A variant of Yarrowia lipolytica lipase with improved activity and enantioselectivity for resolution of 2-bromo-arylacetic acid esters

Article abstract of DOI:10.1016/j.tetasy.2008.06.009

A variant of Lip2p lipase from Yarrowia lipolytica yeast was used for the resolution of 2-bromophenyl and o-tolyl acid esters, an important class of chemical intermediates for the pharmaceutical industry. In comparison with wild-type Lip2p, this variant, which contains one single amino acid change in the active site of the enzyme, V232A, displayed an enantioselectivity enhanced by one order of magnitude for the resolution of 2-bromo-phenylacetic acid ethyl ester (E-value increased from 5.5 to 59 for wild-type and V232A, respectively) and by fourfold for the resolution of 2-bromo-o-tolylacetic acid ethyl ester (going from an E-value of 27 to 111 for the wild-type and V232A, respectively). A remarkable increase in reaction velocity was also observed for both compounds, as a result of a significant gain in reactivity towards the favoured (S)-enantiomer (3- and 16-fold increase for 2-bromo-phenylacetic and -o-tolylacetic acid ethyl esters, respectively). These results demonstrate the key role of the V232 amino acid in enantiomer recognition and selectivity.

Full text of DOI:10.1016/j.tetasy.2008.06.009

Tetrahedron: Asymmetry 19 (2008) 1608–1612
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Tetrahedron: Asymmetry
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A variant of Yarrowia lipolytica lipase with improved activity and
enantioselectivity for resolution of 2-bromo-arylacetic acid esters
Miguel Cancino a,b,c, Philippe Bauchart a,b,c, Georgina Sandoval a,b,c, , Jean-Marc Nicaud d, Isabelle André a,b,c
,
Valérie Dossat a,b,c, Alain Marty a,b,c,
*
a Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France
b INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France
c CNRS, UMR5504, F-31400 Toulouse, France
d Laboratoire de Microbiologie et Génétique Moléculaire AgroParisTech, CNRS UMR2585, INRA UMR1238, 78850 Thiverval-Grignon, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A variant of Lip2p lipase from Yarrowia lipolytica yeast was used for the resolution of 2-bromophenyl and
o-tolyl acid esters, an important class of chemical intermediates for the pharmaceutical industry. In com-
parison with wild-type Lip2p, this variant, which contains one single amino acid change in the active site
of the enzyme, V232A, displayed an enantioselectivity enhanced by one order of magnitude for the res-
olution of 2-bromo-phenylacetic acid ethyl ester (E-value increased from 5.5 to 59 for wild-type and
V232A, respectively) and by fourfold for the resolution of 2-bromo-o-tolylacetic acid ethyl ester (going
from an E-value of 27 to 111 for the wild-type and V232A, respectively). A remarkable increase in reac-
tion velocity was also observed for both compounds, as a result of a significant gain in reactivity towards
the favoured (S)-enantiomer (3- and 16-fold increase for 2-bromo-phenylacetic and -o-tolylacetic acid
ethyl esters, respectively). These results demonstrate the key role of the V232 amino acid in enantiomer
recognition and selectivity.
Received 6 May 2008
Accepted 6 June 2008
Available online 16 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
In this field, lipases are the most frequently used biocatalysts.
The reasons for such interest lie in their high stability, their non-
The market for drugs sold in single-enantiomer form is expand-
ing rapidly (13% increase in 2005) and represented $205 billion
worldwide in 2005.1 It concerns the fields of pharmaceutical, agri-
cultural and synthetic organic chemistry. Enantiopure carboxylic
acids are important building blocks for the synthesis of many phar-
maceuticals, pesticides and natural compounds such as phero-
mones. 2-Arylpropionic acids have the properties of non-steroid
anti-inflammatory drugs (Ibuprofen, Naproxen, Ketoprofen)2–5
whereas 2-halogeno-carboxylic acids are important intermediates
found in synthetic pathways of a number of drugs, such as prosta-
glandin, prostacyclin, semi-synthetic penicillin and thiazolium
salts.6–12 For instance, ethyl ester derivatives of 2-bromo-o-tolyl-
acetic acid are used as precursors for the synthesis of analgesics
and non-peptide angiotensin II-receptor antagonists.6–8
requirement for co-factors, and mostly their synthetic activity in
organic solvents. Moreover, lipases are also capable of catalyzing
reactions with non-natural substrates.
In a previous paper,13 we demonstrated that Lip2p lipase, which
is extra-cellularly produced by the non-pathogenic lipolytic Yarr-
owia lipolytica yeast, is an efficient enzyme for the resolution of
2-halogeno-carboxylic ethyl esters via transesterification with 1-
octanol in n-octane. The resolution of 2-bromo-p-tolylacetic acid
ethyl ester using Lip2p revealed an enantioselectivity of 28, com-
parable to the value obtained with Burkholderia cepacia lipase (E-
value = 30), the most efficient lipase described so far in the litera-
ture.6,7 More interestingly, Y. lipolytica Lip2p was shown to be the
only known enzyme able to catalyze the resolution of 2-bromo-o-
tolylacetic acid ethyl ester with an E-value of 27. While Lip2p indi-
cated a clear preference for the (S)-enantiomer, B. cepacia lipase
was rather selective for the (R)-enantiomer. In addition, a remark-
able increase in velocity was observed using Y. lipolytica Lip2p
compared to B. cepacia lipase (69- and 26-fold higher for para-
and ortho-substituted substrates, respectively). Nonetheless, in
spite of the interesting performances of Y. lipolytica Lip2p, E-values
remain insufficient for industrial requirements and thus the need
for a more selective catalyst appeared crucial. The evolution of
Lip2p was considered using site-directed mutagenesis to search
Over the past decades, enzymes have become important tools
for racemic resolution and have found more and more use, in com-
petition with chemical asymmetric synthesis, stereoselective crys-
tallization and chiral chromatography.
* Corresponding author. Tel.: +33 (5) 61 55 94 39; fax: +33 (5) 61 55 94 00.
E-mail address: alain.marty@insa-toulouse.fr (A. Marty).
Present address: Unidad de Biotechnologia, CIATEJ, Av. Normalistas 800, Guad-
alajara, Jal., Mexico.
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.06.009

M. Cancino et al. / Tetrahedron: Asymmetry 19 (2008) 1608–1612
1609
Br
Br
Br
OEt
OEt
OEt
Lipase
biphasic medium
+
C2H5OH
+
O
O
O
water /decane (v/v)
25 oC
R1
R1
R1
(R)
(S)
(R,S)
2-bromo-phenylacetic acid ethyl esters: R1 = H
2-bromo-o-tolylacetic acid ethyl esters: R1 = ortho-CH3
Scheme 1.
for variants with enhanced activity and selectivity towards the res-
olution of 2-halogeno-carboxylic esters.
the enzyme expression system and thus enabling a comparison of
enzyme activities. In comparison to wild-type Lip2p, variant
V232A displayed an activity reduced to about 80% of the wild-type
Herein we report the characterization of one particular variant
of Y. lipolytica Lip2p isolated from a previous library screen,14 for
the resolution of 2-halogeno-carboxylic esters. In comparison to
wild-type Lip2p, this variant contains one single amino acid change
in the active site, V232A, which remarkably led to an increase in
enantioselectivity for the resolution of 2-bromo-phenylacetic acid
ethyl ester.
Lip2p activity (66 and 83 l
mol minÀ1 mLÀ1 for V232A and wild-
type Lip2p, respectively). Nonetheless, results indicate that the
replacement of Val232 with a smaller hydrophobic alanine amino
acid did not significantly affect the enzyme activity.
2.2. Resolution of (R,S)-2-bromo-phenylacetic acid ethyl ester
Both enzymes, wild-type Lip2p and its V232A variant, were
then evaluated for their hydrolytic activity on a racemic mixture
of 2-bromo-phenylacetic acid ethyl ester in a biphasic medium
(water/decane v/v) at 25 °C (Fig. 1). Results indicate a remarkable
10-fold increase in enantioselectivity for the V232A variant, going
from an E-value of 5.5 to 59 for wild-type Lip2p and V232A variant,
respectively (Table 2). In particular, the V232A variant displayed a
2.8-fold enhanced activity towards the (S)-enantiomer whereas its
activity towards the (R)-enantiomer is about 3.9 times lower than
results obtained using wild-type Lip2p. While an increase in
enzyme selectivity is often accompanied by a decrease in velocity,
no such effect has been observed in the case of the V232A variant.
Indeed, the resolution of (R,S)-2-bromo-phenylacetic acid ethyl
ester using the V232A variant is obtained within a shorter time
that is needed when using wild-type Lip2p, which is an advantage
for the use of this enzyme in an industrial process. It should be
noted that the enantioselectivity determined for the hydrolysis
reaction using the wild-type enzyme is equivalent to the value
previously determined for the transesterification reaction.13 Such
results might thus indicate that the selectivity is controlled by
the first step of the catalysis, namely the formation of the tetra-
hedral intermediate. Enantiomeric excesses of the substrate and
the product are, respectively, 99.9% and 84% at 54.3% total conver-
2. Results and discussion
Performances of wild-type Y. lipolytica Lip2p and its variant
V232A were first investigated from the point of view of their
hydrolytic activity towards para-nitrophenol butyrate and then
for their enantioselectivity towards the hydrolysis of 2-bromo-
phenyl- and 2-bromo-o-tolyl- acetic acid esters in a biphasic sys-
tem (water/organic solvent) (Scheme 1).
2.1. Production and quantification of Lip2p and its V232A
variant
Given the absence of crystallographic data on the Lip2p lipase, a
three-dimensional model of Lip2p was predicted by homology
modelling using as templates the X-ray structures of very similar
lipases.14 On the basis of this model, five amino acids forming
the hydrophobic substrate binding site of Lip2p were targeted by
site-directed mutagenesis. Among the library of Lip2p variants
which were constructed, one particular variant V232A revealed
very promising properties. This variant, together with the wild-
type Lip2p, was first screened for their hydrolytic activity of the
para-nitrophenol butyrate. Results obtained for both enzymes are
shown in Table 1. Three independent clones of the two strains
were cultivated to produce the wild-type enzyme and its variant.
Standard deviations of a comparable order were obtained for all
three clones of each enzyme, indicating that all clones own a single
copy of the lipase gene, introduced at the zeta docking platform at
the LEU2 locus in the genome,15 leading to good reproducibility of
25
20
15
10
5
Table 1
Comparison of wild-type Y. lipolytica lipase and its variant V232A during hydrolysis of
para-nitrophenol butyrate at 25 °C
Lipase
Clones
Initial ratea
Average initial rate
(l
mol minÀ1 mLÀ1
)
(
l
mol minÀ1 mLÀ1
83.2 4.7
)
0
Wild-type
1
2
3
87.1 6.0
80.3 1.9
82.2 3.5
0
1
2
3
4
5
6
7
8
9
10
11
time (h)
Variant V232A
1
2
3
68.0 3.6
64.0 2.0
67.0 3.0
66.3 3.1
Figure 1. Hydrolysis kinetics versus R,S-2-bromo-phenylacetic acid ethyl ester in a
biphasic medium (water/decane v/v) at 25 °C. Wild-type lipase activity versus
S- (e) and R- (s) enantiomers. Variant V232A lipase activity versus S- (Ç) and R- (d)
enantiomers. Thick lines represent initial velocity.
a
Activity assays were realized in triplicates.

1610
M. Cancino et al. / Tetrahedron: Asymmetry 19 (2008) 1608–1612
Table 2
Comparison of activity and selectivity of wild-type Y. lipolytica Lip2p lipase and its variant V232A during hydrolysis of (R,S)-2-bromo-phenylacetic acid ethyl ester racemate
b
c
(S)-initial rate (
l
mol hÀ1 mLÀ1
)
(R)-initial rate (
l
mol hÀ1 mLÀ1
)
E-value (viR/viS)a
Conversion (%)
ees (%)
eep (%)
Wild-type lipase
Variant V232A
7.24
20.3
1.32
0.34
5.5
59
55.1 (4.5 h)
54.3 (6 h)
61.9
99.9
50.5
84
a
viR, viS: initial rates.
Substrate enantiomeric excess.
Product enantiomeric excess.
b
c
sion after 6 h of reaction. At this point in time, 91.3% of the (R)-
enantiomer can be recovered with a purity of 99.0%, and 97.1% at
92.0% purity for the (S)-enantiomer.
observed for the resolution of (R,S)-2-bromo-phenylacetic acid
ethyl esters in which there is no substitution on the aromatic ring.
As observed for (R,S)-2-bromo-phenylacetic acid ethyl esters, the
non-substituted substrate, the change of valine 232 into an alanine
also led to a 10-fold increase in activity towards the preferred (S)-
enantiomer of 2-bromo-o-tolylacetic acid ethyl ester. The velocity
ratio between the two substrates was drastically reduced from
25.6 to 4.5 (comparison of values given in Tables 2 and 3). For
the transesterification reaction with octanol, an E-value of 27
was found for the wild-type enzyme.13 A similar E-value is also ob-
tained for the hydrolysis reaction, suggesting again that discrimi-
nation between the (R,S)-enantiomers might occur during the
first catalytic step of formation of the tetrahedral intermediate.
The enantioselectivity of the variant V232A is increased four times
(going from 27 to 111). In this case, improvement is exclusively
due to the better catalysis of the preferred (S)-enantiomer (16
times higher), although catalysis of poorly recognized R-enantio-
mer is also found to be increased by fourfold. After 18.5 h, the con-
version is 51.3% with enantiomeric excesses of 96.1 and 91.2 for
substrate and product, respectively. Also, 95.6% of the (R)-enantio-
mer can be recovered with a purity of 98.1%, and 98.1% at 95.6%
purity for the (S)-enantiomer.
2.3. Resolution of (R,S)-2-bromo-o-tolylacetic acid ethyl ester
A comparable study was carried out for the resolution of a race-
mic mixture of 2-bromo-o-tolylacetic acid ethyl esters using wild-
type Lip2p lipase and its V232A variant. Kinetics are shown in Fig-
ure 2 while activity and selectivity parameters are given in Table 3.
The presence of a methyl group at the ortho-position of the aro-
matic ring, causes steric hindrance, clearly inhibiting the kinetics.
The activity of wild-type Lip2p was 25.6 times lower for the reso-
lution of (R,S)-2-bromo-o-tolylacetic acid ethyl esters than results
30
25
20
15
10
5
2.4. Influence of the ester group on the activity and selectivity
For both substrates, the influence of the nature of the ester
chain on the activity and enantioselectivity of variant V232A was
investigated (Tables 4 and 5). It is noteworthy that results obtained
for the ethyl- and the octyl-esters of 2-bromo-phenylacetic acid
are quite similar. Indeed, for both substrates, an increase was
observed in the catalysis of the preferred (S)-enantiomer while a
decrease was observed for the catalysis of the poorly reacting
(R)-enantiomer, thus leading to a slight augmentation of the
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
time (h)
Figure 2. Hydrolysis kinetics versus 2-bromo-o-tolylacetic acid ethyl esters in
a biphasic medium (water/decane v/v) at 25 °C. Wild-type lipase activity versus
S- (e) and R- (s) enantiomers. Variant V232A lipase activity versus S- (Ç) and R- (d)
enantiomers. Thick lines represent initial velocity.
Table 3
Comparison of activity and selectivity of wild-type Y. lipolytica lipase and its variant V232A during hydrolysis of (R,S)-2-bromo-o-tolylacetic acid ethyl ester racemate
mol hÀ1 mLÀ1
)
(R)-initial rate (
l
mol hÀ1 mLÀ1
)
E-value (viR/viS)a
Conversion (%)
ees (%)
eep (%)
b
c
(S)-initial rate (
l
Wild-type lipase
Variant V232A
0.282
4.5
0.0105
0.040
27
111
51.3 (18.5 h)
96.1
91.2
a
viR, viS: initial rates.
Substrate enantiomeric excess.
Product enantiomeric excess.
b
c
Table 4
Comparison of the hydrolysis kinetic of ethyl-, octyl- and benzyl-esters of 2-bromo-phenylacetic acid by the variant V232A
b
c
(S)-initial rate (
l
mol hÀ1 mLÀ1
)
(R)-initial rate (
l
mol hÀ1 mLÀ1
)
E-value (viR/viS)a
Conversion (%)
ees (%)
eep (%)
Ethyl ester
Octyl ester
Benzyl ester
20.3
23
39
0.34
0.32
2.14
59
72
18
54.3 (6 h)
54.6 (7 h)
55.6 (1.33 h)
99.89
99.94
98.2
84
83.1
78.4
a
viR, viS: initial rates.
Substrate enantiomeric excess.
Product enantiomeric excess.
b
c

M. Cancino et al. / Tetrahedron: Asymmetry 19 (2008) 1608–1612
1611
Table 5
Comparison of the hydrolysis kinetic of ethyl, octyl and benzyl esters of 2-bromo-o-tolylacetic acid by the variant V232A
b
c
(S)-initial rate (
l
mol hÀ1 mLÀ1
)
(R)-initial rate (
l
mol hÀ1 mLÀ1
)
E-value (viR/viS)a
Conversion (%)
ees (%)
eep (%)
Ethyl ester
Octyl ester
Benzyl ester
4.5
4.9
10.3
0.04
0.10
0.88
111
49
12
51.3 (18.5 h)
54 (22 h)
55.6 (1.33 h)
96.1
95.9
98.2
91.2
81.8
78.4
a
viR, viS: initial rates.
Substrate enantiomeric excess.
Product enantiomeric excess.
b
c
enantioselectivity value. Conversely, the enantioselectivity was
reduced for esters of 2-bromo-o-tolylacetic acid, due to a better
catalysis of the (R)-enantiomer. On the other hand, the use of
benzyl-esters of both 2-bromo-phenylacetic acid and 2-bromo-o-
tolylacetic acid led to increased rates of hydrolysis (Tables 4 and
5). Indeed, a velocity increased by twofold was obtained for the
(S)-enantiomer of both substrates, whereas catalysis of the (R)-
enantiomer is increased by 6.5-fold for the bromo-phenylacetic
acid (Table 4) and by 10-fold for 2-bromo-o-tolylacetic acid (Table
5). As a consequence, the enantioselectivity was lowered to a value
of 18 and 12 for 2-bromo-phenylacetic acid ester and 2-bromo-o-
tolylacetic acid ester, respectively. The same results were obtained
with the wild-type enzyme (data not shown).
6.8). Stock preparation of oleic acid (200 g of oleic acid/L, 5 g of
Tween 40/L) is subjected to sonication three times for 1 min on
ice for emulsification purposes. After centrifugation and 0.2
tration, the enzyme is directly used for enzymatic assays.
lm fil-
4.2. Chemical reagents
Peptone, tryptone and yeast extract were purchased from Difco
(Difco, Paris, France).
All chemicals were of commercial quality and were purchased
from Sigma/Aldrich. n-Decane was dried over molecular sieves
(3 Å) before use.
4.3. General procedure for the preparation of 2-bromo
carboxylic acid esters
3. Conclusion
The procedure for the preparation of ( )-2-bromo-phenylacetic
acid ethyl and octyl ester, ( )-2-bromo-o-tolylacetic acid ethyl oct-
yl and benzyl esters was described in a previous paper.13
In conclusion, we have demonstrated that variant V232A of Y.
lipolytica lipase is a very active and selective catalyst for the hydro-
lysis of 2-bromo-phenyl and tolyl acetic acid ethyl esters. The
change of a single amino acid located in the active site of the en-
zyme led to a drastic 10-fold increase in enantioselectivity for
the resolution of (R,S)-2-bromo-phenylacetic acid ethyl ester race-
mates. Enantioselectivity was also increased for the resolution of
(R,S)-2-bromo-o-tolylacetic acid ethyl ester racemates, reaching a
value of 111. Moreover, the gain in enantioselectivity for both sub-
strates is accompanied by an increase in the catalysis rate of the
preferred (S)-enantiomer.
The activity and enantioselectivity displayed by the variant
V232A of Y. lipolytica lipase are compatible with the industrial
use of this enzyme for the production of pharmaceutical drugs.
We are currently working on the determination of the crystallo-
graphic structure of this lipase in order to understand, on a
molecular level, the structural determinants controlling Lip2p
enantioselectivity and the effects induced by V232A mutation on
the enantio-discrimination of 2-bromo phenyl and o-tolyl acid
esters. In parallel, saturation mutagenesis techniques, consisting
of changing the residue with all the others amino acids, will be
used to help better understand the enantiopreference pheno-
menon and to improve further performances of this promising
enzyme.
4.4. Procedure for enzymatic reactions
4.4.1. Hydrolysis of p-nitrophenyl butyrate
The lipase activity in the culture supernatant was determined
by monitoring the hydrolysis of p-nitrophenyl butyrate (pNPB) into
butyrate and p-nitrophenol.16 The method was optimized using 2-
methyl-butan-2-ol (2M2B) as solvent to solubilize p-nitrophenyl
butyrate. Lipase activity was measured in 96-well microplates with
20
l
L of the supernatant containing Lip2, 175
lL of a 100 mM
phosphate buffer, pH 7.2, 100 mM NaCl and 5
lL of pNPB 40 mM
in 2M2B. Activity was measured by following absorbance at
405 nm at 25 °C for 10 min using the VersaMax tunable microplate
reader apparatus (Molecular Devices, Rennes, France). One unit of
lipase activity was defined as the amount of enzyme releasing
1 lmol of fatty acid per min at 25 °C and pH 7.2.
4.4.2. Hydrolysis of 2-bromo phenyl and tolyl acetic acid esters
Hydrolysis was carried out in 1.5 mL eppendorf tubes contain-
ing a biphasic medium composed of 0.5 mL dried decane contain-
ing the ester (100 mM) and 0.5 mL of the aqueous enzymatic
solution. The mixture was shaken in a Vortex Genie 2 (D. Dutscher,
Brumat, France). Reactions were realized at 25 °C. At regular time
intervals, the progress of the reaction was followed by taking sam-
4. Materials and methods
4.1. Biological reagents
ples after phase separation by centrifugation (100
1 mL hexane).
lL diluted in
Lip2p lipase from Y. lipolytica and its variant were produced
from a strain of Y. lipolytica (JMY 1212) as described elsewhere.15
The oleic acid-inducible POX2 promotor was used to drive trans-
cription of the LIP2 gene. Protein secretion is directed by the
wild-type lipase targeting sequence. For growth in a tube, 5 mL
YPD medium (yeast extract 10 g/L, peptone 10 g/L and glucose
10 g/L) is used. This pre-culture is used to inoculate a 250 mL erlen
flask containing 50 mL YTO medium (yeast extract 10 g/L, peptone
20 g/L and oleic acid 20 g/L) in a 100 mM phosphate buffer (pH
4.5. HPLC analysis
The HPLC device was equipped with a chiral column: Chiralpack
OJ (25 cm  4.6 mm) (Daicel Chemical Industries Ltd, Japan) con-
nected to a UV detector (at 254 nm). A flow rate of 1.0 mL/min
and a 40 °C column temperature were used. The mobile phase
was composed of a mixture of n-hexane/isopropanol (70:30 v/v)
for all the esters, except for ( )-2-bromo-o-tolyl octyl acetate
where a mixture of n-hexane/isopropanol (98:2 v/v) was used.

1612
M. Cancino et al. / Tetrahedron: Asymmetry 19 (2008) 1608–1612
3. Ceynowa, J.; Rauchfleisz, M. J. Mol. Catal. B: Enzym. 2003, 23, 43–51.
4. Choi, G. S.; Kim, J. Y.; Kim, J. H.; Ryu, Y. W.; Kim, G. J. Prot. Expres. Purif. 2003, 29,
85–93.
4.6. Determination of the enantiomeric excess (ee), conversion
and enantioselectivity (E)
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.
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.
12. Ahmed, S. N.; Kazlauskas, R. J.; Morinville, A. H.; Grochulski, P.; Schrag, J. D.;
Cygler, M. Biocatalysis 1994, 9, 209–225.
From HPLC results, enantiomeric excess (ee) was calculated as
defined below: ees = {[R] À [S]}s/{[R] + [S]}s (s = substrate) and the
conversion: C = 1 À [(R À S)t/(R À S)t = 0] Ã 100.
The enantioselectivity value was the ratio of the initial rate of
(S)-enantiomer production (viR) to the initial rate of (R)-enantio-
mer production (viS): E = (viS/viR). The initial rates were deter-
mined by linear regression, which is indicated in Figures 1 and 2.
Acknowledgement
13. Guieysse, D.; Sandoval, G.; Faure, L.; Nicaud, J. M.; Monsan, P.; Marty, A.
Tetrahedron: Asymmetry 2004, 15, 3539–3543.
14. Cancino, M.; Bordes, F.; Dossat, V.; Croux, C.; Nicaud, J. M.; André, I.; Marty, A.
ChemBioChem., submitted for publication.
15. Bordes, F.; Fudalej, F.; Dossat, V.; Nicaud, J. M.; Marty, A. J. Microbial. Meth.
2007, 70, 493–502.
Miguel Cancino thanks CONACYT for financial support.
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