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

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 37^oC. 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 90^oC and pH 7.5 at 37^oC 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 37 ^oC 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 37 ^oC 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

Abstract

CHAPTER ONE: INTRODUCTION

1.1       Carica papaya

1.1.1    Origin of C. papaya

1.1.2    Taxonomy

1.1.3    Morphology

1.1.4    Importance of C. Papaya

1.2       Enzymes

1.2.1    Enzyme structure

1.2.2    Enzyme nomenclature

1.7       Proteases (hydrolases) EC 3.4.

1.8       Cysteine proteases (3.4.22)

1.9       Papain (EC 3.4.22.2)

1.10 Mechanism of action of papain

1.11 Mechanism

1.12 Localisation and biosynthesis

1.2.5.3 Stability of papain

1.2.5.4 Papain specificity

1.2.5.5 Papain activators

1.2.5.6 Papain inhibitors

1.2.5.7 Applications of papain

1.3       Tiger nut (Cyperus esculentus var sativa)

1.3.1    Taxonomy of tiger nut

1.3.2    Origin of tiger nut Cyperus esculentus

1.3.3    Morphology

1.3.4    Phytochemical composition of tiger nuts

1.3.5    Economic importance of tiger nut

1.4       Protein

1.4.1    Classification of amino acids

1.4.2    Plant protein

1.4.2.1 Importance of plant protein

1.4.2.2 Enzymatic hydrolysis of protein

1.5 Tiger nut milk

1.6 Aim and objective of the study

1.6.1    Aim of the study

1.6.2    Specific objectives of the study

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials

2.1.1    Carica papaya

2.1.2    Equipment

2.1.3    Chemicals

2.2       Methods

2.2.1    Preparation of tiger nut sample

2.2.2    Preparation of tiger nut (C. esculentus) protein homogenate

2.2.3    Extraction of papain from C. papaya latex

2.2.4    Proximate analysis

2.2.4.1 Determination of moisture content

2.2.4.2 Determination of crude protein

2.2.4.3 Determination of crude fat

2.2.4.5 Determination of ash content

2.2.4.5 Determination of crude fibre

2.2.4.6 Carbohydrate or nitrogen free extract (NFE)

2.2.5    Protein estimation

2.2.6    Purification of protein

2.2.7    Assay of enzyme activity

2.2.8    Purification of precipitated enzyme

2.2.8.1 Sephadex G50 gel purification

2.2.8.2 Sephadex G200 gel purification

2.2.9    Studies on the purified enzyme

2.2.9.1 Effect of temperature change on papain activity

2.2.9.2 Effect of pH change on papain activity

2.2.9.3 Effect of substrate concentration on papain activity

2.2.10 Hydrolysis of tiger nut protein by papain

CHAPTER THREE: RESULTS

3.1 Proximate composition of tiger nut milk-like suspension

3.2 Ammonium sulphate precipitation profile

3.3 Studies on crude papain

3.4 Purification table

3.5 Enzyme characterization

3.5.1    Effect of pH change on papain activity

3.5.2    Effect of temperature change on papain activity

3.5.3    Determination of kinetic parameters

3.5.4    Effect of substrate on papain activity

CHAPTER FOUR: DISCUSSION

4.1       Discussion

4.2       Conclusion

REFERENCES

APPENDICES

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 16^th 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, 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)......

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