Carica papaya by-products as new biocatalysts for the synthesis of oleic acid esters

Article abstract of DOI:10.3109/10242422.2015.1101239

Carica papaya lipase is a versatile biocatalyst that is employed for many biotechnological purposes. Its lipase activity was first observed to be tightly linked to the insoluble fraction of latex. Nevertheless, recent studies have shown that this activity is also present in the fruit peel and seeds, suggesting that the lipase activity occurs in other parts of the plant. In the present work, the hydrolytic activity on trioctanoin was determined in various plant by-products, including latex, leafs, petioles, meristems, fruits, and the stem. The most hydrolytic activity was found in the latex (11 U/mL), followed by the petioles (1.7 U/mL). The hydrolytic selectivity was determined using triacetin, tripropionin, tributyrin, and trioctanoin. The enzymes present in the latex showed a higher rate of hydrolysis of tributyrin, while those present in the petioles had a preference for tripropionin, possibly indicating the occurrence of at least two different triacylglycerol hydrolases. Five self-immobilized biocatalysts were obtained: lyophilized latex (LL), lyophilized petioles (LP), bagasse from petioles (BP), and, after a simple cold water washing treatment, treated lyophilized latex (TLL), and treated lyophilized petioles (TLP). This procedure yielded a 5- and 10-fold increase in the latex and petiole activity, respectively, on tributyrin. The selected biocatalysts, TLL and BP, were tested for the synthesis of oleic acid esters (OAE), reaching conversions over 80%. Unexpectedly, only BP preferentially synthesized dodecyl oleate (DO) and showed the highest thermostability. Therefore, BP was further assayed for DO synthesis in a packed bed reactor (PBR), achieving 96% conversion over 40 h. This study shows the great potential of C. papaya by-products, particularly BP, as biocatalysts for the synthesis of OAE.

Full text of DOI:10.3109/10242422.2015.1101239

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Carica papaya by-products as new biocatalysts for
the synthesis of oleic acid esters
Mariana ArmendáRiz-Ruiz, Eduardo Mateos-Díaz, Jorge Alberto Rodríguez-
González, Rosa Camacho-ruiz, Antonia Gutiérrez-Mora, Georgina Sandoval-
Fabian, Santiago Gallegos-Tintoré & Juan Carlos Mateos-Díaz
To cite this article: Mariana ArmendáRiz-Ruiz, Eduardo Mateos-Díaz, Jorge Alberto Rodríguez-
González, Rosa Camacho-ruiz, Antonia Gutiérrez-Mora, Georgina Sandoval-Fabian, Santiago
Gallegos-Tintoré & Juan Carlos Mateos-Díaz (2015): Carica papaya by-products as new
biocatalysts for the synthesis of oleic acid esters, Biocatalysis and Biotransformation, DOI:
10.3109/10242422.2015.1101239
To link to this article: http://dx.doi.org/10.3109/10242422.2015.1101239
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Date: 11 November 2015, At: 07:17

Biocatalysis and Biotransformation, 2015; Early Online: 1–8
RESEARCH ARTICLE
Carica papaya by-products as new biocatalysts for the synthesis of
oleic acid esters
1
MARIANA ARMENDARIZ-RUIZ , EDUARDO MATEOS-DIAZ
1
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´
1
JORGE ALBERTO RODRIGUEZ-GONZALEZ , ROSA CAMACHO-RUIZ ,
1
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2
ANTONIA GUTIERREZ-MORA , GEORGINA SANDOVAL-FABIAN ,
1
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3
SANTIAGO GALLEGOS-TINTORE & JUAN CARLOS MATEOS-DIAZ
1
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1
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Department of Industrial Biotechnology, Centro de Investigacion y Asistencia en Tecnologıa y Diseno del Estado de
2
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Jalisco A.C., Guadalajara, Jalisco, Mexico, Department of Plant Biotechnology, Centro de Investigacion y
Asistencia en Tecnologıa y Diseno del Estado de Jalisco A.C., Guadalajara, Jalisco, Mexico, and Faculty of
3
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Chemical Engineering, Campus of Exact Sciences and Engineering, Autonomous University of Yucatan,
Merida, Mexico
Abstract
Carica papaya lipase is a versatile biocatalyst that is employed for many biotechnological purposes. Its lipase activity was first
observed to be tightly linked to the insoluble fraction of latex. Nevertheless, recent studies have shown that this activity is also
present in the fruit peel and seeds, suggesting that the lipase activity occurs in other parts of the plant. In the present work,
the hydrolytic activity on trioctanoin was determined in various plant by-products, including latex, leafs, petioles, meristems,
fruits, and the stem. The most hydrolytic activity was found in the latex (11 U/mL), followed by the petioles (1.7 U/mL). The
hydrolytic selectivity was determined using triacetin, tripropionin, tributyrin, and trioctanoin. The enzymes present in the
latex showed a higher rate of hydrolysis of tributyrin, while those present in the petioles had a preference for tripropionin,
possibly indicating the occurrence of at least two different triacylglycerol hydrolases. Five self-immobilized biocatalysts were
obtained: lyophilized latex (LL), lyophilized petioles (LP), bagasse from petioles (BP), and, after a simple cold water washing
treatment, treated lyophilized latex (TLL), and treated lyophilized petioles (TLP). This procedure yielded a 5- and 10-fold
increase in the latex and petiole activity, respectively, on tributyrin. The selected biocatalysts, TLL and BP, were tested for
the synthesis of oleic acid esters (OAE), reaching conversions over 80%. Unexpectedly, only BP preferentially synthesized
dodecyl oleate (DO) and showed the highest thermostability. Therefore, BP was further assayed for DO synthesis in a packed
bed reactor (PBR), achieving 96% conversion over 40 h. This study shows the great potential of C. papaya by-products,
particularly BP, as biocatalysts for the synthesis of OAE.
Keywords: Biocatalyst, byproducts, Carica papaya, esters, lipase, petioles
Introduction
120 million tons of by-products are generated each
year, including leaves, meristem, petioles, stem, and
unripe fruits, all of which have no commercial value.
In particular, the unripe fruits are rich in latex-
producing cells called laticifers. Latex is a white
thixiotropic fluid, the main function of which is
believed to be a defensive role against pathogens and
insects (el Moussaoui et al. 2001). It contains a wide
range of hydrolytic enzymes, including, but not
limited to, cysteine proteases, chitinases, esterases,
Carica papaya is a palm-like plant in the Caricaceae
family that possesses a hollow, unbranched stem that
sometimes reaches 20 m height (el Moussaoui et al.
2001). It is cultivated in tropical and sub-tropical
regions around the world for its commercialized
fruit. Worldwide, C. papaya production reached 12.4
million tons of fruit in 2012. It should be noted that
the planting cycle lasts for only two years (FAO
2012). As a result of this production, approximately
´ ´ ´
˜
Correspondence: Juan Carlos Mateos-Dıaz, Department of Industrial Biotechnology, Centro de Investigacion y Asistencia en Tecnologıa y Diseno del Estado
de Jalisco A.C., Guadalajara, Jalisco, Mexico. Tel: +52 33 3345 5200. E-mail: jcmateos@ciatej.mx
(Received 5 December 2014; accepted 15 September 2015)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2015 Taylor & Francis
DOI: 10.3109/10242422.2015.1101239

´
M. Armendariz-Ruiz et al.
2
and lipases. Latex is composed of a water-miscible
phase that contains water soluble enzymes (cysteine
proteases and chitinases); while the hydrophobic
enzymes, mainly esterases and lipases, are tightly
bound to the solid polyisoprenoid matrix. To date,
their separation, purification, and molecular charac-
terization have been impossible (Giordani et al.
1991; el Moussaoui et al. 2001; Abdelkafi et al.
2009).
employed this biocatalyst for the resolution of a
(R,S)-naproxen-2,2,2-trifluoroethyl ester (Chen and
Tsai 2005). There are only a few reports available
regarding the use of the by-products of C. papaya.
Paques et al. carried out a partial purification and
characterization of the lipase activity found in the
seeds and fruit peels (Paques et al. 2008). In previous
work performed in our laboratory, Galindo demon-
strated that the proteolytic activity is found in
different proportions in C. papaya by-products
(e.g., leaves, petiole, and stem) (Galindo-Estrella
et al. 2009). Thus, the esterase/lipase activity might
also differ in various by-products, and therefore, the
by-products can be used as non-toxic and cheap
biocatalysts. Furthermore, the high intrinsic hydro-
phobic character of C. papaya esterases and lipases
suggests that these enzymes may be strongly attached
to the matrices of the by-products, and thus, they can
be used as naturally immobilized biocatalysts for the
synthesis of several esters.
Esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3)
are carboxylic ester hydrolases that hydrolyze soluble
short chain (5C4) and non-soluble long chain (4C8)
triglycerides (TAGs), respectively (Lortie 1997).
Both are known for their regio-, topo-, and enantios-
electivity, as well as for the ability to accept a wide
range of natural and synthetic substrates (Schmid
and Verger 1998). Many esterases and lipases are
highly active in the presence of organic solvents
(Bornscheuer and Kazlauskas 2006). In the absence
of water, they can perform esterification, transester-
ification, and other reactions. All of these treatments
make esterases and lipases the most employed
biocatalysts in organic chemistry, leading to the
synthesis of a wide range of valuable products, such
as esters (Schmid and Verger 1998). Short acyl chain
esters (5C4) are used as additives in the food
industry (Liu et al. 2004), while large acyl chain
esters (4C8) are widely used in the cosmetic,
pharmaceutical, and lubricant industries (Villa
et al. 2003; von Scala et al. 2005). Immobilized
esterases or lipases are ideal biocatalysts because it is
possible to reuse them, and they can be used in
packed bed reactors (PBRs), which considerably
reduces the process cost (Malcata et al. 1992; Chen
and Wu 2003). This type of biocatalyst can be
obtained from C. papaya.
In this work, for the first time, it is shown that
esterase/lipase activity is present in other C. papaya
by-products (i.e., petioles). Moreover, it was easy to
obtain different biocatalysts from latex and C. papaya
by-products that are useful for the synthesis of oleic
acid esters, in particular dodecyl oleate (DO).
Finally, bagasse from petioles (BP), the most
thermostable biocatalyst, was successfully used in a
PBR for the synthesis of DO, demonstrating that BP
is a very interesting and attractive by-product that
should be considered for industrial processes invol-
ving the continuous synthesis of long chain esters.
Materials and methods
Latex from C. papaya has been widely studied. Its
hydrolytic activity, which generates medium-long
chain TAGs, such as tributyrin, were formerly
attributed to one lipase, which is commonly referred
to as C. papaya Lipase (CPL). Currently, it is known
that there is more than one enzyme responsible for
this activity on the polymeric network; there is at
least one esterase and one lipase (Abdelkafi et al.
2009; Dhouib et al. 2011). The insoluble fraction of
latex has been largely employed as a biocatalyst in the
organic synthesis because its hydrolytic enzymes are
considered to be naturally immobilized (Giordani
et al. 1991; Gandhi and Mukherjee 2000, 2001).
Previous publications in which CPL was employed
mainly focused on its strict sn-1,3 selectivity and
enantioselectivity. Giordani and Gandhi used CPL
for the synthesis of TAGs, which possess short acyl
chains at the sn-1,3 positions and are considered to
be hypocaloric (Giordani et al. 1991; Gandhi and
Mukherjee 2001). Moreover, Chen successfully
Materials
Triton X100, sodium hydroxide (NaOH), sodium
chloride (NaCl), hydrochloric acid (HCl), oleic acid,
triacetin (TC2), tripropionin (TC3), tributyrin
(TC4), trioctanoin (TC8), methanol, n-propanol,
dodecanol, tert-butanol, diisopropyl ether, toluene,
isooctane, molecular sieves (nominal pore diameter 3
Õ
˚
A), and casein were obtained from Sigma-Aldrich .
Tris(hydroxymethyl aminomethane) was purchased
from Research OrganicÕ. Dibasic sodium phosphate
and pyridine were procured from KaralÕ, while
copper acetate was purchased from CaledonÕ.
Anhydrous ethanol and n-butanol were obtained
from the best quality local suppliers.
Sample collection
Fresh latex was extracted directly from the unripe
fruits from the plants, which were donated by Papaya

Carica papaya by-products
3
´
´
Peninsular S.A. Yucatan, Mexico, by making four
vertical incisions and collecting the fluid in 500-mL
polyethylene-terephthalate jars. Then, the different
parts of the plant were separated into the by-products
mentioned above. All of the samples were immedi-
ately stored in sealed polyethylene bags at ꢀ20 ꢁC
until treatment.
was performed at 25 ꢁC in a thermostated vessel
containing 2.5 mM Tris–HCl buffer and 150 mM
NaCl at pH 7. The rate of spontaneous hydrolysis
of the substrate was recorded 5 min prior to sample
injection. A 500 mL of the different liquid extracts
or 100 mL of a suspension of lyophilized biocatalyst
powder at 100 g/L was used for the assay. The
esterase/lipase activity was expressed in IU per
milliliter of liquid extract or milligram of lyophi-
lized powder. One IU corresponds to 1 mmol of
fatty acid released per minute. Each assay was
performed in triplicate.
The treatment of samples to obtain biocatalysts
The latex samples were treated using a method
similar to that described by Giordani et al. (1991).
The most hydrophilic compounds were removed by
washing them thoroughly from the solid fraction of
the crude latex with distilled water at 4 ꢁC. The
mixture was shaken vigorously with a vortex at a ratio
of 1:7 (v:v) crude latex:water and centrifuged at
8600g and 4 ꢁC for 20 min. The esterase/lipase
activity was measured in both the supernatant and
sediment during the treatment. This treatment was
repeated until no significant protease activity was
detected in the supernatant.
The synthesis of oleic acid esters
The synthesis of oleic acid esters was performed in
1.5 mL vials with an equimolar (200 mM) solution of
oleic acid and the corresponding alcohol in 1 mL of
isooctane. All the reactions contained molecular
sieves and 25 mg of biocatalyst, except those in
which BP (37.5 mg) was used. The samples were
agitated at 300 rpm and 50 ꢁC. Each assay was
performed in triplicate.
The liquid extracts were obtained by milling each
by-product with an industrial disk mill (Exmex). The
solid residues or bagasses were dried at 25 ꢁC,
milled, and sieved (sieve #30). The liquid extract
from the petioles received the same treatment as
described above for the latex. All of the solids
obtained after this washing treatment with cold water
were lyophilized and labeled as ‘‘Treated
Lyophilized’’. The powders obtained were desig-
nated as ‘‘biocatalysts’’ and preserved at ꢀ20 ꢁC.
Monitoring of the synthesis reactions
The concentration of oleic acid was used to calculate
the initial rate and progress of the synthesis reactions.
A spectrophotometric method based on the forma-
tion of a colored copper-fatty acid complex was
employed to quantify the concentration of oleic acid
using an oleic acid standard curve (Kwon and Rhee
1986). A 50 mL of the reaction mixture was diluted
10-fold in isooctane and 250 mL of a 5% (w/v) copper
acetate–pyridine solution pH 6.1 was added to the
diluted sample and mixed in a vortex for 30 s and
was centrifuged at 15,900g for 5 min. A 200 mL of the
organic phase was carefully collected and placed in a
96-well microplate, and the optical density was
measured at 690 nm in an X-Mark Bio RadÕ
spectrophotometer. Each assay was performed in
triplicate.
Protease activity
The proteolytic activity was measured on casein
using the spectrophotometric method reported by
Anson et al. (1938) that was modified for use in a 96-
well microplate format. A 100 mL of the different
liquid extracts or 100 mL of a suspension of
lyophilized biocatalyst powder at 100 g/L was used
for the assay. The protease activity was expressed in
IU per mL of liquid extract or mg of lyophilized
powder. One IU corresponds to 1 mmol of tyrosine
equivalents released per minute. Each assay was
performed in triplicate.
The effects of various reaction solvents and alkyl chain
lengths on the synthesis reactions
The effects of tert-butanol, diisopropyl ether, tolu-
ene, and isooctane on the synthesis of ethyl oleate
were evaluated. The effect of the alkyl chain length
on esterification was determined by carrying out the
synthesis of methyl, ethyl, propyl, butyl, and dodecyl
oleate. All of the reactions were performed as
described in the ‘‘Synthesis of oleic acid esters’’
section.
Esterase/lipase activity assay
The esterase/lipase activity was assayed by measur-
ing the fatty acids released from mechanically
stirred TC2 (354 mM), TC3 (208 mM), TC4
(114 mM), and TC8 (39 mM) emulsions, using 0.1
N
NaOH with
a
pH-Stat GPT Titrino
(MetrohmÕ) set at a pH end point. Each assay

´
M. Armendariz-Ruiz et al.
4
Determination of the thermostability of the C. papaya
biocatalysts
However, even though the petioles have a moderate
weight proportion in plants (3%HM), their hydro-
lytic activity on TC8 was the highest out of all the by-
products (1.67 U/mL). Thus, petioles were selected
as the most promising by-product because it contains
a greater proportion of lipase activity than latex, as
observed by the relative activity towards TC8 with
respect to TC4, which was 3-fold higher in the
petioles (Table 1). Furthermore, the petioles contain
a low specific protease activity (0.63 U/mg protein)
compared to the other by-products (1.22–10 U/mg
protein), as described by Galindo-Estrella et al.
(2009). This activity facilitates its removal from the
insoluble fraction. Therefore, latex and petioles were
selected for the generation of biocatalysts with
esterase/lipase activity because they are the most
promising by-products in terms of their hydrolytic
activity and availability.
Each biocatalyst was incubated for 4 h at 45 ꢁC. After
the incubation period, the thermostability was
evaluated by measuring the residual hydrolytic
activity on TC8 at 30 ꢁC and pH 7.0. Each assay
was performed in triplicate.
Continuous dodecyl oleate synthesis
The bagasse from petioles was used in a PBR for the
continuous synthesis of DO. The PBR (500 mL,
2.5 cm diameter, and 25 cm high) was filled with 72 g
of bagasse. The substrates, oleic acid and dodecanol,
were mixed in an equimolar (200 mM) solution with
isooctane. The flow rate and the temperature were
kept at 1 mL/min and 50 ꢁC, respectively. The
progress of the synthesis reaction was determined
in triplicate.
The esterase/lipase activity of C. papaya biocatalysts
The lyophilized latex (LL), treated lyophilized latex
(TLL), lyophilized petiole (LP), and treated lyophi-
lized petiole (TLP) biocatalysts were obtained by
washing away most of the soluble compounds,
including proteases (see ‘‘Materials and methods’’
section). Bagasse obtained by a petiole milling
process was dried, and its capacity as a biocatalyst
(Bagasse from Petioles: BP) was evaluated. The
activities of the five biocatalysts towards triglycerides
with different chain lengths are shown in Figure 1.
The latex biocatalysts showed higher hydrolytic
activity (up to 517-fold, Figure 1A) in all of the
substrates tested compared to those attained from
petioles (Figure 1B and C). It should be noted that
the maximum activity of the latex biocatalysts was
with tributyrin, as previously observed by the Verger
group (Giordani et al. 1991), whereas in the case of
petiole biocatalysts, the highest hydrolysis rate was
measured with tripropionin. These results strongly
suggest that the esterases and lipases present in the
latex biocatalysts are different than those present in
petioles. After a cold wash, the activity per gram of
the biocatalyst powder towards the partially soluble
substrates, TC2 through TC4, was increased
approximately 5- and 10-fold in TLL and TLP,
respectively. In no case, esterase/lipase activity was
found in the washing water.
Results and discussion
Proportion of C. papaya by-products and esterase/
lipase activity of the liquid extracts
The proportion in humid weight (%HW) and the
hydrolytic activity on TC4 and TC8 for the latex and
liquid extracts from each C. papaya by-product are
shown in Table 1. Although, latex was very difficult
to collect and was obtained in a very small amount
(0.1%HW), its hydrolytic activity was the highest
among the by-products (TC4: 98.76; TC8: 10.88 U/
mL). The stems were by far the most abundant
(89%HW) among the by-products, followed by the
fruits (5%HW). Nonetheless, the hydrolytic activity
on TC8 (lipase activity) of the stems and fruits
combined with the meristems and leaves was less
than 1 U/mL (Table 1). For this reason, we decided
to discard these by-products from further studies.
Table 1. Proportion of Carica papaya by-products and hydrolytic
activity on tributyrin and trioctanoin.
By-products
Proportion in
humid weight
(%HW)
TC4
TC8
Relative
activity
towards
TC8 (%)a
Latex
0.1
0.9
2
3
5
89
98.76
19.37
2.15
4.95
2.00
0.16
10.88
0.67
0.02
1.67
0.02
0.01
11
3
1
34
1
6
Leaves
Meristems
Petioles
Fruits
As previously discussed, the relative activity
towards TC8 with respect to TC4 was higher in
petioles than in latex. Thus, during the processing of
the petiole biocatalyst, the proportion of lipase
activity was evaluated (Table 2). The petiole juice
has the highest proportion of lipase activity.
However, in ester synthesis it is necessary to
Stems
Esterase/lipase activity (U/mL) was measured at 25 ꢁC and pH 7.0,
using tributyrin (TC4) and trioctanoin (TC8) as substrate.
Values are the average of three independent assays with SD less
than 5%.
aExpressed as percentage with respect to TC4.

Carica papaya by-products
5
remove water from the reaction mixture. After
lyophilization, the proportion of lipase activity was
reduced 6-fold in LP. When the drying treatment
was performed at room temperature, as is the case
for generating BP, the proportion of the lipase
activity was reduced only 1.7-fold compared to the
lipase activity of the petiole juice. These results
suggest that the reduction in the lipase activity is due
to freezing. This phenomenon has already been
reported for other enzymes (Privalov 1990). In TLP,
both activities were reduced between 52% and 98%.
This result suggests that the hydrolases responsible
for each activity in petioles were possibly stabilized
by metal ions (Mg2+, Ca2+, Mn2+, Fe2+, Cu2+, and
Zn2+) that could have been removed by washing
(Migliaccio et al. 2010). Similarly, it has been
reported that cationic metals, particularly divalent
cations, can modulate the lipase activity in some
plants. For example, the lipase from a C. papaya
close relative, Carica pentagona, is dependent on
Ca2+ (Dhouib et al. 2011). Similarly, plant lipases
from different classes found in the seed of Prunus
dulcis and latex from Ficus carica increase their
hydrophobicity, heptane (Log P 4.3), was used in
the alcoholysis of different oils, using latex as a
biocatalyst (Su and Wei 2014). Thus, the synthesis of
different alkyl chain length oleic acid esters using
TLL and BP as biocatalysts was studied (Figure 2).
As shown in Figure 2(A), TLL showed a 3- to 44-
fold higher synthesis activity than BP (Figure 2B) in
all of the tested alcohols. Indeed, the average
productivity of TLL (1357 mmol/g h) is higher than
those values calculated using the reported data for
the synthesis of ethyl (441 mmol/g h) and butyl oleate
(603 mmol/g h) and 4-fold less for the synthesis of
propyl (5723 mmol/g h), employing the Rhizopus sp.,
Table 2. Variation of esterase/lipase units during petioles process.
Relative
activity
towards
TC8
TC4 (U)
TC8 (U)
(%)a
Petioles (juice)
1981
24,849
2139
941
668
4938
118
16
34
20
5.5
1.7
Bagasse from petioles
Lyophilized petioles
Treated lyophilized petioles
activity in the presence of Ca2+, Mn2+, Fe2+, Co2+
,
From a petioles kg U (total units) of esterase/lipase activity
measured at 25 ꢁC and pH 7.0, using tributyrin (TC4) and
trioctanoin (TC8) as substrate, respectively.
aExpressed as percentage with respect to TC4.
and Ca2+, Cu2+, respectively (Yes¸ilog˘lu and Bas¸kurt
2008; Lazreg-Aref et al. 2012). We selected BP for
further studies because its lipase/esterase activity
proportion was the greatest and obtaining it was the
most cost effective and easiest to acquire of those
collected. There is a large potential for the applica-
tion of BP in organic synthesis reactions.
Table 3. Effect of different reaction solvents using TLL and BP as
biocatalysts in the ethyl oleate synthesis.
(%) conversion of ethyl oleate
Kinetics of the synthesis of oleic acid esters
Solvent
Log P
TLLa
BPb
Ethyl oleate synthesis was used as a reaction model
to evaluate the effect of solvent hydrophobicity on
the reaction yield (Table 3). The best reaction
solvent was isooctane, a solvent with high hydropho-
bicity (Log P 4.4). This result was consistent with
previous work in which a solvent with similar
Isooctane
Toluene
Diisopropyl ether
tert-Butanol
4.4
2.5
1.5
0.8
82.3 ꢂ 2.8
12.0 ꢂ 2.0
15.0 ꢂ 2.0
7.7 ꢂ 2.1
80.5 ꢂ 0.2
11.6 ꢂ 0.1
15.0 ꢂ 1.5
6.7 ꢂ 0.7
TLL, treated lyophilized latex; BP, bagasse from petioles. % of
conversion after 1 ha and 48 hb. Reaction was performed at
200 mM of oleic acid and ethanol, 50 ꢁC and 300 rpm.
Figure 1. Esterase/lipase activities of the biocatalysts on homogenous triglycerides with different acyl chain lengths. (A) Black bars:
lyophilized latex. Grey bars: treated lyophilized latex. (B) Dark gray bars: lyophilized petioles. White bars: treated lyophilized petioles. (C)
White striped bars: bagasse from petioles. These experiments were carried out at 25 ꢁC in 2.5 mM Tris–HCl buffer (30 mL, final volume)
containing 150 mM of NaCl at pH 7.

´
M. Armendariz-Ruiz et al.
6
Candida antarctica (Novozym 435), and Rhizomucor
miehei (Lipozyme IM) lipases, respectively (Habulin
et al. 1996; Salis et al. 2005; Martinez-Ruiz et al.
2008). Nonetheless, all these productivity data were
obtained at different conditions and comparison
should be taken carefully.
obtained for TLL, indicating that BP is better suited
as a biocatalyst for the synthesis of long chain alkyl
esters in organic solvents than it is for their hydroly-
sis. Furthermore, it should be noted that bagasse, as
a fibrous by-product, has the ability to absorb the
reaction solvent, generating serious diffusion prob-
lems and limiting the amount of the biocatalyst that
could be used in batch systems. Therefore, it was not
possible to reduce the reaction times in batch
systems (48 h). However, BP was active during a
long reaction time (Figure 2B), suggesting that this
biocatalyst is more resistant to thermal inactivation
and better suited for use in continuous systems. An
attractive system is a PBR because the catalyst bed is
positioned with a fixed height after a substrate
mixture is fed into it at a controlled flow rate,
leading to the continuous recovery of a product with
a high conversion yield at the reactor outlet. Thus,
we postulate that a continuous PBR system could be
a better option for the synthesis application of BP.
The kinetic profile for the synthesis of oleic acid
esters using TLL does not show a clear alkyl chain
preference (Figure 2A) compared to that observed by
Caro et al. during the alcoholysis of trilaurin with
primary alcohols (C1–C12; Caro et al. 2004). In
contrast to the literature, TLL does not show
significant inhibition when methanol and ethanol
were used as alkyl chain donors (Caro et al. 2004; Su
and Wei 2014). Therefore, it should be considered to
be a robust biocatalyst with a potential application
for the synthesis of short chain alkyl esters.
Nevertheless, BP showed a synthesis preference
towards medium-long chain esters (C4–C12;
Figure 2B). The initial rate ratio for dodecyl and
methyl oleate (C12/C1) revealed that dodecyl oleate
(C12) was synthesized three times faster than methyl
oleate (C1) by BP. These outcomes demonstrate that
BP has a preferential activity regarding long chain
alcohols, a typical behavior of classic lipases in
organic synthesis (Habulin et al. 1996). Moreover,
this suggests that the binding interactions between
lipase activity and the petiole solid matrix are
stronger than the interaction with other hydrolases,
such as proteases and esterases, present in this by-
product. The latter would explain why the extraction
process (see ‘‘Materials and methods’’ section)
generates an enriched lipase activity biocatalyst, as
demonstrated in the previous section.
Evaluation of the thermostability of the biocatalysts
from C. papaya
The thermostability of a biocatalyst is important to
assess because thermal enzymatic denaturation is
one of the critical factors for the successful operation
of a PBR. Accordingly, the thermostability of the
papaya biocatalysts (LL, TLL, TPL, and BP) was
evaluated after incubation at 45 ꢁC, and the residual
activity was determined using TC8 as the substrate
(Figure 3). As expected, the thermal inactivation
profiles followed first-order kinetics. The petiole
biocatalysts showed a higher thermostability, with a
half-life approximately 7-fold higher than the latex
biocatalysts. This result could also be due to an effect
of the matrix or that the lipase activity of the petiole
corresponds to a different enzyme than that presents
in the latex and is, presumably, more stable. For the
To evaluate the ability of the biocatalysts to
perform synthesis versus hydrolysis, the ratio of
DO synthesis (C12S) and TC8 hydrolysis (TC8H)
was calculated for TLL and BP. The C12S/TC8H
ratio for BP (0.46) was 3-fold higher than that
Figure 2. Kinetics for the synthesis of oleic acid esters. (A) Treated lyophilized latex, TLL; (B) bagasse from petioles, BP. Synthesis was
performed at 50 ꢁC using isooctane as the reaction solvent. *37.5 mg BP/mL. MO – methyl oleate; EO – ethyl oleate; PO – propyl oleate;
BO – butyl oleate; DO – dodecyl oleate.

Carica papaya by-products
7
latex biocatalysts, the washing treatment slightly
improved the biocatalyst thermostability, leading to
a 1.6-fold increase in its half-life (26 min), probably
due to the lack of proteases in the TLL following the
washing treatment. Likewise, among petioles bioca-
talysts, BP was the most thermostable, with a half-life
of 3-fold higher than TLP, suggesting that the
support (bagasse) confers enhanced stability to the
enzyme responsible for the lipase activity (Cao
2006). In general, plant lipases exhibit a lower
thermostability than microbial lipases; some, such
as Thermomyces lanuginosus and type A from C.
antarctica, have half-lives of 20 and 1400 h at 60 and
70 ꢁC, respectively (Heldt-Hansen et al. 1989).
However, the residual activity of BP after one hour
of incubation at 45 ꢁC (65%) was similar to that
reported for other plant lipases, such as wheat germ
(70%), rice bran (90%), or a precipitate of C. papaya
seeds and fruit peel (80%) (Bhardwaj et al. 2001;
Kapranchikov et al. 2004; Paques et al. 2008). BP
could be a good candidate for the continuous
synthesis in a PBR.
The continuous synthesis of dodecyl oleate in a PBR
The continuous synthesis of dodecyl oleate was
carried out in a PBR using BP as a biocatalyst
(Figure 4). The PBR reached the steady-state only
after 2 h of operation, and the reaction conversion
(96%) remained stable for at least 40 h. After this
time, the conversion gradually declined to 65% at 60
h, and this result could be a consequence of water
accumulation in the bed of the reactor during the
reaction progress. Although, the steady-state BP–
PBR (40 h) was not as long as reported for the
transesterification synthesis of butyl oleate (150 h)
using C. papaya latex in a PBR (Su and Wei 2014),
we must emphasize that the amount of petioles in the
plant is much greater than the amount of latex in the
plant. Additionally, the process that is used to obtain
biocatalysts from the petioles is much simpler than
the process that is used to obtain them from latex.
Consequently, BP is a much more attractive bioca-
talyst than latex.
Moreover, the productivity attained in the steady-
state BP–PBR was 11,202 mol/h, which is 23 times
greater than the productivity achieved by the
immobilized lipase of R. miehei (LipozymeÕ) during
the transesterification synthesis of butyl oleate using
a similar system and 20 mM as the initial substrate
concentration (Dossat et al. 1999). This result could
be attributed to the higher substrate concentration
employed in our experiment (200 mM). Therefore,
the BP–PBR system seems to be a competitive
alternative choice for the synthesis of additional
valuable organic compounds, such as dodecyl oleate,
which is widely used in the cosmetics industry.
Figure 3. Kinetics of the thermal inactivation of the Carica papaya
biocatalysts. The optimal conditions for each biocatalyst were
determined by the residual hydrolytic activity on trioctanoin at
45 ꢁC for four hours. The data are the means ꢂ SD (n ¼ 3).
Conclusions
In this work, it was demonstrated, for the first time,
that it is possible to obtain new biocatalysts with
lipase/esterase activity from the by-products of
C. papaya that have different properties for hydroly-
sis and synthesis than those previously reported for
latex (mainly thermostability and selectivity). The
latter suggests the presence of different enzymes in
the C. papaya by-products. To confirm this, more
studies are required. However, we proved that these
biocatalysts can be obtained, leading to a new
outlook for catalytic studies in other C. papaya by-
products, especially petioles.
Figure 4. The continuous synthesis of dodecyl oleate in a packed
bed reactor. The synthesis was catalyzed by bagasse from petioles
using isooctane as the reaction solvent and a 0.2 M final substrate
concentration (molar ratio 1:1) at 50 ꢁC.

´
M. Armendariz-Ruiz et al.
8
´
Galindo-Estrella T, Hernandez-Gutierrez R, Mateos-Dıaz J,
Sandoval-Fabian G, Chel-Guerrero L, Rodrıguez-Buenfil I,
´
´
Acknowledgements
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We would like to thank the Peace Corps volunteer
Tina Coop for revisions made to the language this
paper.
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Declaration of interest
´
´
M. Armendariz and E. Mateos-Dıaz acknowledge
the scholarships received from CONACYT. This
research was supported by the FOMIX-
CONACYT-Gobierno del Estado de Jalisco, project
2012-07-190698.
The authors report no conflicts of interest.
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