

The results of the proximate analysis of C. esculentus milk-like suspension are presented in Table 2. After the proximate analysis of tiger nut, it was found that it contains 80.94% of moisture, 2.33% of crude fat, 1.49% of crude protein, 1.37% ash, 0.63% and 13.24% total carbohydrate.

Table 2: Proximate Analysis of C. esculentus milk-like Suspension Constitutents Proximate composition (%) Moisture 80.94 Crude fat 2.33 Crude protein 1.49 Ash 1.37 Fibre 0.63 Total carbohydrate 13.24

3.2 Ammonium Sulphate Precipitation Profile The enzyme was precipitated with varying percentages of ammonium sulphate (20 – 90%) saturation as represented in Fig 6. At 20% ammonium sulphate saturation, papain activity was found at 148 U/ml. At 30%, the point dropped to 109 U/ml but increased to 238 U/ml at 40% saturation.The activity also increased 287 U/ml at 50% saturation and was drastically decreased at 60% saturation of ammonium sulphate. At 70% saturation, there was gradual increase in the activity of papain to 306 U/ml. This increase continued at 80% saturation to give the highest activity of 384 U/ml. Hence, eighty percent saturation of ammonium sulphate gave the highest yield.

Fig. 6: Ammonium sulphate precipitation profile for papain from C.papaya latex

3.3 Studies on Crude Papain

Figure 7 represents the different protein concentrations of papain after some purification steps. Protein concentration of the crude enzyme was 158µg/ml. After ammonium sulphate precipitation, the protein concentration increased to 199 μg/ml, the protein concentration was 117μg/ml and 53 μg/ml after purifying with sephadex G50 and G200 respectively. PURIFICATION STEPS

200

180

160

140

120

100

0 G200

CRUDE ENZYME AMMONIUM SEPHADEX G50 SEPHADEX G50 SULPHATE

Fig. 7: Protein concentration at different purification steps

3.3 PURIFICATION TABLE

Table 3 shows the protein concentration, activity, specific activity, total activity and the yield of papain at different purification steps of ammonium sulphate, sephadex G50 and G200 respectively.

Table 3: The purification table for purified papain

PURIFICATION VOL.(ml) PROTEIN ACTIVTY SPECIFIC TOTAL % STEPS CONC. (U/Min) ACTIVITY ACTIVIT YIEL (µg/ml) (U/mg) Y D (U) CRUDE ENZYME 1000 136 179 1.28 179000 100 AMMONIUM 300 188 240 1.31 72000 94.70 SULPHATE PRECIPITATION SEPHADEX G50 65 109 361 1.15 10335 13.59 FILTRATION SEPHADEX G200 30 59 531 1.48 4830 6.36 FILTRATION

3.4 ENZYME CHARACTERISATION

3.4.1 Effect of pH Change on Papain Activity

Figure 8 shows that increase in changing values of the pH was accompanied by increasing enzyme activity up to the optimum pH of 7.5, after which the enzyme activity dropped steadily as shown by the plot. The enzyme has highest activity of 129U/min at pH 7.5.

Fig 8: Effect of pH on papain activity

3.4.2 Effect of Temperature Change on Papain Activity.

The optimum temperature for papain was determined over temperature range of 40 – 110C. The activity of papain increased from 40 C̊ to 90̊C but the activity decreased at 100̊C and 110̊C respectively. Temperature optimum was observed at 90oC as shown in Fig 9.

Fig. 9: Effect of temperature on papain activity

3.4.3 Determination of Kinetic Parametres

Kinetic parameters, Vmax and Km were calculatedfrom Lineweaver – Burk plot as shown in by Fig 10 below. The Vmax and Km values of the curve were found to be1.133U/min/ml and 0.217µg/min/ml respectively.

Fig. 10: Lineweaver-Burk plot of papain from C.papaya latex

3.4.4 EFFECT OF SUBSTRATE CONCENTRATION ON PAPAIN ACTIVITY

The optimum substrate concentration was determined over the range of 0.1 – 1.0g of casein, optimum concentration was observed at 0.6g/ml of the substrate.

Fig. 11: Effect of substrate concentration on papain activity 46

Fig .12: Percentage yield of papain at different purification steps

The optimum pH for papain was 7.50, optimum temperature was 90C̊ at substrate concentration of 0.60g/ml. The maximum velocity (Vmax) was 1.133U/min/ml while the Michealis Menten constant, Km was 0.487μg/min/ml. 47

Fig 13: Sephadex G50 gel filtration graph

Fig. 14: Sephadex G200 gel filtration graph

Figure 16 shows papain activity at different purification steps of ammonium sulphate precipitation, sephadex G50 and G200 respectively.

Fig. 16: Papain activity at different purification steps

Figure 16 shows the degree of protein hydrolysis by papain and OPA at different time intervals of 0.1g/ml protein concentration.

Fig 16: Degree of Hydrolysis against time at 0.1g/ml substrate concentration

Fig 17: Degree of hydrolysis against time at 0.2g/ml substrate concentration 52

Fig 18: Degree of hydrolysis against at 0.3g/ml substrate concentration 53

Fig 19: Degree of hydrolysis against time at 0.4g/ml substrate concentration

Fig 20. Degree hydrolysis against time at 0.5g/ml substrate concentration

Fig 21: Degree hydrolysis against time at 0.6g/ml substrate concentration

Fig 22: Degree hydrolysis against time at 0.7g/ml substrate concentration 57

Fig 23: Degree of hydrolysis against time at 0.8g/ml substrate concentration 58

Fig 24: Degree of hydrolysis against time at 0.9g/ml substrate concentration 59

Fig 25. Degree of hydrolysis against time at 1.0g/ml substrate concentration

CHAPTER FOUR

DISCUSSION

In this study, papain was extracted from the latex of unripe fruits of C. papaya for a period of 14 days. Harvested latex weighed 373.06g. The latex yielded 100% of the crude enzyme at pH 7.2, 37oC and incubation time of 10min using a modified method of Arnon (1970). Percentage yield of the enzyme decreased as the purification steps increased. The crude enzyme yield was 100% and after 80% ammonium sulphate precipitation step, the yield was 94.70%. Carrying out further purification steps using sephadex G50 and G200 gel filtrations, their percentage yields decreased to 13.59% and 6.36% respectively. The successive decline of the percentage yield after gel filtration indicated that sephadex G50 and G200 gels have the potential of removing bio molecules bound to the enzyme; hence a more purified enzyme was obtained after the successive purification steps. During these purification steps, protein estimation was carried out (crude enzyme, ammonium sulphate precipitation, sephadex G50 and G200 gel filtrations). Values of 136, 188, 109 and 59µg/ml respectively were obtained indicating that the purification steps removed some unwanted protein substances bound to the enzyme. The highest protein concentration value of 188µg/ml obtained after 80% ammonium sulphate saturation indicated that the salt could precipitate papain at high concentration.

Arnon (1970), Monti et al., (2000) and Nitsawang et al., (2006) had earier reported the use of ammonium sulphate in concentrating or precipitating papain. Ammonium sulphate precipitation profile was generated by assaying for the proteolytic activity of pellets extracted from the precipitation of crude papain isolated from C. papaya latex at 20% - 90% ammonium sulphate saturation.Eighty percent (80%) ammonium sulphate saturation gave the highest precipitate and hence, was used in the extraction of papain in this study. It was recognized that among the different precipitants, the most widespread was ammonium sulphate (Bodzon-Kulakowska et al, 2007). The addition of high amounts of this salt into a protein solution provokes an increase of protein interactions followed by protein aggregation and finally precipitation (Martinez-Maqueda et al, 2013). In this study, the highest enzyme precipitation was observed at 80% ammonium sulphate saturation as shown in Fig. 7. However, 40% ammonium sulphate saturation of papain from C. papaya latex was reported by Arnon (1970). Similary, Nitsawang et al (2006) observed 45% ammonium sulphate saturation and that ammonium sulphate enhanced protein aggregation caused by increase in the number of interactions between the surface hydrophobic groups as the protein concentration was increased. The aggregation of papain at lower ammonium sulphate 61 saturation of 40% and 45% as reported above may be attributable to the findings of Monti et al (2000). They noted that for papaya trees growing on damp soil, the latex obtained was highly diluted, with low papain concentration suggesting that soil texture and structure affect the quality of papain obtained from such trees. The papaya used in this study was got from a loam and well-drained soil and its latex contained concentrated papain as obtained from the results.

Increase in protein concentration after 80% ammonium sulphate precipitation as observed in this study is in accordance with the report of Copeland (2000), that protein can be precipitated by high concentration of ammonium sulphate saturation of 80%. This may reflect the reason ammonium sulphate is commonly used for the separation of cysteine proteases; this high salting out effect of ammonium sulphate on papain is connected to its higher surface hydrophobicity when compared to other proteases. Hence, the result of these findings is in line with the report (Copeland, 2000). After 80% ammonium sulphate precipitation, sephadex G50 and G200 filtrations, their specific activities were 1.28U/mg, 3.31U/mg and 9.0U/mg respectively.

The purified enzyme was characterized based on effects of pH change, temperature change and substrate concentrations on papain activities. The optimal pH of 7.5 was observed in this study for papain extracted from C. papaya latex with an optimal temperature of 90oC as shown in figures 9 and 10 respectively.

The effect of pH on papain activity was studied by conducting the experiments at varying pH values of pH3.5 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, keeping other conditions of temperature and substrate concentrations constant at 37oC and 1ml of 1% casein. Figure 9 shows that as the pH increased from pH 3.5 to pH 7.5, papain activity was found to increase, but further increase of pH beyond pH 7.5 resulted in a very sharp decrease of activity. This decrease at higher pH values toward alkaline may be attributed to the preference of neutral or near neutral pH environment above which the enzyme (papain) would be inactivated. Papain being isolated from a living organism prefers the maintenance of the same condition that prevails in the plants environment or very close to be able to act. Enzymes, as proteins contain varying degrees of ionizable groups, acidic and basic groups mainly positioned at the surface. The charges on these groups are different, but correspond to the pH of their environment. Any change in temperature or pH affects the charge distribution by these ionizable groups thereby interrupting the enzyme tertiary structure which possesses the activeness of the enzyme, thereby inactivating the enzyme. 62

Harton et al (2002) reported optimum pH in the range of 6.0-7.0. Similarly, Chu chi Ming et al (2002) analysed the effect of pH of papain covering both acidic and alkaline regions and reported that the fastest time for the milk clotting assay was in neutral region of pH 7.0. Furthermore, they observed that the activity was lower when in either acidic or alkaline region. Also, Ding et al, (2003) reported optimum pH of 7.0 using casein as substrate and incubating at 70oC for 120 min. Papain (from Carica papaya) bromelain (from Ananas comorus), ficin (ficus spp) are some of the neutral protease of botanical origin as reported by Sumantha et al., (2006). Maximum papain activity of 129U/min was obtained after incubation time of 30 min at pH 7.5 and 37oC, which led to the choice of pH 7.5 as the optimum pH.

The effect of temperature on papain production was carried out using casein as substrate by conducting the experiment at varying temperatures of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, and 110oC and keeping all other parameters constant at the incubation time of 30min. Figure 9 shows that as temperature increased from 40oC to 90oC, papain activity increased and maximum activity of 531µg/ml was obtained at 90oC but further increase in temperature at 100oC produced a sharp decline in the activity of papain. Therefore, optimum temperature was obtained at 90oC. The decrease observed at higher temperature 100oC-110o could be as a result of enzyme denaturation.

In this study, temperature optimum of 90oC was observed. This could be attributed to environmental conditions and climatic influences. This indicates that papain isolated from Edem-Ani, Nsukka can withstand high temperature (90oC) and could be used in different industries where high thermophilic enzymes are needed. The optimum temperature within the range of 65-80oC was reported by Horton et al (2002), but Chu Chi Ming et al (2002) observed optimum temperature of 50oC. Papain was reported by Schechter and Berger (1968) to be thermophilic. Also, Sumantha et al., (2006) reported optimum temperature between the ranges of 40-55 for cysteine protease.

The activity of papain was evaluated at different substrate concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0g/ml casein by incubating the enzyme with the substrate for 30min 37oC and pH 7.5. Figure 11 shows that as substrate concentration increased from 0.1g/ml to 0.4g/ml, papain activity increased progressively and maximum activity of 194U/min was obtained at 0.6gml, but subsequent increase in substrate concentration (0.7- 0.9g/ml) showed a decrease in the enzyme activity.The decrease observed could be as a result 63 of substrate inhibition or low enzyme to substrate ratio, where substrate molecules inhibit the activity of the enzyme.

The kinetic parameters Km and Vmax were evaluated using Lineweaver –Burk double reciprocal plot. Their values were found to 0.487µg/min/ml and 1.133mg/min/ml respectively. The values obtained from this study suggest that papain has high affinity for casein.

Hydrolysis of tiger nut protein homogenate was carried out by monitoring the degree of protein hydrolysis by O-pthaldialdehye (OPA) (a standard hydrolyzing reagent made of 7.628g of Di-sodium borate decahydrate, 200mg of sodium dodecyl sulphate, 176mg of dithreiotol in 200ml distilled water) and papain, a proteolytic enzyme. The OPA reaction was monitored in casein and tiger nut protein homogenate, while papain was only added to the tiger nut protein. The degree of hydrolysis by OPA in tiger nut protein was compared with that of the hydrolysis by papain in tiger nut protein homogenate at varying concentrations of the tiger nut sample and incubation time of 0, 10, 30, 60, and 120min 37oC and pH 7.5.

In Fig. 16, tiger nut concentration (0.1g/ml) of protein hydrolysis by papain at incubation time of 10mins was observed to be higher when compared to other incubation time. At 0.2g/ml concentration, the degree of hydrolysis by papain for 10min of incubation at 37oC and pH 7.5 was equally observed to be highest as presented in Fig 18. The degree of hydrolysis at 0 min by papain when incubated with 0.3g/ml of the sample was 9.33% and OPA was 8.96%, at 10min incubation, papain produced 9.61% while OPA 9.04% as obtained from Fig 19 where there was a noticeable decrease of degree hydrolysis as time increased. Decrease in degree of hydrolysis with increasing time of incubation was evident at all figures of tiger nut concentrations of 0.4, 0.5 to 1.0g/ml. Incubation time of 10min gave the highest degree of hydrolysis as represented in Figs 20,21, 22, 23, 24, 25, and 26 respectively.

From the results of this study, it was observed that papain hydrolysed tiger nut homogenate more than O-pthaldialdehyde (OPA).It was equally observed that the best incubation time for papain to hydrolyse tiger nut protein homogenate was 10min.

The result also suggest that papain, a proteolytic enzyme has more hydrolytic activity compared to OPA in hydrolyzing tiger nut protein homogenate. OPA, a standard hydrolyzing reagent was expected to give higher yield but the reverse was the case. This may be attributed to the fact that OPA contains a strong detergent, sodium dodecyl sulfate (SDS), which according to Eze et al. (2012) can affect the force of interaction within a protein and unfold 64 the protein slightly until a certain point is reached where the protein is denatured. It may be that it denatured completely the protein content of tiger nut as the time of incubation with the reagent increased. Similarly, from the report of Arnon (1970), the optimum incubating time for papain was 10min. On the other hand, papain was reported to have broad substrate specificity by Arnon (1970), therefore the result of this study is in accordance with earlier findings on papain. A high degree of hydrolysis may result in many free amino acids released from the terminal amino acid residue, reducing the number of hydrolysed peptide bonds in the longer peptide chain (Calderon de la Barca et al., 2000).

High values of hydrolysis by a proteolytic enzyme (papain) imply increase in the number of free amino acids in the solution (Nielsen et al., 2001) after their works on soybean, they observed increase in the value of the major soybean components, oil, and protein and antioxidant activity and suggested that disrupting the integrity of the plant cell wall component network by using cell –wall degrading enzyme might increase the extractability of those components. Some studies on enzyme-assisted hydrolysis of other plant materials have shown increase in the yield of protein and oil from material such as soybean, corn germ, rice bran and sunflower. The report of Suphamityotin (2011) equally indicated the activity of hydrolytic enzyme in hydrolyzing components such as protein, fats and other essential materials of the plant, and that incubation time significantly affects the amount of amino acid released during hydrolysis. Calderon de la Barca et al. (2000) reported optimum hydrolysis of 12hr and 50oC for soy protein. Yust et al. (2003) equally studied the improvement of protein extraction from sunflower meal by hydrolysis with alcalase and observed increase in the degree of hydrolysis with increasing concentration of substrate at alkaline pH 10 and 60min.

The optimum time (10min) for the hydrolysis of tiger nut protein homogenate by papain as observed from the present study is relatively short and would be advantageous in large scale industrial processes for protein hydrolysis.

4.2 Conclusion Papain was extracted from C. papaya latex, purified at 80% ammonium sulphate saturation on sephadex G50 and G200 filtrations and used in hydrolyzing tiger nut protein at 37oC and pH 7.5. The degree of hydrolysis by papain was monitored at varying incubation times of 0, 10, 30, 60 and 120 minutes and compared using O-phthaldialdehyde (OPA) as a standard hydrolyzing agent. The results obtained from this study shows that papain extracted from the latex of unripe C. papaya from a well drained soil possesses high proteolytic activity at near neutral pH 7.50 and high temperature of 90ºC. Papain also showed more proteolytic activity 65 in hydrolysing tiger nut protein homogenate than O-pthaldialdehyde, a standard hydrolysing reagent. Maximum incubation time of 10 min was observed from this study, which suggests that papain hydrolyses tiger nut protein homogenate in a very short time when compared to other proteolytic enzymes such as alcalase. 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 fortification of tiger nut milk-like beverage with enriched amino acids derived from tiger nut protein homogenate.

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