Pseudomonas cepacia lipase catalyzed esterification and transesterification of 3-(furan-2-yl) propanoic acid/ethyl ester: A comparison in ionic liquids vs hexane

Article abstract of DOI:10.1016/j.molcatb.2010.01.032

A comparison of the Pseudomonas cepacia lipase (lipase PS) catalyzed esterification of 3-(furan-2-yl) propanoic acid and transesterification of ethyl 3-(furan-2-yl) propanoate with six straight chain alcohols (propanol to octanol) in ionic liquids and hexane was carried out. The ionic liquids selected, [Bmim]BF₄, [Bmim]PF₆, and [Bmim]Tf₂N, consisted of an identical cation and different anions. This is the first report on the biocatalyzed synthesis of these esters. In all the media, lipase PS catalyzed esterification of 3-(furan-2-yl) propanoic acid resulted in high yields of the esters compared to the transesterification of ethyl 3-(furan-2-yl) propanoate. [Bmim]Tf₂N proved to be the best; yielding 98-67% of the product by lipase PS catalyzed esterification. The lipase PS-[Bmim]Tf₂N and lipase PS-[Bmim]PF₆ mixture was recycled five times without any decrease in the yields of the products and was found to be operationally stable up to 10 months at room temperature.

Full text of DOI:10.1016/j.molcatb.2010.01.032

Journal of Molecular Catalysis B: Enzymatic 65 (2010) 68–72
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Pseudomonas cepacia lipase catalyzed esterification and transesterification of
3-(furan-2-yl) propanoic acid/ethyl ester: A comparison in ionic liquids vs hexane
P. Vidyaa, Anju Chadhaa,b,∗
a Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
b National Center for Catalysis Research, Indian Institute of Technology Madras, Chennai 600036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 February 2010
A comparison of the Pseudomonas cepacia lipase (lipase PS) catalyzed esterification of 3-(furan-2-yl)
propanoic acid and transesterification of ethyl 3-(furan-2-yl) propanoate with six straight chain alcohols
(propanol to octanol) in ionic liquids and hexane was carried out. The ionic liquids selected, [Bmim]BF4,
[Bmim]PF6, and [Bmim]Tf2N, consisted of an identical cation and different anions. This is the first report on
the biocatalyzed synthesis of these esters. In all the media, lipase PS catalyzed esterification of 3-(furan-
2-yl) propanoic acid resulted in high yields of the esters compared to the transesterification of ethyl
3-(furan-2-yl) propanoate. [Bmim]Tf2N proved to be the best; yielding 98–67% of the product by lipase
PS catalyzed esterification. The lipase PS–[Bmim]Tf2N and lipase PS–[Bmim]PF6 mixture was recycled five
times without any decrease in the yields of the products and was found to be operationally stable up to
10 months at room temperature.
Keywords:
Ionic liquids
Esterification
Transesterification
Pseudomonas cepacia lipase
3-(Furan-2-yl) propanoic acid ester
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
advantages of ionic liquids is the biocatalytic preparation of com-
pounds that can be used as food additives or in pharmaceutical and
The use of biocatalysis in non-aqueous environments has the
advantages of increasing the solubility of organic substrates, the
possibility of carrying out processes which are thermodynamically
unfavorable in water (e.g. esterification and transesterification)
and facilitating enzyme and product recovery [1]. Among these
non-conventional media, organic solvents are widely used with
enzymes to improve solubility of hydrophobic reactants and/or
products and to shift reaction equilibrium from hydrolysis toward
synthesis [2]. In recent years ionic liquids (ILs) have attracted atten-
tion because, as opposed to organic solvents, they are non-volatile,
non-flammable and have high thermal stability [2–4]. Ionic liquids
are substances that are completely composed of ions and are liq-
uids at or close to room temperature [4]. One of the most promising
cosmetic formulations because of their reduced toxicity. Toxico-
logical tests of a series of ionic liquids with imidazolium cations
and various anions (including PF6−, TF2N− and BF4−) showed that
most of the ionic liquids were less toxic than organic solvents by
several orders of magnitude [5]. Enzymatic reactions carried out in
ILs have shown high activity, good enantioselectivity, high reaction
rates, high thermal and operational stability [6–9]. Another impor-
tant feature of ionic liquids is that their solvent properties can be
finely tuned by selecting appropriate combinations of cations and
anions [2] thus allowing ionic liquids to be specifically designed for
different reaction conditions. For the same cation, properties of ILs
such as hydrophobicity, miscibility with organic solvents, capacity
to dissolve organic and inorganic solutes vary widely depending
on the anion [10–14]. The use of ILs as reaction media can thus
greatly expand the repertoire of enzyme catalyzed transformations.
Consequently, a number of potential applications of enzymes that
are either impossible or marginal in organic media become quite
feasible and commercially attractive in these media [2].
As part of our ongoing research on enzyme catalysis in non-
conventional media, we used ionic liquids as a reaction medium
for lipase PS catalyzed synthesis of 3-(furan-2-yl) propanoic acid
esters, which are used as flavors for food and in the fragrance indus-
try [15]. The chemical methods to synthesize these esters have the
common disadvantage of high temperature requirements, harsh
conditions and the use of carcinogenic organic compounds [16].
In this paper we report the lipase PS catalyzed esterification of
3-(furan-2-yl) propanoic acid and transesterification of ethyl 3-
Abbreviations: ILs, ionic liquids; [Bmim]BF4, 1-butyl-3-methylimidazolium
tetrafluoroborate; [Bmim]PF6, 1-butyl-3-ethylimidazolium hexafluorophos-
phate; [Bmim]Tf2N, 1-butyl-3-methyl imidazolium triflamide; lipase PS,
Pseudomonas cepacia lipase; ser, serine; his, histidine; asp, aspartic acid; glu,
glutmic acid; gln, glutamine; CAL-B, Candida antarctica lipase B; [Emim]Tf2N,
1-ethyl-3-methylimidazolium bis(trifluoromethyl-sulfonyl)imide; [Bm3N]Tf2N,
butyl trimethyl ammonium bis(trifluoromethyl-sulfonyl)imide; EDCl, 1-[3-
(dimethylamino)propyl]3-ethylcarbodiimide hydrochloride; DCC, dicyclohexyl
carbodiimide.
∗
Corresponding author at: Laboratory of Bioorganic Chemistry, Department of
Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India.
Tel.: +91 44 2257 4106; fax: +91 44 2257 4102.
E-mail address: anjuc@iitm.ac.in (A. Chadha).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.01.032

P. Vidya, A. Chadha / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 68–72
69
Table 1
(furan-2-yl) propanoate with a variety of alcohols in ionic liquids
Esterification of 1 in ILs/hexane at 50 ◦C catalyzed by lipase PS.
and hexane. A thorough literature survey showed that there is no
‘best’ ionic liquid for performing biotransformation just as there is
no ‘best’ organic solvent in general for carrying out biocatalysis. As
in our previous study [9] in order to evaluate the influence of anions
on the catalytic performance of lipase PS, three ILs, [Bmim]BF4,
[Bmim]PF6 and [Bmim]Tf2N, with an identical cation [Bmim] and
Entry
ROH
Yield % of ester formed in
[Bmim]BF4
[Bmim]PF6
[Bmim]Tf2N
Hexane
1
2
3
4
5
6
CH3(CH2)2OH
CH3(CH2)3OH
CH3(CH2)4OH
CH3(CH2)5OH
CH3(CH2)6OH
CH3(CH2)7OH
9
8
7
7
8
7
58
56
48
22
17
28
98
98
96
82
73
67
52
57
60
51
45
49
different anions [BF4−, PF6 and Tf2N−] were selected as reaction
−
media. To the best of our knowledge this is the first report on
the biocatalytic synthesis of these esters. In order to understand
the role of ILs, the impact of their physicochemical properties on
the following: (a) stabilization/desatbilization effect on the transi-
tion state, (b) micro-aqueous environment of the enzyme and (c)
charged groups of that enzyme can be taken into account. In this
paper we discuss the high yields of esterification formed over trans-
esterification in hydrophobic ILs, taking into account the impact of
ILs on all the above-mentioned factors, especially on the structure
and function of lipase PS. The long-term stability of the enzyme
in hydrophobic ILs up to a period of 10 months was also exam-
ined. The recyclability of lipase PS–hydrophobic IL mixture was also
investigated.
NMR (CDCl3, 400 MHz) ı ppm: 7.22 (s, 1H, CH), 6.22 (s, 1H, CH), 5.94
(s, 1H, CH), 4.02–3.99 (t, 2H, CH2, J = 6.8 Hz), 2.90–2.87 (t, 3H, CH2,
J = 7.2 Hz), 2.59–2.55 (t, 3H, CH2, J = 7.2 Hz), 1.54–1.51 (m, 2H, CH2),
1.22–1.19 (m, 6H, CH2), 0.81–0.80 (t, 3H, CH2, J = 6.4 Hz); 13C NMR
(CDCl3, 100 MHz) ı ppm: 172.5, 154.1, 141.1, 110.1, 105.2, 64.7,
32.7, 31.3, 28.3, 25.5, 23.4, 22.4, 13.9; IR (neat, cm−1): 2986, 1736,
1507, 1479, 1163; HRMS(EI): calculated for C13H20O3Na (M+Na)+,
247.1310; observed 247.1310.
Heptyl 3-(furan-2-yl) propanoate was obtained as a colorless
liquid in 85% yield after silica gel column purification using hex-
ane as solvent. 1H NMR (CDCl3, 400 MHz) ı ppm: 7.34 (s, 1H, CH),
6.31 (s, 1H, CH), 5.06 (s, 1H, CH), 4.14–4.11 (t, 2H, CH2, J = 6.8 Hz),
3.03–2.99 (t, 3H, CH2, J = 7.2 Hz), 2.71–2.68 (t, 3H, CH2, J = 7.2 Hz),
1.75–1.64 (m, 2H, CH2), 1.35–1.33 (m, 8H, CH2), 0.94–0.92 (t, 3H,
CH2, J = 6.8 Hz); 13C NMR (CDCl3, 100 MHz) ı ppm: 172.5, 154.1,
141.1, 110.1, 105.1, 64.6, 32.7, 31.6, 28.8, 28.5, 25.8, 23.4, 22.5, 13.9;
IR (neat, cm−1): 2927, 1735, 1467, 1161; HRMS(EI): calculated for
2. Experimental procedures
2.1. Materials
ILs [Bmim]BF4, [Bmim]PF6 and [Bmim]Tf2N were prepared as
reported earlier [17]. They were purified according to a published
procedure [18] by filtration through silica gel to remove halide
impurities and then washed with sodium carbonate to remove
acidic impurities. Lipase PS was bought from Amano Pharmaceu-
ticals Co., Nagoya, Japan. All other chemicals were obtained from
commercial sources locally and were of analytical grade. The con-
versions were determined by HPLC analysis using a Jasco PU-1580
liquid chromatograph with a PDA detector using a Sil-C18 col-
umn (size 4.6 mm × 250 mm). The eluent used was acetonitrile and
water (85:15) at a flow rate of 1 ml min−1. Column chromatogra-
phy was done on silica gel (100–200 mesh). 1H NMR and 13C NMR
spectra were recorded in CDCl3 on a JEOL GSX-400 MHz spectrom-
eter. Chemical shifts are expressed in ppm values using TMS as an
internal standard. IR spectra were recorded on a Shimadzu IR 470
Instrument. Mass spectra were recorded on a Q TOF micromass
spectrometer. TLC was done using Kieselgel 60 F254 aluminium
sheets (Merck 1.05554). The water content in the ILs was deter-
mined with an 831 Metrohm Karl–Fischer coulometer.
C
14H23O3 (M+H)+, 239.1644; observed 239.1647.
Octyl 3-(furan-2-yl) propanoate was obtained as a colorless liq-
uid in 87% yield after silica gel column purification using hexane
as solvent. 1H NMR (CDCl3, 400 MHz) ı ppm: 7.32 (s, 1H, CH),
6.29 (s, 1H, CH), 6.04 (s, 1H, CH), 4.12–4.09 (t, 2H, CH2, J = 6.8 Hz),
3.01–2.97 (t, 3H, CH2, J = 7.2 Hz), 2.69–2.65 (t, 3H, CH2, J = 7.2 Hz),
1.65–1.62 (m, 2H, CH2), 1.46–1.30 (m, 10H, CH2), 0.93–0.90 (t, 3H,
CH2, J = 7.2 Hz); 13C NMR (CDCl3, 100 MHz) ı ppm: 172.5, 154.1,
141.1, 110.1, 105.1, 64.7, 32.7, 31.8, 31.5 29.4, 29.6, 28.5, 14.03; IR
(neat, cm−1): 2985, 1736, 1505, 1479, 1162; HRMS(EI): calculated
for C15H25O3 (M+H)+, 253.1796; observed 253.1804.
The conversion was determined by HPLC using a Sil-C18 column
by comparing the area under the peak of the product obtained with
that of the reactant, 3-(furan-2-yl) propanoic acid or ethyl 3-(furan-
2-yl) propanoate. The retention time (in minutes) for the acid and
esters were as follows: 3-(furan-2-yl) propanoic acid, 2.80; ethyl
3-(furan-2-yl) propanoate, 4.10; propyl 3-(furan-2-yl) propanoate,
4.54; butyl 3-(furan-2-yl) propanoate, 4.93; pentyl 3-(furan-2-yl)
propanoate, 5.69; hexyl 3-(furan-2-yl) propanoate, 6.85; heptyl 3-
(furan-2-yl) propanoate, 7.68; and octyl 3-(furan-2-yl) propanoate,
9.80.
2.2. Preparation of standard esters
3-(Furan-2-yl) propanoic acid was synthesized according to the
published procedure [19] and esterified as per reported procedure
for esterification [20] replacing DCC with EDCl. All the 3-(furan-2-
yl) propanoic acid esters, except hexyl, heptyl, and octyl (Table 1
entries 4, 5 and 6) are reported earlier. These were characterized
by 1H and 13C NMR and compared with literature data [21]. Hexyl
3-(furan-2-yl) propanoate was obtained as a colorless liquid in 90%
yield after silica gel column purification using hexane as solvent. 1H
2.3. Typical procedure for lipase PS catalyzed
esterification/transesterification
To screw capped test tubes of 3 ml capacity 3-(furan-2-
yl) propanoic acid (20 mg, 0.14 mmol) or ethyl 3-(furan-2-yl)
Scheme 1. (a) R = H, lipase PS catalyzed esterification of 3-(furan-2-yl) propanoic acid (1) in IL/hexane (b) R = CH3CH2, lipase PS catalyzed transesterification of ethyl-3-(furan-
2-yl) propanoate (2) in IL/hexane.

70
P. Vidya, A. Chadha / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 68–72
Table 2
Transesterification of 2 in ILs/hexane at 50 ◦C catalyzed by lipase PS.
Entry
ROH
Yield % of ester formed in
[Bmim]BF4
[Bmim]PF6
[Bmim]Tf2N
Hexane
1
2
3
4
5
6
CH3(CH2)2OH
CH3(CH2)3OH
CH3(CH2)4OH
CH3(CH2)5OH
CH3(CH2)6OH
CH3(CH2)7OH
5
4
7
5
6
5
6
9
8
10
12
9
18
21
22
20
22
16
2
2
6
4
2
2
propanoate (20 mg, 0.12 mmol), alcohol (0.42 mmol), 20 mg of
lipase PS and 500 ␮l of IL/hexane were added. This mixture was
kept in an orbital shaker at 50 ◦C at 200 rpm for 48 h (Scheme 1). At
intervals of 8 h, aliquots of 20 ␮l were taken in 1 ml of hexane–ethyl
acetate (90:10). For, ILs this biphasic mixture was vortexed for
2 min to extract all the reactants and products to the organic layer.
The organic layers were analyzed by HPLC to monitor the time
course of the reaction (extraction efficiency was >99%). After the
completion of the reaction, the reaction mixture was extracted into
hexane–ethyl acetate (90:10, 2 ml × 3), concentrated, purified by
column chromatography and characterized as given in Section 2.2.
Fig. 1. Time course of 3-(furan-2-yl) propanoic acid conversion by lipase PS to pro-
duce esters with propanol (ꢀ), pentanol (ꢁ), and octanol (ꢂ) in [Bmim] PF6 (—),
[Bmim] Tf2N (- -) and hexane (. . .).
hypothesized that the more polar transition state formed by the
3-(furan-2-yl) propanoic acid is stabilized by polar nature of ionic
liquids. For esterification, one of the products, water can act as a
nucleophile and can compete with the alcohol as a nucleophile
for the acyl enzyme intermediate. The attack of water as a nucle-
ophile results in hydrolysis (backward reaction) forming the acid
(substrate) and the attack by the alcohol leads to the ester prod-
uct (forward reaction). As water is a better nucleophile than all
the alcohols tried, hydrolysis could be expected to dominate over
esterification. But contrary to this, high yields were obtained for
esterification (Table 1). A reasonable explanation is that the bio-
transformation is conducted at 50 ◦C (Section 2.3) in 3 ml screw
capped vials. Most of the water formed (0.14 mmol or 2.52 mg in
case of 100% biotransformation as in Section 2.3) as a byproduct
during the esterification may evaporate into the free space of the
vial during the course of time, and is thus unattainable to act as a
nucleophile for the reverse reaction to occur, leading to high yields
of the product ester. The possibility of evaporation of low boiling
alcohols can be excluded since as discussed in Section 2.3, excess
alcohol (acid:alcohol, 1:3 molar ratio) was used for the biotransfor-
mation.
2.4. Lipase PS–IL/hexane mixture long-term stability and
recyclability for transesterification
To screw capped test tubes of 3 ml capacity, 20 mg of lipase PS
and 500 ␮l of [Bmim] PF6, [Bmim]Tf2N or hexane were added. The
resulting mixture was vortexed for 2 min and kept at room tem-
perature. At selected intervals of time, i.e. every 60 days up to 10
months, a fresh set of substrates (1 and alcohol) were added to
each lipase PS–IL/hexane mixture and the esterification reaction
was carried out as given in Section 2.3.
3. Results and discussion
3.1. A comparison of the lipase PS catalyzed esterification and
transesterification
The results for lipase PS catalyzed esterification of 3-(furan-
2-yl) propanoic acid with six alcohols (propanol to octanol) in
ionic liquids and hexane are summarized in Table 1 and that
for the transesterification of ethyl 3-(furan-2-yl) propanoate are
summarized in Table 2. The yields for the lipase PS catalyzed ester-
ification of 3-(furan-2-yl) propanoic acid were found to be 98–67%
for [Bmim]Tf2N, 58–17% for [Bmim]PF6, 9–7% for [Bmim]BF4 and
60–45% for hexane (Table 1), while for the lipase PS catalyzed trans-
esterification of ethyl-3-(furan-2-yl) propanoate the yields were
found to be quite low in all the media (22–16% for [Bmim]Tf2N,
12–6% for [Bmim]PF6, 7–4% for [Bmim]BF4 and 6–2% for hexane
as in Table 2). In both the media, ILs and hexane, for the lipase PS
catalyzed synthesis of 3-(furan-2-yl) propanoic acid esters, ester-
ification is a better choice than transesterification. The yields in
[Bmim]Tf2N were the highest while in [Bmim]PF6 and hexane were
lower but comparable, while [Bmim]BF4 showed very low yields.
Fig. 1 shows the time course of overall conversion of 3-(furan-2-yl)
propanoic acid catalyzed by lipase PS with propanol, pentanol and
octanol in [Bmim]PF6, [Bmim]Tf2N, and hexane. Fig. 2 shows over-
all conversion of 3-(furan-2-yl) propanoic acid catalyzed by lipase
PS with butanol, hexanol and heptanol in the above said media.
As found for the whole family of serine proteases a highly polar
tetrahedral intermediate (an oxyanion) is formed in the transition
state of this reaction [22]. The transition state formed with the 3-
(furan-2-yl) propanoic acid would be relatively more polar than
that formed with ethyl 3-(furan-2-yl) propionate. Hence it can be
Fig. 2. Time course of 3-(furan-2-yl) propanoic acid conversion by lipase PS to
produce esters with butanol (ꢀ), hexanol (ꢁ), and heptanol (ꢂ) in [Bmim]PF6 (—),
[Bmim]Tf2N (- -) and hexane (. . .).

P. Vidya, A. Chadha / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 68–72
71
3.2. Role of reaction medium on biotransformation
hydrophilic nature lacks this stabilization effect on the hydropho-
bic inside surface of active site. The nucleophilicity of these ions
are in the order of F4− > PF6− > Tf2N−. Another reason for the high
activity in [Bmim]Tf2N could be that, the enzyme compatible anions
exhibit lower nucleophilicity and thus would show lower tendency
to change the enzyme’s conformation by interacting with the pos-
itively charged sites in the enzyme structure [35].
The yields for lipase PS catalyzed esterification (Table 1) in all
the three ILs and hexane were found to be higher than the yields of
tranesterification (Table 2). The results for the lipase PS catalyzed
esterification of 3-(furan-2-yl) propanoic acid (Table 1), demon-
strate that there is a remarkable dependence of the nature of the
reaction media on the yields of the products formed. Among all the
three ILs tried, [Bmim]BF4, [Bmim]PF6, and [Bmim]Tf2N, the high-
est yields were obtained in [Bmim]Tf2N (98–67%). Lipase PS was
found to be catalytically more active in the ionic liquids containing
1-alkyl-3-methylimidazolium cation in combination with anions
It is reported that lipase PS needs calcium ion for its activity
and stability [36,37]. There are six oxygen atoms from six calcium
ion ligands which stabilize the calcium binding [38]. Ionic liquids,
in particular their anions that form strong hydrogen bonds with
these ligands may result in loss of calcium binding and thus desta-
bilize the enzyme structure thereby reducing its catalytic activity.
Since ability of these anions to form hydrogen bonds are in the
order, BF4− > PF6− > Tf2N−, [Bmim]BF4 due to its strong hydrogen
bonding ability may be reducing the calcium binding thus leading
to poor activity. BF4− can also react readily with the internal hydro-
gen bonds of the enzyme thus reducing its activity [9,39]. Moreover,
this esterification reaction was carried out under anhydrous condi-
tions (since, only IL was used as the reaction medium, without any
addition of water as a co-solvent), it is possible that [Bmim]BF4 due
to its high water affinity removes the tightly bound water from the
enzyme molecule causing its deactivation while the hydrophobic
ILs preserve the critical water molecules in the microenvironment
of the enzyme, thus preventing its deactivation due to the loss of the
essential water shell [40]. All these stabilizing/destabilizing effects
are absent in hexane since it is a very apolar molecule.
The experiments conducted (Section 2.4) clearly demonstrated
that while the lipase PS–[Bmim]Tf2N and lipase PS–[Bmim]PF6
were operationally stable up to 10 months at room temperature,
the lipase PS–hexane mixture was found to be completely inac-
tive after 2 months. Recyclability of the lipase PS–hydrophobic
IL/hexane mixture (Section 2.4) showed that both the lipase PS–IL
mixtures can be reused five times without any decrease in the
yields. In accordance with our observation earlier, [Bmim]PF6 and
[Bmim]Tf2N function as excellent stabilizing agents for lipase PS [9].
It should be kept in mind, however, that the hydrophobic effect,
which significantly contributes to protein stabilization, does not
exist in organic solvents [4]. The water content in the ILs for this
transesterification reaction, as determined by Karl–Fischer titra-
tion was found to be in the range 0.72–0.77% for [Bmim]Tf2N and 1.
09–1.13% for [Bmim]PF6. Hence lipase PS in these water-immiscible
ILs at saturation of water (‘wet IL’) can be considered as existing as
an aqueous solution, in which there are free water clusters which
preserve the critical water molecules in the microenvironment of
the enzyme, thus preventing its deactivation due to the loss of the
essential water shell [2,9]. The thermal stability of CAL-B at 50 ◦C
over 4 days was found to be 3–4 times higher in [Emim]Tf2N and
[Bm3N]Tf2N than in water or hexane [41]. The storage stability of
␣-chymotrypsin in the ionic liquid [EmIm]Tf2N was compared with
that in water, 3 M sorbitol, and 1-propanol. The enzyme’s lifetime
in [EmIm]Tf2N at 30 ◦C was more than two and six times as long as
those in 3 M sorbitol and water, respectively, and 96 times longer
than that in 1-propanol [2]. The activity of thermolysin was well-
retained after incubation in [BmIm][PF6] at 37 ◦C for 144 h, whereas
the same treatment in ethyl acetate resulted in the loss of almost
half of the enzyme activity [42].
such as PF6 and Tf2N− [10,11,23–26]. The yields for the esterified
−
products were comparable in hexane and [Bmim] PF6 the yields
were found to be very low in [Bmim]BF4.
Room temperature ILs (molten salts) are simply a composi-
tion of ions and remain liquids because the anions and cations
do not pack well [27]. In this context it is important to mention
that the effect of individual ions of inorganic salts on protein stabi-
lization is well known which follows a recurring sequence known
as the Hofmeister series [28]. According to Hofmeister phenom-
ena, the ions present in the aqueous solution of a protein induces
changes in the bulk water structure thus affecting the enzyme
performance. Kosmotropic anions and chaotropic cations stabi-
lize proteins, while chaotropic anions and kosmotropic cations
destabilize them [28,29]. The kosmotropicity of the anions in this
study follows the order BF4 > PF6 > Tf2N, but the yields of the esters
formed in this study (Tables 1 and 2) are completely contrary to this.
The enzyme was found to be most active in [Bmim]Tf2N followed
by [Bmim]PF6 and was least active in [Bmim] BF4. Earlier studies
also found the enzyme−to be more active and stable in ILs contain-
ing anions such as PF6 and Tf2N− [10,11,23–26]. It is a matter of
debate whether Hofmeister effects on proteins result from direct
interactions of ions with the enzymes or indirectly with the bulk
water [30]. Hence the results obtained in this study could not be
explained by Hofmeister phenomena.
It is evident that enzymes in ILs basically follow the same cat-
alytic mechanism as in organic solvents [31–33]. Therefore, an
ionic liquid may play the same role as an organic solvent and
depending upon its physicochemical properties, affect the enzyme
performance by (i) stripping off the essential water associated with
the enzyme, (ii) interacting with charged groups of the enzyme
and (iii) stabilize/destabilize the transition state. The crystal struc-
ture determinations of lipase PS has shown that it contains the
␣/␤ hydrolase fold, a structural motif common to a wide variety
of hydrolases [34]. In the ‘closed’ structures, both, a large deep
active site cleft and the catalytic residues are buried underneath
a helical segment, called a ‘lid’ or a ‘flap’. In the case of lipase
PS a relatively large number of residues (37 residues) out of 320
residues, are involved in the opening of the lid. As a consequence of
such a large-scale movement, the active site of lipase PS becomes
highly accessible to the solvent exposing the active site cleft and
the hydrophobic inside surface. The resolved structure reveals a
highly open conformation with solvent-accessible active site. The
loop regions are rearranged in such a way that tight intra molecular
contacts are possible in the new environment after opening of the
lid [34]. It can be suggested that lipase PS will be more active and
stable in a media which stabilizes this loop region. The excellent
activity in [Bmim]Tf2N could be partly attributed to its two oppos-
ing properties, high hydrophobicity and high polarity. Due to its
high hydrophobicity, [Bmim]Tf2N possibly stabilizes the active site
cleft and the hydrophobic inside surface of lipase PS in the active
site, at the same time its high polar nature may be stabilizing the
highly polar tetrahedral intermediate (an oxyanion) formed in the
transition state of this reaction [22]. [Bmim]PF6 and hexane do not
exhibit the same kind of stabilizing effect and [Bmim]BF4 due to its
3.3. Effect of alcohols on the lipase PS catalyzed esterification of 1
The effect of alcohols on conversion yields for lipase PS cat-
alyzed esterification of 3-(furan-2-yl) propanoic acid (Table 1) in
hydrophobic ILs, [Bmim]Tf2N and [Bmim]PF6 present some inter-
esting observations. The yields for all the alcohols are higher in
[Bmim]Tf2N [98–67%] than in [Bmim]PF6 [58–17%] but the pat-
tern was found to be the same. The yields were high for propanol,

72
P. Vidya, A. Chadha / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 68–72
butanol and pentanol in both media, 98–96% in the [Bmim]Tf2N and
58–48% in [Bmim]PF6. The yields decrease as the number of carbon
atoms increases, 82–67% in [Bmim]Tf2N and 22–17% in [Bmim]PF6,
except for octanol in [Bmim]PF6 (28%). This difference in yields due
to change in number of carbon atoms is not found to be much pro-
nounced in hexane, which yielded 60–45% of products with all the
alcohols.
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We have here compared the behavior of lipase PS for the ester-
ification of 3-(furan-2-yl) propanoic acid and transesterification of
ethyl 3-(furan-2-yl) propanoate with six straight chain alcohols
(propanol to octanol) in three ionic liquids and hexane. In order
to evaluate the influence of anions on the catalytic performance
of lipase PS, three ILs, [Bmim]BF4, [Bmim]PF6 and [Bmim]Tf2N,
with identical cation and different anions were selected as reaction
media for this biotransformation. This study showed that for the
preparation of these esters, lipase PS catalyzed esterification is bet-
ter choice than transesterification. [Bmim]Tf2N markedly enhanced
the yields of the products for esterification (98–67%), while in
[Bmim]PF6 and in hexane the yields (60–17%) were comparable.
The lipase PS–hydrophobic IL mixture was found to be operationally
stable up to 10 months and was recycled five times. It has there-
fore become clear that with the advent of ionic liquids the choice
of medium for carrying out biotransformation and thus the chance
to find one that is satisfactory has increased enormously.
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Acknowledgements
One of the authors [P. Vidya], is grateful to the University Grants
Commission, Government of India for financial support and the
Analytical Instrumentation Facility (SAIF); IITM for NMR spectra.
Padmanabhan Vidya was born in Tamil Nadu, India.
Graduated in Organic Chemistry (1991) and completed
post graduation in Chemistry (1994) from Calicut Univer-
sity, India. After a span of years in education sector, she
commenced active research career in the field of “Biocatal-
ysis in Ionic liquids” under the guidance of professor Anju
Chadha, IIT Madras. She was awarded Junior Research Fel-
lowship (2004) and Senior Research Fellowship (2006)
from University Grant Commission, Government of India.
She obtained publications in peer reviewed journals and
presented her works in many national and international
conferences. Her research interest includes biocatalysis
in non-conventional media using isolated enzyme and
whole cells.
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