New efficient lipase from Yarrowia lipolytica for the resolution of 2-bromo-arylacetic acid esters

Article abstract of DOI:10.1016/j.tetasy.2004.09.008

A new extracellular lipase (Lip2p) from the yeast Yarrowia lipolytica was used for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates for the pharmaceutical industry. Its efficiency for the transesterification of racemic mixtures with 1-octanol in n-octane was compared with the most efficient lipases described to date, lipases from Burkholderia cepacia and Rhizomucor miehei. Resolution of 2-bromo-p-tolylacetic acid ethyl ester catalyzed by Y. lipolytica lipase showed an enantiopreference of 28, almost equal to that obtained with B. cepacia lipase (E = 30). Moreover, Y. lipolytica lipase presents a higher catalytic activity and an (S)-enantiopreference, while B. cepacia lipase is (R)-enantiomer selective. The most interesting result is that Y. lipolytica lipase has until now been the only enzyme able to catalyze the resolution of 2-bromo-o-tolylacetic acid ethyl ester (E = 27).

Full text of DOI:10.1016/j.tetasy.2004.09.008

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3539–3543
New efficient lipase from Yarrowia lipolytica for the resolution of
2-bromo-arylacetic acid esters
David Guieysse,a Georgina Sandoval,a Laeticia Faure,a Jean-Marc Nicaud,b
Pierre Monsana and Alain Martya,*
a
´ ´
Laboratoire de Biotechnologie et Bioprocedes, INSA, UMR CNRS 5504, UMR INRA 792-135 Rangueil Av.,
31077 Toulouse Cedex 4, France
´ ´ ´
Laboratoire de Microbiologie et Genetique Moleculaire CNRS-INRA-INAPG UMR 2585, 78850 Thiverval-Grignon, France
b
Received 30 July 2004; accepted 6 September 2004
Abstract—A new extracellular lipase (Lip2p) from the yeast Yarrowia lipolytica was used for the resolution of 2-bromo-arylacetic
acid esters, an important class of chemical intermediates for the pharmaceutical industry. Its efficiency for the transesterification of
racemic mixtures with 1-octanol in n-octane was compared with the most efficient lipases described to date, lipases from Burkholde-
ria cepacia and Rhizomucor miehei. Resolution of 2-bromo-p-tolylacetic acid ethyl ester catalyzed by Y. lipolytica lipase showed an
enantiopreference of 28, almost equal to that obtained with B. cepacia lipase (E = 30). Moreover, Y. lipolytica lipase presents a
higher catalytic activity and an (S)-enantiopreference, while B. cepacia lipase is (R)-enantiomer selective. The most interesting result
is that Y. lipolytica lipase has until now been the only enzyme able to catalyze the resolution of 2-bromo-o-tolylacetic acid ethyl ester
(E = 27).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
cides and natural compounds such as pheromones. The
most studied compounds are 2-arylpropionic acids,
which have the properties of non-steroid anti-inflamma-
tory drugs (Ibuprofen, Naproxen, Ketoprofen, etc.).2–5
The resolution of another important group of molecules,
2-haloarylacetic acids, has been sadly neglected.6–12
These compounds are important intermediates found
in the synthetic pathways of a number of drugs, such
as prostaglandin, prostacyclin, semi-synthetic penicillin
and thiazolium salts. In particular, ethyl ester deriva-
tives of 2-bromo-o-tolylacetic acid are used as precur-
sors for the synthesis of analgesics and non-peptide
angiotensin II-receptor antagonists. Recently, it has
been demonstrated that lipases from Burkholderia cepa-
cia and Rhizomucor miehei are the most efficient for the
resolution of these molecules.6,7 Using free B. cepacia
lipase, E-values higher than 50, with an (R)-enantio-
preference, can be achieved during transesterification
in n-octane of 2-bromo-m/p-tolyl- or 2-bromophenylace-
tic acid esters. Unfortunately, a low enantioselectivity
was observed (E = 6) with the ortho derivatives. For
the resolution from this last substrate, the best enzyme
was the lipase for R. miehei immobilized on polypropyl-
ene (E = 11.3).6 However, this enantioselectivity degree
is not acceptable for industrial purposes and the need
for a more selective catalyst is crucial.
The need for enantiomerically pure compounds, espe-
cially in the pharmaceutical industry, has grown since
legislation required investigation on the pharmacologi-
cal effect of each enantiomer. The market for drugs sold
in their single-enantiomer form was worth $160billion
worldwide in 2002.1
Since classical ways to obtain pure enantiomers, that is,
chemical asymmetric synthesis, stereoselective crystalli-
zation and chiral chromatography, are often expensive,
enzymatic procedures have become attractive. In this
field, lipases are the most frequently used catalysts.
The reasons for this interest lie in their high stability,
their non-requirement of cofactors, their synthetic activ-
ity in organic solvents and mainly in the wide range of
substrate specificities. They are also very capable of cat-
alyzing reactions on non-natural substrates.
Enantiopure carboxylic acids are important building
blocks for the synthesis of many pharmaceuticals, pesti-
*
Corresponding author. Tel.: +33 5 6155 9439; e-mail: marty@
insa-tlse.fr
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.09.008

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D. Guieysse et al. / Tetrahedron: Asymmetry 15 (2004) 3539–3543
Herein, a new lipase is evaluated for the resolution of 2-
bromoarylacetic acids. This lipase, LIP2p is produced
by the non-pathogenic lipolytic yeast, Yarrowia lipoly-
tica, and has recently been characterized.13 It is a
secreted lipase with an apparent molecular mass of
38.5kDa. The yeast Y. lipolytica was proposed as an
alternative host organism for heterologous protein pro-
duction.14,15 It combines the facility of single cell use,
high secretion abilities and tools for post-translational
modifications.14 Strains and vectors for protein expres-
sion and secretion have been developed.16,17 The host
strain was made auxotrophe for uracile while protease-
encoding genes were deleted. A gene amplification sys-
tem was developed for efficient lipase expression in
Y. lipolytica.14 The oleic acid-inducible POX2 promotor
was used to drive the transcription of the LIP2 gene.
Protein secretion is directed by the wild-type lipase tar-
geting sequence. After NotI digestion of plasmid JMP6,
the bacterial part was eliminated and only the expres-
sion cassette was integrated within the genome. A mu-
tant URA3 allele, ura3d4, was used for the selection
for multicopy integration into the genome. In this
way, multicopy integrants containing 3–16 copies of
the lipase gene were obtained.14,16 The extracellular
lipase represents the main enzyme in the fermentation
broth.
most efficient lipases already described in the literature,
lipases from B. cepacia and R. miehei. Comparisons
were based both on activity and enantioselectivity.
2. Results and discussion
The performance of Y. lipolyticaÕs lipase enantioselectiv-
ity towards the esters of 2-bromo-phenyl and tolyl acetic
acids was studied during the transesterification in n-oc-
tane using 1-octanol as the alcohol (Scheme 1). In a pre-
vious paper,7 it was demonstrated that the presence and
position of the methyl substitution on the aromatic ring
were crucial for the enzyme enantioselectivity. For this
reason, four substrates were tested, the 2-bromo-phenyl-
acetic acid ethyl ester (substrate 1) and the 2-bromo-o/m/
p-tolylacetic acid ethyl esters (substrates 2–4) (Table 1).
Activity and enantioselectivity efficiency of Y. lipoly-
ticaÕs lipase were compared with those obtained with
the lipases from B. cepacia (Bcl) and R. miehei (Rml),
the most efficient lipases described to date.6,7
2.1. Lipase activity
Whatever the acyl donor, the enzyme from Y. lipolytica
was more active than the two other enzymes (Table 1).
For the non-substituted substrate, this increase in activ-
ity was roughly 10-fold. It was highest for the para-sub-
stituted substrate (69 and 26 times higher when
The objective of this work was to compare performances
obtained with this lipase to those obtained with the two
Br
Br
Br
Lipase
OR
OR
OC8H17
C8H17OH
+
+
ROH
octane, 30oC
+
O
O
O
R1
R1
R1
(R)
(S)
(RS)
1: R1= H, R=C2H5
2: R1= ortho-CH3, R=C2H5
4: R1= para-CH3, R=C2H5
3: R1= meta-CH3, R=C2H5
Scheme 1.
Table 1. The enantioselectivity of free Y. lipolytica, B. cepacia and R. miehei lipases in the transesterification between substrates 1–4 and 1-octanol in
n-octane
Substrate
Lipasea
Initial rate (lmolminÀ1 gÀ1
)
% Conv.
5.1 (
Eb(À)c
S)
69.0( R)
1.4 (S)
%Eep
1
Y. lipolytica
B. cepacia
R. miehei
21.045 (48h)
50
93
10
3.2
2.1
37 (22h)
58 (22h)
2
3
4
Y. lipolytica
B. cepacia
R. miehei
2.6
0.1
38 (56h)
27.0( S)
2.1 (R)
2.5 (S)
84
0.23
Y. lipolytica
B. cepacia
R. miehei
50.0
52 (11h)
55 (72h)
1.1 (R)
R)
1.9 (S)
(
h)
25.90.406 (120
2.5
87
9
Y. lipolytica
B. cepacia
R. miehei
40.0
0.6
46 (20h)
30(43h)
56 (18h)
28.0 (S)
30( R)
3 (S)
82
92
34
1.5
a Y. lipolytica lipase (1mg/mL), B. cepacia lipase (5mg/mL), R. miehei lipase (5mg/mL).
b E = ratio of initial rates.
c Fast reacting enantiomer.

D. Guieysse et al. / Tetrahedron: Asymmetry 15 (2004) 3539–3543
3541
Table 2. The protein content and different activities (pNPB, tributyrin and triolein hydrolysis, esterification of oleic acid with ethanol in n-hexane) of
free Y. lipolytica, B. cepacia and R. miehei lipases
Y. lipolytica
B. cepacia
R. miehei
a
Protein content (%)
pNPB hydrolysis (lmolminÀ1 mgÀ1
Tributyrin hydrolysis (lmolminÀ1 mgÀ1
Triolein hydrolysis (lmolminÀ1 mgÀ1
Oleic acid esterification (lmolminÀ1 mgÀ1
52
41
12
504.4
287
15
)
)
177
114020 274
1.16
429
)
9
)
0.45
0.28
a Determined by the micro-Bradford method using BSA as standard.
Table 3. Effect of temperature on Y. lipolytica lipase activity and
enantioselectivity
compared with Bcl and Rml, respectively). For the
ortho-substituted substrate, the increases were 26- and
11-fold compared with Bcl and Rml, respectively. How-
ever, the methyl group at the ortho-position of the aro-
matic ring inhibits the kinetics whatever the enzyme
used, with the most effective enzyme being the Y. lipoly-
tica enzyme. This effect can be attributed to the ortho
substitution steric hindrance. Reasons for the kinetic
efficiency of the lipase from Y. lipolytica can be found
either in better enzyme preparation purity or in higher
intrinsic catalytic activity. The protein content of the
Y. lipolytica enzyme was three times higher than for
the two other enzymes (Table 2). This was confirmed
by SDS polyacrylamide gel electrophoresis (data not
shown). The three enzyme preparation activities were
tested towards the hydrolysis of para nitro phenyl buty-
rate, tributyrin and triolein, and, during a synthesis, the
esterification of oleic acid with ethanol in n-hexane
(Table 2). Y. lipolytica enzyme preparation appeared
the most active during hydrolysis of triolein (5.5- and
4.2-times higher compared with Bcl and Rml, respec-
tively) and during the esterification (2.6- and 4.1-times
higher when compared with Bcl and Rml, respectively).
If these activities are expressed versus the protein con-
tents, they are globally the same. On the contrary, its
activity during the hydrolysis of tributyrin is lower
(1.6- and 2.4-times lower compared with Bcl and Rml,
respectively). It is a remarkable fact to have an activity
higher on triolein than on tributyrin. Y. lipolytica lipase
presents a high specificity for long chain glycerides,
which underlines its high level of lipase character.
Taking into account enzyme purities, Y. lipolytica lipase
is about 3- to 20-fold more active than B. cepacia lipase
depending on the substrate.
T (ꢁC)
Initial rate
(mmolminÀ1 gÀ1
E
)
201.18
302.56
403.13
504.67
33
27
23
16
different to each other to explain difference in enantio-
selectivity.
The lipase from Y. lipolytica gave no enantioselectivity
for 2-bromo-phenylacetic acid ethyl ester and 2-bromo-
m-tolylacetic acid ethyl ester (E = 5 and 1, respectively).
For the 2-bromo-p-tolylacetic acid ethyl ester, Y. lipoly-
tica lipase gave an enantioselectivity similar to the one
obtained with B. cepacia lipase (E = 28). The most inter-
esting result concerned the 2-bromo-o-tolylacetic acid
ethyl ester. Indeed, no free enzyme was able to efficiently
resolve this racemic mixture.6 Y. lipolytica lipase gave an
enantioselectivity of 27. This result is better than that
observed with the lipase from R. miehei immobilized
on polypropylene (E = 11), which was the best result
found in the literature.6
For the most interesting substrate, the 2-bromo-o-tolyl-
acetic acid ethyl ester, the influence of the temperature
was studied (Table 3). As usually observed with an in-
crease in temperature, activity was higher and enantio-
selectivity lower.
3. Conclusion
2.2. Lipase enantioselectivity
Herein, it has been demonstrated that the lipase from Y.
lipolytica is a very active and selective catalyst in the
transesterification of 2-bromo-phenyl and 2-bromo-tolyl
acetic acid ethyl esters. The most interesting result is that
this lipase is the first described lipase able to resolve the
racemic mixture of 2-bromo-o-tolyl acetic acid ethyl
ester (E = 27), an industrial pharmaceutical building
block. However, this enantioselectivity value is still not
high enough. In order to understand and improve the
enantioselectivity, it would be necessary to obtain the
three-dimensional structure of this protein. While wait-
ing for crystallographic data, Lip2p 3D structure is
being modelled by homology modelling using the
R. miehei lipase as a template. The final objective will
be to design variants of the enzyme by site-directed
mutagenesis.
From the point of view of selectivity, Y. lipolytica and
R. miehei lipases give an opposite enantiopreference to
the lipase from B. cepacia. In fact, the (S)-enantiomer
is preferred by these two lipases, whereas the lipase from
B. cepacia prefers the (R)-enantiomer. This is the result
of the differences in the structure of their active sites.
˚
The binding pocket of the B. cepacia lipase is a 17A long
funnel, whereas the active site of R. miehei lipase is at
the surface of the protein protected by a lid. The three
dimensional structure of the Y. lipolytica lipase is
unknown. However, one of closest lipases at the level
of the primary structure is the R. miehei lipase (30% of
amino acids in common after Blast alignment). It can
be assumed that both active sites are similar, thus
explaining the same enantiopreference, but sufficiently

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D. Guieysse et al. / Tetrahedron: Asymmetry 15 (2004) 3539–3543
4. Materials and methods
The enantioselectivity value was the ratio of the initial
rates of the two enantiomers.
4.1. Biological reagents
4.7. Determination of the protein content of enzyme
preparation
Commercial lipases were purchased from Roche Diag-
nostics (Germany). Chirazyme L-9, lyo., (free lipase
from R. miehei); Chirazyme L-1, lyo., (free lipase from
B. cepacia).
The enzyme solution was prepared in a phosphate
buffer 0.05M pH7 at 100, 25 and 25mg/L for R. miehei,
Y. lipolytica and B. cepacia, respectively. The pro-
tein content was determined with the micro-Bradford
method with BSA as standard.
Lipase Lip2p from Y. lipolytica was produced on a min-
eral medium. The strain JMY329 used in this study con-
tained multiple integrated copies of the LIP2 gene (ꢀ16
copies) in the genome. It was obtained by gene amplifi-
cation as previously described.17 Cells were eliminated
by centrifugation. The enzyme was precipitated by the
addition of an equivolume of cold acetone (À20ꢁC).
Centrifugation enabled the enzyme to be recovered.
The enzyme was used after lyophilization.
4.8. Determination of enzyme activities
Triolein hydrolysis: The enzyme solution was prepared
in a phosphate buffer 0.05M pH7 at 2, 0.5 and 0.6g/L
for R. miehei, Y. lipolytica and B. cepacia, respectively.
The reaction mixture contained 5mL of olive oil emul-
sion (Sigma, 800b), 2mL of trizma buffer (Sigma,
800b) and 20lL of enzyme solution. Hydrolysis was
run for 20min at 37 ꢁC. The reaction was stopped by
the addition of a mixture of acetone/ethanol (v/v) con-
taining three drops of thymolphthalein (0.9% (w/v) in
ethanol). The titration was realized using a 0.05M
sodium hydroxide solution.
4.2. Chemical reagents
All reagents were of commercial quality and purchased
from Sigma/Aldrich. n-Octane was dried over molecular
˚
sieves (3A) before use.
4.3. General procedure for the preparation of 2-bromo
carboxylic acid esters
p-Nitro phenol butyrate hydrolysis: The enzyme solu-
tion was prepared in a phosphate buffer 0.05M pH7
at 20, 5 and 6mg/L for R. miehei, Y. lipolytica and B.
cepacia, respectively. The reaction mixture contained
175lL of a phosphate buffer 0.05M pH6.8, 5lL of a
10mM p-nitro phenol butyrate in 2-methyl 2-butanol,
20lL of enzyme solution. Activity was measured follow-
ing absorbance at 405nm.
The procedure for the preparation of ( )-2-bromo phen-
ylacetic acid ethyl and octyl ester, ( )-2-bromo-o/p/m-
tolylacetic acid ethyl and octyl ester has been described
in a previous paper.7
4.4. Procedure for the enzymatic transesterification
Transesterification was carried out in 5mL tubes con-
taining the ester (50mM) and octanol (150mM) in dried
octane. The temperature was maintained at 30ꢁC. The
mixture was shaken at 1250rpm. Addition of the free
enzyme marked the zero time of the reaction. At regular
time intervals the progress of the reaction was followed
by taking samples (100lL diluted in 1mL of a mixture
hexane/isopropanol 99.8:0.2, v/v).
Esterification: 200mM oleic acid and 300mM ethanol
were dissolved in n-hexane. The enzyme (10–30mg)
was then added. The reaction mixture was incubated
at 40ꢁC and agitated with magnetic stirring. For each
sample, n-hexane was evaporated and the solutes redis-
solved in the HPLC solvent. Oleic acid and ethyl oleate
concentrations were determined by an HPLC system
equipped with a Kontron 420pump, a Varian R14
refractive index monitor (Varian Associates, Orsay,
France) and a Spherisorb ODS2 C18, 5lm column
(250 · 4.0mm). Elution was conducted at 50 ꢁC with
methanol/acetic acid (99.7:0.3, v/v) at a flow rate of
1mL/min.
4.5. HPLC analysis
The HPLC device was equipped with a chiral column:
Chiralpack OJ (25cm · 4.6mm) (Daicel Chemical
Industries Ltd, Japan) connected to a UV detector (at
254nm). A flow rate of 1.0mL/min was used. The mo-
bile phase was composed of a mixture of n-hexane/iso-
propanol [80:20 v/v for ( )-2-bromo phenyl ethyl and
octyl acetate and ( )-2-bromo-p-tolyl ethyl and octyl
acetate and 98/2 v/v for ( )-2-bromo-o/m-tolyl ethyl
and octyl acetate].
References
1. Rouhi, A. M. Chem. Eng. News 2003, 81, 45–55.
2. Brady, D.; Steenkamp, L.; Skein, E.; Chaplin, J. A.;
Reddy, S. Enz. Microb. Technol. 2004, 34, 283–291.
3. Ceynowa, J.; Rauchfleisz, M. J. Mol. Catal. B: Enzym.
2003, 23, 43–51.
4.6. Determination of the enantiomeric excess (ee),
conversion and enantioselectivity (E)
4. Choi, G. S.; Kim, J. Y.; Kim, J. H.; Ryu, Y. W.; Kim, G.
J. Prot. Expres. Purif. 2003, 29, 85–93.
From HPLC results, the enantiomeric excess (ee) was
calculated as defined below: ees = {[R] À [S]}s/{[R] +
[S]}s (s = substrate) and the conversion: C = 1 À
5. Steenkamp, L.; Brady, D. Enzyme Microb. Technol. 2003,
32, 472–477.
6. Guieysse, D.; Salagnad, C.; Monsan, P.; Remaud-Simeon,
M. Tetrahedron: Asymmetry 2001, 12, 2473–2480.
[(R À S)t/(R À S)t=0] 100.
*

D. Guieysse et al. / Tetrahedron: Asymmetry 15 (2004) 3539–3543
3543
7. Guieysse, D.; Salagnad, C.; Monsan, P.; Remaud-Simeon,
M. Tetrahedron: Asymmetry 2003, 14, 317–323.
8. Guieysse, D.; Salagnad, C.; Monsan, P.; Remaud-Simeon,
M. Tetrahedron: Asymmetry 2003, 14, 1807–1817.
9. Antoine Overbeeke, P. L.; Jongejan, J. A. J. Mol. Catal.
B: Enzym. 2003, 21, 89–91.
10. Haughton, L.; Williams, J. M. J. Synthesis 2001, 6, 943–
946.
11. Jones, M. M.; Williams, M. J. Chem. Commun. 1998,
2519–2520.
13. Pignede, G.; Wang, H.; Fudalej, F.; Gaillardin, C.; Seman,
M.; Nicaud, J. M. J. Bacteriol. 2002, 182, 2802–
2810.
14. Pignede G.; Fudalej F.; Nicaud J. M.; Gaillardin C.;
Seman M. French patent 98,899, 1998.
15. Barth, G.; Gaillardin, C. In Biotechnology. A Handbook;
Wolf, K., Ed.; Springer-Verlag: Berlin, 1996; p 313.
16. Nicaud, J.-M.; Madzak, C.; van den Broek, P.; Gysler, C.;
Duboc, P.; Niederberger, P.; Gaillardin, C. FEMS Yeast
Res. 2002, 2, 371–379.
12. Ahmed, S. N.; Kazlauskas, R. J.; Morinville, A. H.;
Grochulski, P.; Schrag, J. D.; Cygler, M. Biocatalysis
1994, 9, 209–225.
17. Pignede, G.; Wang, H.; Fudalej, F.; Seman, M.; Gaillar-
din, C.; Nicaud, J. M. Appl. Environ. Microbiol. 2000, 66,
3283–3289.