Bioprocess Biosyst Eng
DOI 10.1007/s00449-014-1299-x
ORIGINAL PAPER
Improvement of catalytic activity of lipase in the presence
of calix[4]arene valeric acid or hydrazine derivative
Enise Akoz • Serkan Sayin • Selcuk Kaplan •
Mustafa Yilmaz
Received: 11 August 2014 / Accepted: 30 September 2014
Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Sol–gel encapsulation is a simple but powerful
method to enhance the enantioselectivity of lipase-cata-
lyzed transformations in an isooctane/aqueous buffer
solution. Candida rugosa lipase was encapsulated accord-
ing to a sol–gel procedure in the presence and absence of
calix[4]arene hydrazine or carboxylic acid derivatives with
Fe3O4 magnetic nanoparticles as an additive. The activity
of the encapsulated lipases was evaluated for the enantio-
selective hydrolysis of racemic Naproxen methyl ester and
the hydrolysis of p-Nitrophenylpalmitate. The results
indicate that the encapsulated lipase without calix[4]arene
derivative has lower conversion and enantioselectivity
compared to the encapsulated lipase with calix[4]arene
derivative. It was found that the calix[4]arene hydrazine
and carboxylic acid-based encapsulated lipases have
excellent activity and enantioselectivity (E [300) com-
pared to encapsulated lipase without the calix[4]arene
derivatives.
Keywords Nanoparticles  Fe3O4  Lipase  Calixarene 
Enantioselectivity
Introduction
Lipases are enzymes that are widely used in synthetic
organic chemistry and can catalyze an extensive range of
regio- and enantioselective reactions such as esterifications,
hydrolysis, transesterifications and ammoniolysis [1].
Because of their regio- and enantioselectivity, lipases are
E. Akoz  S. Sayin  S. Kaplan  M. Yilmaz (&)
Department of Chemistry, Selcuk University,
42031 Konya, Turkey
e-mail: myilmaz42@yahoo.com
important biocatalysts in several applications, such as the
synthesis of useful chemical compounds for pharmaceuti-
cal, flavor, fragrances, vitamins and other fine chemicals
and agrochemical derivatives [2]. In particular, Candida
rugosa lipase is an important industrial lipase, and due to
its broad substrate specificity, it is successfully used in a
variety of esterification and hydrolysis reactions [3, 4].
Several methods have been recommended to improve the
performance of lipase-catalyzed reactions for optical res-
olution. These include the optimization of reaction condi-
tions, modification of substrate, optimization of the nature
of the solvent [5] and of the water content [6], chemical
and non-covalent modifications of enzymes, and the use of
an additive that regulates lipase reactivity. Among these,
the additive method is the most advantageous [7]. It is
simple to use, but only a few compounds have been
reported as an additive to enhance the enantioselectivity of
lipase reactions [8, 9]. Several macrocyclic compounds,
such as cyclodextrins [10] and crown ether derivatives
have been used [11].
The calix[n]arenes are a fascinating class of macrocyclic
molecules that have been used extensively in supramolec-
ular chemistry [12], together with cyclodextrins [13] and
crown ethers [14]. The highly ordered structures of calix-
arenes offer not only boundless possibilities for chemical
modification but also make them extremely useful in the
study of molecular recognition and supramolecular pro-
cesses. Calixarenes derived from phenol–formaldehyde can
undergo chemical modification by the introduction of
functional moieties at the phenolic –OH groups. In addi-
tion, the easy removal of tert-butyl groups attached to
phenyl rings makes it possible to attach a large variety of
functional groups to the upper rim of calixarenes. Their
rigid conformation enables calixarenes to act as host mol-
ecules because of their preformed hydrophobic cavities.
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 Bioprocess Biosyst Eng

15 % NaOH, EtOH,
ref lux

O HO OH O O HO OH O

O O O O
O OH HO
O

C[4]-C4-(COOH) 2

K2 CO3, Acetone,
Ethyl 5-bromovalerate
reflux

OH HO OH HO

K2 CO3, Acetone,
Methyl bromoacetate
reflux 15 % NaOH, EtOH,
ref lux
O HO OH O

O O
HO OH
DCM/MeOH,
C[4]-(COOH) 2
O HO OH O Hydrazine,
reflux
O O
H3 CO OCH3

O HO OH O

O O
HN NH
NH2 H2N

C[4]CO-(NHNH 2) 2

Scheme 1 A schematic representation of the synthesis of calix[4]arene-based additive

Eventually, they find applications as carriers and selective
binders, and as model structures for biomimetic studies
[15].
Previous studies showed that encapsulated lipases in the
presence of different additives such as derivatives of
cyclodextrin, 18-crown-6, or calixarene show higher
activity and enantioselectivity [16, 17]. The 18-crown-6
may also form complexes with the 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 complexa-
tion and showed lower availability for salt bridge forma-
tion. Therefore, the formation of intra- and inter-molecular
salt bridges of ether–lysine complexes might be reduced
[18–20]. It was demonstrated that the crown ether deriva-
tives have the potential to enhance both the reaction rate
and enantioselectivity of the lipase-catalyzed hydrolysis of
2-cyano-1-methylethyl acetate [14]. These crown com-
pounds cannot change the original enantioselectivity of the
enzyme, but they can enhance its potential ability to a level

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Bioprocess Biosyst Eng
at which the reaction can be used practically [14]. More-
over, 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 [21].
In our previous work, first-time calixarene derivatives
were used as additives for lipase immobilization by the
sol–gel method [12]. The encapsulated lipase was then
used in the enantioselective hydrolysis reaction of racemic
Naproxen methyl ester. S-Naproxen is a non-steroidal anti-
inflammatory drug that belongs to the family of 2-aryl
propionic acid derivatives, it works by reducing hormones
that cause inflammation and pain in the body. The physi-
ological activity of the S form of Naproxen is 28-fold that
of the R form [22]. However, by introducing magnetic
properties to organic molecules or to biomolecules,
researchers can make separation and reusable processes
easy tasks due to magnetic speciation.
Very recently [22, 23], to observe the role of the cal-
ixarene binding site on lipase stability, activity, and e-
nantioselectivity, we reported upper-rim substitute
calix[n]arene carboxylic acid and N-methylglucamine-
based calix[4]arene derivative-grafted magnetic nanopar-
ticles that were used as additives in the sol–gel encapsu-
lation process, and we explored the influence of the
material on the hydrolysis and enantioselectivity of race-
mic Naproxen methyl ester. The magnetic calix[4]arene-
based encapsulated lipases showed excellent conversion
(50 %) and enantioselectivity (E = 460) compared to the
free enzyme. We consider that it could be taken interesting
results if lipase immobilization presence of the calixarene
derivatives with acidic or basic groups as well as magnetic
nanoparticles using sol–gel process was maintained.
Therefore, formation of calixarene–lysine complexes might
reduce the formation of inter- and intra-molecular salt
bridges. This phenomenon might be attributed to some
distortion on the protein conformation, reducing the overall
flexibility of the enzyme molecules generated from the
interactions between the enzyme and the supports during
the immobilization. They may interact with certain sites of
the lipase, as suggested in some proteins, thereby activating
the lipase and changing its enantioselectivity and improv-
ing the catalytic activity of the CRL.
The purpose of this study was to use the sol–gel
encapsulation in the presence of two carboxylic acid
derivatives of calix[4]arene (C[4]–(COOH)2, C[4]–C4–
(COOH)2), and a hydrazine-amide (C[4]–(NHNH2)2) of
calix[4]arene as the new additives to produce immobilized
lipase. These immobilized derivatives were used as a cat-
alyst in both the enantioselective hydrolysis reaction
of racemic Naproxen methyl ester and the hydrolysis of
p-Nitrophenylpalmitate (p-NPP).
Materials and methods
Materials
Bovine serum albumin, p-NPP, and C. rugosa lipase (Type
VII) were supplied from Sigma-Aldrich; octyltriethoxysi-
lane (OTES), tetramethoxysilane (TMOS), and the solvents
were supplied from Fluka, Merck, or Aldrich and were
standard analytical grade and used without further purifi-
cation. All commercial-grade solvents were distilled and
stored over molecular sieves. Racemic Naproxen was
produced in the laboratory by the racemization of optically
pure S-Naproxen as described [24].
Synthesis
The p-tert-butylcalix[4]arene hydrazine (C[4]–(NHNH2)2)
and carboxylic acid derivatives C[4]–(COOH)2, C[4]–C4–
(COOH)2 were synthesized according to the known pro-
cedures [25–27].
Lipase sol–gel encapsulation within calixarene
derivative
The encapsulation with and without the calixarene deriv-
ative was prepared by adapting a known procedure [11]. A
mixture of CRL (245 mg) was placed in a 50-mL Erlen-
meyer together with PBS (1.56 mL; 0.05 M; pH 7.0),
which was gently stirred on a shaker. Then, C[4]–
(COOH)2, C[4]–C4–(COOH)2, and C[4]-(NHNH2)2
(100 mg) with Fe3O4 nanoparticles as an additive (100 mg)
were added to the mixture, and 400 lL of aqueous poly-
vinyl alcohol (PVA) (4 % w/v), aq. NaF (200 lL of a 1 M
solution), and i-PrOH (400 lL) were added and homoge-
nized using a shaker. Next, OTES (2.5 mmol, 3,144 lL)
and TMOS (0.5 mmol; 460 lL) were added, and the
mixture was agitated once more for 10–15 s. The gel was
washed with distilled water (10 mL) and isopropyl alcohol
(10 mL), and then lyophilized. The resulting encapsulated
lipase was stored at 4 °C prior to use.
Activity of the sol–gel encapsulated lipases
Activities of the free and immobilized lipases were
measured by the hydrolysis of p-NPP in an aqueous
phosphate buffer solution (0.05 M PBS buffer, pH 7.0).
The concentration of the hydrolysis product, i.e., p-nitro-
phenol (p-NP), was measured using a spectrophotometry
method. UV/Vis spectra were measured with a Perkin
Elmer Lambda 25 spectrophotometer; the absorbance
increase at 400 nm produced by the release of p-NP in the
enzymatic hydrolysis of (p-NPP) was measured [28, 29].
One unit of enzyme activity was defined as the amount of
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enzyme that liberates 1 lmol p-nitrophenol min-1. By
dividing total activity (U) by the amount of lipase bound
to sol–gel polymers, the specific activity was calculated
and expressed as U/mg protein. In these experiments, the
substrate solution was added to the sol–gel encapsulated
lipase at 35 °C on a shaker at 110 rpm for 5 min. After
collecting nanoparticles with a magnet, the supernatant
was removed to measure the amount of p-NP. For the free
lipase, the same amount of lipase was mixed with the
substrate solution. All measurements were performed at
least three times, and the experimental error rate was less
than 3 %.
The protein content of the encapsulated lipases was
calculated by measuring the initial and final concentration
of protein in the immobilization medium using the Brad-
ford protein assay method [30].
Effect of temperature and pH on activity
The reaction temperature and the optimum pH of immo-
bilized and free lipases were determined as the relative
activity under the variety of temperatures (30–60 °C) and
pH (0.05 M PBS for pH 4.0–9.0). The relative activities
were recorded as the ratio of the activity of encapsulated
lipases after incubation to the activity at the temperature
and optimum reaction pH [12].
General procedures for encapsulated lipase-catalyzed
enantioselective hydrolysis of racemic Naproxen
methyl esters
The hydrolysis reactions were conducted in an aqueous
phase/organic solvent batch reaction system. A solution of
racemic methyl ester (20 mM) in 2 mL of isooctane was
added to a 2 mL buffer solution (pH 7.0, 50 mM PBS)
containing encapsulated lipases (5–50 mg, depending on
the activity). The reactions were carried out inside a shaker
at 150 rpm at 35 °C, and after 24 h, samples from the
isooctane phase were collected.
HPLC was used to calculate the enantioselectivity and
conversion and the results were given as the enantiomeric
ratio (E) estimated from the enantiomeric excess of the
substrate (ees) and the product (eep) and the conversion (x)
using the equation [31].
ln½ð1  xÞð1  ees Þ
E¼
ln½ð1  xÞð1  ees Þ
ees CR  CS CS  CR
x¼ ees ¼ eeP ¼
ees þ eep CR þ CS CS þ CR
E, ees, eep, x, CR and CS indicate enantiomeric ratio for
irreversible reactions, enantiomeric excess of substrate,
enantiomeric excess of product, racemate conversion,
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Bioprocess Biosyst Eng
concentration of R-enantiomer and concentration of S-
enantiomer, respectively.
Results and discussion
X-ray diffraction (XRD) clearly shows characteristic peaks
of Fe3O4 nanoparticles (Fig. 1) in the obtained product
from sol–gel media. Peaks of amorphous silica as well as
calixarene units from 2h = 10o to 27o can also be seen. By
the appearance of those peaks in XRD, it can be concluded
that the encapsulated lipase contains both Fe3O4 nanopar-
ticles and calixarene derivative.
Sol–gel encapsulation of Candida rugosa lipase (CRL)
using calix[n]arene
In the literature [15], it has been reported that di-substituted
calix[4]arene derivatives acted as receptors for molecules,
ions, and effective binding sites. In view of these properties
of calixarene derivatives, some of the calix[4]arene-based
compounds carrying acidic (C[4]–(COOH)2 and C[4]–C4–
(COOH)2), and basic (C[4]–(NHNH2)2) functional groups
have been used as additives for the encapsulation of the
lipase via sol–gel method. Moreover, all encapsulated
lipases with or without calixarene derivatives were driven
as bio-catalyst in the enantioselective hydrolysis reaction
of racemic Naproxen methyl ester.
The hydrolytic activity of the encapsulated lipases was
evaluated in the hydrolysis of (p-NPP). Table 1 shows the
specific activities and protein amounts of encapsulated
lipases with p-NPP. However, the encapsulated lipase with
C[4]CO–(NHNH2)2 was found to be more efficient than the
carboxylic acid derivatives of calix[4]arene (C[4]–
(COOH)2, C[4]–C4–(COOH)2), with respect to the
expression of immobilized lipase activity.
This is not a surprising result because the carboxylic
acid derivatives of calix[4]arene (C[4]–(COOH)2, C[4]–
C4–(COOH)2) containing –COOH groups are highly
effective complexing agents [15], which means some car-
boxyl groups interact with the active center of lipase by a
combination of hydrogen bonding and electrostatic inter-
actions. Most probably, the lipase is not only physically
encapsulated, but there is also additional multipoint inter-
action through hydrogen bonding, ionic, and hydrophobic
interactions. Moreover, this could be explained by the
modification in the three-dimensional structure of the
enzyme, which leads to a conformation change of the
active center. The presence of the hydrazine derivative
C[4]CO–(NHNH2)2 hinders the accessibility of substrate to
the enzyme-active site and limits mass transfer of substrate
and product.
Bioprocess Biosyst Eng

Fig. 1 XRD pattern of Fe3O4 nanoparticles and the sol–gel product with C[4]–C4–(COOH)2 and Fe3O4 nanoparticles. The bottom rows of tick
marks with blue color imply the standard magnetite pattern

Table 1 Yields of protein loading and activity of the calix[4]arene- as additive was 6.0. After immobilization, the optimum pH
encapsulated lipases for reactions catalyzed by free lipase was slightly shifted
Protein Protein Lipase Specific toward acidic values. The immobilized lipase offered better
loading loading activity (U/ activity (U/ pH stability and, hence, resistance to acidic environments
(mg/g yield (%) g support) mg protein) than did free lipase. After immobilization, variations of
support)
optimum pH range on hydrolysis activity have been reported
Enc-lipasea 28.5 58 95 3.3 by some researchers, and these depend on the methods of
C[4]CO- 20.4 47.5 96 9.4 immobilization and the secondary interactions (hydrophilic
(NHNH2)2 and hydrophobic) between the enzyme and support [32], as
C[4]-C4- 19.9 46 54 2.7 well as when the enzymes are immobilized onto polycationic
(COOH)2 supports [33].
C[4]- 11.6 27 72 6.2 At pH 7.0, the temperature dependence of the p-NPP
(COOH)2
hydrolysis reaction catalyzed by encapsulated and free lipa-
a
Encapsulated free lipase without calix[4]arene derivatives. pH 7.0 ses was studied from 30 to 60 °C, and these results are given
and temperature 35 °C in Fig. 3. The observed optimum temperature for the
encapsulated lipase (Enc-lipase) and C[4]–(COOH)2 was
pH and temperature effect on the activity
approximately 35 °C, while it shifted to nearly 45 °C for the
of encapsulated lipases
C[4]–C4–(COOH)2 and (C[4]CO–(NHNH2)2). This could be
explained by either creation of conformational limitations on
Because the enzyme activity is markedly influenced by
the enzyme movements as the results of hydrophobic inter-
environmental conditions, it was seen especially that pH is
action between the enzyme and the support or an improved
the most efficient parameter altering enzymatic activity in an
resistance of the protein to thermal denaturation [6].
aqueous medium. Figure 2 illustrates the effect of pH on the
activity of the free and immobilized lipases in the hydrolysis
of p-NPP, determined by altering the reaction medium pH Enantioselective hydrolysis of racemic Naproxen
from 4 to 9. As shown in Fig. 2, the optimum pHs of the methyl ester with the encapsulated lipases
carboxylic acid derivative of calix[4]arene C[4]–(COOH)2
and C[4]–C4–(COOH)2 as additives were 5.0, and the Enantiopure compounds are currently achieved by chiral
hydrazine derivative of calix[4]arene (C[4]CO–(NHNH2)2) chemical synthesis using metal catalysts or by enzymatic

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 Bioprocess Biosyst Eng

Fig. 2 Effect of pH on relative 100

activity of encapsulated lipases
80

Relave Acvity (%)

60 enc lipase
C[4]CO-(NHNH2)2
40
C[4]-C4-(COOH)2

20 C[4]-(COOH)2

0
4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
pH

Fig. 3 Effect of reaction 100

temperature on the relative
activity of encapsulated lipases
80
Relave Acvity (%)

60 enc lipase

C[4]CO-(NHNH2)2
40
C[4]-C4-(COOH)2

C[4]-(COOH)2
20

0
30 35 40 45 50 55 60
Temperature (oC)

kinetic resolution of racemic mixtures. The kinetic reso-
lution depends not only upon the degree of enantioselec-
tivity but also on the activity and the possibility of
recycling and reusing the lipase. Candida rugosa lipase is
widely used in biotransformation due to its high activity in
hydrolysis as well as synthesis [2].
The crystal structure of C. rugosa lipases shows two
known conformations [34]. In one, called the ‘‘closed
form,’’ the helical surface loop partially covers the
hydrophobic crevice containing the active site, while in the
other conformation, called the ‘‘open form,’’ the surface
loop moves and the space is uncovered [20, 35].
We used the calix[4]arene derivatives (C[4]–(COOH)2,
C[4]–C4–(COOH)2, and C[4]CO–(NHNH2)2) as additives
for the sol–gel encapsulation of lipase to study the possible
effects of the calix[4]arene derivatives in the enantiose-
lective hydrolysis reaction of racemic Naproxen methyl
ester.
It was reported that a 2-propanol treatment of C. rugosa
lipase caused modification of enantioselectivity; 2-propa-
nol treatment was proposed to convert the closed form of
this lipase to the open form, thereby enhancing the
enantioselectivity [14, 36]. In view of that point, in the
current study some of the calixarene derivatives substituted
with carboxylic acid and hydrazine groups would be a
challenge for enhancing not only the enantioselectivity but
also the activity of the lipase in the hydrolysis reaction of
racemic Naproxen methyl ester. Table 2 shows known the
conversion (x), enantiomeric excess (ee) and enantiomeric
ratio (E) in the course of racemic Naproxen methyl ester
hydrolyzed by the encapsulated lipases in an aqueous buffer
solution/isooctane reaction system at pH 7 and optimum
pH, at 35 °C. The enantioselective hydrolysis of racemic
Naproxen methyl ester by encapsulated lipases was studied
in an aqueous buffer solution/isooctane reaction system.
From the results (Table 2), it has been revealed that the sol–
gel free encapsulated lipase without calixarene (Enc-lipase)
has a high enantioselectivity (E) and conversion (x) com-
pared with the free lipase (Non-Enc-lipase).
On the hydrolysis of racemic-Naproxen methyl ester
(Fig. 4), however, using C[4]CO–(NHNH2)2 shows a high
enantioselectivity at pH 7 as well as at the optimum pH,
whereas the resolution reactions with C[4]–(COOH)2 and
C[4]–C4–(COOH)2 exhibited higher enantioselectivity

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Bioprocess Biosyst Eng

Table 2 The enantioselective hydrolysis of racemic Naproxen only at their optimum pHs (Table 2). The reason for
methyl ester of using sol–gel improvement of enantioselectivity by immobilization is not
x (%) ees (%) eep (%) E yet well understood. However, some literature [37] repor-
ted that this phenomenon might be attributed to some
Non-Enc-lipasea 11 24 [98 125
b
distortion on the protein conformation, reducing the overall
Enc lipase 20 25 [98 137 flexibility of the enzyme molecules generated from the
C[4]CO–(NHNH2)2 (pH = 5) 49 92 [98 328 interactions between enzyme and the supports during the
C[4]CO–(NHNH2)2 (pH = 7) 48 90 [98 307 immobilization. Calixarenes represent one of the most
C[4]–C4–(COOH)2 (pH = 6) 49 95 [98 371 important macrocyclic host molecules in supramolecular
C[4]–C4–(COOH)2 (pH = 7) 17 20 [98 121 chemistry. In the literature, it was shown that calixarene
C[4]–(COOH)2 (pH = 5) 47 87 [98 283 derivatives form complexes with cationic lysine [21]. After
C[4]–(COOH)2 (pH = 7) 21 26 [98 128 complexation it may form complexes with cationic lysine
Encapsulated lipases as catalysts. The enantioselective hydrolysis of residue of enzymes. Hence, formation of ether–lysine
racemic Naproxen methyl ester of using sol–gel encapsulated lipases complexes might reduce the formation of inter- and intra-
as catalysts. Enantiomeric excess (ee) as determined by Chiral HPLC, molecular salt bridges. Moreover, researchers observed that
Agilent 1200 Series-chiral column (Chiralcel OD-H); n-hexane/2-
propanol/trifluoroacetic acid (100/1/0.1, v/v/v) as mobile phase; time,
the activation is clearly a macrocyclic effect.
24 h; concentration of substrate, 20 mM; (Optimum pH 5.0 for It was also found that calix[4]arene hydrazine and car-
C[4]CO–(NHNH2)2; pH 6.0 for C[4]–C4–(COOH)2; pH 5.0 for C[4]– boxylic acid-based additives have important effects on the
(COOH)2) and temperature, 35 °C stability of CRL. All the results showed that the catalytic
a
Non-encapsulated free lipase activity of the CRL might have improved due to the sol–gel
b
Encapsulated free lipase without calix[4]arene derivatives processes with the calix[4]arene carboxylic acid

Fig. 4 Schematic illustration of

the enantioselective hydrolysis
of (R/S)-Naproxen methyl ester

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 Bioprocess Biosyst Eng

C[4]CO-(NHNH2)2 A
A 50
C[4]CO-(NHNH2)2
50 40

40 30

X%
30 20 pH=5
X%

pH=7
20 10

10 0
1 2 3 4 5
0 Reuse
35 45
Temperature (oC)
B 50

40
B 50
30 C[4]-C4-(COOH)2
40

X (%)
C[4]-C4-(COOH)2 20
30
X (%)

10
20
0
10
1 2 3 4 5
0 Reuse (pH=6)
35 45
Temperature (oC) C 50

40 C[4]-(COOH)2
C 50
30
X (%)

40
20
30 10
X (%)

20 C[4]-(COOH)2
10
0
35 40
Temperature (oC)
Fig. 5 Effect of temperature on conversion in the hydrolysis of
racemic Naproxen methyl ester with C[4]CO–(NHNH2)2 (a), C[4]–
C4–(COOH)2 (b) and C[4]–(COOH)2 (c)
derivatives used as 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.
To see the temperature dependence of the percentage of
conversion (x) of the hydrolysis reaction that is catalyzed
by encapsulated lipases, we performed our experiments at
35 °C as well as at their optimum pH. The results indicate
that the conversion degree increased at their optimum
temperature values (Fig. 5).
The reusability of immobilized enzymes is very
important to their application. Therefore, we applied the
encapsulated lipases to reusability studies. After each run,
the encapsulated lipases were washed with PBS, and all
0
1 2 3 4 5
Reuse (pH=5)
Fig. 6 Reusability on the conversion (x) in the hydrolysis of racemic
Naproxen methyl ester with C[4]CO–(NHNH2)2 (a), C[4]–C4–
(COOH)2 (b) and C[4]–(COOH)2 (c)
conversion ratios were determined at 24 h after initiation of
the reaction. Figure 6 shows that the encapsulated lipases
still retained 36 and 35 % of their conversion ratios for
(C[4]CO-(NHNH2)2) after the fifth reuse at a pH of 6 and 7,
respectively. Furthermore, the immobilized lipases also
retained 35 and 38 % of their conversion ratios when
encapsulated with C[4]–(COOH)2 and C[4]–C4–(COOH)2)
after the fifth reuse at their optimum pH, respectively.
These results are due to the inactivation of the enzyme
caused by the denaturation of the protein and the leakage of
the protein from the supports upon use.
Conclusions
Candida rugosa lipase (CRL) was immobilized by the sol–
gel encapsulation procedure in the presence and absence of

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Bioprocess Biosyst Eng
calix[4]arene hydrazine and carboxylic acid derivative with
Fe3O4 magnetic nanoparticles (C[4]CO–(NHNH2)2, C[4]–
(COOH)2, and C[4]–C4–(COOH)2) as additive. The cata-
lytic activity of the encapsulated lipases was assessed into
model reactions, i.e., the hydrolytic hydrolysis of p-NPP
and the enantioselective hydrolysis reaction of racemic
Naproxen methyl ester. The immobilization of calix[4]ar-
ene hydrazine derivative with Fe3O4 as an additive resulted
in high conversion and enantioselectivity at pH 7 as well as
at pH 6. Indeed, the encapsulated lipases have an excellent
rate of enantioselectivity, with E values as 283 (for C[4]–
(COOH)2 at pH 5), 371 (for C[4]–C4–(COOH)2 at pH 5),
and 328 (for C[4]CO–(NHNH2)2 at pH 6), as compared to
the free enzyme (E = 137).
In general, these results simply show that calix[n]arene-
based magnetic particles can efficiently be used for the
immobilization of lipase to develop an enzymatic system
with improved loading capacity, recoverability, and stabil-
ity properties. Developers can very easily make economical
encapsulated lipase due to its magnetic property. These
properties are important factors when selecting an appro-
priate enzymic system for different biotechnological
applications. The current study shows that using calix[-
n]arene carboxylic acid derivative to immobilize enzymes
for biphase reaction synthesis could provide appreciable
improvements in the manufacture of pharmaceutical inter-
mediates, fine chemicals, and functional food ingredients.
Acknowledgments We would like to thank the Scientific and
Technological Research Council of Turkey (TUBITAK Grant Num-
ber 111T027) and the CMST COST Action No: CM1005 and the
Research Foundation of Selcuk University (BAP) for financial sup-
port of this work.
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