Papain, a plant enzyme of biological importance: A review

Abstract

Papain is a plant proteolytic enzyme for the cysteine proteinase family cysteine protease enzyme in which enormous progress has been made to understand its functions. Papain is found naturally in papaya (Carica papaya L.) manufactured from the latex of raw papaya fruits. The enzyme is able to break down organic molecules made of amino acids, known as polypeptides and thus plays a crucial role in diverse biological processes in physiological and pathological states, drug designs, industrial uses such as meat tenderizers and pharmaceutical preparations. The unique structure of papain gives it the functionality that helps elucidate how proteolytic enzymes work and also makes it valuable for a variety of purposes. In the present review, its biological importance, properties and structural features that are important to an understanding of their biological function are presented. Its potential for production and market opportunities are also discussed.

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American Journal of Biochemistry and Biotechnology, 2012, 8 (2), 99-104
ISSN: 1553-3468
© 2012 Amri and Mamboya, This open access article is distributed under a Creative Commons Attribution
(CC-BY) 3.0 license
doi:10.3844/ajbbsp.2012.99.104 Published Online 8 (2) 2012 (http://www.thescipub.com/ajbb.toc)
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PAPAIN, A PLANT ENZYME OF BIOLOGICAL IMPORTANCE:
A REVIEW
Ezekiel Amri
and Florence Mamboya
Department of Science and Laboratory Technology,
Dar es Salaam Institute of Technology (DIT), P. O. Box 2958, Dar es Salaam, Tanzania
Received 2012-05-13; Revised 2012-05-20; Accepted 2012-06-02
ABSTRACT
Papain is a plant proteolytic enzyme for the cysteine proteinase family cysteine protease enzyme in which
enormous progress has been made to understand its functions. Papain is found naturally in papaya (Carica
papaya L.) manufactured from the latex of raw papaya fruits. The enzyme is able to break down organic
molecules made of amino acids, known as polypeptides and thus plays a crucial role in diverse biological
processes in physiological and pathological states, drug designs, industrial uses such as meat tenderizers and
pharmaceutical preparations. The unique structure of papain gives it the functionality that helps elucidate
how proteolytic enzymes work and also makes it valuable for a variety of purposes. In the present review,
its biological importance, properties and structural features that are important to an understanding of their
biological function are presented. Its potential for production and market opportunities are also discussed.
Keywords: Proteolytic enzyme, cysteine protease, papain, structure, hydrophobic
1. INTRODUCTION
Papain (EC 3.4.22.2) is an endolytic plant cysteine
protease enzyme which is isolated from papaya (Carica
papaya L.) latex. Papain is obtained by cutting the skin
of the unripe papaya and then collecting and drying the
latex which flows from the cut. The greener the fruit,
more active is the papain. Papain enzyme belongs to the
papain superfamily, as a proteolytic enzyme, papain is of
crucial importance in many vital biological processes in
all living organisms (Tsuge et al., 1999). Papain shows
extensive proteolytic activity towards proteins, short-
chain peptides, amino acid esters and amide links and is
applied extensively in the fields of food and medicine
(Uhlig, 1998). It preferentially cleaves peptide bonds
involving basic amino acids, particularly arginine, lysine
and residues following phenylalanine (Menard et al.,
1990). The unique structure of papain gives its
functionality that helps to understand how this
proteolytic enzyme works and it’s useful for a variety
of purposes. This review addresses structural features
of enzyme, the biological importance and processes in
which papain participates and its potential for
production market opportunities.
1.1. Properties, Structure and Features of Papain
The globular protein, the papain PDB accession
number 1CVZ is a single chain protein with molecular
weight of 23,406 DA and consists of 212 amino acid
with four disulfide bridges and catalytically important
residues in the following positions Gln19, Cys25, His158
and His159 (Mitchel et al., 1970; Robert et al., 1974;
Tsuge et al., 1999). The graphical representation of the
amino acid composition of papain is shown in Fig. 1.
Papain is a cysteine hydrolase that is stable and active
under a wide range of conditions. It is very stable even at
elevated temperatures (Cohen et al., 1986). Papain is
unusually defiant to high concentrations of denaturing
agents, such as, 8M urea or organic solvent like 70%
EtOH. Optimum pH for activity of papain is in the
range of 3.0-9.0 which varies with different substrate
(Edwin and Jagannadham, 2000; Ghosh, 2005).
Papain enzyme as cysteine proteases in papain
superfamily is usually consisting of two well-defined
domains which provide an excellent system for studies
in understanding the folding-unfolding behavior of
proteins (Edwin et al., 2002).

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Fig. 1. Graphical representation of the amino acid composition of papain
Fig. 2. Papain structure (MMDB protein structure summary,
1CVZ)
Fig. 3. Hydrophobic amino acid of papain (1CVZ). Colored gray
in a space fill model are the
backbone oxygen and nitrogen
of the residues with the hydrophobic side chain (Source:
http://www.oocities.org/bramsugar/intro4.html).
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 (Edwin
and Jagannadham, 2000; Tsuge et al., 1999). Its
three-dimensional structure consists of two distinct
structural domains with a cleft between them. This cleft
contains the active site, which contains a catalytic diad
that has been likened to the catalytic triad of
chymotrypsin. The catalytic diad is made up of the
amino acids-cysteine-25 (from which it gets its
classification) and histidine-159. Aspartate-158 was
thought to play a role analogous to the role of aspartate
in the serine protease catalytic triad, but that has since
then been disproved (Menard et al., 1990).
Papain molecule has an all-α domain and an
antparallel β-sheet domain (Kamphuis et al., 1984;
Madej et al., 2012). The conformational behavior of
papain in aqueous solution has been investigated in the
presence of SDS and reported to show high α-helical
content and unfolded structure of papain in the presence of
SDS is due to strong electrostatic repulsion (Huet et al.,
2006). In the molten globule state (pH 2.0), papain show
evidence of substantial secondary structure as ß-sheet
and is relatively less denatured compared to 6 M
Guanidium Hydroc8hloride (GnHCl), the enzyme also
exhibits a great tendency to aggregate at lower
concentrations of GnHCl or a high concentration of salt
(Edwin and Jagannadham, 2000). Papain apart from
being most studied plant cysteine proteases, further
researches in understanding the specificity, the structural
the effect brought by inhibitors, low pH, metal ions and
fluorinated alcohols has been identified as of critical
importance (Huet et al., 2006; Naeem et al., 2006).
Figure 2 shows the structure of papain from Molecular
Modeling Database (MMDB), the structure is shown
with all-α domain and an antparallel ß-sheet domain.

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1.2. Hydrophobicity of Papain
It is often useful to examine the relative
hydrophobicity or hydrophilicity values of the amino
acids in a protein sequence. Since hydrophobic residues
tend to be more buried in the interior of the molecule and
hydrophilic residues are more exposed to solvent, a
profile of these values can indicate the overall folding
pattern. The hydrophobic interactions have the major
influence in protein conformation and the most
hydrophobic of the amino acid side chains are those of
alanine, Valine, leucine, methionine and Isoleucine
which vary in degrees of hydrophobic. The hydrophobic
-hydrophilic interaction of papain amino acids in the side
chain seems to be the major thermodynamic forces
which drive protein folding. Investigation of the
formation of the intermediate state of papain through
inducing n-alkyl sulfates including sodium octyl sulfate,
SOS; sodium decyl sulfate, SDeS; and sodium dodecyl
sulfate, SDS at different concentrations has exhibited
that hydrophobic interactions play an important role in
inducing the two different intermediates along the two
various thermodynamic pathways (Chamani et al.,
2009). Catalytic activity of papain involves hydrolysis of
proteins with broad specificity for peptide bonds, but
preference for an amino acid bearing a large hydrophobic
side chain at the P2 position while does not accept Val in
P1 (Kamphuis et al., 1985). The enzyme has been
reported to be generally more stable in hydrophobic
solvents and at lower water contents and can catalyze
reactions under a variety of conditions in organic
solvents with its substrate specificity little changed from
that in aqueous media (Stevenson and Storer, 1991). In
general, native proteins have a hydrophobic core and a
charged and/or polar group on the surface. The
hydrophobic core helps to stabilize the tertiary structure of
the protein by hydrophobic interaction
while the outer
polar surfaces preferentially interact with the exterior
aqueous medium (Wang et al., 2006). Figure 3 shows
hydrophobic amino acid of papain in space fill model.
Hyrophobicity of papain using carbon distribution
profile along the sequence of papain is shown in Fig. 4.
The graph indicates that carbon content is maintained at
31.45% of carbon all along the sequence. Some regions
along the sequences have values above 31.45%, these are
considered to be higher hydrophobic regions as it has
previously been reported when using carbon content
distribution profile (Rajasekaran and Vijayasarathy, 2011).
Thus, the overall hydrophobicity of papain enzyme being
maintained at 31.45% of carbon all along the sequence
contribute to stability of protein as previous been reported
that stable and ordered proteins maintain 31.45% of
carbon all along the sequence (Jayaraj et al., 2009).
1.3. Mechanism, Biological Importance and
Functions
1.4. Mechanism of Functions
The mechanism in which the function of papain is
made possible is through the cysteine-25 portion of the
triad in the active site that attacks the carbonyl carbon in
the backbone of the peptide chain freeing the amino
terminal portion. As this occurs throughout the peptide
chains of the protein, the protein breaks apart. The
mechanism by which it breaks peptide bonds involves
deprotonation of Cys-25 by His-159. Asparagine-175
helps to orient the imidazole ring of His-159 to allow
this deprotonation to take place. Although far apart
within the chain, these three amino acids are in close
proximity due to the folding structure. It is though these
three amino acids working together in the active site that
provides this enzyme with its unique functions. Cys-25
then performs a nucleophilic attack on the carbonyl
carbon of a peptide backbone (Menard et al., 1990;
Tsuge et al., 1999). In the active site of papain, Cys -25
and His -159 are thought to be catalytically active as a
thiolate-imidazolium ion pair. Papain can be efficiently
inhibited by peptidyl or non-peptidyl N-nitrosoanilines
(Guo et al., 1996; 1998). The inactivation is due to the
formation of a stable S-NO bond in the active site (S-
nitroso-Cys
25
) of papain (Xian et al., 2000).
1.5. Papain in Medical Uses
Papain acts as a debris-removing agent, with no
harmful effect on sound tissues because of the enzyme’s
specificity, acting only on the tissues, which lack the α1-
antitripsine plasmatic antiprotease that inhibits proteolysis
in healthy tissues (Flindt, 1979). The mechanism of
biochemical removal of caries involves cleavage of
polypeptide chains and/or hydrolysis of collagen cross-
linkages. These cross-linkages give stability to the
collagen fibrils, which become weaker and thus more
prone to be removed when exposed to the papain gel
(Beeley et al., 2000). Papain-based gel has also been
reported as a potential useful in biochemical
excavation procedures for dentin (Piva et al., 2008).
Papain has advantages for being used for
chemomechanical dental caries removal since it does
not interfere in the bond strength of restorative
materials to dentin (Lopes et al., 2007).
Papain enzyme has a long history of being used to
treat sports injuries, other causes of trauma and allergies
(Dietrich, 1965). Fortunately papain has a proven track
record in managing all of these conditions with clinical
evidence of significant benefits for use of papain
protease enzyme in cases of sports injury.

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Fig. 4. Carbon distribution profile along the sequence of papain (1CVZ)
It has previously been reported that minor injuries healed
faster with papain proteases than with placebos.
Furthermore, athletes using papain protease supplements
were able to cut recovery time from 8.4 days to 3.9 days
(Trickett, 1964; Dietrich, 1965). Papain also has been
successfully used to overcome the allergies associated
with leaky gut syndrome, hypochlorhydria (insufficient
stomach acid) and intestinal symbiosis like gluten
intolerance. Papain has previously been reported to have
significant analgesic and anti-inflammatory activity against
symptoms of acute allergic sinusitis like headache and
toothache pain without side effects (Mansfield et al., 1985).
1.6. Papain Uses in Drug Design
Papain shares many features with physiologically
important mammalian cysteine proteases and show nearly
identical folding patterns especially around the active site
which has been useful for drug design (Meara and Rich,
1996). The X-ray coordinate system for papain solved at 1.7
A resolutions is a representative example of the structure of
a covalent ligand-bound cysteine protease complex
particularly in the papain superfamily (Tsuge et al., 1999).
Thus, papain is reported to be useful as an experimental
model structure to understand the inhibition mechanism of
newly developed specific inhibitors of cathepsin L, the
papain superfamily and its an antioxidant properties can be
useful in preventing certain types of illnesses (Tsuge et al.,
1999; Gayosso-Garcia et al., 2010). Since most of the
amino acid residues that are involved in the binding to
papain are conserved in cathepsin L, this publicly available
high resolution structure has provided an excellent model
for the successful design of highly active and specific
cathepsin L inhibitors (Katunuma et al., 1999). Papain is
also reported to be used as a surrogate enzyme in a drug
design effort to obtain potent and selective inhibitors of
cathepsin K, a new member of the papain superfamily of
cysteine proteases that is selected and highly expressed
in osteoclasts (LaLonde et al., 1998). Papain is also
reported to be useful as catalyzed (co) oligomerization of
α-amino acids (Schwab et al., 2012).
1.7. Industrial Uses and Pharmaceutical
Preparations
Papain is used in meat tenderizers; the major meat
proteins responsible for tenderness are the myofibrillar
proteins and the connective tissue proteins. Protease
enzymes are used to modify these proteins and papain has
been extensively used as a common ingredient in the
brewery and in the meat and meat processing (Khanna and
Panda, 2007). Papain importance as tenderizers in the food
industry is similar to collagenases, which have application
in the fur and hide tanning to ensure uniform dying of
leather. Papain also can act as a clarifying agent in many
food industry processes. As a protein digestant, papain is
used in combating dyspepsia and other digestive disorders
and disturbances of the gastrointestinal tract (Huet et al.,
2006). Papain has for quite a long time been used in
pharmaceutical preparations of diverse food manufacturing
applications as the production of high quality kunafa and
other popular local sweets and pastries. Papain has been
reported to improve meltability and stretchability of Nabulsi
cheese with outstanding fibrous structure enhancing
superiority in the application in kunafa, pizza and pastries
(Abu-Alruz et al., 2009). Also as pharmaceutical products
in gel based a proteolytic cisteine enzyme, papain presents
antifungal, antibacterial and anti-inflammatory properties
(Chukwuemeka and Anthoni, 2010).
1.8. Potential for Production and Market
Opportunities
Papain enzyme is extracted from Carica papaya
which is a tropical and a herbaceous succulent plant that
possess self supporting stems which grows in all tropical
countries and many sub-tropical regions of the world
(Jaime et al., 2007). Moreover, there is no limitation due
to seasonality as the papaya is available almost round the

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year. Consequently, there is a need to facilitate the
entrepreneurs in understanding the potential of papaya
production and the importance of setting up a unit of
papain. A well managed papaya production has recorded
higher papain yield of 8.17 g per fruit and highest papain
of 686.29 g per plant in a period of 6 months
(Kamalkumar et al., 2007; Reddy et al., 2012). Papain is
used in many industries such as breweries,
pharmaceuticals, food, leather, detergents, meat and
fish processing for a variety of processes. Therefore,
the end use segments are many in signifying that
papain has high export demand. Since there are good
prospects for papain market, the papaya production
and extraction of papain can be a high source of
income even for small farmers.
2. CONCLUSION
Papain has revealed to be an enzymatic protein of
significant biological and economic importance. It is
through the unique structure of papain that provides
functionality and helps explain how this proteolytic
enzyme works and also makes it valuable for a variety of
purposes. Further researches on papain enzyme in
understand the specificity, the structural the effect
brought various thermodynamic pathways is of critical
importance. Papain is found naturally in papaya which is
a versatile plant having number of uses and enzymatic
properties. Since the papaya grows in a wide range of
climate, papaya production for extraction of papain can
be a source of earning a high income to farmers.
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