Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of α-substituted esters

Article abstract of DOI:10.1021/ja100593j

A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of α-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45-276, which is a large improvement compared to 2-20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95-99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates.

Full text of DOI:10.1021/ja100593j

Published on Web 05/07/2010
Directed Evolution of an Enantioselective Lipase with Broad
Substrate Scope for Hydrolysis of r-Substituted Esters
Karin Engstro¨m, Jonas Nyhle´n, Anders G. Sandstro¨m, and Jan-E. Ba¨ckvall*
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm UniVersity,
SE-106 91 Stockholm, Sweden
Received January 22, 2010; E-mail: jeb@organ.su.se
Abstract: A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of R-substituted
p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45-276,
which is a large improvement compared to 2-20 for the wild type. The broad substrate scope of this enzyme
variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess
between 95-99%. A 30-fold increase in activity was also observed for most substrates. The developed
enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for
most substrates.
Introduction
We have previously demonstrated the advantage of using the
episomally replicating pBGP18 vector in Pichia pastoris for
heterologous expression in directed evolution experiments.9 The
latter technique was used to improve the enantioselectivity of
Candida antarctica lipase A (CalA) toward p-nitrophenyl
2-methylheptanoate. The enzyme variants obtained from this
screening showed a very narrow substrate scope, and we
therefore decided to screen for more interesting analogues such
as 2-arylpropionic acids (profens). In this paper we report on a
triple mutant with a remarkably broad substrate scope10 and
excellent enantioselectivity.
2-Arylpropionic acids are an important class of pharmaceu-
ticals that belong to the so-called “profens”, a group of
nonsteroidal anti-inflammatory drugs (NSAIDs).1 These drugs
are used to treat pain, fever, inflammation, and stiffness, and
examples of substances from this class are Naproxen, Ibuprofen,
and Flurbiprofen.2 Methods for the preparation of enantiomeri-
cally pure “profens” are highly desirable since FDA requires
that most chiral drugs are marketed as single enantiomers today.3
Enzymes are nature’s catalysts working with great efficiency
and selectivity. Unfortunately, the substrate scope of enzymes
is usually quite narrow. However, by applying the tools of
protein engineering, enzymes can be modified to accept un-
natural substrates and to evolve enantioselectivity. Directed
evolution is a strategy which mimics nature’s evolution in the
laboratory.4 This is achieved by performing iterative rounds of
(i) generation of a mutated gene library, (ii) expression of the
library of mutated proteins, and (iii) screening of the mutated
proteins for the desired property. The best protein variant found
in the first round is used as a template in the next round of
mutagenesis. Directed evolution has successfully been used to
improve specific qualities of enzymes.5-7
In the present study the aim was to improve the enantiose-
lectivity of CalA toward the R-substituted esters. CalA not only
shows a modest enantioselectivity toward these substrates (E
values 2-20) but also has a very low activity, and therefore
the enzyme is of limited use in synthetic applications. The first
objective was to improve the activity of the enzyme using
p-nitrophenyl 2-phenylpropanoate (1) as a model substrate.
Successful improvement of the activity of Pseudomonas aerugi-
nosa lipase toward R-methyl substituted carboxylic acids by the
use of combinatorial active-site saturation test (CAST) was
(6) (a) Reetz, M. T.; Wu, S. J. Am. Chem. Soc. 2009, 131, 15424–15432.
(b) Lewis, J. C.; Bastian, S.; Bennett, C. S.; Fu, Y.; Mitsuda, Y.; Chen,
M. M.; Greenberg, W. A.; Wong, C.-H.; Arnold, F. H. Proc. Natl.
Acad. Sci. U.S.A. 2009, 106, 16550–16555.
(1) Evans, A. M. Eur. J. Clin. Pharmacol. 1992, 42, 237–256.
(2) (a) Davies, N. M.; Anderson, K. E. Clin. Pharmacokinet. 1997, 32,
268–293. (b) Davies, N. M. Clin. Pharmacokinet. 1995, 28, 100–114.
(c) Davies, N. M. Clin. Pharmacokinet. 1998, 34, 101–154.
(3) Beck, G. Synlett 2002, 6, 837–850.
(7) (a) DeSantis, G.; Wong, K.; Farwell, B.; Chatman, K.; Zhu, Z.;
Tomlinson, G.; Huang, H.; Tan, X.; Bibbs, L.; Chen, P.; Kretz, K.;
Burk, M. J. J. Am. Chem. Soc. 2003, 125, 11476–11477. (b) Landwehr,
M.; Hochrein, L.; Otey, C. R.; Kasrayan, A.; Ba¨ckvall, J.-E.; Arnold,
F. H. J. Am. Chem. Soc. 2006, 128, 6058–6059.
(4) (a) Chen, K.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
5618–5622. (b) Reetz, M. T.; Zonta, A.; Schimossek, K.; Jaeger, K.-
E.; Liebeton, K. Angew. Chem., Int. Ed. 1997, 36, 2830–2832. (c)
Arnold, F. H. Acc. Chem. Res. 1998, 31, 125–131. (d) Zhang, J.-H.;
Dawes, G.; Stemmer, W. P. C. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 4504–4509.
(8) Lee, C. C.; Williams, T. G.; Wong, D. W.; Robertson, G. H. Plasmid
2005, 54, 80–85.
(9) Sandstro¨m, A. G.; Engstro¨m, K.; Nyhle´n, J.; Kasrayan, A.; Ba¨ckvall,
J.-E. Protein Eng., Des. Sel. 2009, 22, 413–420.
(10) For previous examples of extended substrate scope, see: (a) Wada,
M.; Hsu, C.-C.; Franke, D.; Mitchell, M.; Heine, A.; Wilson, I.; Wong,
C.-H. Bioorg. Med. Chem. 2003, 11, 2091–2098. (b) Mihovilovic,
M. D.; Rudroff, F.; Winninger, A.; Schneider, T.; Schulz, F.; Reetz,
M. T. Org. Lett. 2006, 8, 1221–1224. (c) Reetz, M. T.; Bocola, M.;
Wang, L.-W.; Sanchis, J.; Cronin, A.; Arand, M.; Zou, J.; Archelas,
A.; Bottala, A.-L.; Navoryta, A.; Mowbray, S. L. J. Am. Chem. Soc.
2009, 131, 7334–7343.
(5) (a) Arnold, F. H.; Georgiou, G. Directed Enzyme EVolution: Screening
and Selection Methods; Humana Press: Totowa, NJ, 2003; Vol. 230.
(b) Otten, L. G.; Quax, L. G. Biomol. Eng. 2005, 22, 1–9. (c) Turner,
N. J. Nat. Chem. Biol. 2009, 5, 567–573. (d) Lutz, S.; Bornscheuer,
U. T. Protein Engineering Handbook; Wiley-VCH: Weinheim,
Germany, 2009; Vols. 1 and 2. (e) Reetz, M. T. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 5716–5722.
9
7038 J. AM. CHEM. SOC. 2010, 132, 7038–7042
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Directed Evolution of an Enantioselective Lipase
A R T I C L E S
reported by Reetz et al.11 In this approach amino acids
surrounding the active site are varied pairwise which accounts
for synergistic effects. The CAST method has also been
successfully used to improve enantioselectivity.12-14
Results and Discussion
Library Design. Based on the crystal structure of CalA,15
residues situated close to the active site with side chains pointing
toward the substrate cavity were selected for the CASTing
libraries. Since the enzyme was crystallized in a closed
conformation we first had to create a model of the enzyme in
an open conformation; this was done by using Moloc compu-
tational software.16 A molecular dynamics equilibration of the
enzyme revealed that the amino acid region 425-440 (active
site flap) was moving quite substantially, and an equilibrium
conformation was reached after approximately 5 ns. The
equilibrium conformation has a substantially more open entry
to the active site. For the selection of amino acid residues the
covalently bound p-nitrophenyl 2-phenylpropanoate (1) had been
modeled into the structure. The carboxylic acid part of the
substrate was positioned in the model guided by the tetraethylene
glycol already available in the X-ray structure.
Figure 1. Amino acids selected for the CASTing libraries: library FI
(Phe149 and Ile150) and library FG (Phe233 and Gly237).
For the mutagenic libraries, amino acids surrounding the
nucleophilic serine (Ser184) with the side chains pointing toward
the substrate were chosen, since it is believed that these residues
have a large impact on the enantioselectivity. Furthermore,
according to the CASTing methodology, an approach of
targeting two amino acid positions at the same time was used.
The amino acids at these two positions were randomized
simultaneously. Two libraries were considered to be the most
relevant: (i) library FI (Phe149 and Ile150) with side chains
directed toward the R-methyl group of the substrate and (ii)
library FG (Phe233 and Gly237) with side chains pointing
toward the acyl pocket of the active site (Figure 1).
Table 1. Results of the Directed Evolution for Increased
Enantioselectivity of CalA toward 2-Phenylpropanoate 1
Entry
Enzyme
Library origin
E value
1
2
wild type
F233G
-
Lib FG
20 (S)
>200
(259) (R)
>200
(276) (R)
3
YNG
Lib FG/Lib FI
showed a large improvement in activity. Highly active variants
were subsequently used in a kinetic resolution of rac-1 to
determine their enantioselectivity, and several of the variants
showed a great improvement of the enantioselectivity. The best
enzyme variant found, with the mutation Phe233Gly (F233G),
had an E value of 259 (Table 1, entry 2). The F233G variant
showed (R)-selectivity, which is reversed compared to wild type
CalA.
To elucidate if it was possible to improve this enzyme variant
further, a new pair of amino acids in the vicinity of the active
site, Phe149 and Ile150 (library FI), was targeted using the
F233G variant as a template. In this round the focus was on
improving the enantioselectivity, not the activity. Therefore the
screening was performed with single enantiomers of the
substrate separately, and the ratio between the rates of hydrolysis
of the two enantiomers was used to estimate the enantioselec-
tivity (Eapp).17 The enzyme variants with the best ratio were
subsequently used in a kinetic resolution of the p-nitrophenyl
ester that was analyzed by chiral GC to determine the E value.
With the model substrate, this library gave only a slight
improvement in enantioselectivity, the triple mutant Phe149Tyr/
Ile150Asn/Phe233Gly (YNG) had an E value of 276, with
preference for the (R)-enantiomer (Table 1, entry 3).
Reduced libraries were employed to decrease the theoretical
number of enzyme variants. NDT degeneracy, which codes for
12 amino acids (CDFHGILNRSVY), was chosen for the created
libraries. This decreased the colony sampling requirements
significantly. The number of picked colonies was enough to
obtain at least 95% theoretical codon coverage; 600 colonies
were picked for each of the libraries. Mutated variants of CalA
were expressed in P. pastoris and screened using a spectro-
photometric assay, where the release of p-nitrophenol by the
hydrolysis with CalA was followed.
Library Screening. Since a previous study showed that a
library containing Phe233 and Gly237 had a large impact on
the enantioselectivity toward p-nitrophenyl 2-methylheptanoate,9
a library containing these residues was the first to be evaluated
(library FG). The main focus of the initial screening was to
improve the activity of the enzyme since the wild type has very
low activity toward this substrate; screening could therefore be
performed with rac-1. Several enzyme variants from this library
(11) (a) Reetz, M. T.; Bocola, M.; Carballeira, J. D.; Zha, D.; Vogel, A.
Angew. Chem., Int. Ed. 2005, 44, 4192–4196. (b) Reetz, M. T.;
Carballeira, J. D.; Peyralans, J.; Ho¨benreich, H.; Maichele, A.; Vogel,
A. Chem.sEur. J. 2006, 12, 6031–6038.
(12) (a) Reetz, M. T.; Sanchis, J. ChemBioChem 2008, 9, 2260–2267. (b)
See also ref 9 and ref 10c.
Substrate Scope. To determine the substrate scope, similar
substrates were used in the kinetic resolution with the two
enzyme variants F233G and YNG. For the 2-phenylpropanoate
1 the two extra mutations in YNG (Phe149Tyr and Ile150Asn)
(13) Bartsch, S.; Kourist, R.; Bornscheuer, U. T. Angew. Chem., Int. Ed.
2008, 47, 1508–1511.
(14) Liang, L.; Zhang, J.; Lin, Z. Microb. Cell Fact. 2007, 6, 36.
(15) Ericsson, D. J.; Kasrayan, A.; Johansson, P.; Bergfors, T.; Sandstro¨m,
A. G.; Ba¨ckvall, J.-E.; Mowbray, S. L. J. Mol. Biol. 2008, 376, 109–
119.
(16) Gerber, P. R. J. Comput.-Aided Mol. Des. 1998, 12, 37–51.
(17) Janes, E.; Kazlauskas, R. J. J. Org. Chem. 1997, 62, 4560–4561.
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Table 2. Results of the Hydrolytic Kinetic Resolution of p-Nitrophenyl Esters of R-Substituted Carboxylic Acids with Wild Type CalA (wt),
Single Mutant Phe233Gly (F233G), and Triple Mutant Phe149Tyr/Ile150Asn/Phe233Gly (YNG)a
a Reaction conditions: p-nitrophenyl ester (1.25 mL, 2 mg/mL in acetonitrile), enzyme solution (20 µL, 10 µg/µL), potassium phosphate buffer (8.5
c
mL, 100 mM, pH 8.0). b Mean value of 2-4 reactions; results for the separate reactions are available in the Supporting Information. Determined by H
1
NMR. d Determined by chiral GC.
seemed to play a minor role since the single mutation
(Phe233Gly) gave, within experimental error, the same enan-
tioselectivity. However, the two extra mutations were of crucial
importance for a broad substrate selectivity. Thus, the YNG
variant gave high to excellent E values for all substrates in Table
2, whereas the F233G variant completely failed for the 2-ben-
zylpropanoate 5 (entry 14) and gave a moderate E value of 17
for the 2-methylheptanoate 6 (entry 17). For the 2-ethylhex-
anoate 7 the F233G variant was slightly better than the YNG
variant (entries 20 and 21).
A methyl group in the para-position of the phenyl (2) was
accepted with a high enantioselectivity (Table 2, entry 6).
However, with a larger substituent, such as isobutyl (ibuprofen),
both the F233G and the YNG variant showed low reactivity
and gave an unselective reaction. Larger substituents in the
R-position, ethyl and propyl, were accepted with good enanti-
oselectivity (entries 9 and 12). An aliphatic chain in exchange
for the 2-phenyl substituent was also accepted (entry 18). The
broad substrate scope of the YNG variant is summarized in
Table 2.
The reaction rates for the YNG variant were increased
dramatically compared to wild type CalA. An increase of more
than 30-fold was observed for most substrates. This increase in
reaction rate improves the synthetic utility of the enzyme
significantly, as shorter reaction times and lower enzyme
loadings can be used.
Less Reactive Esters as Substrates. For synthetic purposes it
is of interest to use other esters than the p-nitrophenyl esters.
The latter esters are highly reactive, and changing to an alkyl
ester or a simple phenyl ester will lead to lower reaction rates.
In particular, CalA is known to be very slow in catalyzing
hydrolysis of alkyl esters.18 Three different esters 8, 9, and 10,
the ethyl, nonyl, and phenyl ester analogues, respectively, of
the p-nitrophenyl ester 1, were used as substrates in the kinetic
resolution catalyzed by the YNG variant. The results of the
kinetic resolution show that the enantioselectivity is maintained
for all three esters (Table 3), and for the nonyl and phenyl ester
the E values were even higher than those for the p-nitrophenyl
ester (Table 3, entries 2 and 3 vs Table 2, entry 3). As expected
the hydrolysis of the ethyl and nonyl ester was slower than that
of the phenyl ester but much faster than the corresponding
hydrolysis of alkyl esters by wild type CalA.18,19
Active Site Models. Active site models of the enzyme variants
F233G and YNG were created and compared with a model of
the wild type enzyme to rationalize the improved rate and
selectivity (Figure 2). By examining the models it seems that
substitution of the large phenylalanine (Phe233) for a small
glycine creates more space in the active site. The increased space
in the active site can be used to accommodate the substrate,
and this would explain the observed increase in activity.
Furthermore, the modeling indicates that the space created is
(18) Barbayianni, E.; Fotakopoulou, I.; Schmidt, M.; Constantinou-Kokotou,
V.; Bornscheuer, U. T.; Kokotos, G. J. Org. Chem. 2005, 70, 8730–
8733.
(19) The hydrolysis of related alkyl esters catalyzed by CalA in ref 18
employed a much higher concentration of enzyme (50×) and a higher
concentration of substrate (10×), and still the reactions typically took
24-48 h.
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Directed Evolution of an Enantioselective Lipase
A R T I C L E S
Table 3. Results of Kinetic Resolution of Esters with Different
Alcohol Side Chains by YNG, a Triple-Mutated Variant of CalA
(Phe149Tyr/Ile150Asn/Phe233Gly)a
substrate scope that can be used to produce R-substituted acids
in 95-99% enantiomeric excess from ester hydrolysis. Fur-
thermore, a 30-fold increase in activity was observed for most
substrates. The developed enzyme variant shows (R)-selectivity,
which is reversed compared to the wild type enzyme that is
(S)-selective for most substrates.
Time
(h)
Conv.b
(%)
eepc
(%)
Experimental Section
Entry
R
E
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. GC
analyses were performed using an IVADEX-1 chiral column.
Plasmid Library Construction. The construction of the pBGP1-
CalA vector has been described previously.9 Mutagenic libraries
were created by site-directed mutagenesis using degenerate primers.
The PCRs were performed with the following protocol; a prein-
cubation was performed at 95 °C for 10 min, after which a high-
fidelity DNA polymerase was added. The cycle of denaturation at
98 °C for 10 s, annealing at 58-68 °C for 30 s, and elongation at
72 °C for 3 min was repeated 30 times. A 10 min elongation at 72
°C ended the reaction. The PCR products were purified using a
Cycle-pure kit followed by DpnI treatment to remove the wild type
template. The nicked plasmid was transformed into E. coli DH5R,
and a large fraction of the transformed cells were grown overnight
for a plasmid preparation. In parallel, a fraction of the transformed
cells were plated to prepare for a sequencing assay. All pBGP1-
carrying DH5R were selected using carbenicillin (50 µg/mL) in
LB and LB-agar plates.
1
2
3
Et
Nonyl
Ph
3
3
0.5
14
21
22
98.9
99.6
99.6
>200 (211)
>200 (650)
>200 (657)
a Reaction conditions: Ester (1.25 mL, 2 mg/mL in acetonitrile), enzyme
solution (100 µL (entries 1 and 2) or 20 µL (entry 3), 10 µg/µL), potassium
phosphate buffer (8.5 mL, 100 mM, pH 8.0). b Determined by 1H NMR.
c Determined by chiral GC.
Library Expression in P. pastoris. P. pastoris X33 was made
electrocompetent, and until employed, the cells were stored in 40
mL aliquots at -80 °C without any treatment. Library plasmid
preparation was mixed with thawed cells and electroporated. Cells
were incubated at 30 °C with YPDS (1 mL) for 2 h followed by
plating on YPDS-agar plates containing zeocin (100 µg/mL) and
carbenicillin (100 µg/mL). Plates were incubated at 30 °C for 3
days.
Single colonies were picked and inoculated in conical deep 96-
well plates. Each well contained YPD (800 µL), zeocin (100 µg/
mL), and carbenicillin (100 µg/mL). The deep well plates were
shaken at 250 rpm for 5 days at 29 °C, leaning approximately 30°.
The shaking amplitude was 2.5 cm. After the overexpression, the
yeast was pelleted by centrifugation. The supernatant was harvested
by aspiration and used directly for optical screening or stored at
4 °C. The protein concentration of the harvested supernatants was
on average 0.2 mg/mL. Master plates containing the pelleted cells
were stored at -80 °C until further analysis or cultivation.
Optical Screening for Improved Activity. Screening buffer
(165 µL, 100 mM potassium phosphate, 10% v/v acetone, 4% v/v
Triton X-100, 0.2% w/v gum arabic, pH 8.0)20 and rac-1 (10 µL,
2 mg/mL in acetonitrile) were premixed before dispension. Yeast
library supernatant (25 µL) and the premixed buffer and substrate
solution (175 µL) were dispensed into a microtiter plate, and
absorbance was measured at 410 nm for 15 min. Wild type CalA
was used as reference. Variants that showed high activity were
selected for a kinetic resolution experiment to determine the
enantioselectivity (E).
Figure 2. Models of the active site for (a) wild type CalA, (b) Phe233Gly
(F233G) variant, and (c) Phe149Tyr/Ile150Asn/Phe233Gly (YNG) variant.
In all cases (R)-p-nitrophenyl 2-phenyl propanoate ((R)-1) is covalently
bound to the enzyme. Hydrogen bonds are indicated with black lines.
Optical Screening for Improved Enantioselectivity. Separate
enantiomers of the substrate were hydrolyzed in parallel reactions.
Screening buffer (165 µL, 100 mM potassium phosphate, 10% v/v
acetone, 4% v/v Triton X-100, 0.2% w/v gum arabic, pH 8.0)20
and (R or S)-1 (10 µL, 2 mg/mL in acetonitrile) were premixed
before dispension. Yeast library supernatant (25 µL) and the
premixed buffer and substrate solution (175 µL) were dispensed
into a microtiter plate, and absorbance was measured at 410 nm
for 15 min. The ratio between the initial rates of hydrolysis of the
(S)- and the (R)-enantiomer was calculated. Variants that showed
only of advantage for the (R)-enantiomer, which rationalizes
the large effect on the enantioselectivity. The increased enan-
tioselectivity for the triple mutant (YNG) could be explained
by the increase in steric bulk introduced; the elongation of the
Phe149 side chain by a hydroxyl group (Phe149Tyr) creates
congestion with the R-methyl group, which disfavors the (S)
configuration. In addition, two new hydrogen bonds are found
in the YNG variant.
Conclusions
In conclusion, we have used a directed evolution approach
to develop a triple-mutated variant of CalA with a broad
(20) Schulz, N.; Hobley, T. J.; Syldatk, C. Biotechnol. Lett. 2007, 29, 365–
371.
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Engstro¨m et al.
high ratio and acceptable activity were selected for a kinetic
resolution experiment to determine the enantioselectivity (E). All
samples were measured in duplicates.
the reaction had reached 10-40% conversion. The reaction mixture
was acidified by addition of HCl (1 mL, 1 M), extracted twice with
DCM, and concentrated. Conversion was determined by 1H NMR,
and the enantiomeric excess was determined by chiral GC. Optical
rotation was measured, and absolute stereochemistry was deter-
mined by comparison with literature values.
Cultivation of Enzyme Variants. The most interesting enzyme
variants were produced in larger batches to yield more enzyme that
could be used for further analysis. Master plates were used to
inoculate shaking flasks containing YPD (50 mL), zeocin (100 µg/
mL), and carbenicillin (100 µg/mL), which were incubated at 250
rpm for 96 h at 29 °C. Supernatant containing the enzyme was
harvested after centrifugation. The enzyme solution was concen-
trated; at the same time the buffer was exchanged to Tris-HCl (25
mL, 20 mM, pH 7.8), to a final enzyme concentration of 10 µg/
µL.
Acknowledgment. Financial support from the Swedish Research
Council, the Swedish Foundation for Strategic Research, and the
K&A Wallenberg Foundation is gratefully acknowledged.
Supporting Information Available: Experimental procedures
and characterization data of all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
Kinetic Resolution. Potassium phosphate buffer (8.5 mL, 100
mM, pH 8.0), the racemic p-nitrophenyl ester (1-7) (1.25 mL, 2
mg/mL in acetonitrile), and enzyme solution (20 µL, 10 µg/µL in
20 mM Tris-buffer, pH 7.8) were shaken at room temperature until
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