Improvement of catalytic activity of Candida rugosa lipase in the presence of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified iron oxide nanoparticles

Article abstract of DOI:10.1016/j.bioorg.2015.12.001

In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through sol-gel encapsulation. The catalytic activities and enantioselectivities of the two encapsulated lipases in the hydrolysis reaction of (R/S)-naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were assessed. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 246, respectively, compared to the free enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix-P) showed good loading ability and little loss of enzyme activity, and the stability of the catalyst was very good; they only lost 6-11% of the enzyme's activity after five batches.

Full text of DOI:10.1016/j.bioorg.2015.12.001

Accepted Manuscript
Improvement of catalytic activity of Candida rugosa lipase in the presence of
calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified
iron oxide nanoparticles
Elif Ozyilmaz, Mevlut Bayrakci, Mustafa Yilmaz
PII:
S0045-2068(15)30039-0
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.12.001
YBIOO 1862
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 September 2015
25 November 2015
1 December 2015
Please cite this article as: E. Ozyilmaz, M. Bayrakci, M. Yilmaz, Improvement of catalytic activity of Candida
rugosa lipase in the presence of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified iron
oxide nanoparticles, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.12.001
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Improvement of catalytic activity of Candida rugosa lipase in the presence
of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes
modified iron oxide nanoparticles
a
Elif Ozyilmaz , Mevlut Bayrakci and Mustafa Yilmaz *
b
a
a
Department of Chemistry, Selcuk University, Konya-42075, Turkey.
b
Karamanoglu Mehmetbey University, Department of Bioengineering, Karaman-70200, Turkey
Abstract
In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation
method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of
calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase
was then directly immobilized onto the modified nanoparticles through sol–gel encapsulation.
The catalytic activities and enantioselectivities of the two encapsulated lipases in the
hydrolysis reaction of (R/S)-Naproxen methyl ester and (R/S)-2-phenoxypropionic acid
methyl ester were assessed. The results showed that the activity and enantioselectivity of the
lipase were improved when the lipase was encapsulated in the presence of calixarene-based
additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an
excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-
phenoxypropionic acid methyl esters, with E=350 and 246, respectively, compared to the free
enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix–P) showed good
loading ability and little loss of enzyme activity, and the stability of the catalyst was very
good; they only lost 6-11% of the enzyme’s activity after five batches.
Keywords: Calix[4]arene; Magnetite nanoparticles, Enantioselective; Lipase immobilization.
_
___________________________________________________________________
∗
To whom correspondence should be addressed. Tel: +90 332 2233873 ; fax: +90 332 2412499
E-mail: myilmaz42@gmail.com (M.Yilmaz).

1
. Introduction
Lipase is a very important enzyme that catalyzes a large number of processes such as
hydrolysis, esterification, and transesterification reactions [1,2]. Therefore, this enzyme is
among the most sought biocatalysts by the food industry, chemistry, and the pharmaceutical
industry [3,4]. Among others, Candida rugosa lipase (CRL) has been widely studied for its
selectivity with respect to specific functional groups and to stereo- and regioselectivity
because of its high activity and broad specificity [5,6]. However, using a native enzyme as a
biocatalyst presents several disadvantages, such as poor stability under operational conditions,
difficulty of product recovery, high cost of operation, and the impossibility of multiple reuse
in industrial processes [7]. The immobilization of free lipase onto solid supports, which have
many advantages including easy recycling and reusing; large, specific surface areas; catalytic
and operational stability; easy dispersion; reduction of cost; etc., via covalent binding[8-10],
entrapment, [11-13] or adsorption [14-18] can overcome those problems [19].
The sol–gel encapsulation method is particularly easy for preparation without any
covalent modification compared to the other immobilization matrices. It also offers several
advantages. It can entrap a large amount of enzymes, is chemically and thermally stable, and
acts as an excellent catalyst in the kinetic resolution of chiral aromatic alcohols and
carboxylic acids [20-26]. However, researchers would experience difficulty separating the
nanoparticles from the solution. This problem can be resolved by using the magnetic property
[
3 4
3,27]. Fe O nanoparticles have been considered suitable for the immobilization of enzymes
because of their multifunctional characteristics, including small size, low toxicity, easy
separation from the reaction medium, reusability, and, indirectly, lower processing costs
[
3,28,29]. Furthermore, with magnetic separation, there is no need for expensive and time-
consuming techniques such as chromatography, centrifugation, and filtration.

Several methods have been recommended to improve the performance of lipase-
catalyzed reactions for optical resolution. These methods include the optimization of reaction
conditions, modification of substrate, optimization of the nature of the solvent [30] and of the
water content [31], chemical and non-covalent modifications of enzymes, and the use of an
additive that regulates lipase reactivity. Among these options, the additive method is the most
advantageous [32]. However, while it is simple to use, only a few compounds have been
reported to enhance the enantioselectivity of lipase reactions as an additive [33,34]. Several
macrocyclic compounds such as cyclodextrins [35], crown ethers [36] and calix[n]arene
derivatives have been used in this capacity [37, 38].
Calixarenes are important building blocks in supramolecular chemistry. They can be
selectively functionalized at both the phenolic OH groups (lower rim) and the para positions
of the phenol rings (upper rim) to achieve desirable goals [39]. Regarding the immobilization
of lipase via the sol–gel method, we have reported the use of calixarene derivatives as
additives [40,41]. The results showed that the calix[n]arene-based encapsulated lipases had
higher enantioselective conversion as compared to the sol–gel-free lipase in the hydrolysis
reaction of (R/S)-naproxen methyl ester [42-46]. By introducing magnetic properties to
organic molecules or to biomolecules, researchers can make the tasks of separation and
reusable processes easy due to magnetic speciation.
Within this framework, there have been several works about the synthesis and
characterization of the lower rim functionalized calix[4]arene derivatives grafted onto
magnetic nanoparticles and evaluated for the enantioselective hydrolysis reactions
[
19,37,38,45,46]. However, up to this time, there has been no other reported study about upper
rim functionalized calix[4]arene derivatives grafted onto magnetic nanoparticles and their
catalytic activities towards enantioselective hydrolysis reactions. Therefore, the aim of the
present study was to synthesize two upper rim-functionalized calix[4]arene derivatives

bearing iminoacetic acid and/or phosphonate groups and immobilize them onto magnetic
nanoparticles as the new additives for the sol–gel encapsulation of Candida rugosa lipase.
Furthermore, the activity and enantioselectivity of the encapsulated lipases were also
evaluated under different conditions such as temperature and pH influences.
2
. Materials and methods
2
.1. Materials
Lipase from Candida rugosa (type VII), bovine serum albumin used as the standard for
protein assay, and p-nitrophenylpalmitate (p-NPP) used as the substrate to estimate the
enzyme activity were supplied by from Sigma-Chemical Co. and OTES and TMOS were
obtained by Merck. Without further purification or drying, high-performance liquid
chromatography (HPLC) grade organic solvents were used as a mobile phase. All other
chemicals used in this work were of reagent grade or analytical grade and were obtained from
various commercial sources. IR spectra were performed on a Perkin Elmer spectrum 100 FT-
IR spectrometer (ATR). UV/Vis spectra were measured with a Perkin Elmer Lambda 25
spectrometer. High performance liquid chromatography (HPLC) Agilent 1200 Series were
performed using a 1200 model quaternary pump, equipped Diode Array detector. Pure S-
naproxen was provided from TCI. Racemic naproxen was produced in the laboratory by the
racemization of optically pure S-naproxen as described by Wu and Liu [47]. Racemic
naproxen methyl ester has been prepared according to published method [48].
2
.2. Synthesis
The parent compounds (1) and (2) were prepared according to published procedures.
Calix[4]arene (3) containing phosphonate groups at the upper rim, was synthesized by the
reaction with corresponding chloromethylated derivatives of calix[4]arene (2) which was

synthesized according to modified literature procedure [51] and trimethyl phosphite in
chloroform at reflux [52,53] (Scheme 2) while calix[4]arene (4) containing carboxyl groups
was synthesized by the reaction with chloromethylated calix[4]arene (2) and iminodiacetic
3 4
acid. Magnetite nanoparticles (Fe O ) and epoxy groups modification on magnetite
nanoparticles were prepared according to literature procedures [9,12,29]. The p-
iminocarboxylic acid and p-phosphonate derivative of calix[4]arene immobilized magnetite
nanoparticles (Fe-Calix-N(COOH) and (Fe-Calix–P) were herein reported for the first time.
2
.2.1. Synthesis of p-iminocarboxylic acid derivative of calix[4]arene (4)
To a stirred solution of compound 2 (1 mmol) in dry acetonitrile (20 ml), pyridine (12 mmol)
and 2-(carboxymethylamino) acetic acid (4.1 mmol) were added. The color of the mixture
changed to pale purple from the colorless after 30 min. The reaction mixture was refluxed
under nitrogen atmosphere for 24 h and then the solvent was removed under reduced pressure.
The residue was treated with a 1 N HCl solution (50 mL) and extracted with dichloromethane
2
4
x50 mL. The combined organic extracts were washed with water, dried over MgSO and
then the solvent was removed. The residue was treated with ethyl ether and chloroform and
each time the solvent removed under reduced pressure. Product 4 was first obtained by
precipitation with ethyl ether from the dichloromethane solution. The compound 4 was
obtained in 42 % yield as a cream colored solid with m.p. 158-159 °C. IR (ATR) (ν): 1733s
-
1 1
(
C=O) cm . H NMR (CD
and AB system ArCH Ar), 3.53 (d, 4H, J=13.2 Hz, AB system ArCH
ArCH N). Anal. Calc.: C48 20. C, 57.37; H, 5.22; N, 5.58%. Found: C, 57.41; H, 5.13;
N, 5.66%.
3
OD, 400 MHz): 6.98 (s, 8H, Ar-H), 4.23-4.15 (m, 20H, NCH
2
CO
2
2
Ar), 3.21 (s, 8H,
2
52 4
H N O

2
.2.2. Preparation of Fe-Calix-N(COOH) and Fe-Calix–P
The Fe-Calix–P (5) and Fe-Calix-N(COOH) (6) magnetite nanoparticles were prepared by
the ring-opening polycondensation of the epoxy groups of bisglycidyl magnetite nanoparticles
and the phenolic hydroxy groups of calix[4]arenes (3) and/or (4). A solution of corresponding
2 3
calix[4]arene derivative (3) or (4) (1.12 mmol) and K CO (4.5 mmol) was stirred in
anhydrous acetonitrile (20 mL) and then magnetite nanoparticles modified with bisglycidyl
groups (1.5 g) in 30mL anhydrous acetonitrile was added dropwise to this mixture with
continuous stirring at room temperature for about 30 min under nitrogen atmosphere. Then
the reaction mixture was refluxed under a nitrogen atmosphere for 5 days. After magnetic
separation, obtained magnetite nanoparticles were washed in sequence three times with warm
toluene, methanol, acetone, dichloromethane and distilled water. The product was dried under
vacuum at 70 °C for 3 h to give 1.5 g of magnetite nanoparticles modified with calixarene
skeleton (5) and/or (6) and kept in desiccators before use.
2
.2.2.1. Fe-Calix–P (5):
According to the elemental analysis, the bonded calixarene amount onto Fe
3
4
O was found to
-1
be approximately 0.076 mmol of 3/g of Fe-Calix–P. IR (ATR) mmax /cm : 1603; 1475;
1
227; 1196; 1029; 686; elemental analysis, C, 22.67; H, 2.55; P, 0.95 %.
2
.2.2.2. Fe-Calix-N(COOH) (6):
According to the elemental analysis, the bonded calixarene amount onto Fe
be approximately 0.134 mmol of 4/g of Fe-Calix-N(COOH). IR (ATR) mmax/cm : 1613;
479; 1221; 1151, 626; 562, elemental analysis, C, 23.51; H, 3.21; N, 0.75 %.
3 4
O was found to
-
1
1

2
.3. Sol–gel encapsulation of lipases
A modified method of Reetz [36] was carried out in the sol-gel encapsulation of lipase with or
without additives including calixarene derivative immobilized Fe magnetic nanoparticles
5 or 6). Typically, a mixture of CRL-type VII (0.49 g) in 50 mM phosphate buffer (3.12 mL)
adjusted at pH 7.0 was shaken. Calixarene derivative immobilized Fe magnetic
nanoparticles (5 or 6) was added to the mixture, and followed by addition of polyvinyl alcohol
800 µL, 4% w/v), 0.1 M NaF (400 µL), isopropyl alcohol (800 µL), tetramethoxysilane (920
3
O
4
(
3
O
4
(
µL, 0.5 mmol) and octyltrimethoxysilane (6.4 mL, 2.5 mmol), respectively. After 10-15 s, 25
mL of isopropyl alcohol was poured onto the produced white solid. Obtained gel was washed
with 20 mL of ultra pure water and isopropyl alcohol, and it was lyophilized to afford the
encapsulated lipases. The resulting encapsulated lipases were lyophilized and stored at 4 °C
prior to use. The filtrate was collected and the amount of immobilized protein in the
encapsulated lipases was calculated from the protein mass balance among the initial and final
lipase solutions. The protein concentration in solution was determined with Coomassie
Brilliant Blue reagent following Bradford’s method [55]. BSA was used as standard to ensure
that the calibration curve. Each reported value was the mean of three experiments at least, and
the standard deviation was within ca. ±5%.
2
.4. Determination of encapsulated lipase's stability against different pH conditions.
The hydrolysis reaction of p-NPP in 0.05 M of buffer (pH 7.0) was performed to assess the
activities of the encapsulated lipases with or without additives. The concentration of the
corresponding hydrolysis product was determined at 400 nm using a spectrophotometer [54].
The Bradford protein assay method [55] relying on measurement of initial and final
concentration of protein was used to find protein content to the encapsulated lipases.
2
.5 Effect of pH and temperature on the activity

The effect of temperature on the free and immobilized enzyme activity was
determined by incubating in phosphate buffer (50 mM, pH 7.0) at temperatures ranging from
2
5 °C to 60 °C for 20 min, after cooling, the remaining activity was assayed under the
standard conditions and measured. The effect of pH on the activity of the free and
immobilized enzyme was determined by incubating at room temperature in phosphate buffer
(
50 mM) at pH ranging from 4 to 9, followed by determination of enzymatic activity at pH 7.0
as described in activity assay section. The remaining activity of the immobilized lipase was
normalized to the initial value determined at 37 °C, pH 7.0 (the initial activity was defined as
1
00%).
2
.6. Thermal Stability
Free and immobilized lipase preparations were stored in the phosphate buffer solutions
50 mM, pH 7.0) at 60 °C for 2 h, respectively. Samples were periodically withdrawn for
activity assay. The remaining activities were determined as above.
(
2
.7. Reuse of encapsulated lipase
The stability of immobilized lipase under conditions of repeated magnetic isolation
and reuse was studied under the same conditions as described in activity assay section. After
each enzyme run, the immobilized lipase (Fe-Calix–P and Fe-Calix-N(COOH)) were
magnetically isolated and washed twice with isooctane and ultrapure water to remove any
remaining substrate and product species before the next experiment (Scheme 2). The residual
activity of the immobilized lipase after each cycle was normalized to the initial value (the
initial activity was defined as 100%).
Please insert Scheme 2.

2
.8. Enantioselective hydrolysis of racemic arylpropionic acid methyl ester by
encapsulated lipases
R/S)-Naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were
(
hydrolyzed by the encapsulated lipases with or without additives in a mixture of PBS and
isooctane according to the literature procedure [54,55]. The conversion and enantioselectivity
of (R/S)-Naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester by the
encapsulated lipases were expressed as the enantiomeric ratio (E), which was calculated from
the conversion (x) and the enantiomeric excess of the substrate (ee
using HPLC [56].
s p
) and the product (ee ) by
ln[ (1- x) (1-ee ) ]
s
E=
ln[ (1- x) (1+ee ) ]
s
C - C
R
ees
C
R
- C
S
S
ee =
s
ee =
p
x =
C + C
S R
ee + eep
s
C
R
+ C
S
s p R S
E, ee , ee , x, C and C imply enantiomeric ratio for irreversible reactions, enantiomeric
excess of substrate, enantiomeric excess of product, racemate conversion, concentration of R-
enantiomer and concentration of S-enantiomer, respectively.
3
. Results and discussion
In this work, two new magnetite nanoparticles (Fe-Calix–P) and (Fe-Calix-N(COOH)
containing phosphoric and carboxylic acid derivative of calix[4]arenes were prepared. The
synthetic route was given in Scheme 1. The parent compounds (1) and (2) were prepared
according to published procedures.
Please insert Scheme 1.

Calix[4]arene (3) bearing phosphonate groups was synthesized through the reaction of
trimethyl phosphite with corresponding chloromethylated derivatives of calix[4]arene (2),
according to a modified literature procedure [51-53]. Calix[4]arene (4) containing
iminodicarboxyl groups was synthesized by the reaction of chloromethylated calix[4]arene (2)
1
and iminodiacetic acid. All of the structures have been characterized through H NMR, FTIR
and elemental analyses. A typical AB pattern was observed for the methylene bridge
1
Ar protons at 3.53 ppm and 4.20 ppm (J=13 Hz) for 4 in the H NMR. The high field
ArCH
2
doublets at 3.53 ppm for 4 were assigned to the equatorial protons of methylene groups,
whereas the low field signals around 4.20 ppm for 4 were assigned to the axial protons in the
1
H NMR. FTIR spectroscopy was used to elaborate the structure of Fe-Calix–P (5) and Fe-
-1
Calix-N(COOH) (6) (Fig. 1.). The FTIR bands at low wavenumbers (≤700 cm ) come from
-1
vibrations from the Fe–O bonds of iron oxide. The IR peak around 575 cm belongs to the
stretching vibration mode of Fe–O bonds in magnetite nanoparticles. Compared with the IR
spectrum of Fe-Calix–P (5) and Calix-N(COOH) (6), the Calix[4]arene bearing phosphonate
-1
groups possessed peaks around 1227 cm (for 5) attributable to stretching vibrations of the
-
P=O group and 1613 cm (for 6) which are due to stretching vibrations of the carbonyl
1
-
1
-1
groups (C=O). Furthermore, the peaks at 1475 cm for 5 and 1479 cm for 6 are attributed to
the bending vibration of the aromatic C=C bonds of the calix[4]arene skeleton. Additional
-
1
-1
peaks centered at 1029, 686 cm , 626 and 592 cm for compounds 5 and 6 were most
probably due to the symmetric and asymmetric stretching vibration of the framework and
terminal Si-O groups.
Please insert Fig. 1.

Transmission electron microscopy (TEM) analysis was used to obtain more information about
the particle size and morphology of pure Fe nanoparticles (Fig. 2a) Fe-Calix-N(COOH)
Fig. 2b) and (Fe-Calix–P) (Fig. 2c). As shown in Fig. 2a, the nanoparticles form intensive
3
O
4
(
Please insert Fig. 2.
aggregates due to the lack of any repulsive force between the magnetite nanoparticles, which
is due to the nanosize of the pure Fe nanoparticles, which are about 8–10 nm. After
3
O
4
calix[4]arene derivative immobilization, the size of the particles was enlarged to 15–20 nm.
The dispersion of the particles was also greatly improved (see Fig. 2b and 2c), which can be
explained by the electrostatic repulsion force and steric hindrance between the calix[4]arene
on the surface of Fe
3
O
4
nanoparticles.
Please insert Fig. 3.
The thermal properties of (Fe-Calix–P) and (Fe-Calix-N(COOH)) were tested using
thermogravimetric methods (Fig. 3). The indication of the coating formation on the surface of
the magnetic nanoparticles can be obtained using TGA measurement. The main weight loss
0
°
temperature ranges were 280–600 C (20%) for Fe-Calix–P and 150–600 C (26%), for Fe-
Calix-N(COOH). Thermogravimetric results showed a direct relationship between the loss of
mass and the number of calixarene molecules that were bound to the nanoparticle surfaces.
3
.1. Sol–gel procedure for the encapsulation of candida rugosa lipase on additives
In our previous study, carboxylic acid derivatives of calix[n]arene were used as additives in
the sol–gel encapsulation process of lipases in order to estimate its catalytic ability in the
enantioselective hydrolysis of racemic naproxen methyl ester [19-22]. It was found that the
activity and enantioselectivity of the encapsulated lipase with carboxylic acid derivatives of
calix[n]arene were extremely high when compared with the affinity of the encapsulated lipase

without any additive. The calix[n]arenes, cone-shaped molecules with a hydrophobic cavity
that can accommodate a variety of functional groups, have great potential as protein surface
binders[51]. This increase in the activity and enantioselectivity of the lipase was attributed to
a host-guest interactions and the cooperative affinities between the carboxylic acid (Fe-Calix-
N(COOH)) and phosphonic acid (Fe-Calix–P) groups of calix[n]arene with the guanidinyl
group of arginine, the sulfhydryl group of cysteine, the imidazolyl group of histidine, the ε-
amino group of lysine, and the indolyl group of tryptophan[51-53].
The hydrolytic activity of the encapsulated lipases was evaluated in the hydrolysis of p-
nitrophenylpalmitate (p-NPP). Table 1 shows the specific activities and protein amounts of
the encapsulated lipases with p-NPP. It has been observed that the encapsulated lipases with
phosphonic acid (Fe-Calix–P) and iminodicarboxylic acid (Fe-Calix-N(COOH) derivatives
of calix[4]arene were found to be more efficient as compared to the encapsulated free lipase
with respect to the expression of encapsulated lipase activity[49, 50]. This is not a surprising
result because the iminodicarboxylic acid and phosphonic acid of the derivatives of
calix[4]arene containing carboxyl and phosphonyl groups are highly effective complexing
agents [51-53], which means that some of these groups interact with the active center of lipase
by a combination of hydrogen bonding and electrostatic interactions. Most probably, the
lipase is not only physically encapsulated, but there are also additional multi-point
interactions through hydrogen bonding, ionic, and hydrophobic interactions .
3
.2. Effect of pH and temperature on the activity of encapsulated lipase
The catalytic activity of the encapsulated Candida rugosa lipase in the hydrolysis of p-
NPP was investigated at different pH levels (4.0–9.0). This pH range is known to be one of
the most influential parameters for altering enzyme activity in an aqueous system.
Immobilization is mostly mentioned for its result in a conformational change of the enzyme,

which can make a difference in catalytic activity, while localized influences of the matrix
itself can affect properties of enzyme, especially at optimum pH levels. Therefore, an enzyme
needs to be fixed with a support material in order to protect its effective abilities. For this
concept, calixarene immobilized magnetic nanoparticles were used and encapsulated onto
Candida rugosa lipase [42].
As shown in Fig. 4, the optimum pH levels of the encapsulated lipase of magnetic
nanoparticles using phosphonic acid derivatives of calixarene (Fe-Calix–P) or
iminodicarboxylic acid derivatives of calix[4]arene (Fe-Calix-N(COOH) as additives were
5
.0 and 6.0, respectively. Immobilized lipase shows better pH stability and resistance to an
acidic environment than free encapsulated lipase does [25]. After immobilization, the
optimum pH level for reactions catalyzed by free lipase was slightly shifted toward acidic
values. The immobilized lipase offered better pH stability and, hence, better resistance to
acidic environments than the free lipase. After immobilization, variations in the optimum pH
range on hydrolysis activity have been reported by some researchers. These variations depend
on the methods used for immobilization and the secondary interactions (hydrophilic and
hydrophobic) between the enzyme and the support [57], as well as when the enzymes are
immobilized onto poly cationic supports [58].
Please insert Fig. 4.
The effect of temperature in the range 30-60 °C on the activity of encapsulated lipase without
calixarene nanoparticles and phosphonic acid derivative of calixarene (Fe-Calix–P), or
iminodicarboxylic acid derivatives of calix[4]arene (Fe-Calix-N(COOH) with at pH 7.0 is
given in Fig. 5. The observed optimum temperature for the encapsulated lipase without

calix[4]arene derivatives (free lipase) was approximately 35 °C, while this optimum
temperature shifted to nearly 40 °C for Fe-Calix–P and Calix-N(COOH. This difference
could be clarified by either a more rigid structure of the encapsulated enzymes or the
formation of conformational limitations on the lipase movements as the results of improved
resistance of the protein to thermal denaturation [61].
Please insert Fig. 5.
3
.3. Thermal stability on the activity of encapsulated lipases
Fig. 6 shows the thermal stabilities of the encapsulated lipase without calix[4]arene
nanoparticles (free lipase) and the encapsulated lipases (Fe-Calix–P and Calix-N(COOH).
°
The encapsulated lipases were incubated for 2 h at 60 C and the enzyme activity was
measured at various time intervals. It was observed that the free lipase loses its initial activity
°
within around 100 min at 60 C, while the encapsulated lipases retain their initial activities at
about 60% by Fe-Calix-N(COOH and 50% by phosphonic acid derivative (Fe-Calix–P) after
°
20 min of heat treatment at 60 C. Improvement in the thermal stability of encapsulated
1
enzymes typically reflects the interaction between the lipase containing lysine, imidazole,
thiol and indole moieties can be complexed with iminodicarboxylic acid or phosphonic acid
groups of calix[4]arene as well as host–guest interactions and the cooperative affinities of
calix[4]arene derivatives. This aids in preventing conformational transitions of the enzyme at
high temperatures[62].
Please insert Fig. 6.
3
.4. Enantioselective hydrolysis of racemic -naproxen methyl and 2-phenoxypropionic
acid methyl esters with the encapsulated lipases

The pharmaceutical industry has many uses for these enantiopure compounds. Several
studies have demonstrated that using lipases is a more effective strategy for obtaining pure
enantiomers with higher biological activities than using conventional chemical methods. In
fact, the use of lipases recently has been established for the synthesis of chiral drugs [40].
Enantiopure compounds are currently achieved by chiral chemical synthesis using
metal catalysts or by enzymatic kinetic resolution of racemic mixtures. The kinetic resolution
depends not only upon the degree of enantioselectivity but also on the activity of the lipase
and the possibility for recycling and reusing the lipase. Candida rugosa lipase is widely used
3
7
in biotransformation due to its high activity in hydrolysis as well as synthesis. In
supramolecular chemistry, the crown ethers, cyclodextrins, and calixarenes represent the most
important macrocyclic host molecules [37,40]. In our literature review, we observed that
cationic lysine with calix[n]arenes and their derivatives will form complexes [43,45,46]. It has
been known that one characteristic of lipase is the lid structure model, which refers to an
oligopeptide chain around the active site [56,59,60]. In aqueous media, the active site is totally
covered with this lid, so it is isolated from the reaction medium, resulting in a closed and inactive
conformation of lipase. However, when the lid is removed by ionic interaction, hydrogen bonds or
hydrophobic interaction, the lipase experiences a partial equilibrium between the open and closed
conformations, with part of the active site being accessible to substrates and with activity largely
increased [56,59,60].
In view of this point, in the current study some of the calixarene derivatives
substituted with iminodicarboxylic and phosphonic groups would present a challenge for
enhancing not only the enantioselectivity but also the activity of the lipase in the hydrolysis
reaction of racemic Naproxen methyl ester and 2-phenoxypropionic acid methyl ester. From
the results, Table 2 shows the conversion (X), enantiomeric excess (ee) and enantiomeric ratio
(
E) in the course of racemic Naproxen methyl ester and 2-phenoxypropionic acid methyl ester
hydrolyzed by the encapsulated lipases in an aqueous buffer solution/isooctane reaction

system at optimum pH and temperature. For the hydrolysis of racemic-2-phenoxypropionic
acid methyl ester and racemic Naproxen methyl ester (Table 2), however, using Fe-Calix–P
shows a high conversion and enantioselectivity at the optimum pH and temperature. The
enantioselectivity of encapsulated lipases towards the hydrolysis of (R/S)-2-phenoxypropionic
acid methyl ester and (R/S)-Naproxen methyl ester at the optimum pH and temperature is
shown in Table 2. According to the results in Table 2, the resolution reaction with lipase
encapsulated with phosphonic acid derivative (Fe-Calix–P) was analyzed after 24 h at the
optimum pH and temperature. This reaction took place with the ester (R-ester) for the
naproxen methyl ester and the ester (S-ester) for 2-phenoxypropionic acid and corresponding
acids (eep) of >98% at a conversion yield of 48.1% and 44%,designated their
enantioselectivity as E=350 and E=246 respectively.
The enantioselectivity of encapsulated lipases Fe-Calix–P and (Fe-Calix-N(COOH)
against racemic 2-phenoxypropionic acid methyl ester was not very high; therefore, the
changes of the enzyme properties could be easily detected. Table 2 shows that the R-isomer of
2
-phenoxypropionic acid methyl ester was always hydrolyzed quicker with the higher
enantiomeric ratio obtained at pH 7 [60,63]. In contrast, the resolution reactions with
encapsulated lipase without calix[4]arene derivatives gave a corresponding acid (eep) (98%)
at a conversion of 20% with the enantioselectivity (E) being 137. Immobilization led to high
enantioselectivity, high conversion, and fast recovery of the product compared with free
enzymes.
An effort was made to visualize the recovery and reusability capacities of the
3 4
encapsulated lipases regarding their magnetic properties because of the Fe O magnetic
nanoparticles that were used (see Fig. 7a and 7b). Fig. 7a-b shows that Fe-Calix-P and Fe-
Calix-N(COOH) still retained 39% and 35% of their conversion ratios for naproxen methyl
esters after the fifth reuse, respectively. These results demonstrate that the encapsulated

3 4
lipases with calixarene and Fe O magnetic nanoparticles would make them unique candidates
in terms of effective and economical usage of enzymes.
Please insert Fig. 7a.
Please insert Fig. 7b.
After the encapsulations of CRL in the presence of calix[4]arene derivative
3 4
immobilized Fe O magnetic nanoparticles (Fe-Calix-P and Fe-Calix-N(COOH)), they were
used in the reusability studies, where after each run the encapsulated lipases were washed
with PBS. It was found that the percentage of conversion (x) and enantiomeric ratio (E) of the
encapsulated lipase decreased after the second usage. Fig. 7a shows that the encapsulated
lipases were still retained, with 39% and 35% of their conversion ratios for base encapsulated
lipases using naproxen methyl esters as substrate. In contrast, 38% and 36% of conversion
ratios were obtained for the encapsulated lipases using racemic 2-phenoxypropionic acid
methyl ester (Fig. 7b) after the 5th reuse, respectively.
Conclusions
In this study, phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene
3 4
have been synthesized and grafted onto iron oxide nanoparticles. The calixarene grafted Fe O
magnetic nanoparticles (Fe-Calix-N(COOH)) or (Fe-Calix–P) were used as additives for the
encapsulation of Candida rugosa lipase via the sol–gel process to afford two different
encapsulated lipases. Two new encapsulated lipases were employed as catalysts for the
enantioselective hydrolysis reaction of (R/S)-2-phenoxypropionic acid methyl ester and (R/S)-
naproxen methyl ester. It was found that Fe-Calix–P exhibited higher enantioselectivity with

an E value of 350 for (R/S)-2-phenoxypropionic acid methyl ester than did the encapsulated
lipase without an additive, which had an E value of 193. These findings show that phosphonic
acid moieties of calixarene play a substantial role in the lipase encapsulation.
Moreover, due to the low cost of the sol–gel encapsulation process, the excellent
performance of the lipase-immobilization, and its ready recyclability, given the basis of these
results, we recommend immobilized lipases as a prospective preparation.
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Figure Captions
Fig. 1. FTIR spectra of phosphonic acid derivative of calix[4]arene (3), iminoacetic acid
derivative of calix[4]arene (4), Fe-Calix–P and Fe-Calix-N(COOH).
3 4
Fig. 2.TEM micrographs of (a) Fe O -nanoparticles, (b) Fe-Calix-N(COOH) (c) Fe-Calix–P
Fig. 3. TGA curves of Fe-Calix–P and Fe-Calix-N(COOH).
Fig.4. Effect of substrate pH on residual activity of encapsulated lipases
Fig. 5. Effect of reaction temperature on the residual activity of encapsulated lipases
Fig. 6. Thermal stability of encapsulated lipases.
Fig.7a. Reusability on the conversion (x) in the hydrolysis of racemic naproxen methyl ester
Fig.7b. Reusability on the conversion (x) in the hydrolysis of racemic 2-phenoxypropionic
acid methyl ester
Scheme 1. Preparation of Fe-Calix–P and Fe-Calix-N(COOH. Reaction conditions: (i)
NaOH, diphenyl ether, 120 °C; (ii) AlCl
chloroform, rt; (iv) (CH O) P, chloroform, reflux; (v) BTMS, methanol, rt; (vi) iminodiacetic
acid, pyridine, acetonitrile.
3 8 2 4
, phenol, toluene, rt, 3 h; (iii) C H17OCH Cl, SnCl ,
3
3
Scheme 2. Enantioselective hydrolysis of (R/S)-naproxen methyl and (R/S)-2-phenoxy-
propionic acid methyl esters,

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7a.
Fig. 7b.