Lipase from Carica papaya latex presents high enantioselectivity toward the resolution of prodrug (R,S)-2-bromophenylacetic acid octyl ester

Article abstract of DOI:10.1016/j.tetlet.2013.07.151

Besides the well-known papain, lipolytic activity is another interesting enzymatic activity present in latex from Carica papaya. This lipolytic activity is strongly attached to the latex solid phase, resulting in a naturally immobilized biocatalyst. In this work we describe the kinetic resolution of (R,S)-2-bromophenylacetic acid octyl ester by Carica papaya crude latex and two partially purified latex fractions. Several parameters, such as substrate concentration and solvent effects were studied. The best results were obtained using decane as solvent with 50 mM of substrate and 50 mg/mL enzyme/reaction medium; under these conditions, a high enantioselectivity (E >200) was obtained with crude latex. A twofold increase of the initial rate maintaining E >200 was obtained using purified fractions without protease and without esterase. Lipase from Carica papaya latex is the most enantioselective wild-type enzyme ever described for the studied reaction.

Full text of DOI:10.1016/j.tetlet.2013.07.151

Tetrahedron Letters 54 (2013) 5523–5526
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Tetrahedron Letters
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Lipase from Carica papaya latex presents high enantioselectivity
toward the resolution of prodrug (R,S)-2-bromophenylacetic acid
octyl ester
a,b,c,d
, Juan Carlos Mateos , Alain Marty a,b,c, Georgina Sandoval d, , Sophie Duquesne a,b,c, ,
⇑
d
Ivanna Rivera
a
Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France
INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France
CNRS, UMR5504, F-31400 Toulouse, France
b
c
d
CIATEJ, Av. Normalistas 800, Colinas de la Normal, 44270 Guadalajara, Jalisco, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Besides the well-known papain, lipolytic activity is another interesting enzymatic activity present in
latex from Carica papaya. This lipolytic activity is strongly attached to the latex solid phase, resulting
in a naturally immobilized biocatalyst. In this work we describe the kinetic resolution of (R,S)-2-bromo-
phenylacetic acid octyl ester by Carica papaya crude latex and two partially purified latex fractions. Sev-
eral parameters, such as substrate concentration and solvent effects were studied. The best results were
obtained using decane as solvent with 50 mM of substrate and 50 mg/mL enzyme/reaction medium;
under these conditions, a high enantioselectivity (E >200) was obtained with crude latex. A twofold
increase of the initial rate maintaining E >200 was obtained using purified fractions without protease
and without esterase. Lipase from Carica papaya latex is the most enantioselective wild-type enzyme ever
described for the studied reaction.
Received 1 July 2013
Revised 23 July 2013
Accepted 29 July 2013
Available online 6 August 2013
Keywords:
Lipase
Carica papaya latex
Enantioselectivity
2
-Halogenocarboxylic acid
Ó 2013 Elsevier Ltd. All rights reserved.
Introduction
Although microbial lipases are the most studied source of lipases,
such enzymes can also be found in plants, including oil seeds and lat-
Therapeutic application of enantiopure drugs is highly attrac-
tive for pharmaceutical industry. Indeed, pure enantiomers have
improved specificity and biological activity in comparison to race-
mic drugs and their use also avoid potential side effects associated
icifers (plant cells producing a milky fluid constituted by proteins,
alkaloids, and so forth, commonly known as latex). Carica papaya la-
tex (abbreviated here CPLtx) presents an interesting lipolytic activ-
5
ity, which was previously used to produce synthetic cocoa butter,
1
6
7
8
9
to the use of racemic mixtures. Nevertheless, classical methods to
human milk analogs, terpene, biopolymers , and wax esters.
obtain optically enriched compounds, such as chemical synthesis
and chiral chromatography, are often expensive. As an alternative,
several microbial lipases were used over the last years as enantio-
selective biocatalysts. Indeed, these biocatalysts have the capacity
to accept a large variety of substrates, display stability and perfor-
mance in organic solvents, and permit to discriminate enantiomers
from chiral mixtures of pharmaceutical interest, including non-ste-
It has been demonstrated that most of the lipolytic activity of
CPLtx is highly associated to the latex solid phase, thus resulting
1
0
in a stable and naturally immobilized biocatalyst. Many efforts
have been made in order to partially purify CPLtx lipolytic activity,
through eliminating water soluble enzymes from latex (mainly
1
1
proteases). The resulting fraction is abbreviated here as ‘CPL-p’
(Carica papaya latex without protease). Recently, detergents were
used to remove from latex part of the lipolytic activity mainly
2
roidal anti-inflammatory drugs. Amongst such chiral mixtures, 2-
1
2
halogenocarboxylic acid esters are important intermediates for the
synthesis of various chiral drugs such as prostacyclin and prosta-
due to esterase activity. This final preparation is abbreviated here
as ‘CPL-e’ (Carica papaya latex without esterase). CPLtx and CPL-p
have been successfully used for the racemic resolution of different
3
glandin. Therefore, the kinetic resolution of (R,S)-2-bromophenyl-
1
3
acetic acid octyl ester racemates by microbial lipases is of great
non-steroidal anti-inflammatory drugs.
4
interest.
The objective of this work was to optimize the resolution of
(R,S)-2-bromophenylacetic acid esters by CPLtx and to compare
the performance of CPLtx, CPL-p, and CPL-e on the enantioselective
hydrolysis. The effect of substrate concentration, organic solvents,
and pH was studied, using (R,S)-2-bromophenylacetic acid octyl
ester as substrate (Scheme 1).
⇑
Corresponding author. Tel.: +33 561559439; fax: +33 561559401.
E-mail address: Sophie.duquesne@insa-toulouse.fr (S. Duquesne).
These two authors have equally contributed.
0
040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.07.151

5
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I. Rivera et al. / Tetrahedron Letters 54 (2013) 5523–5526
Table 1
Results and discussion
Hydrolytic activities of CPL preparations and ratio of lipase versus protease and
esterase activities
Hydrolytic activities of C. papaya crude and partially purified la-
tex (CPLtx, CPL-p, and CPL-e) were evaluated and their perfor-
mance on the kinetic resolution of (R,S)-2-bromophenylacetic
Hydrolytic activity
Biocatalyst
CPL-p
CPLtx
CPL-e
1
2a
acid octyl esters with optimized conditions was compared.
a
Protease
353 ± 2 U/g
1137 ± 37 U/g
593 ± 1 U/g
1.7
7 ± 0.003 U/g
1248 ± 68 U/g
753 ± 0.0 U/g
108
3 ± 0.001 U/g
486 ± 3 U/g
921 ± 32 U/g
307
b
Esterase
c
Hydrolytic activity of CPL preparations
Lipase
Lipase/protease
Lipase/esterase
0.5
0.6
1.9
First, in order to characterize the enzymatic preparations, prote-
ase, esterase, and lipase activities were evaluated with specific
substrates. Table 1 shows the ratio of these hydrolytic activities.
As expected, a high decrease in proteolytic activity was
achieved during the preparation of CPL-p (98%) and only 0.9% pro-
tease activity remained in CPL-e. In this later, lipase is the predom-
inant activity after removal of 43% esterase activity.
Mean and standard deviation of two independent experiments are presented.
a
Measured by casein hydrolysis, 1 U = 1 equiv tyrosine liberated.
Measured by tributyrin hydrolysis, 1 U = 1
Measured by triolein hydrolysis, 1 U = 1
b
lmol acid liberated/min.
c
lmol acid liberated/min.
Table 2
Effect of pH on the racemic resolution of (R,S)-2-bromophenylacetic acid
In previous works dealing with enantioresolution using C. pa-
paya preparations, only CPLtx and CPL-p were tested. Here we
decided to compare each partially purified fraction, including
CPL-e, to discriminate which of protease, lipase, or esterase activity
is responsible for the observed enantioselectivity.
octyl ester catalyzed by CPLtx
pH
Initial reaction rate for the
E
(r
a
S enantiomer, r
S
S R
/r )
À1
À1
(lmol h g enzyme
)
7
8
8.5
9.0
.0
.0
1.45 ± 0.04
2.02 ± 0.00
2.4 ± 0.10
2.3 ± 0.10
30
168
>200
>200
Kinetic resolution of (R,S)-2-bromophenylacetic acid octyl ester
using CPLtx
5
0 mM Substrate in decane, solvent:buffer 1:1, 50 mg/mL of biocatalyst,
CPLtx’s ability to discriminate between enantiomers in the race-
mic mixture of (R,S)-2-bromophenylacetic acid octyl esters was
evaluated in a biphasic medium (water:decane 1:1). Different reac-
tion parameters were evaluated in order to determine their effects
over activity and enantioselectivity.
25 °C
a
Mean and standard deviation of two independent experiments are
presented.
hydrophobicity was thus performed. The highest initial rate was
obtained using decane, the most hydrophobic solvent tested, prob-
ably due to a good emulsification process where the interface
water/solvent is favorable to lipase catalytic performances
(Table 3).
Previous efforts have been carried out in order to relate CPLtx
catalytic performances (reaction rate and enantioselectivity to-
ward 2-mathylalkanoic acid,
2,2,2-trifluoroethyl thioester ) and the properties of the solvent
used. As in our case, no explicit correlation could be found between
solvent properties and initial rates of reaction.
Determination of the optimal substrate concentration is essen-
tial to obtain high initial rate and E-value. As shown in Table 4,
substrate concentration has only a slight influence on the perfor-
mance of CPLtx in the studied range of concentrations. Neverthe-
less, over 100 mM, a slight decrease in reaction rates is observed,
probably due to substrate inhibition.
1
4
Unlike lipases that operate in acidic or neutral media, CPLtx
was previously shown to operate in basic media.15 This constitutes
a major advantage for applications as detergents, dish washing,
and dry cleaning products.16 High activity in alkaline media might
also be an advantage for substrate solubility in synthesis reac-
1
7
tions. Considering the studied reaction, the highest hydrolysis
activity toward the best-recognized enantiomer was indeed ob-
tained at pH 8.5 and 9.0 (Table 2). These results are in agreement
with those obtained by Abdelkafi et al. on triglycerides,1 where
the highest activity was also obtained between pH 8.5 and 9.0.
pH 8.5 was thus retained for the following experiments.
2
0a
20b
propionic acid,
and naproxen
1
3b
5
At pH 8.5, CPLtx exhibited the highest initial rate
À1
À1
(
2.36 ± 0.10 lmol h g enzyme ) of hydrolysis toward the pre-
ferred (S)-enantiomer and a very high enantioselectivity (>200),
which makes this enzyme the most efficient wild-type lipase for
the resolution of this racemic mixture. Indeed, reported enantiose-
lectivity values of wild-type enzymes were much lower than those
obtained using CPLtx (E = 72 for (S)-selective lipase Lip2 from Yarr-
owia lipolytica and E = 53 for (R)-selective lipase from Burkholderia
Kinetic resolution of (R,S)-2-bromophenylacetic acid octyl ester
by CPL-p and CPL-e
4
c,18
cepacia).
It was necessary to improve these microbial lipases’
Finally, the performances of partially purified fractions CPL-p
and CPL-e were analyzed (Table 5). Results showed that the initial
rate of hydrolysis of the preferred (S)-enantiomer increased when
the partially purified fractions of latex were used, due to an enrich-
ment of these fractions in lipolytic activity (see Table 1). Indeed,
the reaction proceeded 2.1 and 2.4-fold faster when CPL-p and
selectivity by site-directed mutagenesis in order to reach high ini-
tial rates and E-values >200.4
b,18
Selection of a proper reaction medium is a key parameter to
optimize any enzymatic reaction, as it can affect both the initial
rate and enzyme selectivity.19 Screening of solvents with different
Scheme 1. Hydrolysis of (R,S)-2-bromophenylacetic acid octyl ester 1 into (R)-2-bromophenylacetic acid octyl ester 2, (S)-2-bromophenylacetic acid 3, and octyl alcohol 4
provided that lipase is S enantioselective).
(

I. Rivera et al. / Tetrahedron Letters 54 (2013) 5523–5526
5525
Table 3
Solvent effects on the racemic resolution of (R,S)-2-bromophenylacetic acid octyl
ester catalyzed by CPLtx
b
Solvent
LogP)a
Initial reaction rate S-enantiomer, r
S
E
(r
À1
À1
(
(lmol h g enzyme
)
S R
/r )
Cyclohexane (3.2)
Octane (4.0)
Heptane (4.6)
Decane (5.6)
1.2 ± 0.31
1.1 ± 0.23
1.6 ± 0.15
2.4 ± 0.10
>200
>200
>200
>200
5
0 mM Substrate in the solvent, solvent:buffer 1:1, 50 mg/mL of biocatalyst, 25 °C.
LogP (solvent partition coefficient) values taken from Chemspider
a
(
www.chemsider.com).
Mean and standard deviation of two independent experiments are presented.
b
Table 4
Effects of substrate concentration on the racemic resolution of (R,S)-2-bromophen-
ylacetic acid octyl ester catalyzed by CPLtx
a
Substrate
mM)
Initial reaction rate S-enantiomer, r
S
E
(r
À1
À1
(
(lmol h g enzyme
)
S R
/r )
5
00
50
0
2.4 ± 0.10
2.9 ± 0.10
2.5 ± 0.10
>200
>200
>200
1
2
Substrate dissolved in decane, solvent:buffer 1:1, 50 mg/mL of biocatalyst, 25 °C.
a
Mean and standard deviation of two independent experiments are presented.
Table 5
Comparison of the kinetic resolution of (R,S)-2-bromophenylacetic acid octyl ester
catalyzed by CPLtx, CPL-p, and CPL-e
a
Biocatalyst
Initial reaction rate S-enantiomer, r
S
E
(r
À1
À1
(lmol h g enzyme
)
S R
/r )
CPLtx
CPL-p
CPL-e
2.4 ± 0.10
5.0 ± 0.39
5.6 ± 0.30
>200
>200
>200
Figure 1. Time course of kinetic resolution of (R,S)-2-bromophenylacetic acid octyl
ester catalyzed by CPLtx and CPL-p. Assays were performed in the same conditions
as in Table 5. All assays were carried out in duplicate.
5
0 mM Substrate in decane, solvent:buffer 1:1, 50 mg/mL of biocatalyst, 25 °C.
a
Mean and standard deviation of two independent experiments are presented.
Interestingly, enantiomeric excess observed for CPL-e was al-
most the same as that observed for CPL-p; this coincided with
the results obtained when initial reaction rates were evaluated
with both fractions (Table 5).
CPL-e were used respectively, which correlates with a 1.2 and 1.6-
fold increase in the lipase activity assayed on triolein. Moreover,
CPL-p and CPL-e have similar performances, which implies that
the enzyme or enzymes that perform the reaction are still in the
non-soluble fraction of latex. These results are consistent with
the fact that soluble extracts (containing proteases and esterases)
did not show any activity toward the enantiomeric resolutions
tested. In the case of (R,S)-2,2,2-naproxen trifluoethyl thioester,
Ng and Tsai also observed that specific activity was enhanced
when CPL-p was used (compared to non-purified latex), while
enantioselectivity was conserved.1
Figure 1 shows the time course of reactions for both CPLtx and
CPL-p at 4 and 25 °C. When assessed at 25 °C, CPLtx showed an
important decrease in the hydrolysis rate after 6 h reaction, which
was not observed with CPL-p. This is probably due to the degrada-
tion of lipolytic enzymes by proteases. In accordance, this phenom-
enon is less pronounced at 4 °C and for CPL-p (fraction partially
purified where most of proteases have been removed).
Summary & conclusion
There are only few reports of microbial lipases efficient in the
kinetic resolution of (R,S)-2-bromophenylacetic acid octyl ester.
Those mainly include Lip1 and Lip3 from Candida rugosa, Lip2 from
4
Yarrowia lipolytica, and lipase from Burkholderia cepacia. Here we
3c
demonstrate that the catalytic performances of Carica papaya latex
CPLtx and its partially purified fractions CPL-p and CPL-e are more
efficient than those described wild-type microbial lipases. CPLtx is
therefore the most enantioselective wild-type enzyme for the stud-
ied reaction.
CPLtx is an efficient and highly enantioselective biocatalyst for
the kinetic resolution of (R,S)-2-bromophenylacetic acid octyl ester
with preference for the (S)-enantiomer and E >200. In the case of
the partially purified fractions CPL-p and CPL-e, a twofold increase
in the initial rate was obtained, while E >200 was maintained. As
CPL-p and CPL-e have the same performances, but the purification
process for CPL-p is simpler, this is the preferred biocatalyst to con-
tinue further studies.
After 48 h reaction with CPL-p at 25 °C (47.8% total conversion),
enantiomeric excesses of substrate and product are 87.8% and
9
5.8%, respectively. At this time, 98.0% of the (R)-enantiomer can
be recovered with a purity of 93.9%, and 93.6% with 97.9% purity
for the (S)-enantiomer.

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I. Rivera et al. / Tetrahedron Letters 54 (2013) 5523–5526
Experimental
References and notes
1
2
3
.
.
.
Smith, S. W. Toxicol. Sci. 2009, 110, 4–30.
The procedure for the preparation of (R,S)-2-bromophenylacetic
Carvalho, P. d. O.; Contesini, F. J.; Ikegaki, M. Braz. J. Microb. 2006, 37, 329–337.
(a) Holland, G. W.; Jernow, J. L.; Rosen, P. U.S. Patent 4410720, 1983.; (b)
Holland, G. W.; Jernow, J. L.; Rosen, P. U.S. Patent 4246402, 1981.; (c) Holland,
G. W.; Jernow, J. L.; Rosen, P. U.S. Patent 4187381, 1980.; (d) Guieysse, D.;
Salagnad, C.; Monsan, P.; Remaud-Simeon, M. Tetrahedron: Asymmetry 2003,
acid octyl ester, was described previously.3
d
CPLtx collection from fruits of ‘Maradol’ variety was carried out
according to Rivera et al.12a
Partial purification of CPLtx to give a fraction without protease
1
4, 317–323.
(
CPL-p) and without esterase (CPL-e) was also carried out as de-
4. (a) Bordes, F.; Cambon, E.; Dossat-Letisse, V.; Andre, I.; Croux, C.; Nicaud, J. M.;
Marty, A. ChemBioChem 2009, 10, 1705–1713; (b) Cambon, E.; Piamtongkam,
R.; Bordes, F.; Duquesne, S.; Andre, I.; Marty, A. Biotechnol. Bioeng. 2010, 106,
12a
scribed previously by Rivera et al. and lyophilized.
2
1
Proteolytic activity was measured using casein as a substrate.
852–859; (c) Cancino, M.; Bauchart, P.; Sandoval, G.; Nicaud, J. M.; Andre, I.;
Esterase activity in latex was measured by hydrolysis of tribu-
tyrin while lipase activity was measured by hydrolysis of olive
oil. Hydrolysis reactions were performed in a thermostated and
mechanically stirred Envirogenie Scientific Industries (USA) at
Dossat, V.; Marty, A. Tetrahedron: Asymmetry 2008, 19, 1608–1612; (d)
Piamtongkam, R.; Duquesne, S.; Bordes, F.; Barbe, S.; André, I.; Marty, A.;
Chulalaksananukul, W. Biotechnol. Bioeng. 2011, 108, 1749–1756.
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2012, 114, 266–276.
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3
0 °C containing 2.5 mL tributyrin, 1 mL 50 mM Tris–HCl, 50 mM
7
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9
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You, P.; Su, E.; Yang, X.; Mao, D.; Wei, D. J. Mol. Catal. B Enzym. 2011, 71, 152–
NaCl, and 0.2% triton pH 8.5 buffer solution with the corresponding
amount of enzyme (usually 50 mg). Free fatty acids released were
titrated with NaOH and lipase activity was expressed as
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Sandoval, G.; Rivera, I.; Barrera-Rivera, K. A.; Martinez-Richa, A. Macromol.
Symp. 2010, 289, 135–139.
(a) Quintana, P. G.; Sandoval, G.; Baldessari, A. Biocatal. Biotransform. 2011, 29,
1
U = 1
Hydrolysis of racemic substrate was carried out as described by
Piamtongkam et al.4d with the following modifications: substrate
00 mM, buffer Tris–HCl, 100 mM NaCl and 0.2% triton TX100,
lmol free fatty acid (FFA) released per minute.
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1
pH 8.5 (of buffer solution), and 50 mg/mL of enzyme. The mixture
was shaken in a Vortex Genie 2 (Brumat, France). Reactions were
carried out at 25 °C. Progress of the reaction was followed at regu-
lar time intervals by taking samples after phase separation by cen-
trifugation (50 lL diluted in 500 lL hexane).
The enantioselectivity value (E-value) was defined as the ratio
of the initial rate of (S)-enantiomer production to the initial rate
1
1
1
S R
of (R)-enantiomer production (E = r /r ). Enantiomeric excess (ee)
was calculated as defined below: ees = {[R] À [S]}s/{[R] + [S]}s
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(
s = substrate).
Initial rates were determined by linear regression from concen-
1
trations measured by HPLC as follows: the HPLC was equipped
with a chiral column: Chiralpack OJ (25 cm  4.6 mm) (Daicel
Chemical Industries Ltd, Japan) connected to a UV detector (at
1
1
1
2
54 nm). A flow rate of 1.0 mL/min was used. The mobile phase
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was composed of a mixture of n-hexane/isopropanol (80:20 v/v).
Acknowledgments
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Tsai, S. W. Tetrahedron: Asymmetry 2004, 15, 2917–2920.
2
1. Sigma–Aldrich. Universal Protease Activity Assay: Casein as
Accessed December 2012 http://www.sigmaaldrich.com/life-science/
learning-center/lifescience-video/universal-protease.html.
a Substrate.
I.R. thanks CONACYT (Mexico) and projects ECOS-ANUIES M08-
A03, and CB-2008-01-104429 for financial support.