Enantioselective transesterification of racemic phenyl ethanol and its derivatives in organic solvent and ionic liquid using Pseudomonas aeruginosa lipase

Article abstract of DOI:10.1016/j.procbio.2009.07.020

The transesterification of 1-phenyl ethanol has been carried out using lipases from Pseudomonas aeruginosa MTCC 5113, to obtain chirally pure aryl ethanols with good yield and excellent enantioselectivity. The lipase from P. aeruginosa gave good conversion and moderate enantioselectivity (ee) in organic solvents, however, when the catalytic amount of ionic liquids were added in the reaction mixture, excellent enantioselectivity was obtained. Moreover, the change in enantiomer preference was seen in the presence of catalytic amount of ionic liquids. The findings revealed that hydrophobic ionic liquids (two-phase system) were the best solvents and 4-substituted aryl ethanols were the pre-eminent substrates for such type of reactions. The preparative scale (5 g) transesterification of 1-phenylethanol using lipases from P. aeruginosa yielded S-(-)-1-phenyl ethanol with 39% yield and >99% ee in hexane and 46% yield and >99% ee in [BMIm][PF₆].

Full text of DOI:10.1016/j.procbio.2009.07.020

Process Biochemistry 45 (2010) 25–29
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Enantioselective transesterification of racemic phenyl ethanol and its derivatives
in organic solvent and ionic liquid using Pseudomonas aeruginosa lipase
Manpreet Singh a, Ram Sarup Singh b, Uttam Chand Banerjee a,
*
a Biocatalysis Laboratory, Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research (NIPER),
Sector-67, SAS Nagar 160062, Punjab, India
b Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala 147002, India
A R T I C L E I N F O
A B S T R A C T
Article history:
The transesterification of 1-phenyl ethanol has been carried out using lipases from Pseudomonas
aeruginosa MTCC 5113, to obtain chirally pure aryl ethanols with good yield and excellent
enantioselectivity. The lipase from P. aeruginosa gave good conversion and moderate enantioselectivity
(ee) in organic solvents, however, when the catalytic amount of ionic liquids were added in the reaction
mixture, excellent enantioselectivity was obtained. Moreover, the change in enantiomer preference was
seen in the presence of catalytic amount of ionic liquids. The findings revealed that hydrophobic ionic
liquids (two-phase system) were the best solvents and 4-substituted aryl ethanols were the pre-eminent
substrates for such type of reactions. The preparative scale (5 g) transesterification of 1-phenylethanol
using lipases from P. aeruginosa yielded S-(ꢀ)-1-phenyl ethanol with 39% yield and >99% ee in hexane
and 46% yield and >99% ee in [BMIm][PF6].
Received 20 May 2009
Received in revised form 22 July 2009
Accepted 23 July 2009
Keywords:
Phenyl ethanols
Phenyl ethanol derivatives
Transesterification reactions
Lipases
Ionic liquids
ß 2009 Elsevier Ltd. All rights reserved.
1. Introduction
utilizedpredominantlyforthepreparation ofcompounds associated
to pharmaceuticals through stereoselective hydrolysis, esterifica-
Biocatalysts such as lipases, proteases, dehydrogenases and
oxidoreductases were reported to be active in ionic liquids (as pure
reaction media) [1,2], as well as ionic liquids dissolved in
molecular solvents [3,4], and as a mixture of two different ionic
liquids [5]. In all the cases, ionic liquids as reaction media have
been found to improve the solubility of the substrates and/or
products with hydrophobic moieties (e.g. amino acids, nucleo-
sides), providing better enzyme activity and regioselectivity. It is
possible to design two-phase reaction systems that easily permit
product recovery [6,7]. Furthermore, water-immiscible ionic
liquids also have vital stabilizing effect on hydrolases (lipases,
esterases, proteases, etc.) and spectroscopic studies (i.e. fluores-
cence, circular dichroism, etc.) have confirmed the ability of these
neoteric solvents to maintain the secondary structure and the
native conformation of proteins in the face of the usual unfolding
that occurs in non-aqueous environment [8,9].
tion or transesterification [14–16]. The lipase-catalyzed transester-
ification of (R,S) 1-chloro-3(3,4-difluorophenoxy)-2-propanol was
carried out to obtain (R)-alcohol which is an intermediate for the
synthesis of (S)-lubeluzole, an anti-ischemic drug [17].
Enantiomerically pure alcohols are useful building blocks and
chiral auxiliaries for the synthesis of bioactive compounds such as
pharmaceuticals, agrochemicals and natural products [18]. In
biocatalysis, microorganisms and enzymes are employed to perform
thereactions. Lipase-catalyzed reactionshave been appliedtosolvea
number of synthetic problems, one of which is the kinetic resolution
of diastereomeric and enantiomeric mixtures of primary and
secondary alcohols. Lipase-catalyzed transesterification of second-
ary alcohols is a valuable approach as compared to asymmetric
reduction of corresponding prochiralketones using oxidoreductases,
as the later require expensive cofactors such as NAD(P)H.
Recently, a number of microorganisms with lipase activity have
been reported for the stereoselective transesterification of
racemic-1-phenyl ethanols [19]. However, a special catalyst with
broad substrate range must be developed to utilize it on an
industrial scale, unfortunately, no such catalyst is available. It is
well recognized that the screening of a wide variety of micro-
organisms living in the soil is effective and efficient method to
obtain the desired enzyme towards unnatural substrates [20].
Unnatural substrate 3,4-difluorophenol has been utilized as a
model substrate for the isolation of microorganisms having lipase
activity [18,21]. Forty organisms with high lipase activity were
Lipases have been acknowledged as extremely fascinating
catalysts for organic syntheses because of their stability, usability
and broad substrate tolerance [10,11]. Furthermore, they are easily
available from a variety of sources, especially bacteria and fungi
[12,13]. Although lipases have found various uses, their full
prospective has not been completely explored. They have been
* Corresponding author.
E-mail address: ucbanerjee@niper.ac.in (U.C. Banerjee).
1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2009.07.020

26
M. Singh et al. / Process Biochemistry 45 (2010) 25–29
prepared by adding 900
ml hexane and 100 ml isopropyl alcohol (IPA). The
isolated successfully using 3,4-difluorophenol as a sole carbon
source for the enrichment of soil samples. This paper describes the
use of lipase from the strain Pseudomonas aeruginosa MTCC 5113
isolated from soil, for the transesterification of 1-phenyl ethanol
and its derivatives. The reactions were carried out in hydrophobic
solvent (hexane) and ionic liquids (two-phase system). Also the
preparative scale transesterification of 1-phenyl ethanol has been
achieved with good yield and excellent enantioselectivity.
conversion and enantiomeric excess of the substrate and product were analyzed.
In case of ionic liquids, the reaction was carried out in 10 ml round bottom flask
containing 20 mM substrate, 150 mM vinyl butyrate, 1.0 ml ionic liquids
([BMIm][BF4], [BMIm][PF6] and [EMIM][BF4]) as solvent and 4 ml lipase mixture.
Samples were taken at regular interval of time and prepared by filtration through
Whatman filter paper no. 1, employing hexane as filtering solvent.
2.5.2. Preparative scale
The gram scale transesterification of 1-phenyl ethanol was carried out as follows.
The mixture containing lipases from P. aeruginosa was harvested from 500 ml
culture broth by centrifugation. The reaction mixture consisted of 5 g 1-phenyl
ethanol, vinyl butyrate as acyl donor, stirred in a 500 ml flask containing 250 ml
hexane and 250 ml lipase mixture. After the completion (24 h) of reaction, the
solvent layer was removed thrice with equal volume of hexane. The organic solvent
was evaporated under reduced pressure. The oily mass obtained was subjected to
column chromatography using ethyl acetate:hexane (1:10) as eluent.
2. Materials and methods
2.1. Instrumental
The conversion as well as the enantiomeric excess of the transesterification was
monitored using High Pressure Liquid Chromatography (HPLC). Analysis was
performed on a Shimadzu 10AVP instrument equipped with a UV detector. The
conversion and enantiomeric excess of the products formed were determined by
chiral HPLC with a Chiracel-ODH column (0.46 mm ꢁ 250 mm, 5
mm, Diacel) using
3. Results and discussion
hexane:isopropyl alcohol in the ratio of 9:1 at a flow rate of 0.5 ml/min and detected
at 210 nm. The retention times of S-(ꢀ) and R-(+)-1-phenyl ethanol were 11.2 and
12.8 min, respectively, and the corresponding esters come after 6.4 and 7.6 min in
Chiracel-ODH column. To further confirm the products, the extracts of the
biocatalytic reaction were analyzed by GC–MS using Shimadzu QP5000 GC–MS
equipped with a quadrupole mass filter and DB-1 (100% dimethylpolysiloxane)
capillary column (30 mm ꢁ 0.25 mm), ionisation of 70 eV, scan interval 1.5 s and
mass range 40–500. The oven parameters were 150 8C for 5 min with 10 8C increase
per minute to a final temperature of 300 8C for 20 min, and the injector temperature
was kept at 250 8C. The enantiomeric excess (ee (%)) was defined as the ratio of
[S] ꢀ [R]/[S] + [R] ꢁ 100%, where [R] and [S] are the concentrations of (R) and (S)
enantiomers, respectively. 1H NMR and 13C NMR spectra were recorded on a Bruker
Advance DPX 300 NMR spectrometer.
3.1. Enantioselective transesterification of 1-phenyl ethanol and its
derivatives in hexane
The time-course of transesterification revealed that after 24 h,
the conversion of 1-phenyl ethanol to S-ester was 43% with an ee
99% (Fig. 1). With the further increase in reaction time, the
percentage conversion increased, however, a fall in the ee was
observed. The reaction was allowed only for 24 h. In order to
optimize the reaction temperature, transesterification was carried
out at different temperatures ranging from 20 to 50 8C. It was
observed that both conversion and ee declined above 30 8C
(conversion 45.8%, ee > 99%) (Fig. 2). Hence, all the subsequent
experiments were carried out at 30 8C. In order to scale up the
bioprocess, the effect of substrate concentration on the conversion
and enantioselectivity was studied with a fixed amount (25 ml) of
lipase (700 U/ml). The optimum substrate concentration was
found to be 20 mM for 1-phenyl ethanol (Fig. 3). Beyond this, there
was a decrease in conversion as well as enantioselectivity. The
lower yield at higher substrate concentration may be due to the
substrate inhibition.
2.2. Chemicals
1-Phenyl ethanol and different derivatives of acetophenone namely 2-
fluoroacetophenone, 3-fluoroacetophenone, 4-fluoroacetophenone, 4-chloroaceto-
phenone, 4-bromo-acetophenone and 4-iodoacetophenone were obtained from
Lancaster Synthesis Ltd., UK. All other chemicals were of analytical grade and
purchased from local suppliers.
2.3. Chemical synthesis of racemic alcohols from there corresponding ketones
A series of racemic alcohols synthesized using sodium borohydride (NaHB4)
reduction of prochiral compounds. The alcohols thus formed were subjected to
column chromatography to separate the pure alcohol, which were used as
substrates in the transesterification reaction using P. aeruginosa lipases [19].
3.2. Enantioselective transesterification of 1-phenyl ethanol in ionic
liquids (two-phase system)
2.4. Enzyme assay
The assay was performed by measuring the increase in absorbance at 410 nm
produced by the release of p-nitrophenol in the hydrolysis of p-nitrophenyl
palmitate. Winkler and Stuckmann method with slight modification was applied for
the quantification of lipase activity of P. aeruginosa [22]. An aqueous solution
(9.0 ml) was prepared by adding gum arabic (0.11%, w/v) and Triton X-100 (0.44%,
w/v) in Tris–buffer (50 mM, pH 8). The substrate pNPP was dissolved in iso-
propanol (3 mg/ml) and mixed with aqueous solution using intense agitation
(vortex mixture) to form emulsion. The reaction mixture consisted of emulsion
(0.9 ml) mixed with 1.5 ml Tris–HCl buffer and 0.5 ml CaCl2 (75 mM). After 5 min of
Three ionic liquids used for the present study [BMIm][PF6],
[BMIm][BF4] and [EMIM][BF4] are distinctly different in their
incubation at 60 8C, 100
ml appropriately diluted in Tris–HCl buffer pH 8.0, enzyme
solution was added to it. Incubation was continued for a further 10 min. The
reaction was stopped by putting the test tubes in ice and the optical density was
measured spectrophotometrically. Enzyme activity was defined as the amount of
enzyme required to hydrolyze
conditions.
1 mmol pNPP/min (IU) under standard assay
2.5. Transesterification of 1-phenyl ethanol and its derivatives using extracellular
lipases from Pseudomonas aeruginosa MTCC 5113
2.5.1. Analytical scale
Transesterification of 1-phenyl ethanol and its derivatives was carried out using
lipases from P. aeruginosa in solvents and ionic liquids. In a typical experimental
procedure containing 1:1 ratio of lipase mixture and hexane (25 ml) as solvent,
20 mM 1-phenyl ethanol and its derivatives in separate reactions containing
150 mM vinyl butyrate were stirred (ꢂ1000 rpm) in 50 ml capacity round bottom
flask (RBF). The constant temperature (30 8C) was maintained by incubating the
reaction mixture in an incubator (Kuhnur, Switzerland). At regular time interval,
Fig. 1. Time-course of conversion and enantioselectivity of 1-phenyl ethanol using
Pseudomonas aeruginosa lipase at 30 8C in hexane as solvent. [The transesterification
was carried out using 5 mM substrate and 150 mM vinyl butyrate at 30 8C
(ꢂ1000 rpm) for 36 h. Samples were withdrawn after regular interval (6 h) of time].
500
ml sample was taken by adding 500 ml hexane each time for 6 h. This was done
to maintain the volume, till the end of the reaction. Hexane was evaporated from
the samples on rotavapor (Buchi, Switzerland). Samples for chiral HPLC were

M. Singh et al. / Process Biochemistry 45 (2010) 25–29
27
Table 1
Comparison of lipase-catalyzed transesterification reaction of phenyl ethanol in
three ionic liquids ([BMIm][PF6], [BMIm][BF4], [EMIm][BF4]).
Ionic liquids
Conversion (%)
ee (%)
[BMIm][PF6]
[BMIm][BF4]
[EMIm][BF4]
42.4 ꢃ 0.56
28.6 ꢃ 0.96
12.1 ꢃ 1.64
98.3ꢃ 0.23
98.4ꢃ 0.39
95.2ꢃ 0.36
Therefore, all the subsequent reactions were carried out up to
6 h. Interesting results were observed when the reaction was
carried out at different temperatures (20–50 8C) using ionic
liquid as solvent system (Fig. 5). Previously, it has been reported
that in two-phase system (hexane + [BMIm][PF6]) the reaction
rate increased with the increase in temperature because the
stability of enzyme was maintained by the presence of ionic
liquid in the reaction medium at higher temperatures, the
increased stability in ionic liquids may be the reason for
temperature tolerance [25].
Fig. 2. Effect of reaction temperature on the transesterification of 1-phenyl ethanol
in hexane. [The reaction was carried out using 5 mM 1-phenyl ethanol and 150 mM
vinyl butyrate (acyl donor) for 24 h. The temperature was varied from 20 to 50 8C in
an incubator shaker].
3.3. Effect of substitution on the biocatalytic transesterification
reaction in solvent and ionic liquid (two-phase system)
properties, such as, polarity, hydrophobicity, anion nucleophilicity,
hydrogen-bond basicity and viscosity. Catalytic amount of these
ionic liquids was used for the transesterification reaction. It was
found that out of three ionic liquids taken for this study the best
solvent was [BMIm][PF6] (Table 1). The hydrophobic nature of
[BMIm][PF6] may have influenced the catalytic properties of lipase
[23,24]. It has already been established that the [BMIm][PF6] is the
best co-solvent for the transesterification of [R,S]-1-chloro-3(3,4-
difluorophenoxy)-2-propanol using lipases mixture from Pseudo-
monas aeruginosa [25].
The time-course of conversion and enantiomeric excess for
the transesterification of 1-phenyl ethanol in ionic liquid is
shown in Fig. 4. The reaction was catalyzed by lipase mixture
from P. aeruginosa in a two-phase system composed of ionic
liquid [BMIm][PF6]. The reaction was allowed to proceed for 24 h
and samples were processed as described in materials and
methods. The conversion of alcohol to ester increased with the
time up to 6 h, while the ee (%) was initially maximum and then
decreased as commonly observed in such biotransformation.
The biocatalytic potential of lipases from this microorganism
was assessed using a series of 1-phenyl ethanol derivatives. In all
the reactions, good to excellent conversion and enantiomeric
excess were attained in almost all the cases signifying that the
lipases of Pseudomonas aeruginosa MTCC 5113 is predominately S-
specific.
3.3.1. Transesterification in hexane
Primarily, the effect of the position of the substituents on the
biocatalytic transesterification was examined in hexane (Table 2).
The transesterification of the para-substituted 1-phenyl ethanol
was more favoured in comparison to meta- and ortho-substituted
phenyl ethanol. This may perhaps due to the decrease in steric-
hindrance as we progress from ortho- to para-position. The nature
of the substituents on transesterification reaction was monitored
by carrying out the reaction with 1-phenyl ethanol derivatives
with different kinds of para-substitutions present on the phenyl
ring. The effect of the bulkiness of the substituents on the
Fig. 4. Time-course of conversion and enantioselectivity of 1-phenyl ethanol using
lipases from Pseudomonas aeruginosa at 30 8C in ionic liquid [BMIm][PF6]. [The
transesterification was carried out in 1 ml ionic liquid, 4 ml lipase mixture and 5 ml
hexane. Using 5 mM substrate and 150 mM vinyl butyrate at 30 8C (ꢂ1000 rpm) for
12 h. Samples were withdrawn after regular interval (2 h) of time].
Fig. 3. Effect of substrate concentration on the transesterification of 1-phenyl
ethanol using P. aeruginosa lipase in hexane. [The reaction was carried out in a flask
containing 5–30 mM of 1-phenyl ethanol and keeping vinyl butyrate concentration
constant (150 mM). The reaction temperature was maintained at 30 8C for 24 h].

28
M. Singh et al. / Process Biochemistry 45 (2010) 25–29
bromo- and iodo-substituted phenyl ethanols the lipase from P.
aeruginosa become R-selective, however, the conversion and
enantioselectivity were very low. The change in enantioselectivity
may be due to the hydrophilic nature of IL molecules which might
be coordinating with the active site, interfering differently with the
substrate enantiomers [26]. Reversal of enantioselectivity for
different substrates by changing the solvent conditions is reported
previously using lipase from Candida rugosa, Pseudomonas cepacia
and for protease [27–29]. Owing to the hydrophilic nature
(presence of both cation and anion) of the ionic liquids, the
enzyme might have distorted in such a way that (R)-enantiomer
got the preference.
4. Conclusion
The synthesis of optically active alcohols using biocatalysts
under mild reaction conditions is of immense importance. Since
these synthetic substrates are poor substrates for natural
microorganisms or enzymes, screening for a novel microorganism
or enzyme is one of the most powerful tools for finding biocatalytic
system that displays high activity and desired enantioselectivity
[20]. In the present study, soil isolated bacterial strain Pseudomo-
nas aeruginosa catalyses the enantioselective transesterification of
1-phenyl ethanol and its various derivatives with absolute
enantioselectivity. It is evident from the time-course study of
the transesterification that within 24 h of exposure of 1-
phenylethanol to the extracellular lipase in hexane, the reaction
achieved good conversion (>43%) along with excellent ee (>99%).
However, in the presence of catalytic amount of ionic liquids (two-
phase), good conversion (>48%) and almost absolute ee (>99) was
achieved in 6 h of reaction. The results revealed that presence of
catalytic amount of hydrophilic ionic liquids in a two-phase
reaction system decreases the reaction time tremendously.
Furthermore, ionic liquids in the presence of organic solvent
increase the temperature dependence of lipase and in the present
study higher activity was observed at 50 8C in ionic liquids in
comparison to neat organic solvent.
Fig. 5. Effect of reaction temperature on the transesterification of 1-phenyl ethanol
in [BMIm][PF6]. [The reaction was carried out using 5 mM 1-phenyl ethanol and
150 mM vinyl butyrate (acyl donor) for 6 h. The temperature was varied from 20 to
50 8C in an incubator shaker].
transesterification efficiency of the lipases from Pseudomonas
aeruginosa was also studied. It was observed that as the size of
the substituents increased, the transesterification efficiency
decreased. A very high conversion (48%) was observed in the case
of fluoro-1-phenyl ethanol that decreased drastically to mere 12%
in case of iodo-1-phenyl ethanol. This might be due to the steric-
hindrance of the substituents for the enzyme active site.
3.3.2. Transesterification in ionic liquid
The results were similar where fluoro-1-phenyl ethanol was
used as substrate, higher conversion rate (49.3%) and excellent
enantioselectivity (>99%) for the S-enantiomer has been achieved
(Table 3). The reaction containing para-substituted ethanols
showed higher conversion and ee in contrast to ortho- or meta-
substituted compounds.
Interesting results were obtained when ionic liquids (two-
phase system) were used as solvent, the selectivity of the enzyme
changed with the increase in bulkiness of the substituent. In case of
In conclusion, a simple and efficient process has been developed
for the preparation of varieties of aryl alcohols, with good
enantiomeric excess, by transesterification of the corresponding
racemic alcohols using lipase from Pseudomonas aeruginosa.
Table 2
Effect of 1-phenyl ethanol and its derivatives on the enantioselective transesterification reaction using P. aeruginosa lipases in hexane.
Substrate
Converion (%)
ee (%)
Configuration
Yeild (g/l)
1-Phenyl-ethanol
48.5 ꢃ 1.13
12.6 ꢃ 0.16
29.3 ꢃ 0.64
43.7 ꢃ 1.34
38.9 ꢃ 1.74
37.8 ꢃ 0.97
28.4 ꢃ 0.92
99.1 ꢃ 0.22
72.9 ꢃ 0.27
83.4 ꢃ 0.64
98 ꢃ 0.35
S
S
S
S
S
S
S
0.6 (0.27)
0.78 (0.09)
0.78 (0.17)
0.78 (0.32)
0.86 (0.32)
1.1 (0.32)
1.3 (0.30)
1-(2-Fluoro-phenoxy)-ethanol
1-(3-Fluoro-phenoxy)-ethanol
1-(4-Fluoro-phenoxy)-ethanol
1-(2-Chloro-phenoxy)-ethanol
1-(4-Bromo-phenoxy)-ethanol
1-(4-Iodo-phenoxy)-ethanol
92.4 ꢃ 1.6
91.7 ꢃ 0.85
88.3 ꢃ 0.98
Table 3
Lipase-catalyzed transesterification of phenyl ethanol derivatives in ionic liquid.
Substrate
Conversion (%)
ee (%)
Configuration
1-Phenyl-ethanol
49.3 ꢃ 1.13
20.4 ꢃ 0.87
32.7 ꢃ 0.94
49.1 ꢃ 1.9
25.3 ꢃ 2.13
20.5 ꢃ 1.68
6.9 ꢃ 1.66
>99 ꢃ 0.45
88.4 ꢃ 0.77
90.2 ꢃ 0.75
>99 ꢃ 1.02
95.3 ꢃ 0.92
90 ꢃ 0.95
Nil
S
1-(2-Fluoro-phenoxy)-ethanol
1-(3-Fluoro-phenoxy)-ethanol
1-(4-Fluoro-phenoxy)-ethanol
1-(2-Chloro-phenoxy)-ethanol
1-(4-Bromo-phenoxy)-ethanol
1-(4-Iodo-phenoxy)-ethanol
S
S
S
R
R
Nil

M. Singh et al. / Process Biochemistry 45 (2010) 25–29
29
References
[16] Patel RN. Microbial/enzymatic synthesis of chiral pharmaceutical intermedi-
ates. Current Opinion in Drug Discovery Development 2003;6:902–20.
[17] Singh M, Banerjee UC. Enantioselective transesterification of (RS)-1-chloro-3-
(3,4-difluorophenoxy)-2-propanol using Pseudomonas aeruginosa lipases. Tet-
rahedron Asymmetry 2007;18:2079–85.
[18] Faber K. Biotransformations in Organic Chemistry. Berlin: Springer-Verlag;
2000.
[19] Goswami A, Goswami J. DMSO-triggered enhancement of enantioselectivity in
Novozyme[435]-catalyzed transesterification of chiral 1-phenylethanols. Tet-
rahedron Letters 2005;46:4411–3.
[20] Asano Y. Overview of screening for new microbial catalysts and their uses in
organic synthesis—selection and optimization of biocatalysts. Journal of Bio-
technology 2002;94:65–72.
[21] Process for the preparation of (R)-1-chloro-3-(3,4-difluorophenoxy)-2-propa-
nol.
[22] Winkler UK, Stuckmann M. Glycogen, hyaluronate, and some other polysac-
charides greatly enhance the formation of exolipase by Serratia marcescens.
Journal of Bacteriology 1979;138:663–70.
[23] Pringle JM, Golding J, Baranyai K, Forsyth CM, Deacon GB, Scott JL, et al. The
effect of anion fluorination in ionic liquids-physical properties of a range of
bis(methanesulfonyl)amide salts. New Journal of Chemistry 2003;27:1504–
10.
[1] Yang Z, Pan W. Ionic liquids: green solvents for nonaqueous biocatalysis.
Enzyme and Microbial Technology 2005;37:19–28.
[2] van Rantwijk F, Sheldon RA. Biocatalysis in ionic liquids. Chemical Reviews
2007;107:2757–85.
[3] Kaftzik N, Wasserscheid P, Kragl U. Use of ionic liquids to increase the yield and
enzyme stability in the
b-galactosidase catalysed synthesis of N-acetyllacto-
samine. Organic Process Research & Development 2002;6:553–7.
[4] Li N, Ma D, Zong M-H. Enhancing the activity and regioselectivity of lipases for
30-benzoylation of floxuridine and its analogs by using ionic liquid-containing
systems. Journal of Biotechnology 2008;133:103–9.
[5] Lee SH, Ha SH, Hiep NM, Chang W-J, Koo Y-M. Lipase-catalyzed synthesis of
glucose fatty acid ester using ionic liquids mixtures. Journal of Biotechnology
2008;133:486–9.
[6] Itoh T, Nishimura Y, Ouchi N, Hayase S. 1-Butyl-2,3-dimethylimidazolium
tetrafluoroborate: the most desirable ionic liquid solvent for recycling use of
enzyme in lipase-catalyzed transesterification using vinyl acetate as acyl
donor. Journal of Molecular Catalysis B Enzymatic 2003;26:41–5.
[7] Abe Y, Kude K, Hayase S, Kawatsura M, Tsunashima K, Itoh T. Design of
phosphonium ionic liquids for lipase-catalyzed transesterification. Journal
of Molecular Catalysis B Enzymatic 2008;51:81–5.
[24] Kosmulski M, Gustafsson J, Rosenholm JB. Thermal stability of low tempera-
ture ionic liquids revisited. Thermochimica Acta 2004;412:47–53.
[25] Singh M, Singh RS, Banerjee UC. Stereoselective synthesis of (R)-1-chloro-
3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aerugi-
[8] Lozano P, de Diego T, Gmouh S, Vaultier M, Iborra JL. Criteria to design green
enzymatic processes in ionic liquid/supercritical carbon dioxide systems.
Biotechnology Progress 2004;20:661–9.
[9] De Diego T, Lozano P, Gmouh S, Vaultier M, Iborra JL. Understanding structure–
stability relationships of Candida antartica lipase B in ionic liquids. Biomacro-
molecules 2005;6:1457–64.
[10] Benjamin S, Pandey A. Candida rugosa lipases: molecular biology and versa-
tility in biotechnology. Yeast 1998;14:1069–87.
[11] Faber K. Biotransformations in Organic Chemistry. Berlin: Springer; 2004.
[12] White JS, White DC. Source Book of Enzymes. New York: CRC Press; 1997.
[13] Jaeger KE, Reetz MT. Microbial lipases form versatile tools for biotechnology.
Trends in Biotechnology 1998;16:396–403.
nosa in ionic liquid-containing system. Journal of Molecular Catalysis
B
Enzymatic 2009;56:294–9.
[26] Hirose Y, Kariya K, Sasaki I, Kurono Y, Ebiike H, Achiwa K. Drastic solvent effect
on lipase-catalyzed enantioselective hydrolysis of prochiral 1,4-dihydropyr-
idines. Tetrahedron Letters 1992;33:7157–60.
¯
[27] Ueji S, Fujino R, Okubo N, Miyazawa T, Kurita S, Kitadani M, et al. Solvent-
induced inversion of enantioselectivity in lipase-catalyzed esterification of 2-
phenoxypropionic acids. Biotechnology Letters 1992;14:163–8.
[28] Tawaki S, Klibanov AM. Inversion of enzyme enantioselectivity mediated by
the solvent. Journal of the American Chemical Society 1992;114:1882–4.
[29] Bornscheuer U, Herar A, Kreye L, Wendel V, Capewell A, Meyer HH, et al.
Factors affecting the lipase catalyzed transesterification reactions of 3-
hydroxy esters in organic solvents. Tetrahedron Asymmetry 1993;4:1007–16.
[14] Margolin AL. Enzymes in the synthesis of chiral drugs. Enzyme and Microbial
Technology 1993;15:266–80.
[15] Jaeger KE, Eggert T. Lipases for biotechnology. Current Opinion Biotechnology
2002;13:390–7.