Enzyme and Microbial Technology 39 (2006) 11031107

Purication of papain from Carica papaya latex: Aqueous two-phase

extraction versus two-step salt precipitation
Sarote Nitsawang
a
, Rajni Hatti-Kaul
b,
, Pawinee Kanasawud
a
a
Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
b
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, SE-221 00 Lund, Sweden
Received 4 January 2006; received in revised form 10 February 2006; accepted 13 February 2006
Abstract
Purication of papain from wet Carica papaya latex by extraction in aqueous two-phase system was studied and compared with the traditional
procedure involving a two-step salt precipitation. The papain obtained by the latter method was usually contaminated with other proteins, and its
purity was dependent on the initial protein concentration in the material used for processing. Highly pure papain was obtained in a much shorter
processing time directly from unclaried latex with the use of an aqueous two-phase system consisting of 8% (w/w) polyethylene glycol and 15%
(w/w) ammonium sulfate.
2006 Elsevier Inc. All rights reserved.
Keywords: Carica papaya latex; Papain; Aqueous two-phase extraction; Two-step salt precipitation
1. Introduction
The latex of Carica papaya is a rich source of the cysteine
endopeptidases, including papain, glycyl endopeptidase, chy-
mopapain and caricain, which constitute more than 80% of the
whole enzyme fraction [1]. Papain (EC3.4.22.2) is a minor con-
stituent (58%) among the papaya endopeptidases [13]. The
enzyme is used widely as meat tenderizer, and has also sev-
eral other applications, e.g. for debrinating wounds, treatment
of edemas, shrink proong of wool, etc. Purication of papain
from papaya latex has traditionally been achieved by precipita-
tion methods [46], however, the puried enzyme still remains
contaminated with other proteases. An alternative purication
strategy has involved the use of various chromatographic tech-
niques including ion exchange, covalent, or afnity chromatog-
raphy [1,710], but here the initial processing of the latex is
essential before samples can be applied on a chromatography
column.
Aqueous two-phase systems (ATPS), made up of two poly-
mers or one polymer and a salt in water, have shown interesting
potential for downstream processing of proteins, especially in
view of providing integrated clarication, concentration and

Corresponding author. Tel.: +46 46 222 4840; fax: +46 46 222 4713.
E-mail address: Rajni.Hatti-Kaul@biotek.lu.se (R. Hatti-Kaul).
purication of the target product in one unit operation [11]. They
have been successfully applied for large-scale enzyme separa-
tion and purication [1214]. A desired partition of proteins
in such systems can be obtained by manipulating a variety of
system parameters [15,16]. The polymersaltwater systems
have the advantage of low cost and low viscosity compared
to polymerpolymerwater systems. The most frequently used
among the former systems has been the polyethylene glycol
(PEG)phosphate [13,17], however other salts with multivalent
anions have also been used [18]. Use of PEGphosphate system
has earlier beenreportedfor separationandpuricationof papain
from papaya latex [19]. The study showed that the separation of
papain was negatively affected by the presence of chymopapain,
however the use of PEG modied with Procion Blue enhanced
the partition of papain to the PEG rich upper phase.
This paper reports the use of ATPS composed of PEG and
ammonium sulfate for purication of papain from C. papaya
latex and compares it with the conventional method based on
precipitation.
2. Materials and methods
2.1. Materials
The papaya fruit, C. papaya grown locally (Chiang Mai, Thailand) was
used as starting latex material. Standard papain, polyacrylamide, bis-acrylamide,
0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2006.02.013
1104 S. Nitsawang et al. / Enzyme and Microbial Technology 39 (2006) 11031107
ammonium persulfate and PEG 6000 were purchased from SigmaAldrich (St.
Louis, USA), while casien (Hammarsten) was obtained from BDH (Poole, Eng-
land).
2.2. Isolation of latex from C. papaya
Fresh latex was collected from locally grown C. papaya. Initially, four to
six longitudinal incisions were made on the unripe fruit using a stainless steel
knife. The exuded latex was allowed to run down the fruit and drip into collecting
devices attached around the trunk. Following collection, the latex was transferred
to a plastic bottle and stored at 20

C.
2.3. Purication of papain from latex by two-step salt
precipitation
The procedure used was modied from that reported earlier by Kimmel and
Smith [4] and Baines and Brocklehurst [6]. Thawed latex was mixed with 40 mM
cysteine at a ratio of 3:1 (w/v) and the suspension was adjusted to pH 5.6 using
6 M HCl and then stirred for 15 min at 4

C. The mixture was ltered and pH

of the ltrate was adjusted to 9.0 using 6 M NaOH. The insoluble material was
removed by centrifugation at 9000 g for 30 min at 4

C. The protein content in

the supernatant was determined and then adjusted with water before precipitation
with (NH
4
)
2
SO
4
at 45%saturation. The salt-enriched solution was slowly stirred
at 4

C for 30 min. The precipitate was collected by centrifugation as above, and

dissolved using 20 mM cysteine. The solution was kept at 4

C before adding
sodium chloride (10%, w/v). The mixture was slowly stirred for 30 min before
separating the precipitated papain by centrifugation. The enzyme was dissolved
in water and dialyzed overnight at 4

C against three changes (1 l each) of water.

The dialysate was nally lyophilized (in a freeze-dryer, LY5FM-ULE, Snijders
Scientic BV, Tilburg, The Netherlands) to obtain puried papain powder.
2.4. Purication of papain by extraction in aqueous two-phase
system
The thawed latex was mixed with water and pH adjusted using 6 M HCl.
For extraction in aqueous two-phase system, dened amounts of solid PEG and
(NH
4
)
2
SO
4
were added to the latex preparation (30 g), and the total mixture was
made up to 50 g with water. When studying the effect of pH on protease/papain
partitioning, the latexwas adjustedtothe desiredpHusing6 MHCl or 6 MNaOH
solutions prior to mixing with the phase components. The mixture was then
gently shaken for 15 min. The two phases were separated by centrifugation at
9000 g for 30 min at 4

C. Aliquots of the phases were taken for determination

of protein concentration and protease activity. For calculation of protease activity
inthe twophases, volumes of the respective phases were takenintoconsideration.
The presence of papain was veried by cathodic gel electrophoresis and FPLC.
For analysis of papain purity by FPLC, the top phase (6 ml) froman aqueous
two-phase systemwas dialyzedthree times at 4

Cagainst 50 mMsodiumacetate
buffer at pH5. The dialyzed solution was subjected to ion-exchange chromatog-
raphy for separation of the enzyme from PEG [20]. The solution was loaded on
a CM-cellulose column (1.5 cm2 cm) equilibrated with 50 mMacetate buffer,
pH 5 and after washing the enzyme was eluted from the column with the buffer
containing 1 M NaCl. Papain was largely eluted at the front of the two buffers.
The fractions containing protease activity were pooled, dialyzed and lyophilized
to obtain puried papain powder.
2.5. Protease activity determination
The proteolytic activity was determined according to the procedure of Arnon
[21] with slight modication. The reaction mixture contained 200 l of 50 mM
cysteine20 mMEDTA(disodiumsalt), pH8.0, 700 l 50 mMTrisHCl buffer,
pH 8.0 and 100 l enzyme solution. The mixture was incubated at 37

C for
5 min before starting the reaction by adding 1 ml of 1% (w/v) casein solution.
After 10 min, the reaction was stopped by adding 3 ml of 5%(v/v) trichloroacetic
acid (TCA) and then cooled for 1 h. The reaction mixture was centrifuged, and
absorbance of the supernatant was measured at 275 nm. The reading was cor-
rected for a blank in which the enzyme was added after addition of TCA.
One unit of protease activity was dened as the amount of enzyme giving sol-
uble digestion product providing an increase in absorbance at 275 nmequivalent
to 1 mol of tyrosine/min under the assay conditions.
2.6. Determination of protein content
The protein content in the samples during purication was determined by
Bradford method [22].
2.7. Cathodic electrophoresis
The experiment was carried out according to the method of Reisfeld et al.
[23] with some modication, using a slab gel consisting of a resolving gel
(pH 4.3, 15%, w/v, acrylamide) and a stacking gel of 4% acrylamide (pH 6.7).
The upper and lower chamber electrode buffer (pH 4.5) consisted of 0.36 M
beta-alanine0.14 M acetic acid. The protein sample was diluted (1:1, v/v) with
stacking buffer (pH 6.7) containing 10% sucrose and 0.002% basic fuchsin
(used as a tracking dye) prior to loading onto the gel. Electrophoresis was run at
a constant current of 40 mA. The protein samples migrated towards the cathode
during electrophoresis. The relative concentration of the papain was quantied
by measuring the absorbance of the protein bands at 595 nm using CS-9301 PC
densitometer (Shimadzu) after staining with Coomassie Brilliant Blue R-250.
The in situ proteolytic activity was determined on the gel after cathodic
electrophoresis [24]. The gel was rinsed twice with 50 ml of 0.1 M TrisHCl
buffer, pH 8.0, and then a solution containing 0.5% (w/v) agarose and 1.8%
(w/v) casein in the same buffer was applied on the gel surface. The gel was
incubated at 37

C between 12 and 48 h to visualize the clear zone of proteolytic

activity.
2.8. Purity analysis by fast protein liquid chromatography (FPLC)
Purity of the puried papain was also evaluated by ion-exchange chromatog-
raphy on FPLC (Pharmacia-Biotech, Sweden). Twenty-ve microliter solution
of the puried papain (25 g protein) in 20 mM glycineNaOH buffer, pH 10.6
was applied on a Mono Q HR 5/5 (1 ml) column pre-equilibrated with the same
buffer. The column was washed with 5 ml buffer. Papain and other proteins were
subsequently eluted using a programmed linear gradient of sodiumchloride from
0 to 0.5 M (total volume of about 24 ml) at a ow rate of 1.0 ml/min. Fractions
of 1 ml were collected. Chromatographic plots of A
280
and gradient composition
versus elution volumes were recorded. The elution peak of papain was conrmed
by using standard papain. The percentages of peak areas of papain and other
proteins were obtained from an automatic integrator. The purity of papain was
specied as the percentage peak area of papain with respect to the total peak area.
3. Results and discussion
The fresh milky latex extracted from 23 samples of Thai
C. papaya contained 40 13 mg protein and 529 162 units
protease activity/g wet latex. As papain is a protease of broad
specicity and no specic synthetic substrate is available, casein
was used as a substrate to determine the total protease activity
present in the latex while the purity of papain was determined
by electrophoresis and chromatography. On cathodic gel elec-
trophoresis, the proteins in the latex separated as ve bands
(Fig. 1, lane 2). One of these proteins was identied as papain
according to its mobility that was equal to that of the standard
papain and its in situ proteolytic activity on polyacrylamide gel.
The electrophoretic analysis of the latex protein also indicated
that papain constituted about 8% of the total protease. This cor-
responds to the earlier reports dealing with papain fromdifferent
sources of the latex [1,2,4]. The other components in the latex
were chitinase (22%) and three cysteine proteases including
chymopapain, glycyl endopeptidase and caricain, which com-
S. Nitsawang et al. / Enzyme and Microbial Technology 39 (2006) 11031107 1105
Fig. 1. Cathodic polyacrylamide gel electrophoresis of papain during purica-
tion by two-step salt precipitation and extraction in aqueous two-phase system
consisting of 8% PEG15% (NH
4
)
2
SO
4
. The samples in the different lanes
represent: standard papain (lane 1, 6 g), crude latex extract (lane 2), puried
papain from a two-step salt precipitation (lane 3), and from top phase of the sys-
tem consisting of 8% (w/w) PEG15% (w/w) (NH
4
)
2
SO
4
(lane 4), and bottom
phase of the same two-phase system (lane 5) after partitioning of latex.
prised about 20, 40 and 12%, respectively, of the total protease
(unpublished data).
3.1. Purication of papain by two-step salt precipitation
A modication of a two-step precipitation method proposed
by Baines and Brocklehurst [6] was used for purication of
papain from the claried latex. The rst precipitation was
performed using ammonium sulfate while sodium chloride
was used for the second precipitation step. As shown in
Table 1, increase in protein concentration used during each
precipitation step resulted in an increase in the amount of
protease activity (including papain) recovered in the precipitate,
but a lower purity of papain. This may be explained by
enhanced protein aggregation caused by increase in number
of interactions between the surface hydrophobic groups as the
protein concentration is increased. A maximum of about 39%
of the protease activity present in the latex was precipitated
with ammonium sulfate at an initial protein concentration of
40 mg/ml. The second precipitation step provided signicantly
lower recovery of protease activity but with a relatively enriched
papain fraction. The maximum purity of papain (>89%) was
achieved by limiting the protein concentration during the
second precipitation to 6 mg/ml (Table 1).
The electrophoresis patterns indicated that papain still con-
tained the contaminating proteases, chymopapain and chitinase
after the two-step salt precipitation procedure (Fig. 1, lane 3),
as also observed earlier by Kimmel and Smith [4] and Baines
and Brocklehurst [6]. The processing time of this experiment
was however faster than that in the earlier reports. Earlier stud-
ies have shown that chymopapain-free papain could be obtained
by crystallization after the two-step salt precipitation, however
only at a dened concentration of latex used for purication [6].
3.2. Purication of papain by aqueous two-phase
extraction
As ammonium sulfate is commonly used for separa-
tion of cysteine proteases, a two-phase system composed
of PEGammonium sulfate was evaluated for partitioning of
papain. According to the phase diagram of PEG(NH
4
)
2
SO
4
system by Salabat [25], the system comprising 12% (w/w) PEG
600015% (w/w) (NH
4
)
2
SO
4
provides two separated phases.
These were the maximum concentrations of the phase compo-
nents that could be used for purication of papain from papaya
latex. The systemcontaining more than 12%(w/w) PEGresulted
in a highly viscous mixture, whereas the system consisting of
more than 15%ammoniumsulfate provoked the precipitation of
protein from the latex.
Studies on partitioning of total protease activity and of papain
by varying different parameters (initial protein concentration,
Table 1
Inuence of protein concentration on purication of papain from 30 g papaya latex by two-step salt precipitation
Experiment Steps Initial protein
concentration
(mg/ml)
Total protease
activity
a
(Units)
Recovery of
protease activity
(%)
Relative papain
amount
b
(%)
Papain recovery
b
(%)
Latex Claried solution 43.5 30534 100 8.2 100
A 1st step
c
20 3511 11.5 36.2 51
2nd step
d
6 824 2.7 89.8 30
14 1374 4.5 78 43
B 1st step
c
30 7359 24.1 30.8 91
2nd step
d
6 1099 3.6 86.3 38
12 1496 4.9 75.8 45
14 1191 3.9 87.5 42
C 1st step
c
40 11969 39 20 96
2nd step
d
6 1344 4.4 89.6 49
14 1618 5.3 83.1 53
a
The amount of protease activity recovered in the precipitate was measured by assaying with casein.
b
Determined by FPLC.
c
Precipitation by 45% saturated ammonium sulfate.
d
Precipitation by 10% (w/v) sodium chloride.
1106 S. Nitsawang et al. / Enzyme and Microbial Technology 39 (2006) 11031107
Table 2
Effect of (a) concentration of phase components and (b) protein concentration and pH, on the total protease and papain recovery from 30 g papaya latex
a
by aqueous
two-phase extraction in PEGammonium sulfate system
X% PEG15% (NH
4
)
2
SO
4
b
12% PEGX% (NH
4
)
2
SO
4
b
PEG (X%) Protease activity
in top phase (U)
Papain recovery in
top phase
c
(%)
(NH
4
)
2
SO
4
(X%) Protease activity
in top phase (U)
Papain recovery in
top phase
c
(%)
Part (a)
4 1515 78.8 9 1708 35.5
6 1611 83.7 12 1627 84.6
8 1708 88.8 15 1642 85.3
10 1653 85.9
12 1659 86.2
12% PEG15% (NH
4
)
2
SO
4
Protein concentration
d
(mg/ml) Protease activity
in top phase (U)
Papain recovery in
top phase
c
(%)
pH
e
Protease activity
in top phase (U)
Papain recovery in
top phase
c
(%)
Part (b)
10 1443 75.0 3 313 16.3
20 1515 78.7 4 1154 60.0
30 1443 75.0 5 1659 86.2
40 1515 78.7 6 1563 81.2
50 1347 70.0 7 1563 81.2
60 794 41.3 8 1515 78.7
70 625 32.5 9 1419 73.8
a
The protease activity in 30 g latex was 24,050 units, 8% of which was papain.
b
Two-phase extraction was done at pH 5 and 40 mg protein/ml.
c
Papain recovery was estimated by FPLC. The protease activity recovered in the top phase of all the two-phase systems tested was constituted by papain only,
with the exception of the 12% PEG9% (NH
4
)
2
SO
4
where papain was 40% of the protease activity.
d
The two-phase system was maintained at pH 5.
e
Protein concentration in the latex was 40 mg/ml.
pH, concentration of phase components) showed that extremely
low levels of total protease activity were recovered in the PEG
rich upper phase, which contained the major fraction of papain
present in the latex (Table 2). This may reect a higher salting out
effect of ammoniumsulfate on papain owing to its higher surface
hydrophobicity as compared to the other proteases [18]. Conse-
quently, papain is preferentially partitioned to the top phase. This
is also in accordance with the relatively higher papain recovery
during ammonium sulfate precipitation reported in Table 1. As
seen in Table 2, increase in ammonium sulfate concentration
used to make the two-phase system increased only the recovery
of papain in the top phase but not that of total protease activity.
On the other hand, PEG concentration did not have any signif-
icant effect on protease partitioning in general. Partitioning at
protein concentrations ranging between 10 and 40 mg protein/ml
in 12% PEG15% (NH
4
)
2
SO
4
revealed no notable variation in
protease partition to the top phase. A signicant decrease in
partition was indeed observed at higher protein concentrations
(60 mg/ml and above) probably due to the limiting solvating
effect of PEG. Highest recovery of protease (including papain)
activity was achieved at pH 5; lowering the pH had a more neg-
ative effect than increase in pH, and was attributed more to the
inactivation of the proteases than any direct effect on partition.
Accordingtoelectrophoresis results, all the papaininthe latex
was moved into PEG phase (Fig. 1), which represented 8%
of the total proteolytic activity in the papaya latex (Table 2).
All the PEG(NH
4
)
2
SO
4
systems except the system of 12%
PEG9% (NH
4
)
2
SO
4
provided pure papain in the top phase.
Interference with chymopapain was thus not noticed, as was the
case in PEGphosphate system [19]. The remaining proteolytic
activity corresponding to chymopapain, glycyl endopeptidase
and caricain remained in the salt rich (bottom) phase (Fig. 1,
lane 5). The optimal conditions for extraction turned out to be
a two-phase system composed of 8% (w/w) PEG15% (w/w)
(NH
4
)
2
SO
4
and papaya latex containing 2040 mg protein/ml
at pH 5 (Table 2). Furthermore, the pure papain was obtained in
a more concentrated form since the top phase volume was about
1/4th the volume of the whole system (top phase volume =6 ml;
bottom phase volume =25.5 ml).
A comparison between the optimal precipitation and two-
phase systemshowed that the latter provided signicantly higher
recovery (88% versus 49%) and purity (100% versus 89%)
of papain from the papaya latex.
4. Concluding remarks
This study demonstrates the high resolving power of aque-
ous two-phase extraction for separating closely related enzymes.
The technique provided a convenient means of obtaining pure
papain that was free of any contaminating protease activities. It
also allowed the direct use of papaya latex without removal of
insoluble material, which makes for a faster and simpler pro-
cess compared to other purication procedures that are limited
by the necessity to remove insoluble material from latex. High
S. Nitsawang et al. / Enzyme and Microbial Technology 39 (2006) 11031107 1107
selectivity in separation was easily achieved by a suitable choice
of phase components and partitioning conditions. The fast and
simple operation and easy scalability of the two-phase systems
are additionally important features that favor the large-scale use
of this technique for the purication of papain.
Acknowledgements
The authors gratefully acknowledge The National Science
and Technology Department Agency (Thailand) and Mae Fah
Luang University for the doctoral scholarship.
References
[1] Azarkan M, Moussaoui AE, van Wuytswinkel D, Dehon G, Looze Y.
Fractionation and purication of the enzymes stored in the latex of
Carica papaya. J Chromatogr B 2003;790:22938.
[2] Baines BS, Brocklehurst K. Characterization of papaya peptidase A as
a cysteine proteinase of Carica papaya L. with active center prop-
erties that differ from those of papain by using 2,2

-dipyridyl disul-
de and 4-chloro-7-nitrobenzofurazan as reactivity probes. Biochem J
1982;205:20511.
[3] Barrett AJ, Rawlings ND, Woessner JF. Introduction: cysteine pepti-
dases and their clans. In: Handbook of proteolytic enzymes. San Diego:
Academic Press; 1998. p. 54566.
[4] Kimmel JR, Smith EL. Crystalline papain: preparation, specicity and
activation. J Biol Chem 1954;207:51531.
[5] Finkle BJ, Smith EL. Crystalline papain: number and reactivity of thiol
groups; chromatographic behavior. J Biol Chem 1958;230:66990.
[6] Baines BS, Brocklehurst K. A necessary modication to the preparation
of papain from any high-quality latex of Carica papaya and evidence for
the structural integrity of the enzyme produced by traditional methods.
Biochem J 1979;177:5418.
[7] Fukal L, Kas J. Chromatographic separation of papain evaluated by
immunochemical methods. J Chromatogr 1984;285:36572.
[8] Brocklehurst K, Carlsson J, Kierstan MPJ, Crook EM. Covalent chro-
matography: preparation of fully active papain from dried papaya latex.
Biochem J 1973;133:57384.
[9] Burke DE, Lewis SD, Shafer JA. A two-step procedure for purica-
tion of papain from extract of papaya latex. Arch Biochem Biophys
1974;164:305.
[10] DSouza F, Lali A. Purication of papain by immobilized metal afnity
chromatography (IMAC) on chelating carboxymethyl cellulose. Biotech-
nol Technol 1999;13:5963.
[11] Hatti-Kaul R. Aqueous two-phase systems: a general overview. Mol
Biotechnol 2001;19:26977.
[12] Kula MR, Kroner KH, Hustedt H, Schutte H. Scale-up of protein puri-
cation by liquidliquid extraction. Enzyme Eng 1982;6:6974.
[13] Veide A, Smeds AL, Enfors S-O. A process for large scale isolation of
-galactosidase from E. coli in an aqueous two-phase system. Biotechnol
Bioeng 1983;25:1789800.
[14] Hustedt H, Kroner KH, Papamichael N. Continuous cross-current aque-
ous two-phase extraction of enzymes from biomass: automated recovery
in production scale. Process Biochem 1988;23:12937.
[15] Albertsson P

A. Partition of cell particles and macromolecules. 3rd ed.

New York: Wiley; 1986.
[16] Menge U. Optimization of extractions in aqueous two-phase systems.
In: Aqueous two-phase systems: methods and protocols. NJ: Humana
Press; 2000. p. 23549.
[17] Hustedt H, Kroner KH, Kula M-R. Applications of phase partitioning
in biotechnology. In: Walter H, Brooks DE, Fisher D, editors. Partition
in aqueous two-phase systems: theory, methods, uses and applications
in biotechnology. Orlando: Academic; 1985. p. 52987.
[18] Vernau J, Kula M-R. Extraction of proteins from biological raw mate-
rial using aqueous polyethylene glycolcitrate phase systems. Biotechnol
Appl Biochem 1990;12:397404.
[19] Kuboi R, Wang WH, Komasawa I. Effect of contaminating proteins on
the separation and purication of papain from papaya latex using aque-
ous two-phase extraction. Kagaku Kogaku Ronbunshu 1990;16:7729.
[20] Johansson G. Recovery of proteins and phase components. In: Hatti-
Kaul R, editor. Aqueous two-phase systems: methods and protocols.
NJ: Humana; 2000. p. 35561.
[21] Arnon R. Papain Methods Enzymol 1970;19:22642.
[22] Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye
binding. Anal Biochem 1976;72:24854.
[23] Reisfeld RA, Lewis UJ, Williams DE. Disk electrophoresis of basic
proteins and peptides on polyacrylamide gel. Nature 1962;195:2813.
[24] Moutim V, Silva LG, Lopes MTP, Fernandes GW, Salas CE. Sponta-
neous processing of peptides during coagulation of latex from Carica
papaya. Plant Sci 1999;142:11521.
[25] Salabat A. The inuence of salts on the phase composition in aqueous
two-phase systems: experiments and predictions. Fluid Phase Equilib
2001;187:48998.