Enantioselective hydrolysis of rasemic naproxen methyl ester with sol-gel encapsulated lipase in the presence of sporopollenin

Article abstract of DOI:10.1016/j.molcatb.2009.10.003

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, the Candida rugosa lipase (CRL) was encapsulated within a chemically inert sol-gel support prepared by polycondensation with tetraethoxysilane (TEOS) and octyltriethoxysilane (OTES) in the presence and absence of sporopollenin and activated sporopollenin as additive. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of rasemic Naproxen methyl ester that was studied in aqueous buffer solution/isooctane reaction system. The results indicated that the sporopollenin based encapsulated lipase particularly had higher conversion and enantioselectivity compared to the sol-gel free lipase. In this study, excellent enantioselectivity (E > 400) has been noticed for most lipase preparations (E = 166 for the free enzyme) with an ee value ～98% for S-Naproxen. Moreover, (S)-Naproxen was recovered from the reaction mixture with 98% optical purity.

Full text of DOI:10.1016/j.molcatb.2009.10.003

Journal of Molecular Catalysis B: Enzymatic 62 (2010) 162–168
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enantioselective hydrolysis of rasemic naproxen methyl ester with sol–gel
encapsulated lipase in the presence of sporopollenin
∗
Elif Yilmaz, Mehmet Sezgin, Mustafa Yilmaz
Department of Chemistry, Faculty of Science, Selcuk University, Konya 42075, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2009
Received in revised form
Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with con-
stant chemical structure and has high resistant capacity to chemical attack. In this study, the Candida
rugosa lipase (CRL) was encapsulated within a chemically inert sol–gel support prepared by polycon-
densation with tetraethoxysilane (TEOS) and octyltriethoxysilane (OTES) in the presence and absence
of sporopollenin and activated sporopollenin as additive. The catalytic properties of the immobilized
lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP), and
the enantioselective hydrolysis of rasemic Naproxen methyl ester that was studied in aqueous buffer solu-
tion/isooctane reaction system. The results indicated that the sporopollenin based encapsulated lipase
particularly had higher conversion and enantioselectivity compared to the sol–gel free lipase. In this
study, excellent enantioselectivity (E > 400) has been noticed for most lipase preparations (E = 166 for the
free enzyme) with an ee value ∼98% for S-Naproxen. Moreover, (S)-Naproxen was recovered from the
reaction mixture with 98% optical purity.
2
7 September 2009
Accepted 19 October 2009
Available online 27 October 2009
Keywords:
Candida rugosa lipase
Sporopollenin
Encapsulation
Enantioselective hydrolysis
S-Naproxen
©
2009 Elsevier B.V. All rights reserved.
1
. Introduction
mity is difficult and expensive to achieve in man made products,
especially possessing a large cavity capable of being filled with a
wide range of polar and non-polar materials. Particular encapsu-
lants previously used for macromolecules are relatively expensive
with lower loadings than sporopollenin [5].
Sporopollenin is a natural biopolymer which occurs in the outer
membranes of moss and fern spores and most pollen grains. It has
been shown that spore and pollen membranes have two layers, an
inner one known as intine, and an outer one the exine containing
a material to which the name sporopollenin was given. Sporopol-
lenin is stable and highly resistant to chemicals. It has a constant
chemical structure and exhibits very good stability even after a pro-
long exposure to mineral acids and alkalies [1]. The mechanism of
its synthesis and its consolidation are not yet understood [2]. In the
past two decades modified forms of sporopollenin have been uti-
lized as anion, ligand [3], and as cation exchangers for removal of
heavy metal ions from aqueous solutions [4].
Sporopollenin offers several advantages over man made and
other naturally occurring materials (e.g. acrylic resins, chitosan).
It is easily extracted from its natural source, spores or pollen, using
cheap non-toxic reagents which are used in the food industry.
It forms microcapsules which have a large internal cavity avail-
able for encapsulation with very high loadings. The particles are
monodispersed and, from one species, uniform in size, morphology
and chemical composition, i.e. sporopollenin contains only carbon,
hydrogen and oxygen. It is thus free from any allergens. Such unifor-
Sol–gel encapsulation has proven to be a particularly easy and
effective way to immobilize enzymes [6]. Following isolated reports
describe the specific examples; it was the seminal work of Avnir and
co-workers that led to the generalization of this technique [6–8].
A well-established sol–gel processing technique consists in
hydrolyzing adequate precursors in aqueous solutions to produce
soluble hydroxylated monomers, followed by polymerization and
phase separation to produce a hydrated metal or semi-metal oxide
hydrogel [9]. Removal of water from the wet gel, which is usually
accompanied by changes in the structure of the pores and of the
gel’s network, results in a porous xerogel. The most widely used
precursors are alkyl-alkoxysilanes. These precursors were used
already in the mid-1980s these precursors were used to prepare
organically modified silicates for the successful encapsulation of
antibodies and enzymes [10,11]. Although the final structure of the
material is basically determined by the differences in chain length,
functionality and hydrophobic character of the precursors, it can
be tailored via the addition of a wide range of molecules. Examples
include surfactants [12–14], room-temperature ionic liquids [15],
crown ethers, ␤-cyclodextrins or porous solid supports like Celite
[
16]. The commonly used catalysts are weak acids or bases [9,16].
∗ Corresponding author. Tel.: +90 332 2232774; fax: +90 332 2231620.
E-mail address: myilmaz42@yahoo.com (M. Yilmaz).
Recent research describes the use of other species that include
1
381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.10.003

E. Yilmaz et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 162–168
163
peptides like silaffins [17], polyamines [18] or enzymes such as
hydrolases [19] and silicateins [20].
degasser, and an Agilent Chemstation B.02.01-SR2 Tatch data pro-
cessor.
Candida rugosa is an important industrial lipase due to its wide
application in oil hydrolysis, transesterification, esterification and
enantioselective biotransformation [21,22]. Thus, lipase is becom-
ing one of the most industrially used enzymes due to its high
stereoselectivity, regioselectivity, and low price [23]. (S)-(+)-2-(6-
Methoxy-2-naphthyl)propionic acid (Naproxen) is a non-steroidal
anti-inflammatory drug that belongs to the family of 2-aryl pro-
pionic acid derivatives, it is widely used as a drug for human
connective tissue diseases. The physiological activity of the S form
of Naproxen is 28-fold that of the R form [24]. Hence, only the S form
is used as a drug for humans [25–27]. Of the profen drugs, an impor-
tant family of non-steroidal anti-inflammatory drugs, Naproxen is
now the only member to be sold as a single enantiomer.
In the case of Naproxen, lipase has been used to prepare opti-
cally pure Naproxen by enantioselective hydrolysis of its racemic
esters [28–30]. In addition, the preparation of Naproxen ester pro-
drugs has been accomplished by resolution of racemic Naproxen
mixtures [31–33]. More recently, alcohol and buffer or acetone
treatment method has been developed to improve the enantiose-
lectivity of CRL towards the hydrolysis of racemic Naproxen methyl
ester [34,35]. The changes in the enantioselectivity of CRL for S-
Naproxen with the alterations in its microenvironment by changing
the medium properties [36], by immobilization [37] and by intro-
ducing additives into the reaction medium [38] have also been
reported.
The enantiomeric excess determination was performed with
HPLC (Agilent 1200 Series) by using a Chiralcel OD-H column at
the temperature of 25 C with n-hexane/2-propanol/trifluoroacetic
acid (100/1/0.1, v/v/v). The flow rate of 1 mL/min; the UV detector
was fixed at 254 nm.
The surface morphology of samples was examined by scanning
electron microscope (SEM, Jeol, JSM 5310, Japan).
◦
2.3. Preparation of activated sporopollenin
The sporopollenin exines (25 ␮m) were extracted from L. clava-
tum as follows: raw L. clavatum spores (powdered form, 50 g) were
suspended in acetone (150 mL) and stirred under reflux for 6 h.
The defatted spores were filtered and then treated with 8% (w/v)
potassium hydroxide (150 mL) solution. After that the mixture was
refluxed for 15 h. This base-hydrolyzed sporopollenin was filtered
and washed with hot water and hot ethanol. It was finally sus-
pended in 85% (w/v) ortho-phosphoric acid (150 mL) and stirred
under reflux for 7 days. This acid-hydrolyzed sporopollenin was
filtered, washed with water, acetone, 2 M hydrochloric acid solu-
tion, 2 M sodium hydroxide solution, water, acetone and ethanol
◦
and dried at 60 C until constant weight [39].
2.4. General procedure for sol–gel encapsulation of lipases
To the best of our knowledge, there exists no report on the use
of sporopollenin from Lycopodium clavatum as support for immo-
bilization of lipase. It has been thought that the sol–gel procedure
could be interesting for lipase immobilization. Therefore, we now
report the use of sporopollenin (Spo) and activated sporopollenin
Sol–gel encapsulated lipases were prepared according to a mod-
ified method of Reetz et al. [14]. A commercial lipase powder
lyophilizate) such as CRL type VII (60 mg) was placed in a 50-
mL Falcon tube (Corning) together with phosphate buffer (390 ␮L;
0 mM; pH 7.0) and the mixture was vigorously shaken with a
(
(
Spoact) as additives on lipase immobilization made by sol–gel pro-
5
cess and explore the effect of these materials in the enantioselective
hydrolysis of (RS)-Naproxen methyl ester. The effect of tempera-
ture, pH and thermal/storage stability was also investigated.
Vortex-Mixer. The Spo or Spoact (0.05 g) was included. Then 100 ␮L
of aqueous polyvinyl alcohol (PVA) (4% W/V), aqueous sodium flu-
oride (50 ␮L of 1 M solution) and isopropyl alcohol (100 ␮L) were
added, and the mixture homogenized using a Vortex-Mixer. Then
the alkylsilane (2.5 mmol) and TMOS (0.5 mmol; 74 ␮L; 76 mg)
were added and the mixture agitated once more for 10–15 s. Gela-
tion was usually observed within seconds or minutes while gently
shaking the reaction vessel. Following drying overnight in the
opened Falcon tube, isopropyl alcohol (10 ± 15 mL) was added in
order to facilitate removal of the white solid material (filtration).
The gel was successively washed with distilled water (10 mL), iso-
propyl alcohol (10 mL). The resulting encapsulated lipases were
2
. Materials and methods
2.1. Materials
C. rugosa lipase (CRL) was a commercial enzyme obtained
from Sigma-chemical Co. (St. Louis, MO) used in the immo-
bilization. Lycopodium clavatum with a particle size of 25 ␮m
was purchased from Fluka Chemicals. Bradford reagent, Bovine
Serum Albumin 99% (BSA), p-nitrophenylpalmitate (p-NPP), TEOS
◦
lyophilized and stored at 4 C prior to use.
(
tetraethoxysilane) and OTES (octyltriethoxysilane) were pur-
chased from Sigma-chemical Co. (St. Louis, MO). Pure S-Naproxen,
was purchased from Sigma (USA). The solvents used in HPLC analy-
ses were HPLC grade (Merck, Germany). All aqueous solutions were
prepared with deionized water that had been passed through a Mil-
lipore Milli-Q Plus water purification system. All other chemicals
2
.5. Determination of enzyme activity
Activity of the free and encapsulated lipases were assayed using
4.4 mM p-nitrophenyl palmitate in 2-propanol as substrate. The
1
reaction mixture consisting of 1 mL of 50 mM phosphate buffer
pH 7.0 for immobilized lipase) containing 25 mg of immobilized
(
Merck, Darmstadt, Germany) were of analytical grade and used
(
without further purification.
lipase (or 0.1 mL free lipase) was initiated by adding 1 mL of sub-
strate and mixed for 5 min at 30 C. The reaction was terminated by
◦
2
.2. Instrumentation
adding 2 mL of 0.5 N Na2CO3 followed by centrifuging at 4000 rpm
for 10 min. The increase in the absorbance at 410 nm produced
by the release of p-nitrophenol in the enzymatic hydrolysis of
p-NPP was measured in a Shimadzu UV-160A (Japan) spectropho-
FT-IR spectra were recorded on a PerkinElmer 1605 FT-IR
spectrometer as KBr pellets. UV–vis spectra were obtained on
a Shimadzu 160A UV-vis recording spectrophotometer. High-
performance liquid chromatography (HPLC) Agilent 1200 Series
was carried out using a 1200 model quaternary pump, a G1315B
model Diode Array and Multiple Wavelength UV–vis detector, a
−
1
−1
tometer. A molar extinction coefficient (ε410) of 15,000 M cm
for p-nitrophenol was used [40]. One unit (U) of lipase activity
was defined as the amount of enzyme necessary to hydrolyze
1 ␮mol/min of p-NPP under the conditions of assay. All measure-
ments were performed in triplicate and an average was taken as
final result.
1
200 model standard and preparative auto sampler, a G1316A
model thermostated column compartment, a 1200 model vacuum

1
64
E. Yilmaz et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 162–168
2.6. Protein assay
where
ees
ees + eep
CR − C
CR + C
C − CR
S
S
Protein content was estimated by the method of Bradford [41]
using Bio-Rad protein dye reagent concentrate. Bovine serum albu-
min was used as the standard.
x =
ees =
eep =
C − CR
S
S
where E, ees, eep, x, CS and CR denote enantiomeric ratio for
irreversible reactions, enantiomeric excess of substrate, enan-
tiomeric excess of product, racemate conversion, concentration of
S-enantiomer and concentration of R-enantiomer, respectively.
2
.7. Effect of pH and temperature on activity
The optimum pH and reaction temperature of free and encap-
sulated lipases were determined as the relative activity under the
variety of pH (50 mM phosphate buffer for pH 4.0–10.0) and tem-
perature (25–60 C). Relative activities were calculated as the ratio
3. Results and discussion
◦
of the activity of encapsulated enzyme after incubation to the activ-
ity at the optimum reaction pH and temperature.
3.1. Sol–gel encapsulation procedure using sporopollenin as
additives
2
.8. Thermal stability and reusability of encapsulated lipases
Sporopollenin forms microcapsules which have a large internal
cavity available for encapsulation with very high loadings. Particu-
lar encapsulants previously used for macromolecules are relatively
expensive with lower loadings than sporopollenin. Paunov et al.
[39] showed that alfa-amylase, beta-lactase, anti-AIDS drug or
oligonucleotides ranging in molecular mass from 1 to 116 kDa, are
capable of being encapsulated efficiently into sporopollenin exine
particles.
Moreover, in another study Kazlauskas demonstrated that the
presence of a small amount of isopropyl alcohol is beneficial
[44–46]. Usually interactions of the additive and the lipase on the
basis of hydrogen bonds were postulated, although in some cases
ambiguities as to the origin of the activating effect remain. In pre-
liminary experiments it was shown that the presence of isopropyl
alcohol during the sol–gel process does indeed exert a beneficial
effect [14]. Therefore, isopropyl alcohol and PVA were included in
all experiments. Besides this, considerably higher loadings were
strived for.
Free and encapsulated lipase preparations were stored in phos-
◦
phate buffer solutions (50 mM, pH 7.0) at 60 C for 2 h, respectively.
Samples were periodically withdrawn for activity assay. The resid-
ual activities were determined as above.
The stability of encapsulated enzymes on repeated use was
also examined by measuring the activity towards the hydrolysis
of p-NPP assay. After each activity determination, the encapsulated
enzyme was washed with buffer solution (PBS, 50 mM, pH 7.0) and
reintroduced into a fresh medium, this procedure being repeated
for up to 8 cycles.
2
.9. Storage stability
The stabilities of the free and encapsulated lipase were mea-
sured by calculating the residual activity after long time storage.
Free lipase was stored as a solution of 2 mg/ml in PBS (50 mM, pH
7
◦
Infrared and 13C NMR spectroscopic studies on sporopollenin
derived from pteridophyta and spermatophyta have shown that
sporopollenin has aliphatic, aromatic, hydroxyl, carbonyl/carboxyl
and ether functions in various portions in its polymeric struc-
ture [47]. Therefore, in this study the sporopollenin and activated
sporopollenin were chosen as the suitable adsorbents for C. rugosa
lipase. Sporopollenin was activated according to published proce-
dure [39]. This is evidenced by the aggressive extraction procedure
using strong alkali and acid. After extraction, exines retain the same
shape as the parent spores and as such constitute an empty shell
that can be used for encapsulation.
Most importantly, the encapsulated lipases exhibited enzymatic
activity against the p-NPP substrate. These results demonstrate that
the C. rugosa lipase was encapsulated within a chemically inert
sol–gel support prepared by polycondensation by tetraethoxysi-
lane (TEOS) and octyltriethoxysilane (OTES) in the presence and
absence of sporopollenin and activated sporopollenin as additive.
Scanning electron microscopy (SEM) allowed the verification
of morphological differences between the sporopollenin, activated
sporopollenin and lipase encapsulated on sporopollenin (Spo-E)
.0) and the encapsulated lipase was stored in wet form at 4 C.
Enzyme activity was assayed at regular intervals.
2
.10. Racemic Naproxen methyl ester production
Racemic Naproxen was prepared by the racemization of opti-
cally pure S-Naproxen as described by Wu and Liu [42]. The
1
synthesized Naproxen methyl ester was identified with FT-IR,
H
NMR and C NMR. FT-IR (KBr); 1765 cm (C O, ester), 1H NMR
1
3
−1
(
CDCI ); ı = 1.58 (s, 3H, CHCH ), 3.67 (s, 3H, OCH ), 3.86 (q, 1H,
3
3
3
CHCH ), 3.91 (s, 3H, COOCH ), 7.09–7.17 (m, 2H, Ar–H), 7.37–7.43
3
3
m, 1H, Ar–H), 7.64–7.73 (m, 3H, Ar–H). 13C NMR (CDCI ); ı = 18.81,
(
3
4
1
5.57, 52.27, 55.54, 105.81, 119.21, 126.15, 126.40, 127.39, 129.15,
29.49, 133.91, 135.89, 157.86, 175.36.
2.11. Hydrolysis of racemic Naproxen methyl ester
Hydrolysis reactions were performed in an aqueous
phase–organic solvent batch reaction system.
A solution of
racemic Naproxen methyl ester (20 mM) in 2 mL of isooctane was
added to 2 mL buffer solution (pH 7.0, 50 mM phosphate buffer
solution) containing encapsulated lipases (5–50 mg depending
on the activity), the mixture was treated in an horizontal shaker
at 150 rpm at 30 C and samples drawn from isooctane phase
at 24 h were analyzed by HPLC to calculate the conversion and
enantioselectivity.
(Fig. 1). For the pure support, microcapsule has a large internal
cavity. After the activated process, pore structure of the sporopol-
lenin (Spoact) was decomposed (Fig. 1(b)). After encapsulation, the
surface cavity of the sporopollenin was filled with lipase and the
other materials. Fig. 1(c) shows the rounded structure, which is
presumably protein aggregate.
◦
Table 1 shows the activity of the encapsulated lipases. However,
the encapsulated lipase with sporopollenin (Spo-E) was found to
be more efficient compared to activated sporopollenin (Spoact-E)
with respect to expression of encapsulated lipase activity. The Spo-
E was found to give 71 U/g of support with 88.8% activity yield,
while Spoact-E was found to give 68.4 U/g of support with 61.74%
activity yield.
Enantiomeric excess of the substrate (ees) and the product (eep)
were analyzed by HPLC using Chiral column and the enantiomeric
ratio (enantioselectivity, E) was calculated according to the equa-
tion of Chen et al. [43].
ln[(1 − x)(1 − ees)]
E =
ln[(1 − x)(1 + ees)]

E. Yilmaz et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 162–168
165
Fig. 1. SEM photographs: sporopollenin (a), activated sporopollenin (b) and encapsulated lipase (c).
Table 1
Activity of the free and encapsulated lipases under optimum reactions conditions.
Encap. protein (mg/g)
Encap. protein yield (%)
Lipase activity (U/g support)
Spesific activity (U/mg protein)
Activity yield (%)
100b
88.8
61.74
Free lipasea
Encapsulated lipase (Spo-E)
Encapsulated lipase (Spoact-E)
28.56
24.0
33.3
58.4
57.14
79.33
95.1
71.0
68.4
3.32
2.95
2.05
a
Encapsulated free lipase without sporopollenin.
Activity yield for free lipase was defined as 100%.
b
In our previous work [48], C. rugosa lipase (CRL) on sporopol-
lenin by adsorption method is reported. It has been observed that
under the optimum conditions (enzyme/support: 0.3/1 (w/w)),
the activity yield (%) of the immobilized lipase was 45.8, which
is approximately 2.0 times less than that of the encapsulated
lipase. Besides the enantioselective hydrolysis of rasemic Naproxen
methyl ester that was studied in aqueous buffer solution/isooctane
reaction system. It was observed that non-encapsulated lipase
shows poor enantioselectivity (E: 24.2).
3.2. pH effect on the activity of encapsulated lipases
It is well known that the procedure of enzyme immobiliza-
Fig. 2. Effect of substrate pH on residual activity of free (ꢀ), encapsulated lipase. The
activities of the enzymes were monitored with p-NPP assay at different pH values.
tion on insoluble supports has a variety of effects on the state of
ionization and dissociation of the enzyme and its environment.
Immobilization is likely to result in a conformational change of
the enzyme, which leads to inactivity of the enzyme. The catalytic
activity of the free and encapsulated lipases in the hydrolysis of
p-NPP was investigated at different pH (4.0–10.0). Upon immobi-
lization on sporopollenin, the optimum pH for reactions catalyzed
by free lipase was slightly shifted towards acidic values. Generally,
an acidic shift in the pH optimum is expected when enzymes are
immobilized onto polycationic supports [47].
◦
◦
3
5 C, while it shifted nearly to 40 C for encapsulated lipase (Spo)
◦
and 45 C for encapsulated lipase (Spoact).
Furthermore, the temperature profiles of the immobilized
lipases are broader than that of the free enzyme, which means that
the immobilization methods preserved the enzyme activity over
a wider temperature range. One of the main reasons for enzyme
As shown in Fig. 2 the optimum pH of the encapsulated lipases
was 5.0. Immobilized lipase showed better pH stability and resis-
tance to acidic environments than free lipase. Pereira et al. reported
the optimum pH values as 6.0 and 7.0, respectively, for immobilized
and free lipase [49]. The pH shift depends mainly on the method
of immobilization and the interaction of enzyme and support. A
change of the optimum pH for C. rugosa lipase immobilized on
poly(vinyl alcohol) microspheres was reported by Oh et al. [50].
3.3. Temperature effect on the activity of encapsulated lipases
The effect of temperature on free and encapsulated lipases is
given in Fig. 3. The effect of temperature on the activity of free and
encapsulated lipases for p-NPP hydrolysis at pH 7.0 in the tem-
perature range of 25–60 C is shown in Fig. 3. It was found that
the optimum temperature for the free enzyme was approximately
Fig. 3. Effect of reaction temperature on the residual activity of free (ꢀ), encapsu-
lated lipase (Spo-E) (ꢁ) and encapsulated lipase (Spoact-E) (ꢂ). The activities of the
enzymes were monitored with p-NPP assay at pH 7.0.
◦

1
66
E. Yilmaz et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 162–168
Fig. 4. Thermal stability of free (ꢀ), encapsulated lipase (Spo-E) (ꢁ) and encapsu-
lated lipase (Spoact-E) (ꢂ). The activities of the enzymes were monitored with p-NPP
assay at pH 7.0.
immobilization is the anticipated increase in stability towards var-
ious deactivating forces, due to restricted conformational mobility
of the molecules following immobilization [51–53]. This was either
due to the creation of conformational limitation on the enzyme
movement as a result of electrostatic interaction and hydrogen
bond formation between the enzyme and the support or a low
restriction in the diffusion of the substrate at high temperature.
Thus, the immobilized enzymes showed their catalytic activities at
a higher reaction temperature [54]. In literature, similar changes
in the optimum temperature have also been reported [55]. In our
previous work it was found that the optimum temperature for
immobilized CRL on cyclodextrin based polymer or calix[n]arenes
Fig. 5. Reusability of the encapsulated lipase (Spo-E) and encapsulated lipase
Spoact-E). The activities of the enzymes were monitored with p-NPP assay at pH
.0.
(
7
The activity of immobilized enzymes decreases with the increase
in reuse number.
These results could be explained by the inactivation of the
enzyme by the denaturation of the protein and the leakage of pro-
tein from the support upon use [60].
3.5. Storage stability
◦
based polymers was 45 and 50 C respectively [56,57].
The storage stability of the encapsulated enzymes was clearly
better than the free lipase (Fig. 6).
3.4. Thermal stability and reusability on the activity of
Thus, the support and the technique of immobilization provided
a longer shelf life than that of free counter part [61]. The reten-
tion in activity is usually observed after enzyme immobilization.
This could be explained by the modification in three-dimensional
structure of the enzyme, which leads to conformation change of
the active center. The presence of matrix hinders the accessibility
of substrate to the enzyme active site, and limitation of mass trans-
fer of substrate and product to or from the active site of the enzyme
may also be responsible. This explanation is in agreement with the
results reported [62].
encapsulated lipase
In this work, Fig. 4 shows the thermal stabilities of the free and
encapsulated lipases. Free and encapsulated lipases were incubated
◦
for 2 h at 60 C and the enzyme activity was measured at various
time intervals. It can be observed that the free lipase loses its ini-
tial activity within around 80 min at 60 C, while the encapsulated
lipases retain their initial activities of about 54% by Spo and 58% by
Spoact after 120 min of heat treatment at 60 C.
◦
◦
Utilization of enzymes in processes often encounters the prob-
lem of thermal inactivation of enzyme. At high temperature,
enzyme undergoes partial unfolding by heat-induced destruction
of non-covalent interaction [58,59]. The resistance of immobilized
lipase to temperature at a certain time is an important poten-
tial advantage for practical applications of this enzyme. Thermal
stability of the immobilized enzyme was greatly improved. Ther-
mal stability of lipase is obviously related with its structure [45].
These results indicate that the thermal stability of the immobilized
lipases is much better than that of the free one since the interac-
tion between the enzyme and the support, which could prevent the
conformation transition of the enzyme at high temperature.
Reusability for the immobilized enzyme is very important in
economics, and an increased stability can make the immobilized
enzyme more advantageous than its free counterpart. To investi-
gate the reusability, the enzymes immobilized were washed with
PBS (50 mM, pH 7.0) after one catalysis run and reintroduced into
The ability to be stored for a period of time at a certain tem-
perature is one of the key factors to be considered when using
immobilized lipases. Both lipases obtained show their full activ-
◦
ity (100%) when stored at 4 C. Generally, enzymes are still active
◦
a fresh p-NPP solution for another hydrolysis at 30 C. Fig. 5 shows
that the encapsulated lipase activities were maintained at levels
exceeding 28% and 26% of their original activities for encapsulated
lipase Spo and Spoact after 8 reuses, respectively. This shows that
immobilized lipase using this technique can be used successfully
for industrial applications requiring long-term reaction stability.
Fig. 6. Storage stability of free (ꢁ) and encapsulated lipase (Spo-E) (ꢂ) and encap-
◦
sulated lipase (Spoact-E) (ꢀ). They were stored at 4 C in 50 mM phosphate buffer at
pH value 7.0. The activities of the enzymes were monitored periodically with p-NPP
assay at pH 7.0.

E. Yilmaz et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 162–168
167
Table 2
Kinetic resolution of racemic Naproxen methyl ester using free and encapsulated
a
lipases. .
X (%)
ees (%)
eep (%)
E
Free lipaseb
Encapsulated lipase (Spo-E)
Encapsulated lipase (Spoact-E)
Non-encapsulated lipase
37.9
44.3
23.0
10.8
60.2
78.71
29.0
24.2
>98
>98
>98
>98
166.0
>400
89.7
24.2
a
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) as mobile phase; time, 24 h; concentration of substrate, 20 mM;
◦
pH 7.0; temperature, 35 C.
b
Encapsulated free lipase without sporopollenin.
when kept at low temperature probably because lipases tend to
lock to its original conformation, which is catalytically active. The
free enzyme rapidly loses its activity with a residual value of 15%
after12 days, the decrease in activity occurs more slowly with the
encapsulated lipases, and about 95% (Spo-E) and 93% (Spoact-E) of
their initial activity were recovered after the same period. The sta-
bility achieved was referred to multiple attachment of the enzyme
to the support, preventing any intermolecular process such as pro-
teolysis and aggregation, therefore creating a more rigid enzyme
molecule [63].
Fig. 7. Effect of pH on the conversion (x) in the hydrolysis of racemic Naproxen
methyl ester.
and the enantioselectivity being very high (E > 400). Whereas the
resolution reactions with free encapsulated lipase (free lipaseenc)
gave an unreacted Naproxen methylate (R)-ester and correspond-
ing acid (eep) 98% at conversion of 37.9% and the enantioselectivity
(E) being 166. Immobilization led to high enantioselectivity, high
conversion and fast recovery of product compared to free enzyme.
The result was not surprising because in the literature Tsai et al.
[64] used lipase MY from C. rugosa and catalyzed hydrolysis of
(R, S)-Naproxen esters in water-saturated isooctane as the model
system. They found E value was 510. In addition [65], racemic fluox-
etine was catalyzed with sol–gel immobilized ABL and sol–gel/PVA
immobilized ABL and found E values from 43 to 127–473.
The results indicate that in particular sporopollenin and
activated sporopollenin based encapsulated lipases had higher con-
version and enantioselectivity compared to the sol–gel free lipase.
However during the activated process, because pore structure of
activated sporopollenin was decomposed (Fig. 1). Thus its activity
and enantioselectivity are lower than the non-activated sporopol-
lenin.
3.6. Enantioselectivity of the encapsulated Candida rugosa lipase
with sporopollenin and activated sporopollenin in the kinetic
resolution of racemic Naproxen methyl ester
From an industrial point of view, the quality of a given kinetic
resolution not only depends upon the degree of enantioselectiv-
ity, but also on the activity and the possibility of recycling and
reusing the lipase. We therefore studied all of these factors in a
test reaction involving the hydrolysis reaction-kinetic resolution
of (RS)-Naproxen methyl ester, although complete optimization
was not strived for. All reactions were carried out on a small scale
and stereoselectivity was ascertained by measuring the selectivity
factor E on the basis of the formula of Chen et al. [43].
In chiral resolution using enzyme as a catalyst, it has been
reported that variation of pH might influence chiral selectivity since
the conformation of an enzyme depends on its ionization state [49].
The effects of pH on enantioselectivity of encapsulated lipase were
determined by incubating encapsulated lipase in the presence of
Table 2 shows the results obtained in the resolution of racemic
Naproxen methyl ester catalyzed by encapsulated lipases. It shows
the conversion (x), enantiomeric excess (ee) and enantiomeric ratio
(
E) in the course of (RS)-Naproxen methyl ester hydrolysis by the
◦
encapsulated lipases. The enantioselective hydrolysis of racemic
Naproxen methyl ester by sol–gel encapsulated lipases was studied
in aqueous buffer solution/isooctane reaction system (Scheme 1).
The resolution reaction with encapculated lipase (Spo) was
terminated after 24 h, obtaining Naproxen methylate (unreacted
R-ester) and corresponding acid (eep) 98% at conversion of 44.3%
sporopollenin at different pH (i.e. pH 5.0 and 7.0) and, at 35 C for
24 h. At the end of the incubation time the rate of enzyme reaction
and enantiomeric excess (ee) were determined using HPLC (Agilent
1200 Series) equipped with Chiralcel OD-H column at the temper-
◦
ature of 25 C. The optimum pH values were determined from the
graph of pH plotted against the percentage of conversion (x) (Fig. 7).
Scheme 1. Enantioselective hydrolysis of (R, S)-Naproxen methyl ester.

1
68
E. Yilmaz et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 162–168
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