Enzymatic esterification of fatty acid esters by tetraethylammonium amino acid ionic liquids-coated Candida rugosa lipase

Article abstract of DOI:10.1016/j.molcatb.2012.03.003

Enzymatic production of fatty acid esters from the esterification of oleyl alcohol with various fatty acids was investigated by using two new tetraethylammonium amino acid ionic liquids-coated Candida rugosa lipase (ILs-CRL) as biocatalysts in hexane. Both enzyme derivatives were prepared by mixing Candida rugosa lipase with tetraethylammonium l-histidinate (IL1) and tetraethylammonium l-asparaginate (IL2). The ILs-CRL system containing the equivalent protein concentration as in CRL showed higher esterification activity especially on medium chain fatty acids (C₁₂-C₁₆) as compared to non-coated CRL. Hydrophilicity of ILs may play an important role in hydrogen bonding with enzyme surface and consequently stabilize the ILs-CRL.

Full text of DOI:10.1016/j.molcatb.2012.03.003

Journal of Molecular Catalysis B: Enzymatic 79 (2012) 61–65
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzymatic esterification of fatty acid esters by tetraethylammonium amino acid
ionic liquids-coated Candida rugosa lipase
Mohd Basyaruddin Abdul Rahmana,b,∗, Khairulazhar Jumbria, Nurul Ain Mohd Ali Hanafiaha,
Emilia Abdulmaleka, Bimo Ario Tejoa, Mahiran Basria, Abu Bakar Sallehc
a Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b Structural Biology Research Centre, Malaysia Genome Institute, Jalan Bangi, 43000 Kajang, Selangor, Malaysia
c Laboratory of Industrial Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Enzymatic production of fatty acid esters from the esterification of oleyl alcohol with various fatty acids
was investigated by using two new tetraethylammonium amino acid ionic liquids-coated Candida rugosa
lipase (ILs-CRL) as biocatalysts in hexane. Both enzyme derivatives were prepared by mixing Candida
rugosa lipase with tetraethylammonium l-histidinate (IL1) and tetraethylammonium l-asparaginate
(IL2). The ILs-CRL system containing the equivalent protein concentration as in CRL showed higher
esterification activity especially on medium chain fatty acids (C12–C16) as compared to non-coated
CRL. Hydrophilicity of ILs may play an important role in hydrogen bonding with enzyme surface and
consequently stabilize the ILs-CRL.
Received 1 November 2011
Received in revised form 30 January 2012
Accepted 8 March 2012
Available online 16 March 2012
Keywords:
Amino acid ionic liquids
Candida rugosa lipase
Coated-enzyme
Biocatalyst
© 2012 Elsevier B.V. All rights reserved.
Esterification
1. Introduction
including immobilization of the enzyme onto many supports [3–7],
cross linking [8,9], modification with polyethylene glycol [10], poly-
Fatty acids or derivatives of fatty acids are used in a wide vari-
ety of applications. The demand for fatty acids has been growing
over the past 20 years and the number of it is still increasing. Fatty
acids are consumed in the preparation of various fatty acid esters
by either chemical or enzymatic reaction but the latter is preferred.
The advantage of enzymatic catalytic reaction comparing with con-
ventional methods is that the reaction can be performed under
mild reaction conditions. Therefore, it is more advantageous for
producing high value-added ester products for pharmaceutical and
oleochemicals industry.
Lipase (glycerol ester hydrolase, EC 3.1.1.3) has been widely
employed in many organic reactions. Lipases have gained spe-
cial attention over few decades owing to their ability to act
in micro-aqueous environment and catalyze esterification, trans-
esterification, aminolysis, acidolysis reactions [1]. However, lipase
in non-aqueous media often suffers from reduced activity, selec-
tivity and stability [2]. Efforts have been made for improving lipase
efficiency in organic media through various types of modifications
mers [11], lipid- and surfactant-coated enzymes [12,13], molecular
imprinting [14], lyophilization [15] and salt-mediated activation of
lipases [16,17].
These immobilized or modified enzymes demonstrate bet-
ter activity, selectivity and high thermal stability. However, the
procedure faced a few difficulties such as low reactivity of inter-
mediates and easy cleavage of the modifier protein. Among these
examples, the salt-mediated activation of lipases is interesting as
enzymes could be activated by salts with kosmotropic anions dur-
ing lyophilization [18–20]. The salts of kosmotropic anions are
mainly achieved especially by using ionic liquids.
Ionic liquids (ILs) are known as a salt containing pure ions that
melt below 100 ◦C. From this definition, ILs can be divided into two
groups in which liquid and solid state ILs. They are widely used
in many type of reaction such as extraction and separation tech-
nologies [21,22], electrochemical [23,24], biocatalysis application
[25,26] and etc. Most of ILs is used as a solvent in many kinds of
organic reaction that involved lipase as a biocatalyst. Many of them
showed improved lipase activity especially in esterification reac-
tion and sometimes can denature enzyme’s activity. High solvent
polarity of ILs can have an adverse effect on lipase activity and ILs
appears as unsuitable solvents in view of their often high polarity
[27]. Therefore, less polar ILs appears appropriate for lipase catal-
ysis. Many groups reported the successful use of ILs as reaction
∗
Corresponding author at: Department of Chemistry, Faculty of Science, Univer-
siti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.: +603 8946 6798;
fax: +603 8943 2508.
E-mail address: basya@science.upm.edu.my (M.B. Abdul Rahman).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2012.03.003

62
M.B. Abdul Rahman et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 61–65
O
2. Materials and methods
NH
NH2
NH2
O
N
N
O
N
2.1. Materials
NH2
O
O
Lipase from Candida rugosa (E.C. 3.1.1.3 Type VII) was purchased
from Sigma Chemical Co. (St. Louis, MO). Substrates, solvents and
other chemicals were purchased from Sigma–Aldrich-Fluka Chem-
ical Co, and were of the highest purity analytical grade (AR).
IL2
IL1
Fig. 1. Two different types of new amino acid-based ionic liquids used as a coat-
ing material with Candida rugosa lipase (CRL). Tetraethylammonium l-histidinate
[N2222][his] (IL1) and tetraethylammonium l-asparaginate [N2222][asn] (IL2).
2.2. Synthesis of tetraethylammonium-based amino acids ILs
Two new amino acids-based ionic liquids tetraethylammo-
nium l-histidinate ([N2222][his], IL1) and tetraethylammonium
l-asparaginate ([N2222][asn], IL2) were prepared according to our
previous work [33] (Fig. 1). An aqueous solution of tetraethylam-
monium hydroxide (20%, w/w, 50 mmol) was added to a solution of
excess amino acid (55 mmol). The reaction mixture was stirred for
2 h at room temperature. Then, the crude products were obtained
after being dried at 70 ◦C under vacuum for 1 day. Since the crude
product was miscible with EtOH and amino acid was nearly insol-
uble in EtOH, the crude product was added into EtOH (50 mL) to
precipitate the excess of amino acid. After filtration, the solvent
was evaporated and the product was obtained after being dried
in vacuo for 1 day at 65 ◦C. Both ILs were kept in a desiccator prior
to use.
media, but only few reported the use of ILs as a coating material
with enzyme.
An emerging biotransformation technique is the employment
of coated lipase beads, where the coating is ILs, and uses them
with solvent [28–30] or in solvent free conditions [31]. Lee
and Kim [32] was the first group reported a very interesting
salt-mediated activation of lipase, which they called “ionic liquid-
coated enzyme (ILCE)” by using Pseudomonas cepacia lipase (PCL)
mixed with 1-(3ꢀ-phenylpropyl)-3-methylimidazolium hexafluo-
rophosphorate IL ([PPMIM][PF6]). The preparation of ILCE was
simple and showed enhanced activity, enantioselectivity and reli-
able stability when compared to commercial PCL in toluene, but
no significant modification of the reaction rate was obtained.
Very recently, they come out with the same approach [20]. Their
room temperature solid-phase ionic liquid (RTSPIL) co-lyophilized
enzyme exhibited markedly enhanced activity in organic solvent.
The enzyme co-lyophilized with a dodecyl-imidazolium salt was
660-fold more active compared to its RTSPIL-free counterpart. They
also found that the activity enhancement by RTSPILs was mainly
attributable to the reduced particle sizes and improved dispersion
of enzymes suspended in organic solvent.
Another interesting biocatalyst was studied by Itoh and his
group [28,29]. Their group discovered that extraordinary accel-
eration was accomplished by their “ionic-liquid coating” with
lipase from Burkholderia cepacia in an iPr2O solvent system,
while maintaining excellent enantioselectivity as well as enhanc-
ing enantioselectivity for some substrates. Not only native lipase
have been used, the immobilize enzyme also been used with ILs.
Lozano and his group [31] reported ionic liquid coated immobi-
lized Candida antarctica lipase B (Novozyme 435) was improved the
irreversible transesterification of citronellol by up to 2 fold with
a range of fatty acid vinyl esters. An homologous series of com-
mercially available imidazole-based hexafluorophosphates, such
as [bmim][PF6], [hmim][PF6] and [omim][PF6], were employed as
the ILs coating. Recently, the similar approached using Novozyme
435 has been reported. Coating of immobilized enzymes with ILs
is readily realized by the use of equal quantities of enzyme and ILs.
The results showed positive correlation with increasing polarity ILs
used as liquid-film coating, which was in opposition to the use of
the same ILs as solvent [27].
2.3. Preparation of tetraethylammonium amino acids ionic
liquid-coated Candida rugosa lipase (ILs-CRL)
2.0 g of tetraethylammonium l-histidinate (IL1, m.p. 54 ◦C 1.0)
was melted to its liquid phase by heating above 54 ◦C. Candida
rugosa lipase (CRL, 0.2 g mass equivalent) was added to this liq-
uid phase and the mixtures were stirred to get a heterogeneous
solution. The solution was then allowed to cool to room temper-
ature until the IL1-CRL mixture solidified. The solidified mixture
was ground into powder. The IL1-CRL particles were then used
without any further treatment. The same procedures were repeated
for tetraethylammonium l-asparaginate (IL2, m.p. 58 ◦C 1.0). This
method was modified from Lee and Kim [32].
2.4. Typical enzymatic esterification of fatty acids esters
The enzymatic reaction system consisting of oleyl alcohol
(2.0 mmol), fatty acid (1.0 mmol) and biocatalyst (CRL 15 mg; ILs-
CRL 165 mg containing the equivalent protein concentration as in
15 mg native CRL) was incubated in hexane (5.0 mL) at 50 ◦C for
1 h with continuous shaking at 150 rpm (Scheme 1). The steps
were used for adipic acid (C6), lauric acid (C12), myristic acid (C14),
palmitic acid (C16), stearic acid (C18) and oleic acid (C18:1) with a
1:1 ratio with oleyl alcohol (modified method from Chaibakhsh
et al. [34]). All experiments were done in triplicate and control
experiments were performed without biocatalysts.
Based on the positive results obtained from the literatures and
as an extension of our ongoing research program on enzyme-
catalyzed reactions, we herein report the use of ILs-promoted
activation of lipase. Novel amino acids ionic liquid-coated Can-
dida rugosa lipase (ILs-CRL) was employed in the esterification of
fatty acid esters. Previously we have designed and synthesized sev-
eral new tetraethylammonium-based amino acids ILs and their
physico-chemical properties have been carried out [33]. All of them
showed hydrophilic character due to the amino acids’s used as
anion. Only two formed solid ILs with the melting point below 60 ◦C
meanwhile the rest formed liquid at room temperature. Therefore,
it was assumed that this two solid ILs should be suitable as a coating
material due to their hydrophilic character.
2.5. Analysis and characterization
The reaction was terminated by dilution with 3.0 mL of
ethanol:acetone (50:50, v/v) and the biocatalyst was filtered
through filter paper. A 5 ml sample was taken and the remaining
free fatty acid in the reaction mixture was determined by titra-
tion with 0.1 M NaOH using an Autotitrator (808 Titrando System,
Metrohm) to an end point of pH 10. The activity of biocatalyst was
expressed as percentage of conversion.
(Vcontrol − Vsample
)
Percentage of conversion =
× 100%
(1)
Vcontrol

M.B. Abdul Rahman et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 61–65
63
O
C
O
C
R1
ILs-CRL / non-coated CRL
O
R1 OH
OH
Hexane, 50 o
C
H2O
oleyl alcohol
R1 = Di-C6, C6, C12, C14, C16, C18 and C18-1
Scheme 1. Enzymatic esterification of various fatty acids and oleyl alcohol in the presence of amino acid ionic liquids coated-Candida rugosa lipase (ILs-CRL) and non-coated
CRL.
Vcontrol = volume of 0.1 M NaOH needed to titrate the control;
Vsample = volume of 0.1 M NaOH needed to titrate the sample.
The moles of acid reacted were calculated from the values
obtained for the blank (without biocatalyst) and the test samples.
The ester formed was expressed as equivalent to the conversion
of the acid. The ester formed was monitored by thin layer chro-
matography (TLC) using a mixture of chloroform/hexane (95:5) as
the solvent system. Product was also monitored by GC/MS on a
Shimadzu instrument (model GC 17A, model MS QP5050A; Shi-
madzu Corp., Tokyo, Japan) with a BPX5 column (0.25 mm 30 m,
25 mm). Production of di-oleyl adipate ester was characterized by
can affect the production of ester [35,36]. The effect of chain length
of fatty acids (C12–C16) in esterification with both ILs coated-CRL
catalysts was examined (Fig. 3). On average, both coated-enzymes
enhanced the conversion of medium fatty acids esters up to 0.5
fold than non-coated CRL, with the IL1-CRL system showing slightly
higher activity than IL2-CRL.
From the results obtained, it was found that ILs coated lipase
favored the esterification of fatty acids with medium chain lengths,
especially lauric acid (C12) with the production of lauric oleate up
to 87%. Similar studies on lipase from Candida cylindracea in ester-
ification of citronellol with various chain lengths of acids has been
carried out by Kawamoto et al. [37]. They observed that fatty acids
of longer chain lengths served as excellent acyl donors for esteri-
fication when compared to shorter chain fatty acids. However, the
decrease in enzyme activity with relatively long chain saturated
fatty acid (stearic acid, C18) may be due to the bulky chain moi-
ety. It possibly may restrain the molecule from free rotation in the
enzyme’s acyl binding site or impose a hindrance to attack by the
nucleophile (alcohol) [38]. It can be hypothesized that amino acid
anions from ILs play a specific role in assisting the lid of CRL in
order to adapt the medium chain fatty acid or unsaturated long
fatty acid. Overall, it was demonstrated that both ILs-CRL in most
cases are more effective than their native counterparts for short,
medium and long alkyl chain acids.
FT-IR spectroscopy with absorption bands at 1734 cm−1 for C
O
and 1062, 1169 and 1263 cm−1 for C–O stretching vibrations of
ester (Fig. 2).
3. Results and discussion
3.1. Catalytic activity of lipase by ILs-coated CRL system
Two different lipase preparations IL1-CRL and IL2-CRL were
applied in the esterification of fatty acids esters in order to com-
pare their catalytic activity with native CRL. The catalytic activities
of both ILs-CRL were higher by about one fold than CRL for short
(Di-C6 and C6) and long chain fatty acids (C18) as depicted in Fig. 3.
The conversion of di-oleyl adipate ester (Di-C6) by using IL1-CRL
and IL2-CRL was higher by 71% and 63% respectively as compared
to native CRL. However, for oleyl adipate ester (C6), both coated
enzymes showed similar trends for Di-C6. IL1-CRL presented a
higher conversion rate of about 72% as compared to 64% conver-
sion achieved by using IL2-CRL. Meanwhile, for native CRL, only 40%
conversion of product was obtained after 1 h of reaction at 50 ◦C.
Generally, each enzyme has its own selectivity and substrate
specificity. Length of the hydrocarbon chain of fatty acid, the pres-
ence of branched groups and the configurations of double bonds
As reported, the lyophilization is essential for inorganic salts to
activate the lipase [18,19]. Ueji et al. [39], reported that lyophiliza-
tion of CRL in the presence of some ionic compounds resulted in
Fig. 3. Effect of chain length of fatty acids as acyl donors in the esterification of fatty
acids esters by ILs-coated CRL (IL1-CRL and IL2-CRL) and non-coated CRL.
Fig. 2. FT-IR spectrum of di-oleyl adipate.

64
M.B. Abdul Rahman et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 61–65
dramatic improvements in its activity and enantioselectivity for
the esterification of 2-arylpropionic acid derivatives in an organic
solvent system. It is assumed that the coating procedure might be
essential for the activation and conformation of lipases by ILs.
In an ionic liquid-coated enzyme, the ILs may play an impor-
tant role in stabilizing the CRL in the organic solvent reaction. In
this context, the enzyme CRL was able to enhance its performance
without losing significant activity when it was coated with ILs and
the IL-coated CRL form had satisfactory stability. It was also found
that both IL1-CRL and IL2-CRL did not aggregate and that the coat
was stable and remained on the enzyme’s surface during the reac-
tion. It was hypothesized that the hydrophilic character of these
two new amino acids ILs, especially the anions may interact with
the hydrophilic enzyme’s surface to form intermolecular hydrogen
bonds between the amino acids’ anions and the essential water
layer on the enzyme’s surface.
3.2. Selection of the best ILs-coated CRL system
As mentioned previously, two different types of new ILs-CRL
were screened for their ability in the esterification of fatty acids
esters. Evaluation of their activity was based on the conversion of
esters. As shown in Fig. 3, high catalytic activity was obtained for
IL1-CRL if compared to IL2-CRL. This might be due to the confor-
mational structure of ILs used, as shown previously in Fig. 1. The
higher conversion rate of fatty acids esters produced in the pres-
ence of IL1-CRL is possibly due to the effect of the imidazole ring
in the side chain of l-histidine. As reported by Jarvo [45], both the
carboxylate and the secondary amine in the structure of l-histidine
were found to be essential for catalysis, which may be the reason
for the higher conversion rate of esters with IL1-CRL.
4. Conclusion
Another modifier used with enzyme is polyethylene glycol (PEG)
as reported by Nakashima et al. [10] and Yamasaki et al. [40].
Although PEG-modified enzymes have shown many advantages
such as the increased solubility, activity and stability when used in
organic solvents compared to the native enzymes, some disadvan-
tages still exist. As PEG is very hydrophilic, PEG-modified enzymes
have a strong hydrophilicity and high capacity of adsorbing water,
especially the water layer around enzyme’s surface. Besides, the
hydrophilic nature of PEG may adversely affect the solubility
and thermostability of the modified enzymes in organic solvents.
However, in case of ILs-CRL it was found that tetraethylammonium-
based amino acids ILs used as modifier do not showed strong
hydrophilic character as compared to PEG and also did not affect
the solubility of catalyst especially in hexane. From the results
obtained, it was assumed that the nature of hydrophilic charac-
ter of ILs used was said to be appropriate in order to maintain the
water layer on enzyme’s surface and thus stabilize the catalyst by
the interaction between ILs and surface of enzyme as discussed
above.
In different opinion based on hydrophobicity, Okahata and Mori
[41] observed similar intermolecular hydrogen bonds between the
lipid and enzyme in their lipid-coated enzyme. The presence of
hydrogen bonds with polar and non-polar regions may be respon-
sible for the stability of enzymes coated in ILs that can maintain
their functionality under extreme denaturative conditions. Thus,
both the solvophobic interactions are essential for maintaining
the native structure. The protein’s water shell is preserved by the
“inclusion” of the aqueous solution of free enzyme into the ILs net-
work, resulting in a clear enhancement of the enzyme’s stability
[42]. This is only a preliminary suggestion on how the enzyme and
the ILs bond during coating and further research in characteriz-
ing its physico-chemical properties and molecular modeling are
planned.
In conclusion, it was found that amino acids ILs-coated CRL
system as new biocatalyst caused remarkable catalytic activity of
lipase-catalyzed esterification of long chain fatty acid esters. In par-
ticular, the l-histidine-based served as an excellent partner with
CRL. Both ILs-coated CRL catalysts may be suitable for biotrans-
formation and biotechnology fields and provide new aspects in
theoretical and application of ILs in enzymatic reactions.
Acknowledgements
We are grateful for the financial support from the Genetics and
Molecular Biology Initiative and Universiti Putra Malaysia.
References
[1] B. Joseph, P.W. Ramteke, G. Thomas, Biotechnol. Adv. 26 (2008) 457–470.
[2] M.A. Klibanov, Trends Biotechnol. 15 (1997) 97–101.
[3] A.A. Mendes, H.F.De. Castro, S.D.De. Rodrigues, W.S. Adriano, P.W. Tardioli, E.J.
Mammarella, R.De.C. Giordano, R.De.L.C. Giordano, J. Ind. Microbiol. Biotechnol.
38 (2011) 1055–1066.
[4] K. Solanki, M.N.A. Gupta, Bioorg. Med. Chem. Lett. 21 (2011) 2934–2936.
[5] J. Forde, A. Vakurov, T.D. Gibson, P. Millner, M. Whelehan, I.W. Marison, C.
Ó’Fágáin, J. Mol. Catal. B: Enzym. 66 (2010) 203–209.
[6] M.B. Abdul Rahman, U.H. Zaidan, M. Basri, M.Z. Hussein, R.N.Z.R. Abdul Rahman,
A.B. Salleh, J. Mol. Catal. B: Enzym. 50 (2008) 33–39.
[7] U.H. Zaidan, M.B. Abdul Rahman, M. Basri, S.S. Othman, R.N.Z.R. Abdul Rahman,
A.B. Salleh, Appl. Clay Sci. 47 (2010) 276–282.
[8] N. Mileti, K. Loos, Aust. J. Chem. 62 (2009) 799–805.
[9] D.H. Lee, C.H. Park, J.M. Yeo, S.W. Kim, J. Ind. Eng. Chem. 12 (2006) 777–782.
[10] K. Nakashima, J. Okada, T. Maruyama, N. Kamiya, M. Goto, Sci. Technol. Adv.
Mater. 7 (2006) 692–698.
[11] C.A. Godoy, B. de las Rivas, M. Filice, G. Fernández-Lorente, J.M. Guisan, J.M.
Palomo, Process Biochem. 45 (2010) 534–541.
[12] A. Thakar, D. Madamwar, Process Biochem. 40 (2005) 3263–3266.
[13] B.-D. Song, H. Ding, S.-C. Wang, Biotechnol. Bioproc. E 12 (2007) 121–124.
[14] J.-Y. Yan, Y.-J. Yan, J.-K. Yang, L. Xu, Y. Liu, Process Biochem. 44 (2009)
1128–1132.
[15] Y. Mine, K. Fukunaga, K.-I. Samejima, M. Yoshimoto, K. Nakao, Y. Sugimura, J.
Biosci. Bioeng. 96 (2003) 525–528.
[16] T. Itoh, Y. Abe, T. Hirakawa, N. Okano, S. Nakajima, S. Hayase, M. Kawatsura,
T. Matsuda, K. Nakamura, in: S.V. Malhotra (Ed.), Ionic Liquid Applications:
Pharmaceuticals, Therapeutics, and Biotechnology, Oxford University Press,
Washington, DC, 2010, pp. 155–167.
[17] Y. Abe, T. Hirakawa, S. Nakajima, N. Okano, S. Hayase, M. Kawatsura, Y. Hirose,
T. Itoh, Adv. Synth. Catal. 350 (2008) 1954–1958.
Higher conversion rates in the reaction catalyzed by ILs-coated
CRL may also be explained by the hydrophilic nature of the ILs.
Generally, hydrophobic salts are preferable in organic reactions.
However, it is assumed that the hydrophilic character of ILs is
important since the water formed in the reaction is used by the
ILs and the ester forms a separate phase. Therefore, improved con-
version rates through better product separation can be achieved by
using hydrophilic amino acids ILs.
[18] M.T. Ru, S.Y. Hirokane, A.S. Lo, J.S. Dordick, J.A. Reimer, S.S. Clark, J. Am. Chem.
Soc. 122 (2000) 1565–1571.
Meanwhile, low activity of native CRL could be due to the effect
of high temperatures during the reaction. Lipase is easily denatured
at high temperatures [43] when the peptide bonds and amino acids
side chains become reactive and participate in deleterious reac-
tions [44]. This might be the reason why low conversion rates are
observed with native CRL in reactions at 50 ◦C. However, in both
ILs-CRL, the lipase’s active conformation is protected by ILs against
high temperatures, as both ILs [N2222][his] and [N2222][asn] were
thermally stable up to 170 ◦C [33].
[19] J.P. Lindsay, D.S. Clark, J.S. Dordick, Biotechnol. Bioeng. 85 (2004) 553–560.
[20] J.K. Lee, M.-J. Kim, J. Mol. Catal. B: Enzym. 68 (2011) 275–278.
[21] Y. Polyakova, Y. Jin, J. Zheng, K. Ho Row, J. Liq. Chromatogr. R 29 (2006)
1687–1701.
[22] M. Tian, H. Yan, K.H. Row, Anal. Lett. 43 (2010) 110–118.
[23] F. Endres, S. Zein El Abedin, Phys. Chem. Chem. Phys. 8 (2006) 2101–2116.
[24] J. Zhang, A.M. Bond, Analyst 130 (2005) 1132–1147.
[25] S. Sunitha, S. Kanjilal, P.S. Reddy, R.B.N. Prasad, Biotechnol. Lett. 29 (2007)
1881–1885.
[26] Y. Abe, K. Yoshiyama, Y. Yagi, S. Hayase, M. Kawatsura, T. Itoh, Green Chem. 12
(2010) 1976–1980.

M.B. Abdul Rahman et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 61–65
65
[27] J. Mutschler, T. Rausis, J.-M. Bourgeois, C. Bastian, D. Zufferey, I.V. Mohrenz, F.
Fischer, Green Chem. 11 (2009) 1793–1800.
[28] T. Itoh, S. Han, Y. Matsushita, S. Hayase, Green Chem. 6 (2004) 437–439.
[29] T. Itoh, Y. Matsushita, Y. Abe, S.-H. Han, S. Wada, S. Hayase, M. Kawatsura, Y.
Hirose, Chem. Eur. J. 12 (2006) 9228–9237.
[36] A.B. Salleh, M. Basri, M. Taib, H. Jasmani, R.N.Z.R. Abdul Rahman, M.B. Abdul
Rahman, C.N.A. Razak, Appl. Biochem. Biotech. 102–103 (2002) 349–357.
[37] T. Kawamoto, K. Sonomoto, A. Tanaka, Biocatalysis 1 (1987) 137–145.
[38] S.M. Radzi, M. Basri, A.B. Salleh, A. Ariff, R. Mohammad, M.B. Abdul Rahman,
R.N.Z.R. Abdul Rahman, Electron. J. Biotechn. 8 (2005) 291–298.
[39] S.-I. Ueji, S.-I. Mori, H. Yumoto, N. Hiroshima, Y. Ebara, Chem. Lett. 34 (2005)
110–111.
[30] A.P. de los Ríos, F.J. Hernández-Fernández, D. Gómez, M. Rubio, F. Tomás-
Alonso, G. Víllora, J. Supercrit. Fluids 43 (2007) 303–309.
[31] P. Lozano, R. Piamtongkam, K. Kohns, T. De Diego, M. Vaultier, J.L. Iborra, Green
Chem. 9 (2007) 780–784.
[40] M. Yamasaki, M. Okabe, T. Suzawa, Y. Yokoo, Biotechnol. Tech. 2 (1998)
751–754.
[32] J.K. Lee, M.J. Kim, J. Org. Chem. 67 (2002) 6845–6847.
[33] M.B. Abdul Rahman, K. Jumbri, M. Basri, E. Abdulmalek, K. Sirat, A.B. Salleh,
Molecules 15 (2010) 2388–2397.
[34] N. Chaibakhsh, M.B. Abdul Rahman, M. Basri, A.B. Salleh, R.N.Z.R.A. Rahman,
Biocatal. Biotransform. 27 (2009) 303–308.
[41] Y. Okahata, T. Mori, Trends Biotechnol. 15 (1997) 50–54.
[42] P. Kölle, R. Dronskowski, Inorg. Chem. 43 (2004) 2803–2809.
[43] D. Wei, C. Gu, Q. Song, W. Su, Enzyme Microb. Technol. 33 (2003) 508–512.
[44] C.O. Fágáin, in: C.O. Fágáin (Ed.), Protein Stability and its Measurement, Springer
Verlag Inc., Berlin, 1997, pp. 67–191.
[35] S.S. Othman, M. Basri, M.Z. Hussein, M.B. Abdul Rahman, R.N.Z.R. Abdul Rahman,
A.B. Salleh, H. Jasmani, Food Chem. 106 (2008) 437–443.
[45] E.R. Jarvo, S.J. Miller, Tetrahedron 58 (2002) 2481–2495.