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 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.

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,

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).

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

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

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

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) NUMBER	ENZYME CLASS	REACTION
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;

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).

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