Improvement of catalytic activity of lipase in the presence of wide rim substituted calix[4]arene carboxylic acid-grafted magnetic nanoparticles

Article abstract of DOI:10.1007/s10847-013-0332-z

Candida rugosa lipase immobilized on calix[4]arene carboxylic acid-grafted magnetic nanoparticles using a sol-gel encapsulation technique was tested for activity, which was assessed both in the enantioselective hydrolysis of racemic Naproxen methyl ester and that of p-nitrophenylpalmitate. It has also been noticed that, compared to the free enzyme (E = 137) with an ee value of [98 %, S-Naproxen calix[4]arene carboxylic acid-grafted magnetic nanoparticles based on encapsulated lipase (Calix-1-MN and Calix-2-MN) offer excellent enantioselectivity (E = 373 and E = 381). Moreover, the results indicated that after the fifth reuse in the enantioselective reaction, the encapsulated lipase (Calix-2-MN) still retained about 43 % of its conversion power. Springer Science+Business Media Dordrecht 2013.

Full text of DOI:10.1007/s10847-013-0332-z

J Incl Phenom Macrocycl Chem
DOI 10.1007/s10847-013-0332-z
ORIGINAL ARTICLE
Improvement of catalytic activity of lipase in the presence of wide
rim substituted calix[4]arene carboxylic acid-grafted magnetic
nanoparticles
•
Ezgi Akceylan Ozlem Sahin Mustafa Yilmaz
•
Received: 11 February 2013 / Accepted: 30 April 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Candida rugosa lipase immobilized on
calix[4]arene carboxylic acid-grafted magnetic nanoparti-
cles using a sol–gel encapsulation technique was tested for
activity, which was assessed both in the enantioselective
hydrolysis of racemic Naproxen methyl ester and that of
p-nitrophenylpalmitate. It has also been noticed that,
compared to the free enzyme (E = 137) with an ee value of
[98 %, S-Naproxen calix[4]arene carboxylic acid-grafted
magnetic nanoparticles based on encapsulated lipase
(Calix-1-MN and Calix-2-MN) offer excellent enantiose-
lectivity (E = 373 and E = 381). Moreover, the results
indicated that after the fifth reuse in the enantioselective
reaction, the encapsulated lipase (Calix-2-MN) still
retained about 43 % of its conversion power.
medium or in a harsh conditions such as high temperature
or excessive pH. The stability, catalytic activity, and
reusability of immobilized lipase are improved in contin-
uous operations by the immobilization of CRL on various
supports, providing the separation of products [1–3].
Investigations of the immobilization of CRL on different
carriers have been reported by a series of recent studies [3],
and carriers have included chitosan, amberlite, cyclodex-
trin, and calixarene [4–8]. The calix[4]arene platform in
supramolecular chemistry shows interesting organizational
properties for the construction of ligating sites to recognize
different species, which includes anions, cations, and
neutral molecules [9]. As receptors in supramolecular
chemistry, they have attracted much attention in the last
25 years. The increasing interest in these compounds is due
to the simple large-scale synthesis of calixarenes, and the
various methods by which they can be selectively func-
tionalized either at the wide rim or the narrow rim. The
nature and the number of donor groups and the confor-
mation of the calix[4]arene moiety are highly responsible
for the complexation properties of these molecules [9, 10].
Magnetic supports have been used in enzyme immobi-
lization [11–13] and cell separation [14, 15]. Magnetically
supported immobilized lipases can be recovered more
easily from a reaction with the help of external magnetic
field, in addition to offering the benefits of other solid
support media [16–18]. Nanoparticles of paramagnetic iron
oxides have been used in many applications in the last
decade [19], including for bioseparation [20, 21], tumor
hyperthermia [22], magnetic resonance imaging (MRI)
diagnostic contrast agents [23], magnetically guided site-
specific drug delivery agents [24], and the immobilization
of biomolecules [25–27].
Keywords Lipase ꢀ Enantioselectivity ꢀ Calix[4]arene ꢀ
Magnetic nanoparticles
Introduction
In biotechnology, lipases are the class of enzymes most
widely used in the kinetic resolution of racemic compounds
and organic synthesis [1]. In particular, lipase from Can-
dida rugosa has important industrial uses. It is well known
that Candida rugosa is used in a wide variety of esterifi-
cation reactions and hydrolysis [2]. The activity of Candida
rugosa lipase (CRL) is high and it also has broad speci-
ficity in reaction medium, as compared to free lipase,
which has low activity, and is usually unstable in organic
E. Akceylan ꢀ O. Sahin ꢀ M. Yilmaz (&)
Department of Chemistry, Selcuk University, 42031 Konya,
Turkey
In our previous study [6], the use of calix[n]arenes and
their lower rim substitute amine and carboxyl derivatives
e-mail: myilmaz42@yahoo.com
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J Incl Phenom Macrocycl Chem
as additives for lipase sol–gel encapsulation and the effect
of the derivatives of calix[n]arene on the hydrolysis and
enantioselectivity of racemic Naproxen methyl ester was
reported. Herein, we report about upper rim substitute
calix[4]arene carboxylic acid derivative-grafted magnetic
nanoparticles that were used as additives in the sol–gel
encapsulation process, and explore the influence of the
material on the hydrolysis and enantioselectivity of race-
mic Naproxen methyl ester.
5,11,17,23-Tetrakis[[(isonipecoticacido)methyl]-
25,26,27,28-tetrahydroxycalix[4]arene (3)
To a solution of calix[4]arene (1) (10 mmol) in 90 mL of
THF-DMF were added 11 mL of acetic acid, isonipecotic
acid (50 or 100 mmol), and 37 % aqueous formaldehyde
(50 mmol) and the reaction mixture was stirred for 24 h at
room temperature. The precipitate that formed was
removed by suction filtration. Received product was
washed with water and acetone and dried under vacuum.
Compound
3 was obtained in 79 % yield, m.p.
Materials and methods
328–330 °C. The IR (ATR) spectral data is as cm-1: 1,655
1
(COO). H NMR (400 MHz, CHCl3): d = 1.55 (q, 8H,
Materials
J = 11.1 Hz, NCH2CH2), 1.77 (m, 8H, NCH2CH2),
2.02–2.3 (m, 10H, NCH2CH2, CH2CH), 2.78 (m, 8H,
NCH2CH2), 3.15 (d, 4H, J = 14 Hz, ArCH2Ar), 3.32
(s, 8H, ArCH2N), 4.25 (d, 4H, J = 14 Hz, ArCH2Ar), 6.85
(s, 8H, Ar). Anal. Calcd for C56H68O12N4: C, 68.0; H, 6.93;
N, 5.66 %. Found: C, 68.31; H, 6.99; N, 5.71 %.
Lipase from C. rugosa (E.C.3.1.1.3, Type VII), p-nitro-
phenyl palmitate (p-NPP) used as the substrate to estimate
the enzyme activity, bovine serum albumin (BSA) used as
the standard for protein assay, TEOS (tetraetoxysilane) and
OTES (octyltrietoxysilane) were acquired from Sigma (St.
Louis, MO). HPLC grade organic solvents were used as the
mobile phase without further purification or drying. All other
chemicals used in this work were of analytical or of reagent
grade and became available from various commercial sour-
ces. IR spectra were obtained on a Perkin-Elmer spectrum
100 FTIR spectrometer (ATR). 1H NMR spectra were
recorded on a Varian 400 MHz spectrometer. Reactions
were monitored by TLC on pre-coated silica gel plates
(SiO2, Merck, 60F254). UV–vis. spectra were obtained on a
Shimadzu 160A UV–visible recording spectrophotometer.
High-performance liquid chromatography (HPLC) Agilent
1200 Series were carried out using a 1200 model quaternary
pump, a G1315Bmodel Diode Array and Multiple Wave-
length UV–vis detector, a 1200 model Standard and pre-
parative autosampler, a G1316A model thermostated
column compartment, a 1200 model vacuum degasser, and
an Agilent Chemstation B.02.01-SR2 Tatch data processor.
purchased from Sigma-chemical Co. (St. Louis, MO). Pure
S-Naproxen was purchased from Sigma (USA). Racemic
Naproxen was produced in the laboratory by the racemiza-
tion of optically pure S-Naproxen as described by Wu and
Liu [28]. Racemic naproxen methyl ester has been prepared
according to published method [6, 29].
Preparation of magnetic calix[4]arene derivative
(Calix-1-MN and Calix-2-MN)
A mixture of the compound 2 or 3 (0.6 g), potassium
carbonate (0.5 g) in acetonitrile (30 mL) was stirred for
30 min before adding 0.9 g of EPPTMS-MN and heated
under reflux for 3 days. After magnetic separation, the
resulted compound was washed with DMF (three times) to
remove excess compound 3, then washed with water and
dried under vacuum. The IR (ATR) spectral data of the
Calix-1-MN is as cm-1: 1,624 (C = O), 1,341 (aromatic
C = C), 1,031, 948 and 788 (Si–O) and Calix-2-MN is as
cm-1: 1,651(C = O), 1,578, 1,451 and 1,410 (aromatic
C = C), 1,035, 939 and 783 (Si–O).
Sol–gel encapsulation of lipase
Sol–gel encapsulated lipase with and without the
calix[4]arene grafted magnetic nanoparticles (Calix-1-MN
or Calix-2-MN) were prepared according to a modified
method of Reetz et al. [32]. A mixture of CRL (60 mg)
placed in a 50 mL erlenmeyer together with phosphate
buffer solution (PBS) (390 lL; 0.05 M; pH 7.0), which
was vigorously stirred on a horizontal shaker and a mixture
of Calix-1-MN or Calix-2-MN (50 mg), 100 lL of aque-
ous polyvinyl alcohol (PVA) (4 % w/v), aq. NaF (50 lL of
a 1 M solution) and i-PrOH (100 lL) was homogenized
using a shaker. Then, OTES (2.5 mmol) and TEOS
(0.5 mmol; 120 lL) were added and the mixture was agi-
tated once more for 10–15 s. Gelatin was usually observed
within seconds or minutes while gently shaking the
Synthesis
The syntheses of compounds 1, 2, Fe3O4 nanoparticles, and
[3-(2,3-epoxypropoxy)propyl]-grafted Fe3O4 nanoparticles
(EPPTMS-MN) were carried out according to published
procedures [27, 30, 31]. The other materials (3, Calix-1-MN
and Calix-2-MN) used in this work were prepared according
to the methods given below, as illustrated in Scheme 1.
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Scheme 1 A schematic
representation of the synthesis
of calix[4]arene carboxylic
acid-grafted magnetic
nanoparticles
reaction vessel. The gel was lyophilized and successively
washed with distilled water (10 mL) and isopropyl alcohol
(10 mL). The resulting encapsulated lipase was held at
4 °C prior to use.
[6, 33, 34]. All measurements were performed in triplicate
and an average was taken as final result.
Determination of protein assay
Activity of the sol–gel encapsulated lipases
Protein content was determined by the dye-binding method
of Bradford, using bovine serum albumin as a standard
[35]. The amount of bound protein was determined indi-
rectly from the difference between the amount of protein
introduced into the coupling reaction mixture and the
The catalytic activity, specific activity and the effect of pH
and temperature on activity of the sol–gel encapsulated
lipases were measured according to method in literature
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amount of protein in the filtrates and also in washings after
immobilization. The amount of immobilised enzyme was
calculated by subtracting the amount of unimmobilised
enzyme from the total amount of the lipase used for the
immobilisation.
in THF with secondary amine (piperidyl-4-carboxylic acid)
and formaldehyde to afford the cone conformer a 77 %
yield of 3. The structure of 3 was confirmed by spectro-
scopic and analytical data. The IR spectra of compound 3
1
shows a carbonyl band at 1,655 cm-1. In general, the H
NMR spectra of compound 3 showed singlet of d 3.32 ppm
(8H each) for ArCH2N. The conformational characteristics
of calix[4]arenes were conveniently estimated by the
splitting pattern of the ArCH2Ar methylene protons in the
1H NMR spectrum. The cone conformation of compound 3
was confirmed from 1H NMR spectroscopic data. The
methylene bridge of ArCH2Ar protons was observed by a
typical AB pattern at d 3.15 and 4.25 ppm (J = 14 Hz) for
3, in 1H NMR. The high field doublets at d 3.15 ppm were
assigned to the equatorial protons of methylene groups,
whereas the low field signals at d 4.25 ppm were assigned
to the axial protons in the 1H NMR. According to the
described method, the magnetic nanoparticles of Fe3O4
were prepared by the chemical co-precipitation of Fe2? and
Fe3? ions, with the ratio of their concentration selected in
the stoichiometric ratio of 2:1. By this method the Fe3O4
nanoparticles have a number of hydroxyl groups on the
surface to interact with the aqueous phase. EPPTMS-
modified Fe3O4 nanoparticles (EPPTMS-MN) were formed
by reaction between the hydroxyl groups on the surface of
the magnetite and the EPPTMS. As a result, the nanopar-
ticles were directly modified by [3-(2,3-epoxyprop-
oxy)propyl]-trimethoxysilane (EPPTMS) to introduce
reactive groups on the particle surfaces. Finally, calix[4]-
arene derivatives (2 or 3) were immobilized in the presence
of K2CO3 in acetonitrile to nanoparticles modified by that
surface [27]. The new compounds were characterized by a
combination of FTIR, TGA, elemental analyses, and SEM.
In the FTIR spectra given in Figs. 1 and 2, the appear-
ance of carbonyl bands at 1,624 cm-1 for Calix-1-MN and
at 1,578 cm-1 for Calix-2-MN also offer evidence for the
existence of carboxylate moieties on the nanoparticles. In
addition, the characteristic bands of the silane group at
1,031, 788 cm-1 for Calix-1-MN and at 1,035, 783 cm-1
for Calix-2-MN (Si–O) placed on the FTIR spectra have
confirmed the structure of Calix-1-MN and Calix-2-MN.
Thermal properties of Calix-1-MN and Calix-2-MN
were analyzed by thermogravimetric methods (Figs. 3 and
4). The indication of the coating formation on the magnetic
nanoparticles’ surface can be obtained from TGA mea-
surement. The main weight loss temperature ranges were
250–600 °C (32 %) for Calix-1-MN and 310–570 °C
(39 %), for Calix-1-MN. Thermogravimetric results
showed a direct relationship between the loss of mass and
the number of calixarene molecules bound to the nano-
particle surfaces.
General procedures for encapsulated lipase-catalyzed
enantioselective hydrolysis of esters
Hydrolysis reactions were carried out in an aqueous phase/
organic solvent batch reaction system. To a solution of
2 mL buffer solution (pH = 7.0, 50 mM PBS) containing
encapsulated lipases (5–50 mg depending on the activity)
was added a solution of racemic methyl ester (20 mM) in
2 mL of isooctane. The reactions were performed in a
shaker at 150 rpm at 35 °C and drawn samples from iso-
octane phase after 24 h.
HPLC were used to calculate the conversion and
enantioselectivity being expressed as the enantiomeric ratio
(E) calculated from the conversion (x) and the enantio-
meric excess of the substrat (ees) and the product (eep)
using the equation of Chen et al. [36].
ln½ð1 ꢁ cÞð1 ꢁ eesÞꢂ
E ¼
ln½ð1 ꢁ cÞð1 þ eesÞꢂ
where
ees
CR ꢁ Cs
CR þ Cs
Cs ꢁ CR
Cs þ CR
x ¼
ees ¼
eep ¼
ees þ eep
where E, ees, eep, x, CR and CS denote 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.
Results and discussion
Calix[n]arenes are a very important class of macrocycles
extensively used in supramolecular chemistry. They are
composed mainly of cyclic oligomers built with phenol
units through methylene bridges [9]. It has been suggested
that calixarenes could be regarded as the third generation of
supramolecules, after crowns and cyclodextrins [10].
In this study, we report the synthesis of two calix[4]-
arene carboxylic acid derivatives immobilized onto mag-
netic nanoparticles used as an additive in the
enanatioselective hydrolysis reaction of Naproxen methyl
ester. To achieve the desired goal, the synthesis of the
L-proline derivative of calix[4] arene 2 was carried out
according to the published method [31] and the calix[4]-
arene derivative 3 was conducted in the presence of AcOH
The Calix-1-MN and Calix-2-MN gave also satisfac-
tory analytical data, consistent with the proposed formulas
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Fig. 1 The IR spectrum of
compound 2, magnetic
nanoparticle (EPPTMS-MN)
and Calix-1-MN
Fig. 2 The IR spectrum of
compound 3, magnetic
nanoparticle (EPPTMS-MN)
and Calix-2-MN
integrating residual molecules. According to the elemental
analysis, (Table 1) the resulting Calix-1-MN contains
1.71 %, corresponding to 0.30 mmol, of 2/g of support and
Calix-2-MN contains 1.89 % nitrogen, corresponding to
0.34 mmol of 3/g of support.
irregular surface cavities of the Calix-1-MN and Calix-2-
MN (Fig. 5).
Sol–gel encapsulation of Candida rugosa lipase (CRL)
using calix[4]arene based magnetic nanoparticles
Scanning electron micrographs comparing the sol–gel
encapsulated lipases (Calix-1-MN-E and Calix-2-MN-E)
with the calixarene immobilized nanoparticles (Calix-1-
MN and Calix-2-MN) showed that the sol–gel encapsu-
lated nanoparticles had a porous surface compared with the
The original procedure for the encapsulation of lipases in
sol–gel materials produced by the fluoride-catalyzed
hydrolysis of mixtures of RSi(OCH3)3 and Si(OCH3)4 was
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Fig. 3 The TGA spectrum of
Calix-1-MN
Fig. 4 The TGA spectrum of
Calix-2-MN
Table 1 Results of elemental analysis for EPPTMS, Calix-1-MN
and Calix-2-MN
lipases show higher activity and enhanced enantioselec-
tivity. The mechanistic evidence for organic solvents in the
crown-ether-introduced activation of enzymes was descri-
bed by Reinhoudt et al. [37]. The 18-crown-6 may also
form complexes with cationic lysine group of enzymes, as
it is already known that it has a high affinity for forming
complexes with ammonium groups. The charge of the
lysine groups was screened after complexation, and
showed lower availability for salt bridge formation.
Therefore, the formation of intra- and inter-molecular salt
bridges of ether-lysine complexes might be reduced [1, 37].
C (%)
H (%)
N (%)
EPPTNS-MN
Calix-1-MN
Calix-2-MN
13.20
23.06
29.12
2.61
4.44
5.12
–
1.71
1.87
described by Reetz et al. [32]. Furthermore, this study
showed that in the presence of different additives such as
derivatives of cyclodextrin and 18-crown-6, encapsulated
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J Incl Phenom Macrocycl Chem
Fig. 5 SEM micrographs of a Calix-1-MN, b Calix-2-MN, c Calix-1-MN-E and d Calix-2-MN-E
Itoh et al. [38] also demonstrated that the crown ether
derivatives have the potential to enhance both the reaction
rate and enantioselectivity of the lipase-catalyzed hydro-
lysis of 2-cyano-1-methylethyl acetate. These crown
compounds cannot change the original enantioselectivity of
the enzyme, but enhance its potential ability to a level at
which the reaction can be used practically [38]. Moreover,
they observed that the macrocyclic effect causes the
activation. Like crown ethers, calix[n]arenes are also the
most important macrocyclic host molecules. The calixa-
rene derivatives are known to form complexes with cat-
ionic lysine [39].
We have successfully used carboxylic acid groups on
lower rim calixarene as additives for lipase immobilization
by sol–gel methods in our previous work [6]. The results
showed that, as compared to sol–gel free lipase, in the
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J Incl Phenom Macrocycl Chem
hydrolysis reaction of (R,S)-Naproxen methyl ester, calix-
arene-based encapsulated lipases offered distinctly higher
conversion rates and enantioselectivity. The purpose of this
study was to use the sol–gel encapsulation in the presence
of a chiral proline derivative of calix[4]arene (Calix-1-
MN) and a piperidyl-4-carboxylic acid derivative of
calix[4]arene-grafted magnetic nanoparticles (Calix-2-
MN) as the new additives to produce immobilized lipase.
These immobilized derivatives were used as a cata-
lyst in both the enantioselective hydrolysis reaction
of (R,S)-Naproxen methyl ester and hydrolysis of
p-nitrophenylpalmitate.
immobilized lipase. Generally, compared to free enzymes,
the optimum temperatures of immobilized enzymes are
shifted towards higher temperatures. At pH 7.0, the tem-
perature dependence of the p-NPP hydrolysis reaction
catalyzed by immobilized and free lipases was studied
from 30 to 60 °C, and these results are given in Fig. 7. The
observed optimum temperature for the encapsulated lipase
(enc-lipase) was approximately 35 °C, while it shifted to
nearly 40 °C for the Calix-1-MN and Calix-2-MN.
Enantioselective hydrolysis of racemic Naproxen
methyl ester with the encapsulated lipases
Table 2 shows the specific activities and protein
amounts of encapsulated lipases toward p-NPP. However,
the encapsulated lipase with Calix-2-MN was found to be
more efficient than Calix-1-MN with respect to the
expression of immobilized lipase activity. This result was
not surprising, because Calix-1-MN contains proline
groups, which ease intramolecular hydrogen bonding. In
this case, Calix-1-MN shows weaker interactions with the
enzyme than does Calix-2-MN. However, Calix-1-MN
was found to be less efficient compared to Calix-2-MN.
From an industrial point of view, the quality of a given
kinetic resolution not only depends upon the degree of
enantioselectivity, but also depends on the activity and the
possibility of recycling and reusing the lipase. The crystal
structure of Candida rugosa lipase exhibits two known
conformations [40, 41]. In one, called the ‘‘closed form,’’
the helical surface loop partially covers the hydrophobic
crevice containing the active site, while in the other con-
formation, called the ‘‘open form,’’ the surface loop moves
and the space is uncovered [42].
pH and temperature effect on the activity
of encapsulated lipases
It was recently reported by Kazlauskas et al. [43] that
2-propanol treatment of Candida rugosa lipase caused
modification of enantioselectivity. 2-Propanol treatment
was proposed to convert the closed form of this lipase to
the open form, thereby enhancing the enantioselectivity
[38, 43]. The present study demonstrated that the calixa-
rene carboxylic acid derivatives have the potential to
enhance both the enantioselectivity and reaction rate of the
lipase-catalyzed hydrolysis of rasemic-Naproxen methyl-
ester. These calixarene-based compounds cannot change
the basic enzymatic enantioselectivity, but increase its
potential ability to a level at which the reaction can be used
practically [6].
Because environmental conditions also affect enzymatic
activity, it was observed that when altering enzymatic
activity in an aqueous solution, pH is also one of the most
efficient parameters. In this study, the activity and the pH
effect on the free and immobilized lipase in the hydrolysis
of p-NPP were determined by altering the reaction medium
pH from 3 to 9.
As shown in Fig. 6, the optimum pHs of encapsulated
free lipase (enc lipase), Calix-1-MN-E and Calix-2-MN-E
as additives were 7.0, 6.0, and 5.0 respectively. The
immobilized lipase offered better pH stability and hence
resistance to acidic environments than did free lipase.
One of the most important parameters that must be
known to understand how the procedure of immobilization
affects enzymatic activity is the temperature profile of
In this work, to encapsulate CRL within a chemically
inert sol–gel support prepared by polycondensation by
(TEOS) and (OTES), derivatives of calix[4]arene carbox-
ylic acid were used as additives. The reactions were carried
out on a small scale, and on the basis of the formula of
Chen et al. [36] the enantioselectivity was determined by
measuring the selectivity factor E. A high catalytic activity
on the hydrolysis of (R,S)-Naproxen methyl ester was
exhibited by the immobilized CRL. Using the Calix-1-MN
and Calix-2-MN, high enantioselectivity E [ 300 was
achieved (Table 3).
Table 2 Initial specific activities and protein amounts of encapsu-
lated lipases toward p-NPP
Additives used
in sol–gel
process
Protein
loading
(mg/g-sol–gel)
Lipase
activity
(U/g-sol–gel)
Specific
activity
(U/mg-protein)
It was also found that calix[4]arene carboxylic acid
based additives have important effect son the stability of
CRL. All the results showed that the catalytic activity of
the CRL might have improved due to the sol–gel processes
with the calix[4]arene carboxylic acid derivatives used as
Calix-1-MN
Calix-2-MN
Free lipasea
a
27.3
19.1
28.5
42.4
47.5
95.1
1.60
2.48
3.32
Encapsulated lipase without support
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Table 3 The enantioselective hydrolysis of racemic Naproxen
methyl ester of using sol–gel encapsulated lipases as catalysts
Additives used in sol–gel process x (%) ees (%) eep (%)
E
Free-Ea
20.3
43.4
49.2
49.5
48.7
25
75
95
96
93
[98
[98
[98
[98
[98
137
224
373
381
341
Calix-1-MN-E (pH 7)
Calix-1-MN-E (pH 6)
Calix-2-MN-E (pH 7)
Calix-2-MN-E (pH 5)
Enantiomeric excess (ee) as determined by Chiral HPLC, Agilent
1200 Series -chiral column (Chiralcel OD-H); n-hexane/2 propanol/
trifluoroacetic acid (100/1/0.1, v/v/v) for 1 as mobile phase; time,
24 h; concentration of substrate, 20 mM; temperature, 35 °C
a
Encapsulated Candida rugosa lipase without supports
inactivation of the enzyme’s denaturation of protein and
the leakage of protein from the supports upon use. This
indicates that lipase encapsulated Calix-1-MN or Calix-2-
MN could be used in industrial applications.
Fig. 6 Effect of substrate pH on residual activity of encapsulated
lipases
Conclusions
In this work, C. rugosa lipase was immobilized on
calix[4]arene
carboxylic
acid-grafted
magnetic
Fig. 7 Effect of substrate temperature on residual activity of
encapsulated lipases
additives. The calix[4]arene-based nanoparticles employed
may interact with certain sites of the lipase as proposed in
some proteins, thereby activating the lipase and changing
its enantioselectivity.
In order to investigate whether or not variations in pH
affect the chiral selectivity of the enzyme as a catalyst
depending on the ionization state of the lipase, the effects
of pH on the enantioselectivity of lipase encapsulated
Calix-1-MN and Calix-2-MN was determined by incu-
bating at 35 °C at pH 7.0 and at optimum pH. The results
given in Fig. 8 show the optimum pH value was 6.0 for
lipase encapsulated Calix-1-MN and the optimum pH
value was 5.0 for lipase encapsulated Calix-2-MN.
The reusability of immobilized lipase is also important
for economical use of the enzyme. Figure 9 shows that the
ratios of conversion for Calix-1-MN after the fifth reuse of
the encapsulated lipases were 23 % for pH 6 and 13 % for
pH 7. For Calix-2-MN, these ratios were 43 % for pH 5,
and 29 % for pH 7 (Fig. 10). These results are due to the
Fig. 8 Effect of pH on the conversion (x) in the hydrolysis of
racemic naproxen methyl ester with Calix-1-MN-E and Calix-2-MN-
E
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Acknowledgments We would like to thank The Scientific and
Technical Research Council of Turkey (TUBITAK-Grant Number
111T027) and CMST COST Action CM1005 and The Research
Foundation of Selcuk University (BAP) for financial support of this
work.
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