Plant Foods Hum Nutr (2011) 66:3440

DOI 10.1007/s11130-010-0206-0

ORIGINAL PAPER

Carica papaya Lipase: A Naturally Immobilized Enzyme

with Interesting Biochemical Properties
Slim Abdelkafi & Nathalie Barouh & Benjamin Fouquet &
Imen Fendri & Michel Pina & Frantz Scheirlinckx &
Pierre Villeneuve & Frdric Carrire

Published online: 26 January 2011

# Springer Science+Business Media, LLC 2011

Abstract Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms,
whereas very little is known about plant TAG lipases. The
lipolytic activity occurring in all the laticies is known to be
associated with sedimentable particles, and all attempts to
solubilize the lipolytic activity of Carica papaya latex have
been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex
(CPL) were determined from the insoluble fraction of the
latex. The activity was optimum at a temperature of 37C
and a pH of 9.0, and the specific activities of CPL were
found to be 2,000185 and 2568 U/g when tributyrin and
olive oil were used as substrates, respectively. CPL was
found to be active in the absence of any detergent, whereas
many lipases require detergent to prevent the occurrence of
interfacial denaturation. CPL was inactive in the presence
of micellar concentrations of Triton X-100, sodium dodecyl
sulfate (SDS) and tetradecyl trimethylammonium bromide
(TTAB), and still showed high levels of activity in the
presence of sodium taurodeoxycholate (NaTDC) and the
S. Abdelkafi : I. Fendri : F. Carrire (*)
CNRS - Aix-Marseille Universit, Enzymologie Interfaciale et
Physiologie de la Lipolyse UPR9025,
Marseille, France
e-mail: carriere@ifr88.cnrs-mrs.fr
S. Abdelkafi
e-mail: slim.abdelkafi@yahoo.fr
N. Barouh : M. Pina : P. Villeneuve
Ingnierie des Agro-polymres et Technologies Emergentes,
CIRAD - UMR IATE,
Montpellier, France
B. Fouquet : F. Scheirlinckx
Biohainaut, Z A E de Ghislenghien,
Ghislenghien, Belgium

zwitterionic Chaps detergent. The effects of various

proteases on the lipolytic activity of CPL were studied,
and CPL was found to be resistant to treatment with various
enzymes, except in the presence of trypsin. All these
properties suggest that CPL may be a good candidate for
various biotechnological applications.
Keywords Carica papaya latex . Lipase . Stability .
Detergent . Protease

Introduction
Lipases (triacylglycerol hydrolase, EC.3.1.1.3) are enzymes
with a wide range of biotechnological applications, including areas such as the hydrolysis of milk fat in the food
industry, applications in the oleochemical industry, in the
synthesis of structured triglycerides and in manufacture of
polymers and surfactants [14]. During the last few years, a
great deal of research has focused on increasing the use of
lipases on the industrial scale, especially those of plant
origin, which are present in stalks and seeds[5] as well as in
latex [6, 7] with a view to provide an alternative to the
microbial lipases produced using genetic engineering
methods. Among the plant lipases, the most thoroughly
studied are those extracted from cereals and seeds, which
are present in several plant tissues and normally activated
during germination [5], but the extraction process is usually
rather complex and gives a low yield, and the enzymes
recovered are generally rather unstable [8]. All these
drawbacks explain the cause from which the biochemical
properties of plant lipases have been poorly characterized
so far. However, there are other plant sources of lipases
such as the Carica papaya latex which are of considerable
interest because of their potential industrial applications.

Plant Foods Hum Nutr (2011) 66:3440

Papaya (Carica papaya) is one of the main fruit crops in

tropical and subtropical regions around the world. Papaya is
a member of the Caricaceae family, which belongs to the
Brassicales order [9]. The latex of Carica papaya is already
known as a rich source of cysteine endopeptidases,
including papain, chymopapain and caricain [10]. These
proteinases can be extracted in the form of water-soluble
proteins from the latex [11, 12]. The presence of lipase
activity was first reported by Giordani et al. [13], but this
activity was found to be associated with the insoluble
pellet obtained after latex centrifugation [6, 1113]. Until
recently, all attempts to solubilise the enzymatic activity
from this latex fraction were unsuccessful [14]. The lipase
from Carica papaya latex (CPL) [13] has therefore been
traditionally regarded as a naturally immobilized biocatalyst [14, 15]. A dry powder containing lipase activity
can be obtained after washing the latex particles with
water and centrifuging them. The regio-selectivity, stereoselectivity and typo-selectivity of the crude Carica papaya
latex have been studied during both hydrolysis and
acyltransfer reactions. During the process of hydrolysis,
this biocatalyst was found to be 1,3-regioselective with a
slight sn-3 stereo-preference [16].
Although, few authors have performed preliminary
studies on the biochemical characterization of lipases
belonging to the Caricaceae family [1618], biochemical
data on the Carica papaya lipase are still incomplete
because this enzyme could not be purified to homogeneity.
Only a carboxylester hydrolase (CpEst) has been identified
so far in this latex, and some of its biochemical properties
have been determined [19].
In the present study, some further biochemical properties of the lipase present in a crude fraction from
Carica papaya latex were investigated, such as the
optimum pH and temperature for its activity and stability.
The effects of detergents and proteases were also studied,
since lipases have to be active in the presence of these
substances in many food processing and other biotechnological applications.

Material and Methods

Carica papaya Lipase (CPL)
A crude lipase preparation was obtained in the form of a
waste product from the industrial production of papain. The
latex from Carica papaya was dried and roughly crushed in
order to obtain a crude powder called P3. A non-water
soluble cake containing lipase activity was obtained after
partial solubilization of the latex in water prior to the
recovery of papain in the presence of solid additives
followed by various filtration steps [20]. This cake was

35

washed five times in order to completely remove all the

water-soluble part of the latex. In a second step, the washed
cake was lyophilized to obtain a powder containing >95%
dry matter. The complete process led to a mass recovery
rate of about 35% (w/w) from the cake in comparison with
the crude latex initially used in the process, and most of the
lipolytic activity initially present in the latex was recovered
in this material, which will be refer from here as Carica
papaya lipase (CPL) .
Lipase Activity Measurements
A pH-stat (Metrohm 718 Stat Titrino, Zofingen, Switzerland) was used to measure the lipase activities potentiometrically in a thermostated reaction vessel (37C)
containing a mechanically stirred emulsion of either
0.5 ml tributyrin, 0.5 ml trioctanoin or 0.5 ml olive oil.
The olive oil was emulsified immediately before use in
10% gum Arabic solution as described by Abdelkafi et al.
[6]. Each assay was performed in 15 ml of reaction mixture
containing 2.5 mM TrisHCl, 5 mM CaCl2 and 150 mM
NaCl. With all the triglycerides tested, it was checked that
the amount of substrate available for the enzyme was not a
limiting factor during the reaction. Lipase activity was
expressed in international units (1 U=1 mol FFA released
per min). Phospholipase activity was measured as described
by Abousalham and Verger [21]. Cholesterol esterase
activity was measured as desribed by Ben Ali et al. [22].
Effects of pH and Temperature on CPL Activity
and Stability
The effects of the pH on CPL activity were investigated at
various pH ranging from 3 to 10, using an olive oil
substrate. At pH values below 7, the assays were performed
by back titration at pH 9 after a period of incubation at a
given pH ranging from 3 to 7. The pH-stability of CPL was
determined by measuring the residual lipase activity after
enzyme incubation at various pH values.
The effects of the temperature on lipase activity were
studied at various temperatures in the 2560C range at pH
9.0, using TrisHCl buffer and an olive oil substrate. The
reaction was carried out for 5 min using the pH-stat
technique. The thermostability of CPL was tested at various
temperatures ranging from 25C to 60C, for different
incubation times ranging from 0 to 6 h. At each time
interval, the residual lipase activity of each sample was
measured using the optimized assay.
Determination of Protein Concentration
Protein concentration in CPL samples was estimated either
by determining the amino acid composition [23], or using

36

Plant Foods Hum Nutr (2011) 66:3440

Table 1 Carica papaya latex lipolytic activities

Substrates
Olive oil b
Trioctanoinb
Tributyrin b
Phosphatidylcoline
Cholesterol oleate
a

Specific activity (U/g)

2568
98329
2,000185
653
0

Values are means SD (n=3)

Assays with triacylglycerols were performed at 37C in 2.5 mM Tris-HCl

buffer, 150 mM NaCl and at pH 9
c

Assays with phospholipids were performed at 37C in 7.5 mM CaCl2,

13.3 mM NaTDC and at pH 8

Lowrys method [24] with bovine serum albumin (BSA) as

the standard.
Effects of Storage Time
The stability of CPL was investigated after storage of the
lipase at 4C and 20C. The residual lipase activity was
measured at various times during 45 weeks (at 4C) and
30 weeks (20C).
Incubation of CPL with Proteases
The reaction mixtures contained 10% CPL (w/v) and the
protease to CPL ratio (w/w) was 1/5, except in the case of
carboxypeptidase (1/25). All reactions were performed in
Tris-buffer (25 mM, pH 8 for trypsin; 10 mM, pH 7.5 for
protease XXV; 25 mM, pH 9 for proteinase K; 20 mM, pH 6.2
for papain and 25 mM, pH 7.65 for carboxypeptidase) at 37C
in the case of trypsin and protease XXV, at 30C in that of
proteinase K and at 25C in that of papain and carboxypeptidase. Experiments with carboxypeptidase were performed in
the presence of 100 mM NaCl. Control experiments were
performed in the absence of proteases. After the protease
treatment, the reaction mixtures were tested to determine the
residual lipolytic activity after centrifuging them at 2,500 g
at 4C for 10 min in order to separate the supernatant
containing solubilized compounds. The insoluble pellet was
lyophilyzed prior to quantifying the protein content.

the short chain triglyceride tributyrin was described several

years ago [13]. However, tributyrin is partly soluble in
water and some esterases which are active on this substrate
did not show any activity on a true lipase substrate such as
olive oil. It was observed in a more recent study, however,
that CPL could hydrolyze the long chain triglycerides
present in test meals and could therefore be considered as a
source of true lipase activity [6]. It was confirmed here
(Table 1) that CPL is active on olive oil (2568 U/g) as
well as on trioctanoin (98329 U/g). CPL is much more
active on short and medium chain TAGs than on long chain
TAGs, as occurs with most lipases [25]. It is worth noting
here that the specific activity of CPL on olive oil was
similar to that detected in the dry mesocarp of oil palm fruit
(25014 U/g) [25]. CPL was also found to hydrolyze
phosphatidylcholine with a specific activity of 653 U/g,
but showed no activity on cholesterol oleate. Several TAG
lipases have been found to show a dual TAG lipase/
phospholipase A1 activity [26, 27] but only if a pure CPL
enzyme is obtained it will be possible to determine whether
the phospholipase and lipase activities measured with crude
CPL are due to the same enzyme.
Both lipolytic activities were found, however, to be
consistently associated with sedimentable particles of latex,
and conventional protein separation and purification methods are therefore not suitable for isolating and identifying
lipases from the latex of Carica papaya [28]. One possible
explanation for these findings is that lipases may be packed
in rubber particles in the same way as proposed for Hevea
brasiliensis latex [29].
Effects of pH on the Activity and Stability of CPL
When olive oil was used as the substrate, CPL activity was
found to be optimum at pH levels ranging between 9 and
9.5 (Fig. 1), and the kinetics of fatty acid release were linear
for at least 5 min when the pH value was equal to or below
9. At pH values above 9, the kinetics were linear for only
12 min. These data suggest that CPL is less stable at high
pH levels. Optimum conditions for assaying CPL activity

Results and Discussion

Lipolytic Activities in Carica papaya Latex
Using tributyrin as substrate, a high level of lipase activity
reaching 2,000185 U/g of CPL was measured using a
10% w/v dispersion of CPL powder in deionized water
(Table 1). The lipolytic activity of Carica papaya latex on

Fig. 1 Effects of pH on CPL lipase activity on olive oil. The lipase

activity was measured using an olive oil emulsion stabilized with gum
Arabic as described in Material and Methods. Assays were carried out
at 37C. Data are means SD (n=3)

Plant Foods Hum Nutr (2011) 66:3440

37

Effects of Temperature on Activity and Stability of CPL

Fig. 2 Effects of temperature on CPL lipase activity on olive oil. The

lipase activity was measured using an emulsion of olive oil emulsion
stabilized with gum Arabic as described in Material and Methods.
Assays were carried out at pH 9. Data are means SD (n=3)

on olive oil were therefore set at pH 9. No significant

activity could be detected at pH 6 or less (Fig. 1). The
optimum pH range for CPL activity is similar to that
determined in the case of other plant lipases from palm oil
fruit [25] and babaco [16]. When tributyrin and trioctanoin
were used as substrates, the maximum activity of CPL was
recorded at pH 8 and 9, respectively.
CPL was found to be highly stable from pH 4 to 10, and
75% of its activity still persisted after 24 h of incubation at
pH 10 (data not shown). However, CPL was unstable at pH
levels of 2, 3 and 11, giving half-inactivation times of 8, 8.5
and 9 min, respectively. It is worth noting that only a few
microbial lipases, such as that of Thermomyces lanuginosus
(Humicola lanuginosa) [30] have shown similar levels of
resistance so far in a large pH range up to pH 10.

Table 2 Effects of detergents

on CPL and TLL specific
activities on trioctanoin as
substrate. Experiments with
CPL and TLL were performed
at pH 9 and 7, respectively, in
the presence of various
detergent concentrations

Detergents

Detergent concentration
(mM)

No
Nonionic

Triton X-100

Anionic

NaTDC

SDS

Zwitterionic

Chaps

Zwittergent 3-12

Cationic
Values are means SD (n=3)
a

Data from [34]

Lipase activity was tested at temperatures ranging from 25C

to 60C using an olive oil substrate (Fig. 2). The optimum
temperature for CPL activity was found to be 50C. This is
higher than the value (45C) reported in a study on oil palm
seed (Elaeis guineensis Jacp) mesocarpe lipase, where olive
oil was used as the substrate [25]. A similar optimal
temperature of 50C was reported in the case of bay laurel
(Laurus nobilis L.) lipase seeds [31]. Caro et al. [32] also
carried out lipolysis reactions catalyzed by Euphorbia
characias latex lipase at 45C. The kinetics were linear for
at least 5 min when the activity was assayed at temperatures
below 37C. Above this value, the activity decreased after 1
or 2 min, which suggested that this enzyme is less stable at
high temperatures. The activity of CPL was therefore tested
here at a temperature of 37C.
The thermostability of CPL was determined by measuring the residual activity after incubating the lipase at
various temperatures. CPL showed appreciable level of
stability during prolonged incubation at 25 and 37C, with
a maximum stability at 25C (data not shown). The halfinactivation values (t1/2) of CPL at 25 and 37C were >480
and 360 min, respectively. These high t1/2 values show that
CPL is an enzyme with a relatively high level of stability in
solution at room and physiological temperatures. At 50C,
50% of the residual activity was still recorded after a 160min period of incubation. When the enzyme was incubated

TTAB

CPL residual
activity (%)

TLL residual
activity (%)a

0
<CMC
CMC
>CMC
<CMC
CMC
>CMC
<CMC
CMC
>CMC
<CMC
CMC
>CMC
<CMC
CMC
>CMC
<CMC

0.05
0.1
5
0.25
1
8
0.125
7
>7
1.6
4
6.4
0.25
4
16
0.1

1004
984
915
00
922
950.9
926
903
00
00
952.7
855
842.8
930.9
464
238
920

1009
1006
703.6
00
6912
6012
110.9
843.3
00
00
734.7
487.6
3.60.7
640.77
00
00
595.8

CMC
>CMC

0.5
8

568
00

254.4
8.70.6

38

Plant Foods Hum Nutr (2011) 66:3440

in the presence of detergent monomers, which suggests that

these monomers were competing with the lipase to bind at
the interface. Only a few lipases (gastric lipase [35] and
Yarrowia lipolytica LIP2 lipase [34]) are not inhibited by
bile salts such as NaTDC. CPL may therefore be a good
candidate for enzyme replacement therapy on the gastrointestinal tract [6].
The Effects of Storage on Lipase Stability

Fig. 3 Effects of storage time on stability of CPL (4 and 20C)

at 60C, it was rapidly inactivated, giving a half-inactivation

time of 25 min.
Effects of Detergents on CPL and Thermomyces
lanuginosus Lipase Activities
The effects of several classes of detergents having different
chemical compositions and physical properties (i.e., chain
lengths, head group size, and charge) on the enzymatic
activity of CPL were investigated. Lipase activities were
recorded at pH 9 using an emulsified trioctanoin substrate
(completely insoluble substrate), in the presence of various
detergent concentrations in the pH-stat reaction mixture
(Table 2). TC8 was used in these experiments because this
substrate could be emulsified without any gum Arabic,
which has also been described as tensioactive compound
[33]. In addition, the use of TC8 made it possible to
compare the data obtained with CPL with those previously
obtained on the lipase from Thermomyces lanuginosus
(Humicola lanuginosa), a widely used lipase which is
present in some household detergents and is marketed
under the name Lipolase [34]. In the present control
experiments, Thermomyces lanuginosus lipase (TLL) and
CPL were found to be active in the absence of detergent,
and linear lipolysis kinetics were recorded.
The patterns of activity of CPL in the presence of
nonionic (Triton X-100), anionic (sodium dodecyl sulfate
(SDS)) and cationic (tetradecyl trimethylammonium bromide (TTAB)) detergents were similar. Inhibitory effects
were observed when the detergent concentration was
increased above the CMC, which resulted in the complete
loss of activity (Table 2). A similar pattern was observed
with TLL. It is noteworthy, however, that the activity of
CPL did not decrease when another anionic bile salt
detergent (NaTDC) and the zwitterionic detergent (Chaps)
were tested, whereas the TLL activity decreased, and
similar findings were also previously obtained at detergent
concentrations below the critical micelle concentration
(CMC) (Table 2). The concentration range in which these
detergents inhibited TLL indicates that the inhibition started

The storage stability of enzymes is one of the main

parameters which has to be taken into account when
scheduling its application to a particular reaction. The
storage stability of CPL was investigated at temperatures of
4 and 20C (Fig. 3). CPL on olive oil lost about 50% of its
activity after 20 weeks of storage at 20C. The activity of
CPL stored at 4C was measured for more than 40 weeks,
and the residual activity was still found to be greater than
90% after this period.

Fig. 4 Effect of proteases on CPL. a residual CPL activity on olive

oil; b solubilization of total material and proteins after 8 h of
incubation; c changes in residual lipase activity with the solubilization
of proteins after 8 h of incubation

Plant Foods Hum Nutr (2011) 66:3440

Effects of Proteases on CPL Activity and Stability

The effects of proteases on the lipolytic activity of CPL
were investigated with different proteases, and the residual
activity was assayed at various times. The CPL lipolytic
activity decreased only in the presence of trypsin after 8 h
(Fig. 4a). CPL lipolytic activity was found to be highly
stable in the presence of proteinase K, protease XXV from
Streptomyces griseus, papain from Carica papaya and
carboxypeptidase. This is consistent with the previous
finding that CPL activity is stable in Carica papaya latex,
which contains many proteases such as papain, caricain and
chymopapain [15]. The relatively high resistance of CPL to
proteases may be due to the lipase being immobilized on
insoluble latex particles.
It was also investigated whether treatment of a crude
CPL preparation with proteases might result in the release
of proteins or other compounds from the insoluble latex
fraction. The crude CPL preparation (powder) lost around
78% of its total mass in the control experiments (buffer
containing no proteases), whereas all the protease treatments tested increased the solubilization of some of the
components of the CPL powder (Fig. 4b): the highest levels
of solubilization were obtained with trypsin (18.964) and
protease XXV (18.45).
The contribution of proteins to the material solubilized
from the crude CPL preparation was estimated by determining the amino acid composition and concentrations in
the reaction supernatant and performing Lowrys protein
assay. In most cases (trypsin, carboxypeptidase and
protease XXV), both assays gave similar results and
showed that proteins contributed to a large fraction of the
material solubilized from CPL preparation (up to 5.5% of
the total mass with trypsin). Discrepancies were observed,
however, between the results of the two assays in the
control experiments and the experiments performed with
proteinase K and papain (Fig. 4b), and no explanation
seems to be available so far for these differences.
The finding that a protease like protease XXV can be
used to reduce the mass of CPL preparations by about 20%
without affecting the lipase activity is noteworthy. Proteases
could therefore be used to increase the specific activity of
CPL. Alternatively, the results obtained with trypsin
suggest that one or several proteins cleaved by the protease
and partly released from the insoluble fraction may have
been involved in the lipase activity, since there was a
concomitant loss of lipase activity. As shown in Fig. 4c, the
loss of lipase activity increased with the solubilization of
the proteins exposed to protease. This finding could be used
to identify specific peptides resulting from the proteolytic
cleavage of the lipase. Attemps to identify a lipase from
papaya latex using proteomics approach have been recently
successful in the case of Carica pentagona [36].

39

This study yields further insights on the biochemical

properties of the lipase(s) present in Carica papaya latex.
Although obtaining an enzyme purified to homogeneity is
still a challenge due to the insolubility of the latex fraction
containing lipase activity, the data presented here should
provide a useful basis for performing further investigations
and developing applications for the raw/crude latex fraction
showing lipase activity.
Acknowledgments The authors thank Mr. Peter Van Cauwenberghe,
the Managing Director of Biohainaut for his continuous support.
English revision by Dr Jessica Blanc is acknowledged. This study was
performed in the framework of the European EUREKA LIPLANT E!
3818 Project (20072009) with the financial support of Agence
Nationale de Recherche (ANR) in France and the Direction Gnrale
des Technologies, de la Recherche et de lEnergie (DGTRE) de la
Rgion Wallonne in Belgium.

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