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International Journal of Agricultural and Food Science

Universal Research Publications. All rights reserved

ISSN 2249-8516
Original Article
Comparative Analysis of Papain from Different varieties
of Papaya Plant Latex.
Manjunath Hullikere M 1, Chandrashekhar G. Joshi1*, Vijay R1, Mahesh M2
1
Department of Biochemistry, Mangalore University, P.G.Centre, Chikkaaluvara, Kodagu Karnataka, India- 571 232.
2
Azyme Biosciences, Jayanagar 9th Block, Bangalore.
Email address: Josheejoshee@gmail.com;
Mobile- 91-9448446641
Received 07 November 2014; accepted 25 November 2014
Abstract
Background: Papain is a proteolytic enzyme that catalyzes the breakdown of a protein. It shows several biological
functions in plants and animals. It is used in many industries like food, leather, detergents, meat processing and
medical application like blood coagulation, fibrinolysis and d igestion process. Aim: The present study focused on
evaluation of proteolytic activity from lattices of different variety of papaya such as carica papaya, red lady, arca
surya and arca prabath. To study the protein concentration of various papaya lateces. Methods and Materials: Four
different varieties of papaya latex were collected from different parts of Karnataka. Papain was isolated from latex.
Estimation of protein was done by Lowry’s method. Protease enzyme was assessed by Kunitz method. Protease
controlling parameters were studied by assessing Temperature, pH, Time and Substrate concentration. Result: Arka
Surya showed good activity followed by Arka Prabhath, Red Lady and Carica Papaya. Conclusion: An innovative aspect
of the current study helps to determine the protease activity in the different lateces of plant that could have possible
positive effect on local economy.
© 2014 Universal Research Publications. All rights reserved
Key words: Papain, Carica Papaya, Red Lady, Arka Prabhath, Arka Surya, Protease.
compounds among lateces. Many of these compounds
INTRODUCTION provide resistance to herbivores via toxicity or
Papaya belongs to Caricaceae family and the plant is antinutritive effects, whereas others are involved in the
originated from Caribbean Coast of Central America. stickiness that can mire insect herbivores (Agrawal and
This small botanical family, native to tropical and Konno 2009). These defense-related components
subtropical America is represented by 31 species. appearing in latex of distant phylogenetic groups are
(Pantoja, Follett, & Villanueva-Jiménez, 2002) thought to have possible biological effects on
In India only, the production of papain annually is herbivores. Among these compounds are the proteases
around 150 tones. About 35% is BPC grade papain and which have shown toxicological effects on insects.
the rest is purified papain, 55% of the BPC grade papain Proteases from a variety of sources (viruses, bacteria,
is consumed internally and the rest is exported, while fungi, plants, and insects) have toxicity towards insects
90% of the purified papain is exported. The source of (Harrison and Bonning 2010).
the production of papain solely relies on latex. (Dubey et Papain is a proteolytic enzyme from the cysteine
al. 2007) proteinase family. It is manufactured from the latex of
Latex-bearing plants are believed to provide protection raw papaya fruits as papaya is very rich in papain. A
against the attack of herbivores. Latex is known to milky fluid known as latex containing papain oozes out
compose of various kinds of proteins including enzymes of the green papaya. The greener the fruit, more active
which interact with the cellular aspect of the host is the papain.
insects resulting in growth inhibition, physiological Papain enzyme are very important in digestion as they
damages and mortality. This prompted much research breakdown the protein foods to liberate the amino acids
endeavour aiming to provide exact information on the needed by the body. Additionally, proteolytic enzymes
defense mechanisms offered by the constituting have been used for a long time in various forms of
International Journal of Agricultural and Food Science 2014; 4(4): 123-127
123
therapy. Their use in medicine is gaining more and
more attention as several clinical studies are indicating
their benefits in oncology, inflammatory conditions,
blood rheology control, and immune regulation
(Brocklehurst et al. 1981;Apud Thomas 1994; Mellor et
al. 1993), The main function of papain is to degrade or
break down proteins. It does this by breaking the
peptide bonds. This function made possible because of
the structure as it is 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. However,
these enzymes have different protein concentration at
different variety of papaya species. The functional
activation of proteins that are involved in blood
coagulation, fibrinolysis and protein digestion in
biological system.
Therefore they have become the focus of wide range of
industrial and medical application, however the main
aim of the work is to isolate proteolytic enzyme, helpful
to the industries (Caygill 1971; Neidlema 1991) for the
large scale production of papain enzyme.
This study focuses on the comparation of papain
enzymes and identifies its protein concentration in
different varieties of papaya lateces.
MATERIALS AND METHODS
Sample Collection:
Latex of Carica Papaya was collected from Hullikere,
Chikkamangalore, Karnataka, India.
Latex of Red lady Papaya was collected from
Sakarapatna, Chikkamangalore, Karnataka, India.
Latex of Arka Surya and Arka Prabhath Papaya were
collected from Indian Institute of Horticulture research
Campus, Hesaraghatta, Bangalore, Karnataka, India.
Collection of Latex:
Latex is obtained by cutting the skin of the unripe but
almost mature papaya, then collecting and drying the
latex which flows from the cuts, collecting the latex
should start early in the morning. Two or three vertical
cuts (except the first cut) 1-2mm deep are made,
meeting at the base of the fruit. The incisions are made
using a stainless steel razor blade set into a piece of
rubber attached to a long stick. After about 4-6 minutes
the flow of latex ceases. A dish is used to collect the
latex and the latex is then scraped into a polythene lined
box with a close fitting lid; such a box should be stored
in the shade and stored at 4 0 c.
Protein Estimation:
Protein concentration in the enzyme extract was
determined using Folin Ciocalteu reagent as per the
procedure of Lowry et al. (1951), The different aliquots
of protein standard allowed reacting with Folin phenol
reagent, The absorption of the blue colour developed
was measured at 540nm using spectrophotometer.
Isolation of protease:
Clarification of crude latex performed on suitable
dilution of latex in different buffer system with varying
pH conditions. The buffers used were 0.2M glycine-
HCL, 0.2M sodium acetate, 0.2M phosphate buffer,
International Journal of Agricultural and Food Science 2014; 4(4): 123-127
124
Tris-HCL buffer and 0.1M sodium hydroxide. These
were subjected for freezing and thawing to remove
scum, gum until it produce clear latex sera separated by
centrifugation at 10,000 g for 15min at 4 0c the same
was used for protease assay in the study .
Protease Assay:
The protease assay was done according to the method of
Kunitz (1947). The enzyme extract of processed latex
was incubated with 1.0mL of 1% casein substrate
prepared using 0.2 M phosphate buffer at pH 7.6 for 20
min at 37 oc. The reaction was arrested by addition of
3.0 mL of 5% Trichloro acetic acid (TCA). The TCA
soluble fragments and total protein of the enzyme
extract. Mix by swirling and incubate at 37°c for 30
minutes. Remove the vials and allow them to cool to
room temperature. Filter through a 0.45 μm filter
immediately prior to reading. Read the absorbance at
660 nm for each of the vials in suitable cuvettes.
Purification of crude enzyme
Proteins are usually soluble in water because they have
hydrophilic amino acids on their surfaces that attract
water molecules and interact with them. Thus solubility
is a function of the ionic strength and pH of the
solution, Proteins have isoelectric points at which the
charges of their amino acids side groups balance each
other, If the ionic strength of a solution is either very
high or very low, proteins will tend to precipitate at
their isoelectric point. The solubility is also a function
of ionic strength and as we increase the ionic strength
by adding salt, proteins will precipitate, Ammonium
surface is the most common salt used for this purpose
because it is unusually soluble in cold buffers,
Ammonium sulphate fractionation is commonly used is
research laboratories as a first step in protein
purification because it provides some clued purification
of proteins away from non-proteins and also separates
some proteins. Ammonium sulphate also yields
precipitated protein slurry that is usually very stable.
Dialysis is the process in which the solution is put
inside a semi permeable bag and the bag is subjected to
a solution of low ionic strength, Due to diffusion of the
materials from the bag to the hypotonic solution after a
certain amount of time we have the pure material inside
the bag, which is subjected to ion exchange
chromatography for further purification.
Ion-exchange separations are carried out mainly in
columns packed with an ion-exchanger. The
chromatography column packed with DEAE cellulose
was washed using distilled water one to two times. The
column was then washed with solution `A’ (10 ml of 25
mm Tris HCl + 25 mm NaCl). The dialyzed enzyme
sample was poured into the column, The enzymes were
then eluted using solution `B’ (10 ml of 25 mm Tris Hill
+ 50 mm NaCl), The elutants were collected in the
same test tubes, The process of elution was carried out
using solutions C, D, E, F and G which contains the
different concentrations of NaCl.
Parameter controlling protease production:
Incubation temperature
Papain enzyme was growing on production medium and
incubated at different incubation temperatures viz.: 10, chromatography ammonium precipitate and Ion
20, 25, 30, 35, 40, 45, 50, 55 and 60 º c respectively. exchange chromatography Methanol precipitates.
Incubation time In case of Carica papaya ion exchange chromatography
The effect of incubation time was determined by ammonium precipitate showed good specific activity
incubating production medium for different incubation compare to all other extracts. Given in Table no 1.
periods 0, 5, 10, 15, 20, 25, 30 and 35 min at 35 º C. Specific activity of protein in Red lady papaya was more in
Taking other conditions into consideration. Ion exchange chromatography ammonium precipitate
Different pH values: (1.80) followed by Ion exchange chromatography
The different buffers prepared at different pH values Methanol precipitate (1.54) and Dialysis (1.32),
were applied. The production medium was adjusting whereas methanol (0.95) showed the least activity.
using a standard pH meter (model Jenway 3020 pH Crude showed 100% yield. Activity of different
meter). Other conditions were taken into consideration. fractions was showed in Table no 2.
Different substrate (casein) concentrations Ion exchange chromatography ammonium precipitate
The effect of different casein concentrations (g/l) was (3.09) showed highest specific activity in case of Arka
performed using 0, 2, 4, 6, 8, 10, 12 and 14 (w/v)), then Surya papaya followed by dialysis (2) and Ion exchange
incubated for 24 h at 37 º C. chromatography Methanol precipitate (1.72).Activity of
RESULTS Arka surya is given in Table no 3.
In the present study we estimated the protein Ion exchange chromatography ammonium precipitate
concentration, yield and protease activity from the latex (2.02) showed highest specific activity in case of Arka
of four different varieties of papaya. Prabhath papaya followed by Ion exchange
In this study we estimated the protein content of Carica chromatography Methanol precipitate (1.47) and
Papaya, Red lady, Arka surya and Arka prabatha in dialysis (1.42), whereas methanol (0.892) fraction
different extracts such as crude, Ammonium precipitate, showed less activity from all other fractions. Activity of
methanol precipitate, Dialysis, Ion exchange Arka Prabhath is given in Table no 4.
Table No.1: Estimation of protein concentration from Carica papaya.
Total Protein Specific
Fraction Activity(iu/ml) Fold Purification % yield
(mg) Activity
CRUDE 2.8 2.22 1.27 1.00 100
AMM PPT 2.4 2.00 1.23 0.96 85
METHANOL PPT 2.3 2.42 0.95 0.74 82
DIALYSIS 1.7 1.15 1.47 1.15 60
IEC AMM PPT 1.2 0.84 1.55 1.18 42
IEC METHANOL PPT 1.4 1.22 1.16 0.91 50
Table No.2: Estimation of protein concentration from Red lady Papaya.
Activity Total
Fraction Specific Activity Fold Purification %Yield
(iu/ml) Protein(mg)
CRUDE 2.8 2.70 1.03 1.0 100
AMM PPT 2.7 2.01 1.30 1.26 96
METHANOL PPT 2.3 2.40 0.95 0.87 82
DIALYSIS 2.2 1.75 1.32 1.31 78
IEC AMM PPT 2.0 1.15 1.80 1.74 71
IEC METHANOL PPT 2.1 1.43 1.54 1.49 75
Table No.3: Estimation of protein concentration from Arka Surya Papaya.
Activity Total Specific
Fraction Fold Purification %yield
(iu/ml) Protein(mg) Activity
CRUDE 3.15 2.42 1.30 1.00 100
AMM PPT 2.64 3.50 0.75 0.57 83
METHANOL PPT 2.50 3.92 0.64 0.49 79
DIALYSIS 2.43 1.26 2.00 1.53 76
IEC AMM PPT 2.16 0.78 3.09 2.37 68
IEC METHANOL PPT 2.40 1.42 1.72 1.32 75

International Journal of Agricultural and Food Science 2014; 4(4): 123-127

125
Table No.4: Estimation of protein concentration from Arka Prabhath Papaya.
Fraction Activity(iu/ml) Total protein(mg) Specific activity Fold purification % yield
CRUDE 2.52 1.87 1.38 1.0 100
AMM PPT 2.40 2.20 1.11 0.80 96
METHANOL PPT 2.52 2.82 0.892 0.64 99
DIALYSIS 2.00 1.41 1.42 1.02 79
IEC AMM PPT 1.73 0.82 2.02 1.46 68
IEC METHANOL PPT 1.9 1.35 1.47 1.06 75

Fig No. 1: Effect of Temperature on Protease enzyme .

Fig No. 2: Effect of Time on Protease enzyme.

Fig No. 3: Effect of pH on Protease enzyme.

Fig No. 4: Effect of substrate concentration on Protease enzyme

International Journal of Agricultural and Food Science 2014; 4(4): 123-127
126
Figure no 1 shows the thermal stability profile at various
temperatures. The optimum temperature of protease
enzyme was 400 C.
Effect of time on protease enzyme activity increases
with increase in the time in minutes.
The pH dependence of the purified protease enzyme is
shown in Figure no 3. The activity was measured on a
pH range of 0-10.0. The optimum pH was found at pH
7.0.
The effect of substrate concentration on protease
enzyme was maximum at 8ml of substrate is shown in
figure no 4.
DISSCUSSION:
Proteases are a unique class of enzymes; they have
immense physiological as well as commercial importance.
They possess both degradative and synthetic properties.
They occur ubiquitously in plants, animals and microbes.
Crude preparation of the enzyme has a wide specificity due
to the presence of various proteinase and peptidase
isozymes. The activity of the enzyme depends on the plant
source, and the methods used in its extraction and
purification. (Rao et al.1998)
In the present study four different varieties of papaya latex
were used for the papain isolation.
Protein activity of different extracts was studied in which
Ion exchange chromatography ammonium precipitate
showed good activity in all the four different varieties of
papaya latex.
Arka Surya showed good activity followed by Arka
Prabhath, Red Lady and Carica Papaya.
Optimum temperature is an important factor for industrial
enzymes selection because most industrial processes occur
at slightly higher than physiological temperature. The
enzyme had an optimum temperature of 40 oC and was
stable up to the same temperature in a thermostability
studies. This observation is interesting because most
industrial enzymes display optimum activity at 40-50oC
(Devi et al. 2008; Ire et al. 2011). The pH dependence of
the purified protease enzyme was assessed. The activity
was measured on a pH range of 0-10.0. The optimum pH
was found at pH 7.0. Both observations from the pH and
temperature-activity relationships can note that the enzyme
has the capacity to withstand the rigors of industrial
applications such as food and brewing industries where
these variables slightly differ with the physiological
environment.
We observed the increased in Protease enzyme activity
when the time was increased. The Substrate Concentration
of protease enzyme was maximum at 8 mL.
CONCLUSION:
An innovative aspect of the current study helps to
determine the protease activity in the different lateces of
plant that could have possible positive effect on local
economics in relation to garden composting, leather
industries, food technology, breweries, or modification
of functional and nutritional properties of food
proteins through controlled enzymatic hydrolysis.
Source of support: Nil; Conflict of interest: None declared
International Journal of Agricultural and Food Science 2014; 4(4): 123-127
127
ACKNOLWDEGEMENT: Authors are very thankful to
Azyme Biosciences, Bangalore for their Support in
carrying out research work.
REFERENCES:
1. Harrison RL, Bonning BC.2010. Proteases as
Insecticidal Agents. Toxins 2: 935-953.
doi:10.3390/toxins2050935
2. Agrawal, Konno. 2009. Latex: A model for
understanding mechanisms, ecology, and evolution of
plant chemical analysis of Carica papaya L. crude
latex defense against herbivory. Annual review of
ecology, evolution, and systematics 40:311-331.
doi:10.1146/annurev.ecolsys.110308-120307
3. Rao MB, Tanksale AM, Ghatge MS, Deshpande VV.
1998. Molecular and biotechnological protease. Microbiol
& Molecular Biol Reviews. 62 (3):597- 635
4. Devi MK, Banu AR, Gnanaprabhal GR, Pradeep BV,
Palaniswamy M. 2008. Purification, characterization of
alkaline protease enzyme from native isolate Aspergillus
niger and its compatibility with commercial detergents.
Indian Journal of Science and Technology. 1: 1-6
5. Ire FS, Okolo BM, Moneke AA .2011. Purification and
characterization of an acid protease from Aspergillus
carbonarius. African Journal of Food Sciences. 5:695-709
6. Pantoja A, Follett PA, Villanueva-Jiménez JA .2002.
Pests of papaya. In: Tropical fruit pests and pollinators:
biology, economic importance, natural enemies and
control. CAB International. Cambridge USA. 131-156
Brocklehurst K, Baldev S, BAINES J, Paul G,
MALTHOUSE .1981. Differences in the interactions of
the catalytic groups of the active centres of actinidin and
papain. Biochem. J. 197: 739-746
7. Mellor GW et al .1993. Ionization characteristics of the
Cys - 25/His - 159 interactive system and of the
modulatory group of papain: resolution of ambiguity by
electronic perturbation of the quasi - 2 - mercaptopyridine
leaving group in a new pyrimidyl disulphide reactivity
probe. Biochem. J . 290: 289 - 96
8. Caygill JC .1979. Sulfhydryl plant proteases. Enzyme and
Microb. Technology. 1: 233 -42
9. Neidlema SL.1991.Enzymes in the food industry a
backward glance. Food Technology. 45: 88 - 91
10. Thomas MP et al .1994. Structure of chymopapain M
thelate - eluted chymopapain deduced by comparative
modelling techniques and active – centre characteristics
determined by pH - dependent kinetics of catalysis and
reactions with time – dependent inibitors: the Cys -
25/His - 159 ion - pair is insufficient for catalytic
competence in both chymopapain M and papain.
Biochem.J. 300: 805 – 20
11. Kunitz M.1947. Crystalline soybean trypsin inhibitor ii.
General properties. J Gen Physiol. 30(4): 291–310
12. Lowry et al .1951. Protein measurement with the Folin
Phenol Reagent. J Biol Chem. 193:265-275
13. Dubey VK, Pande M, Singh BK, Jagannadham MV.
2007. Papain-like proteases: Applications of their
inhibitors. African Journal of Biotechnology. 6:1077-
1086.