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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 Tris-HCL buffer and 0.1M sodium hydroxide. These
more attention as several clinical studies are indicating were subjected for freezing and thawing to remove
their benefits in oncology, inflammatory conditions, scum, gum until it produce clear latex sera separated by
blood rheology control, and immune regulation centrifugation at 10,000 g for 15min at 4 0c the same
(Brocklehurst et al. 1981;Apud Thomas 1994; Mellor et was used for protease assay in the study .
al. 1993), The main function of papain is to degrade or Protease Assay:
break down proteins. It does this by breaking the The protease assay was done according to the method of
peptide bonds. This function made possible because of Kunitz (1947). The enzyme extract of processed latex
the structure as it is the cysteine-25 portion of the triad was incubated with 1.0mL of 1% casein substrate
in the active site that attacks the carbonyl carbon in the prepared using 0.2 M phosphate buffer at pH 7.6 for 20
backbone of the peptide chain freeing the amino min at 37 oc. The reaction was arrested by addition of
terminal portion. As this occurs throughout the peptide 3.0 mL of 5% Trichloro acetic acid (TCA). The TCA
chains of the protein, the protein breaks apart. However, soluble fragments and total protein of the enzyme
these enzymes have different protein concentration at extract. Mix by swirling and incubate at 37°c for 30
different variety of papaya species. The functional minutes. Remove the vials and allow them to cool to
activation of proteins that are involved in blood room temperature. Filter through a 0.45 μm filter
coagulation, fibrinolysis and protein digestion in immediately prior to reading. Read the absorbance at
biological system. 660 nm for each of the vials in suitable cuvettes.
Therefore they have become the focus of wide range of Purification of crude enzyme
industrial and medical application, however the main Proteins are usually soluble in water because they have
aim of the work is to isolate proteolytic enzyme, helpful hydrophilic amino acids on their surfaces that attract
to the industries (Caygill 1971; Neidlema 1991) for the water molecules and interact with them. Thus solubility
large scale production of papain enzyme. is a function of the ionic strength and pH of the
This study focuses on the comparation of papain solution, Proteins have isoelectric points at which the
enzymes and identifies its protein concentration in charges of their amino acids side groups balance each
different varieties of papaya lateces. other, If the ionic strength of a solution is either very
MATERIALS AND METHODS high or very low, proteins will tend to precipitate at
Sample Collection: their isoelectric point. The solubility is also a function
Latex of Carica Papaya was collected from Hullikere, of ionic strength and as we increase the ionic strength
Chikkamangalore, Karnataka, India. by adding salt, proteins will precipitate, Ammonium
Latex of Red lady Papaya was collected from surface is the most common salt used for this purpose
Sakarapatna, Chikkamangalore, Karnataka, India. because it is unusually soluble in cold buffers,
Latex of Arka Surya and Arka Prabhath Papaya were Ammonium sulphate fractionation is commonly used is
collected from Indian Institute of Horticulture research research laboratories as a first step in protein
Campus, Hesaraghatta, Bangalore, Karnataka, India. purification because it provides some clued purification
Collection of Latex: of proteins away from non-proteins and also separates
Latex is obtained by cutting the skin of the unripe but some proteins. Ammonium sulphate also yields
almost mature papaya, then collecting and drying the precipitated protein slurry that is usually very stable.
latex which flows from the cuts, collecting the latex Dialysis is the process in which the solution is put
should start early in the morning. Two or three vertical inside a semi permeable bag and the bag is subjected to
cuts (except the first cut) 1-2mm deep are made, a solution of low ionic strength, Due to diffusion of the
meeting at the base of the fruit. The incisions are made materials from the bag to the hypotonic solution after a
using a stainless steel razor blade set into a piece of certain amount of time we have the pure material inside
rubber attached to a long stick. After about 4-6 minutes the bag, which is subjected to ion exchange
the flow of latex ceases. A dish is used to collect the chromatography for further purification.
latex and the latex is then scraped into a polythene lined Ion-exchange separations are carried out mainly in
box with a close fitting lid; such a box should be stored columns packed with an ion-exchanger. The
in the shade and stored at 4 0 c. chromatography column packed with DEAE cellulose
Protein Estimation: was washed using distilled water one to two times. The
Protein concentration in the enzyme extract was column was then washed with solution `A’ (10 ml of 25
determined using Folin Ciocalteu reagent as per the mm Tris HCl + 25 mm NaCl). The dialyzed enzyme
procedure of Lowry et al. (1951), The different aliquots sample was poured into the column, The enzymes were
of protein standard allowed reacting with Folin phenol then eluted using solution `B’ (10 ml of 25 mm Tris Hill
reagent, The absorption of the blue colour developed + 50 mm NaCl), The elutants were collected in the
was measured at 540nm using spectrophotometer. same test tubes, The process of elution was carried out
Isolation of protease: using solutions C, D, E, F and G which contains the
Clarification of crude latex performed on suitable different concentrations of NaCl.
dilution of latex in different buffer system with varying Parameter controlling protease production:
pH conditions. The buffers used were 0.2M glycine- Incubation temperature
HCL, 0.2M sodium acetate, 0.2M phosphate buffer, Papain enzyme was growing on production medium and

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


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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

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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
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Figure no 1 shows the thermal stability profile at various ACKNOLWDEGEMENT: Authors are very thankful to
temperatures. The optimum temperature of protease Azyme Biosciences, Bangalore for their Support in
enzyme was 400 C. carrying out research work.
Effect of time on protease enzyme activity increases REFERENCES:
with increase in the time in minutes. 1. Harrison RL, Bonning BC.2010. Proteases as
The pH dependence of the purified protease enzyme is Insecticidal Agents. Toxins 2: 935-953.
shown in Figure no 3. The activity was measured on a doi:10.3390/toxins2050935
pH range of 0-10.0. The optimum pH was found at pH 2. Agrawal, Konno. 2009. Latex: A model for
7.0. understanding mechanisms, ecology, and evolution of
The effect of substrate concentration on protease plant chemical analysis of Carica papaya L. crude
enzyme was maximum at 8ml of substrate is shown in latex defense against herbivory. Annual review of
figure no 4. ecology, evolution, and systematics 40:311-331.
doi:10.1146/annurev.ecolsys.110308-120307
DISSCUSSION:
3. Rao MB, Tanksale AM, Ghatge MS, Deshpande VV.
Proteases are a unique class of enzymes; they have
1998. Molecular and biotechnological protease. Microbiol
immense physiological as well as commercial importance. & Molecular Biol Reviews. 62 (3):597- 635
They possess both degradative and synthetic properties. 4. Devi MK, Banu AR, Gnanaprabhal GR, Pradeep BV,
They occur ubiquitously in plants, animals and microbes. Palaniswamy M. 2008. Purification, characterization of
Crude preparation of the enzyme has a wide specificity due alkaline protease enzyme from native isolate Aspergillus
to the presence of various proteinase and peptidase niger and its compatibility with commercial detergents.
isozymes. The activity of the enzyme depends on the plant Indian Journal of Science and Technology. 1: 1-6
source, and the methods used in its extraction and 5. Ire FS, Okolo BM, Moneke AA .2011. Purification and
purification. (Rao et al.1998) characterization of an acid protease from Aspergillus
In the present study four different varieties of papaya latex carbonarius. African Journal of Food Sciences. 5:695-709
were used for the papain isolation. 6. Pantoja A, Follett PA, Villanueva-Jiménez JA .2002.
Protein activity of different extracts was studied in which Pests of papaya. In: Tropical fruit pests and pollinators:
Ion exchange chromatography ammonium precipitate biology, economic importance, natural enemies and
showed good activity in all the four different varieties of control. CAB International. Cambridge USA. 131-156
papaya latex. Brocklehurst K, Baldev S, BAINES J, Paul G,
Arka Surya showed good activity followed by Arka MALTHOUSE .1981. Differences in the interactions of
Prabhath, Red Lady and Carica Papaya. the catalytic groups of the active centres of actinidin and
Optimum temperature is an important factor for industrial papain. Biochem. J. 197: 739-746
7. Mellor GW et al .1993. Ionization characteristics of the
enzymes selection because most industrial processes occur
Cys - 25/His - 159 interactive system and of the
at slightly higher than physiological temperature. The
modulatory group of papain: resolution of ambiguity by
enzyme had an optimum temperature of 40 oC and was electronic perturbation of the quasi - 2 - mercaptopyridine
stable up to the same temperature in a thermostability leaving group in a new pyrimidyl disulphide reactivity
studies. This observation is interesting because most probe. Biochem. J . 290: 289 - 96
industrial enzymes display optimum activity at 40-50oC 8. Caygill JC .1979. Sulfhydryl plant proteases. Enzyme and
(Devi et al. 2008; Ire et al. 2011). The pH dependence of Microb. Technology. 1: 233 -42
the purified protease enzyme was assessed. The activity 9. Neidlema SL.1991.Enzymes in the food industry a
was measured on a pH range of 0-10.0. The optimum pH backward glance. Food Technology. 45: 88 - 91
was found at pH 7.0. Both observations from the pH and 10. Thomas MP et al .1994. Structure of chymopapain M
temperature-activity relationships can note that the enzyme thelate - eluted chymopapain deduced by comparative
has the capacity to withstand the rigors of industrial modelling techniques and active – centre characteristics
applications such as food and brewing industries where determined by pH - dependent kinetics of catalysis and
these variables slightly differ with the physiological reactions with time – dependent inibitors: the Cys -
environment. 25/His - 159 ion - pair is insufficient for catalytic
We observed the increased in Protease enzyme activity competence in both chymopapain M and papain.
when the time was increased. The Substrate Concentration Biochem.J. 300: 805 – 20
of protease enzyme was maximum at 8 mL. 11. Kunitz M.1947. Crystalline soybean trypsin inhibitor ii.
CONCLUSION: General properties. J Gen Physiol. 30(4): 291–310
12. Lowry et al .1951. Protein measurement with the Folin
An innovative aspect of the current study helps to
Phenol Reagent. J Biol Chem. 193:265-275
determine the protease activity in the different lateces of
13. Dubey VK, Pande M, Singh BK, Jagannadham MV.
plant that could have possible positive effect on local 2007. Papain-like proteases: Applications of their
economics in relation to garden composting, leather inhibitors. African Journal of Biotechnology. 6:1077-
industries, food technology, breweries, or modification 1086.
of functional and nutritional properties of food
proteins through controlled enzymatic hydrolysis.

Source of support: Nil; Conflict of interest: None declared


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