Investigation of synthetic lipase and its use in transesterification reactions

Article abstract of DOI:10.1016/j.polymer.2012.03.021

A novel synthetic polymer selective for p-nitrophenylpalmitate was synthesized by molecular imprinting technique. We have combined the principle of molecular imprinting with the ability of histidine, glutamic acid and serine to form a catalytic cavity that can promote the catalytic degradation of p-nitrophenyl palmitate. For the creation of such catalytic sites we first synthesized appropriate monomers and used p-nitrophenyl palmitate as a template to synthesize the imprinted polymers and the binding characteristics of the polymers were evaluated. The optimum pH was determined by evaluating different pH values and the hydrolytic activity of synthetic lipase was evaluated in the framework of Micheaelis-Menten kinetics. In addition, the values of maximal rate: Vm (0.68 mM/min) and Michaelis-Menten constant, Km (1.4 × 10 ^-2 mM) were obtained from Lineweaver-Burk plots for the imprinted polymeric catalyst.

Full text of DOI:10.1016/j.polymer.2012.03.021

Polymer 53 (2012) 1981e1984
Contents lists available at SciVerse ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Investigation of synthetic lipase and its use in transesterification reactions
a,*
a
a
a
b
Rustem Kecili , Rıdvan Say , Arzu Ersöz , Deniz Hür , Adil Denizli
a
Department of Chemistry, Anadolu University, Yunus Emre Kampusu, 26470 Eskisehir, Turkey
Department of Chemistry, Hacettepe University, 06800 Beytepe, Ankara, Turkey
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 November 2011
Received in revised form
A novel synthetic polymer selective for p-nitrophenylpalmitate was synthesized by molecular imprinting
technique. We have combined the principle of molecular imprinting with the ability of histidine, glu-
tamic acid and serine to form a catalytic cavity that can promote the catalytic degradation of p-nitro-
phenyl palmitate.
27 February 2012
Accepted 8 March 2012
For the creation of such catalytic sites we first synthesized appropriate monomers and used p-
nitrophenyl palmitate as a template to synthesize the imprinted polymers and the binding characteristics
of the polymers were evaluated. The optimum pH was determined by evaluating different pH values and
the hydrolytic activity of synthetic lipase was evaluated in the framework of MicheaeliseMenten
kinetics. In addition, the values of maximal rate: Vm (0.68 mM/min) and MichaeliseMenten constant,
Km (1.4 ꢀ 10ꢁ mM) were obtained from LineweavereBurk plots for the imprinted polymeric catalyst.
Ó 2012 Elsevier Ltd. All rights reserved.
Available online 15 March 2012
Keywords:
Molecular imprinting
Mip
2
Synthetic lipase
1. Introduction
the problems of using enzymes as catalysts is their serious limita-
tions in terms of recovery [29], when enhancing the stability and
Molecular imprinting is a method for making selective binding
sites in synthetic polymers by using a molecular template. Target
molecules can be used as templates for imprinting crosslinked
polymers. After the removal of the template, the remaining poly-
mer is more selective. The selectivity of the polymer depends on
various factors like the size and the shape of the cavity and
rebinding interactions. Covalent interactions [1e4], non-covalent
interactions [5e9], electrostatic interactions [10] and metal ion
coordination [11e13] can be exploited to organise the functional
monomers around the template.
maintaining the activity of the biocatalyst, the choice of the enzyme
support is of extreme importance [30,31]. Lipases have
histidineeglutamic acid and serine trial amino acid group in the
active centre.
On the other hand, Methyl jasmonate (MeJA) is a substance used
in plant defence [32] and also under early research for cancer
treatment in humans. Plants produce jasmonic acid and methyl
jasmonate in response to many biotic and abiotic stresses (partic-
ularly herbivory and wounding), which build up in the damaged
parts of the plant.
The molecularly imprinted polymers (MIPs) have been used for
separations, diagnostic assays, biosensors and biocatalysis. MIPs
have also been employed in producing of synthetic enzyme catal-
ysis applications. A detailed review about this subject [14] and
some applications of MIPs in catalysis [15e27] have been reported.
Lipases (triacylglycerol acylhydrolases) belong to the class of
serine hydrolases and therefore do not require any cofactor. The
usual industrial lipases are special classes of esterase enzymes that
act on fats and oils, and hydrolyse them initially into the substituted
glycerides and fatty acids, and finally on total hydrolysis into
glycerol and fatty acids [28], a wide variety of reactions, such as
hydrolysis, transesterification, and aldol reactions. However, one of
In this study, we have used advantages of high selectivity of
molecular imprinting technique for synthesis of artificial lipase for
catalysis of p-nitrophenyl palmitate and using in synthesis of bio-
diesel and methyl jasmonate.
2. Experimental section
2.1. Chemicals
Candida Rugosa lipase and p-nitrophenyl palmitate were
purchased from Sigma (St Louis, MO, USA). Ethyleneglycol dime-
thacrylate (EDMA) was purchased from Fluka A.G. (Buchs,
Switzerland), distilled under reduced pressure in the presence of
ꢂ
hydroquinone inhibitor and stored at 4 C until use. Azobisisobu-
tyronitrile (AIBN) and Poly (vinyl alcohol) (PVA; MW: 22,000) were
purchased from Aldrich Chem. Co. (Milwaukee, WI, USA). All
*
Corresponding author. Tel.: þ90 222 335 0580/5816; fax: þ90 222 320 4910.
E-mail address: rkecili@anadolu.edu.tr (R. Kecili).
0
032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2012.03.021

1982
R. Kecili et al. / Polymer 53 (2012) 1981e1984
glassware was extensively washed with diluted nitric acid before
CH3
CH3
use. Water used in the experiments was purified using a Barnstead
Ò
O
(
Dubuque, IA) ROpure LP reverse osmosis unit with a high flow
O
cellulose acetate membrane (Barnstead D2731) followed by
Ò
a Barnstead D3804 NANO pure organic/colloid removal and ion
exchange packed-bed system.
O
O
O
NH
CH3
CH OH
2
2
.2. Synthesis of p-nitrophenyl palmitate imprinted polymeric
CH3
OH
O
beads
O
CH3
OH
The MASE, MAH, and MAGA monomers were synthesized
according to previously published procedure [33].
O
O
pNPP
O
O
N
NH
C
An appropriate amino acid (5.52 mmol, 1 g, 1 eq.) was dissolved
in 1 M aqueous solution of NaOH (1 eq.) in a round bottom-flask. A
solution of MA-Bt (5, 52 mmol, 1.033 g, 1eq) in 25 mL of 1,4-dioxane
was slowly added into the amino acid solution. The reaction
mixture was allowed to stir for 10e20 min at room temperature.
Completion of the reaction was monitored by TLC, after the reaction
finished, 1,4-dioxane was evaporated under vacuum. The residue
was diluted with water and extracted with ethyl acetate
O
CH2
HO
CH
C
O
C
O
O
O
HN
OH
O
O
CH3
O
H C
3
CH
(
3 ꢀ 50 mL) to remove 1 H- benzotriazole. Collected water phases
CH3
CH3
were neutralized to pH ¼ 6e7 using 10% water solution of HCl (pH
should keep around 6e7 to prevent possible polymerization of
methacryloyl group in acidic medium). Then, water was removed
via rotary-evaporator to give amino acid monomer in 85e90% yield.
P-nitrophenyl palmitate imprinted beads (PIBs) were synthe-
sized by suspension technique as follows. The dispersion medium
was prepared by dissolving 0.15 g of poly(vinyl alcohol) in 60 mL of
distilled water. Methacryloylamido serine (MASE), meth-
acryloylamido histidine (MAH) and methacryloylamido glutamic
acid (MAGA) monomers (0.1 mmol) and p-nitrophenyl palmitate
CH3
O
O
O
CH3
O
Fig. 1. Schematic representation of p-nitrophenyl palmitate (pNPP)-template recon-
struction in polymeric structure.
(
0.1 mmol) as a template molecule were added into 6.0 mL of
EDMA/12.0 mL of tolueneeacetonitrile mixture (80:20); then
.609 mmol of 2,2-azobisisobutyronitrile (AIBN) was dissolved in
0
methanol were used for esterification using the same amount of
synthetic catalyst. After each batch reaction, the synthetic catalyst
was recovered by filtration, washed with distilled water and dried
at room temperature. Dried synthetic catalyst was used in the next
batch reaction for new substrates. This proves that this catalyst can
be used repeatedly.
the monomer mixture. This solution was then transferred into the
dispersion medium placed in a magnetically stirred (at a constant
stirring rate of 600 rpm) glass polymerization reactor (100 mL) in
a thermostatic water bath. The reactor was flushed by bubbling
nitrogen and then was sealed. The reactor temperature was kept
ꢂ
constant at 75 C for 8 h. Then the polymerization was completed at
ꢂ
9
0
C during 4 h. After polymerization, the PIBs were separated
2.4.2. GCeMS analysis
from the polymerization medium. The template was removed by
washing procedure using 100 mL of 50/50 v/v methanol/water
containing 1.0 M of KOH for 24 h. In Fig. 1, Schematic representation
of p-nitrophenyl palmitate (pNPP)-template reconstruction in
polymeric structure is given.
GC MS analysis was performed by injecting 1 mL of reaction
mixture into a Shimadzu GCMS-QP5050 instrument equipped with
an apolar column (25 ꢀ 0.25 mm id, CP Sil 5 CB). Analysis was
carried out using gradient temperature program running from
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
60 C to 180 C at 2 C/min and 180 C to 240 C at 2 C/min.
ꢂ
Injection and detection temperature were fixed at 250 C using
2.3. Activity assay of synthetic lipase
helium as carrier gas.
Lipase activity was measured by the spectrometric method
using p-nitrophenyl palmitate (p-NPP) as a substrate. The activity
was assayed by measuring the absorbance of liberated p-nitro-
phenol at 405 nm [34].
2.4. Biodiesel production and jasmonic acid esterification by using
synthetic lipase
2.4.1. Transesterification reactions
Transesterification reactions were carried out in 50 ml reactors
containing a mixture of 5 g of olive oil, 6 g of methanol and 0.5%
water (w/w) using 25 mg of synthetic lipase for biodiesel produc-
tion. The pH of reaction medium was adjusted to 8.0 with meth-
ꢂ
anolic NaOH. The reaction mixture was incubated at 50 C and
shaken in an reactor thermomixer. In the second step of the
experiments, it was used 14 mg of jasmonic acid and 14 mL of
Fig. 2. Effect of pH on p-NPP hydrolysis using free and synthetic lipase.

R. Kecili et al. / Polymer 53 (2012) 1981e1984
1983
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
500
1000
Inverse p-NPP concentration( 1/M)
1500
2000
2500
Fig. 3. Effect of temperature on p-NPP hydrolysis using free and synthetic lipase.
Fig. 5. LineweavereBurk plot of kinetics data of hydrolysis of paranitrophenyl
palmitate.
3
. Results and discussion
The hydrolytic activity of synthetic lipase was evaluated with
respect to MichaeliseMenten formula and kinetics. For this
purpose, p-nitrophenyl palmitate substrate concentrations were
varied from 2 mM to 20 mM and initial reaction rates (IRR) of
hydrolysis were determined. Then, 1/IRR versus reciprocal of the
substrate concentration (1/S) for polymeric beads was plotted
which is also known as the LineweavereBurk plot (Fig. 5) and the
values of Vm and Km were obtained from plots as 0.68 mM/min and
3.1. Effect of pH on p-nitrophenyl palmitate (p-NPP) hydrolysis
The pH is one of the important parameters that can affect the
enzymatic activity in aqueous solution. In this study, the optimum
pH was determined by evaluating different pH values in the range
of 6.0e10.0 using 20 mM p-nitrophenylpalmitate as a substrate.
The effect of pH on the time course of p-nitrophenylpalmitate
hydrolysis is shown in Fig. 2. As seen from the figure, the maximum
hydrolytic activity was observed at pH 8.0.
ꢁ
2
1,4 ꢀ 10 mM, respectively.
In MichaeliseMenten kinetics, Km reflects the affinity of an
enzyme for a particular substrate i.e. the lower value of Km the
higher affinity [17]. In the present case Km represents the affinity of
functional groups of mimics lipase for substrate p-nitrophenyl
palmitate in the imprinted material.
3.2. Effect of temperature on p-nitrophenyl palmitate (p-NPP)
hydrolysis
It is known that temperature has a strong influence on the
catalytic activity of a catalyst. Here we have evaluated a tempera-
ture range between 20 Ce50 C. Fig. 3 shows the effect of
temperature on p-nitrophenylpalmitate hydrolysis.
3.4. Biodiesel production and jasmonic acid esterification by using
synthetic lipase
ꢂ
ꢂ
As can be seen from the figure, the optimum temperature is at
approximately 40 C.
In this part of the study, we wanted to evaluate the synthetic
lipase to use in transesterification reactions. The fatty acid methyl
ester and methyl jasmonate conversion are seen in Fig. 6.
FAME conversion of fatty acid in olive oil is 92.97% and the
conversion of jasmonic acid to methyl jasmonate by using synthetic
lipase is 100%.
ꢂ
3
.3. Hydrolytic activity of synthetic lipase
Fig. 4 shows the time course of p-nitrophenyl palmitate
These results show that synthesized biomimetic artificial
enzyme can effectively be used in transesterifaction reactions. The
main advantages of synthetic lipase used in this work are high
thermal and chemical stability, low cost and reusability compared
to natural lipase.
hydrolysis by the synthetic lipase. The kinetic constants were
obtained by fitting the data to equation (1), where V is the initial
rate, Vm is the maximum rate, S is the concentration of substrate,
Km is the Michaelis-Menten constant.
V ¼ Vm$S=ðKm þ SÞ
(1)
2
2
1
1
50
00
50
00
50
0
0
20
40
60
Reaction Time (min)
80
100
120
140
Fig. 4. p-Nitrophenyl palmitate hydrolysis using synthetic lipase at pH 8.0.
Fig. 6. Conversion of fatty acids and jasmonic acid using synthetic lipase.

1984
R. Kecili et al. / Polymer 53 (2012) 1981e1984
Fig. 7. Reusability of synthetic lipase.
3
.5. Reusability of synthetic lipase
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4
. Conclusions
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125e36.
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prepared using MAH, MAGA and MASE monomers. Those were
chosen with the purpose to improve the stability, decrease the cost
and to yield a selective catalyst towards p-nitrophenyl palmitate
hydrolysis and esterification reactions. The preparation of the poly-
mer is simple and straight forward. The results demonstrate that the
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efficient mimic catalyst for the hydrolysis of p-nitrophenyl palmitate.
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