Immobilization of acetylcholinesterase i
Eng. Life Sci. 2010, 10, No. 1, 57–64
57
Atousa Moradzadegan1
Seyed-Omid Ranaei-
Research Article
Siadat1
Azadeh Ebrahim-Habibi2
Immobilization of acetylcholinesterase
in nanofibrous PVA/BSA membranes by
electrospinning
Mohammad BarshanTashnizi1
Rouhollah Jalili3
Seyed-Fakhraddin Torabi4
Khosro Khajeh5
1
NanoBiotechnology
Engineering Lab.,
Department of
Biotechnology, Faculty of
Energy Engineering and
New Technologies, Shahid
Beheshti University, GC,
Tehran, Iran
2
Endocrinology and
Metabolism Research
Center, Tehran University of
Medical Sciences, Dr.
Shariati Hospital, Tehran,
Iran
Electrospinning, a simple and versatile method to fabricate nanofibrous supports,
has attracted continuous attention in the field of enzyme immobilization. In this
study, acetylcholinesterase (AChE) has been successfully immobilized in PVA
nanofibers via electrospinning of a mixture of AChE, BSA as an enzyme stabilizing
additive and PVA. The maximum activity recovery of immobilized AChE was
about 40%. In comparison with free enzyme, the immobilized AChE showed
improved stability while retaining a considerable amount of activity at lower pH
values. Moreover, the immobilized AChE retained 434% of its initial activity
when stored at 301C for 100 days and retained 70% of its initial activity after ten
consecutive reactor batch cycles.
Keywords: Acetylcholinesterase / Electrospinning / Immobilization / Nanofiber / PVA
Received: January 6, 2009; revised: October 21, 2009; accepted: October 27, 2009
DOI: 10.1002/elsc.200900001
3
New Ideas Research
Institute (NIRI), Tehran,
Iran
4
Department of
Biotechnology, University of
Tehran, Tehran, Iran
5
Department of
Biochemistry, Faculty of
Biological Science, Tarbiat
Modares University,
Tehran, Iran
1
Introduction
Enzyme immobilization has been a popular strategy for most
large-scale applications due to the ease in catalyst recycling,
continuous operation, and product purification [1, 2]. The
most explored approach to immobilize enzymes has involved
Correspondence: Dr. Khosro Khajeh (khajeh@modares.ac.ir),
Department of Biochemistry, Faculty of Biological Science, Tarbiat
Modares University, Tehran, Iran.
the use of solid supports via a variety of mechanisms, such as
physical adsorption, covalent bonds, or entrapment/encapsulation [3]. With increasing demand for nanotechnology, electrospinning has become a novel technique for generating
composite nanofibers. Electrospinning is a method for
producing nanofibers from a variety of materials with fiber
diameters ranging from several micrometers down to tens of
nanometers [4–6]. The electrospun nanofibrous membrane
has high specific area and porous structure, so there are
Current Address: Department of Biology, Sciences and Research Branch,
Islamic Azad University, Tehran, Iran
Abbreviations: AChE, acetylcholinesterase;
cholineiodide; GTA, glutaraldehyde
ATChI,
acetylthio-
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Additional corresponding author: Dr. Seyed-Omid Ranaei-Siadat
E-mail: O_ranaei@sbu.ac.ir
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Eng. Life Sci. 2010, 10, No. 1, 57–64
A. Moradzadegan et al.
excellent candidates for filtration, drug delivery carrier, tissue
engineering, wound dressing, nano-sensors, and enzyme
immobilization [7–9]. Immobilization of enzymes on both
natural and synthetic polymeric supports has been widely
reported [10–17]. PVA can be used for enzyme immobilization
by electrospinning since it has good properties in forming
membranes and fibers. Besides, it is a biologically compatible,
nontoxic, hydrophilic, and readily available low-cost polymer
[18–22]. Cellulase loaded at 10% in 200 nm diameter PVA
fibers exhibited 65% of original activity [23]. Immobilization
of lipase in 100–150 nm diameter PVA nanofibrous, enzyme
loading in these biocomposite fibers reached as high as 50%
[24]. Glucose oxidase loaded at 8% in 70–250 nm diameter
PVA fibers and chronoamperometric measurmens demonstrated that electrospun fibrous enzymatic electrodes exhibited
a rapid response (1 s) and a higher response current (mA level)
to glucose in the normal and diabetic level [25] Acetylcholinesterase (AChE) (E.C 3.1.1.7) is a serine hydrolase,
which catalyzes the hydrolysis of the neurotransmitter acetylcholine [26]. Carbamates and organophosphates are potent
inhibitors of AChE, which causes the blocking of the nerve
signal transference into the postsynaptic membrane [27].
Because of the uniform mechanism of signal transduction in
animals, these inhibitors have been used as agricultural pesticides as well as chemical warfare agents (nerve agents) [28].
Immobilized AChE can be used for construction of biosensor
for pesticide determination [29, 30].
Two main methods have been employed to make an
assemblage of enzymes and polymeric fibers via electrospinning: (i) immobilization of the enzyme on the outer surface of
the nanofibers and (ii) mixing enzyme with the polymer
solution and subsequently spinning. In the first approach,
polymeric materials can be used, which are soluble in organic
media. However, enzyme is immobilized only on the outer
surface of the fibers while the interior of the fibers remains
inactive. In contrast to the first approach, water-soluble
polymers such as PVA or dextran can be used as a solid
support in the second approach. However, dissolution of the
fibers in an aqueous environment and subsequent enzyme
leaching would be a problem. To overcome this problem, it was
suggested to use vapor glutaraldehyde (GTA) [24] so that the
nanofibers can be cross-linked to prevent support dissolution.
In the present study, AChE was immobilized using mixed
spinning approach of the enzyme, PVA, and BSA biocomposite
nanofibers with the use of vapor GTA. Kinetic parameters of
both free and immobilized enzymes were determined. Also
storage stability of the immobilized system was compared with
free enzyme and reusable stability of immobilized enzyme was
assayed.
2
Materials and methods
2.1
Materials
AChE (E.C 3.1.1.7) was expressed with the baculovirus system
[31]. GTA 25% was purchased from TAAB Laboratories (England,
UK). BSA, acetylthiocholineiodide (ATChI) and 5,50 -dithiobis(2-nitrobenzoic acid) were obtained from Sigma-Aldrich (MO,
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
USA). PVA 72000 and 498% of degree of hydrolysis and all other
agents used in the experiments were of analytical grade and were
from Merck (Darmstadt, Germany). Aqueous solutions were
prepared in doubly distilled deionized water.
2.2
2.2.1
Methods
Preparation of nanofibrous membranes
PVA aqueous solution (6% w/w) was prepared by dissolving
PVA powder in deionized water at 801C with gentle stirring for
4 h to form homogenous solution after removing air bubbles.
After the solution was cooled to the room temperature,
prepared solutions in different concentrations were placed in a
plastic syringe (1 ml) bearing a metal capillary (0.8 mm
diameter) connected to a high-voltage power supply; the
grounded counter electrode connected to the aluminum foil
collector. Electrospinning was performed at 10–12 kV voltage,
15 cm distance between the needle tip and the collector. The
flow rate of the solution was controlled by a syringe pump to
maintain at 26 mL/h from the needle outlet. It usually takes
45 min to obtain a sufficiently thick membrane that can be
detached from the aluminum foil collector.
PVA/BSA solution was prepared by gently mixing PVA and
BSA solution to yield the spinning solution with final
concentrations of 6% w/w and 10 mg/mL for PVA and BSA in
spinning solution, respectively. The same procedure was
used to prepare PVA/BSA/AChE solution by adding an
aqueous solution of AChE to 25 mM phosphate buffer, pH 7.4,
different ratios of BSA and AChE to PVA were used. An aliquot
of 1 mL of each solution under the mentioned conditions
was electrospun to form the biocomposite nanofibrous
membranes. The electrospinning was performed at room
temperature and the resulting PVA/BSA and PVA/BSA/AChE
biocomposite fibers were collected on the surface of aluminum
foil collector.
Covalent attachment of BSA in nanofibrous PVA
membranes was carried out by cross-linking the electrospun
PVA/BSA membranes at room temperature by GTA vapor for
12 h. PVA/BSA membrane (20 mg) was immersed in glass vials
containing deionized water at room temperature and was
agitated at 300 rpm for 120 min to detach all un-bounded
protein from the membrane. The amount of released protein
was determined by measuring the residual protein present in
the supernatants by Bradford method using coomassie brilliant
blue G-250 and BSA as the standard protein [32]. The amount
of immobilized protein was estimated by subtracting the
amount of protein in the supernatants from the total amount
of protein used in the immobilization procedure. The protein
loading efficiency was determined by dividing the amount of
immobilized BSA (mg) in the membrane to the whole support
membrane mass (g).
In the case of AChE, enzyme immobilization was carried
out by cross-linking the electrospun PVA/AChE membranes at
41C by GTA vapor for 12 h. AChE immobilization was also
studied in presence of BSA to yield the PVA/BSA/AChE
nanofibrous membranes using GTA vapor at different enzyme/
BSA ratio. Finally, a solution of 6% w/w PVA, 10 mg/mL BSA
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Eng. Life Sci. 2010, No. 1, 57–64
and 3 mg/mL AChE was electrospun into biocomposite nanofibrous membranes and used for determination of kinetic
parameters, pH profile, and stability experiments. The
morphology of this nanofibrous PVA/BSA/AChE membrane
was characterized by a Philips XL-30 scanning electronic
microscope after being sputtered with gold.
2.2.2
Catalytic activity determination
The AChE activity was determined according to the ellman
method [33, 34] using ATChI as substrate and 5,50 -dithiobis(2-nitrobenzoic acid) as chromogen and increasing absorbance
at 412 nm was measured for 1 min. For the immobilized
enzyme assay, the electrospun nanofibrous membrane was
removed from the collector and cut into 1 cm 1 cm pieces,
3 mg of nanofibers containing immobilized enzymes were
added to each mixture reaction (1 mL) in vials, vortexed for
1 min, and then centrifuged rapidly at 16 000 g for 1 min.
The product concentration that was proportional to the
hydrolyzed ATChI, therefore, to the intensity of the yellow
solution in the resulting supernatant was determined spectrophotometrically at 412 nm. One unit of enzymatic activity
was defined as the amount of enzyme that catalyzes 1 mmol of
substrate to product per minute. Specific activity was expressed
as units per milligram of protein in the assay medium (1 mL)
and the relative activity was defined as the ratio between the
specific activity of a bound enzyme and free enzyme. All
reactions and measurements were carried out at room
temperature. In order to determine kinetic parameters (Km
and Vmax) for free and immobilized enzyme, several concentrations of ATChI were used ranging from 0.01 to 1 mM. The
activity of free and immobilized enzyme was determined
within the pH range of 4 to 8.5.
The amount of protein in the different samples was estimated
according to the Bradford method using BSA as standard [34].
2.2.3
Immobilization of acetylcholinesterase
59
tion can be electrospun into nanofibers with diameter around
190 nm as shown in Fig. 1A. With the addition of BSA and
AChE, electrospun fibers became irregular but the diameter of
the fibers did not change (Fig.1B). As a protein, it is impossible
for BSA or AChE to be electrospun alone into nanofibers due
to its complicated three-dimentional structures as well
as strong inter- and intra-molecular forces [12]. The blending
of BSA and AChE with PVA can interrupt its complex structure. PVA has a capacity for secondary binding; because of
this, the mixture of PVA and the enzyme can be electrospun
and form nanofibers by electrospinning. On the other
hand, the interaction of PVA and protein resulted in the
reduction of stability of PVA solution and induced the
appearance of irregular [24].
3.2
Covalent immobilization of BSA in PVA
membrane
After co-spinning of different concentration of BSA with PVA,
the nanofibers were incubated with vaporized GTA for 12 h.
The amount of released BSA was measured (Fig. 2). A significant decrease was observed after increasing the concentration
of BSA from 3 to 18 mg/mL. The optimum BSA concentration
for immobilization of AChE in nanofibers was found to be
10 mg/mL. GTA acts as a linker and links amine groups of
lysine residues of BSA and due to the conglutination between
BSA molecules, creates a protein networks onto PVA
membranes. This may be the reason for more immobilization
when high concentration of BSA was used.
Storage stability and reusability assay
Activities of the free and immobilized AChE were determined
after storage in 25 mM phosphate buffer solution (pH 7.4) at 4
and 301C. The measurements were performed at intervals of
2 wk within a period of 100 days. The reusability of bound
AChE was examined by conducting the activity measurement
of bound AChE at time intervals of 15 min. After each activity
measurement, the bound AChE was washed three times with
25 mM phosphate buffer. The supports were then centrifuged,
the supernatant was decanted, and the recycled supports
subjected to the activity assay for the second cycle and so on.
3
Results and discussion
3.1
Morphology of PVA/BSA/AChE electrospinning
composite membrane
The physical morphology of the electrospun samples was
examined using the scanning electron microscope. PVA solu-
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Scanning electron micrographs of the electrospun PVA
(A) and the electrospun PVA/BSA/AChE fibers (B).
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60
Figure 2. Immobilization of several concentration of BSA onto
PVA membranes. For more details please see Section 2.
3.3
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A. Moradzadegan et al.
Enzyme immobilization
AChE was covalently immobilized in modified membranes
using vapor GTA. Because of low concentration of AChE with
respect to BSA, no activity was detected in the supernatant at
the end of immobilization procedure. According to the
Drosophila AChE crystal structure [35] there are more than 20
lysine residues available on the AChE surface for reaction with
GTA.
GTA is highly reactive toward both the amine and hydroxyl
groups and acts as a linker [36]. These mechanisms result in
covalent attachment of AChE in PVA/BSA membranes. The
activity of immobilized AChE with different enzyme loading
(0.2–3.5 mg/mL) was determined (Fig. 3). The relative immobilized AChE activity (activity recovery) was more than 40%.
Decrease in activity is usually observed after enzyme immobilization. Enzyme immobilization via covalent binding will
change enzyme conformation or reduce its flexibility and may
cause reduction in enzyme mobility to induce fit to a substrate
[37] that may lead to a reduction of AChE activities. Moreover,
GTA as a cross-linking agent reacts with hydroxyl groups from
PVA chains and causes the PVA membranes to pack more
densely, thus decreasing surface area of the support as well as
mass transfer through it [23]. Besides, cross-linking can reduce
membrane porosity, which in turn causes limited accessibility
of substrates to active site.
In addition, different concentrations of BSA (0, 3, 6, 10, 14,
and 18 mg/mL) were electrospun into biocomposite
membranes containing 6% w/w PVA and 3 mg/mL AChE. All
samples were subjected to cross-linking with vapor GTA as
mentioned in Section 2. The fibers were washed three times
thoroughly with phosphate buffer solution (25 mM, pH 7.4) to
extract out un-bonded enzymes. Buffer solutions were found
to have no detectable enzyme activity, indicating that there was
negligible enzyme leakage during washing procedure. Therefore, the yield of AChE immobilization was found to be about
100%. The activity of the resulting immobilized AChE was
compared with the free enzyme to determine enzyme activity
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. Effect of several concentrations of enzyme on the
remaining activity of the immobilized enzyme. All experiments
were carried out at pH 7.4 (25 mM phosphate buffer) and room
temperature.
retention after immobilization. Results showed that the
presence of BSA had a significant effect on AChE activity
retention. In comparison to PVA, a significant increase in the
retaining activity of immobilized enzyme (23–40%) was
observed using PVA and BSA (10 mg/mL), simultaneously.
Several speculate could be used to explain this phenomenon.
First, the BSA-modified fibers could create a biocompatible
microenvironment for the immobilized AChE, which usually
leads to high activity retention of the immobilized enzyme
[10]. Second, decreased number of covalent bonds with the
support (number of hydroxyl groups of PVA is very high
compared with NH2 groups of BSA) may cause improvement
in the catalytic efficiency of the immobilized enzyme. Finally,
using BSA as a linker between AChE and the support would be
in favor of enzyme flexibility.
Although the presence of BSA led to a significant increase in
the activity retention of immobilized AChE, increasing loaded
protein from 14 to 18% resulted in decreasing activity retention from 40 to 33%. This may be due to the presence of large
numbers of amino groups on the surface of BSA molecules,
which are potential reaction sites for covalent coupling with
enzyme. Thus, it becomes easier to immobilize one enzyme
molecule through multipoint covalent attachments on the
BSA-modified membrane with higher concentrations of BSA,
which negatively affect the enzymatic activity. Based on
observed results, it has been suggested that enzyme immobilization using 3 mg/mL AChE into biocomposite membrane
loaded with 10% BSA would be the condition to achieve
maximum activity retention.
3.4
pH-activity profile
The pH is one of the important parameters capable of altering
enzymatic activities in aqueous solution. The effect of pH on
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Eng. Life Sci. 2010, No. 1, 57–64
the activity of the free and immobilized AChE was investigated
within range of pH 4.5–8.5 at room temperature. Relative
activity as a function of pH is depicted in Fig. 4. The optimum
pH of immobilized AChE was found to be about 7.5. So, the
three dimensional structure of enzyme’s active site may not be
affected from immobilization procedure and optimum pH of
immobilized enzyme can be observed as free enzyme [38]. The
immobilized enzyme was less sensitive to pH changes at acidic
pHs than alkaline pHs compared with that of free AChE. These
data indicate that the pH stability of AChE could be enhanced
by the immobilization process at low pH values [39].
PVA/BSA/AChE biocomposite contained about 15% w/w
protein and 85% polymer. The isoelectric point of BSA is 4.8
and is positively charged at lower pH values [40]. The presence
of protonated BSA amino groups on the surface of the
membrane might repel protons from the region in the vicinity
of the surface and create a higher pH at the boundary layer
between the support and the bulk solution. This microenvironment with a pH slightly higher than bulk solution may
lead to an increase in pH stability of the immobilized enzyme.
3.5
Immobilization of acetylcholinesterase
61
enzyme, respectively. There was approximately a 1.7-fold
increase in Km value for immobilized enzyme. This alteration
was either due to the conformational changes of the enzyme
resulting in a lower possibility of forming a substrate–enzyme
complex or the lower accessibility of the substrate to the active
sites of the immobilized enzyme caused by the increased
diffusion limitation.
3.6
Storage and reuse stability
In order to investigate the industrial practicability of an
immobilized enzyme, the loss of enzyme activity, known
as storage stability, is an important parameter to be taken
into account. The immobilized AChE was stored in phosphate buffer solution at 4 and 301C separately and activities
were measured periodically over duration of 100 days.
Upon 100 days of storage, the catalytic activity of immobilized
enzyme was retained 490% at 41C and 434% at 301C
(Fig. 6A).
Kinetic parameters
The kinetic parameters of the free and immobilized AChE were
determined using acetylthiocholine iodide as substrate at
constant temperature and pH. The Km and kcat of the immobilized enzyme were calculated from Lineweaver–Burk plot
and the results were compared with those obtained for the free
AChE (Fig. 5). In comparison with the free AChE, the kcat
obtained for immobilized AChE (3500 sec 1) was approximately four times lower as shown in Table 1. The Km values
were estimated as 0.5 and 0.3 mM for immobilized and free
Figure 5. The Michaelis–Menten diagram of soluble and
immobilized form of AChE. Soluble and immobilized enzymes.
Insets: Lineweaver-Burk plots of soluble and immobilized. A
solution of 6% w/w PVA, 10 mg/mL BSA, and 3 mg/mL AChE was
electrospun into biocomposite nanofibrous membranes and
used as immobilized enzyme. For more details see Section 2.
Table 1. Activity recovery and kinetic parameters of soluble and
immobilized AChE
Enzyme
Figure 4. Effect of pH on the activity of free and immobilized
AChE. The reactions were carried out at room temperature under
the variety of pH. A solution of 6% w/w PVA, 10 mg/mL BSA,
and 3 mg/mL AChE was electrospun into biocomposite nanofibrous membranes and used as immobilized enzyme. For more
details see Section 2.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Soluble AChE
Immobilized
AChEb)
Km (mM) Vmax (mmol/min mL) kcat (s 1)
0.3
0.5
0.37
0.27
15 000
3500
R (%)a)
–
40%
a) R is the remaining activity of the immobilized enzyme.
b) A solution of 6% w/w PVA, 10 mg/mL BSA and 3 mg/mL AChE was
electrospun into biocomposite nanofibrous membranes (please see
Section 2).
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A. Moradzadegan et al.
got smaller after several arrays due to the hydrophilicity of PVA
[23].
4
Concluding remarks
PVA/BSA/AChE biocomposite nanofibrous membrane could
be fabricated by electrospinning and the enzyme molecules
could be covalently bound to the nanofiber with GTA. With
the huge specific surface area provided by the nanofiber,
immobilized AChE retains 40% of its initial activity. After
immobilization, there was a 1.7-fold increase in Km value for
immobilized enzyme and in addition, immobilized AChE can
retain most of the activity at lower pH values when compared
with the free enzyme. Immobilized system had good storage
stability and it was shown to have better reusable stability than
previous studies. This immobilized system may have industrial
application for pesticide determination.
Acknowledgements
The authors express their gratitude to Research Center of Basic
Science, Shahed University for the financial support during the
course of this project.
Conflict of interest statement
The authors have declared no conflict of interest.
References
Figure 6. (A) Storage stability of immobilized AChE in 4 and
301C. All experiments were carried out in phosphate buffer (pH
7.4). (B) The influence of the number of reuse on the activity of
immobilized AChE with repeated cycles. All cycles were carried
out at room temperature. A solution of 6% w/w PVA, 10 mg/mL
BSA, and 3 mg/ml AChE was electrospun into biocomposite
nanofibrous membranes and used as immobilized enzyme. For
more details see Section 2.
In addition, reusability of immobilized enzymes that was
important for their practical application was carried out by
measuring the activity of the immobilized enzyme successive
times. As shown in Fig. 6B, the immobilized enzyme maintained more than 70% of its original activity after ten reuses,
which is better than previous study that AChE was immobilized on PANCHI-B membranes, and the enzyme activity loss
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& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Electrospun poly(vinyl alcohol)/glucose oxidase biocomposite membranes for biosensor applications. React. Funct.
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in polyvinyl alcohol (PVA) nanofibrous membranes.
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bonds on the stability of Drosophila melanogaster acetylcholinesterase. BMC Biochem. 2006, 7, 12–18.
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57
Atousa Moradzadegan1
Seyed-Omid Ranaei-
Research Article
Siadat1
Azadeh Ebrahim-Habibi2
Immobilization of acetylcholinesterase
in nanofibrous PVA/BSA membranes by
electrospinning
Mohammad BarshanTashnizi1
Rouhollah Jalili3
Seyed-Fakhraddin Torabi4
Khosro Khajeh5
1
NanoBiotechnology
Engineering Lab.,
Department of
Biotechnology, Faculty of
Energy Engineering and
New Technologies, Shahid
Beheshti University, GC,
Tehran, Iran
2
Endocrinology and
Metabolism Research
Center, Tehran University of
Medical Sciences, Dr.
Shariati Hospital, Tehran,
Iran
Electrospinning, a simple and versatile method to fabricate nanofibrous supports,
has attracted continuous attention in the field of enzyme immobilization. In this
study, acetylcholinesterase (AChE) has been successfully immobilized in PVA
nanofibers via electrospinning of a mixture of AChE, BSA as an enzyme stabilizing
additive and PVA. The maximum activity recovery of immobilized AChE was
about 40%. In comparison with free enzyme, the immobilized AChE showed
improved stability while retaining a considerable amount of activity at lower pH
values. Moreover, the immobilized AChE retained 434% of its initial activity
when stored at 301C for 100 days and retained 70% of its initial activity after ten
consecutive reactor batch cycles.
Keywords: Acetylcholinesterase / Electrospinning / Immobilization / Nanofiber / PVA
Received: January 6, 2009; revised: October 21, 2009; accepted: October 27, 2009
DOI: 10.1002/elsc.200900001
3
New Ideas Research
Institute (NIRI), Tehran,
Iran
4
Department of
Biotechnology, University of
Tehran, Tehran, Iran
5
Department of
Biochemistry, Faculty of
Biological Science, Tarbiat
Modares University,
Tehran, Iran
1
Introduction
Enzyme immobilization has been a popular strategy for most
large-scale applications due to the ease in catalyst recycling,
continuous operation, and product purification [1, 2]. The
most explored approach to immobilize enzymes has involved
Correspondence: Dr. Khosro Khajeh (khajeh@modares.ac.ir),
Department of Biochemistry, Faculty of Biological Science, Tarbiat
Modares University, Tehran, Iran.
the use of solid supports via a variety of mechanisms, such as
physical adsorption, covalent bonds, or entrapment/encapsulation [3]. With increasing demand for nanotechnology, electrospinning has become a novel technique for generating
composite nanofibers. Electrospinning is a method for
producing nanofibers from a variety of materials with fiber
diameters ranging from several micrometers down to tens of
nanometers [4–6]. The electrospun nanofibrous membrane
has high specific area and porous structure, so there are
Current Address: Department of Biology, Sciences and Research Branch,
Islamic Azad University, Tehran, Iran
Abbreviations: AChE, acetylcholinesterase;
cholineiodide; GTA, glutaraldehyde
ATChI,
acetylthio-
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Additional corresponding author: Dr. Seyed-Omid Ranaei-Siadat
E-mail: O_ranaei@sbu.ac.ir
http://www.els-journal.com
58
Eng. Life Sci. 2010, 10, No. 1, 57–64
A. Moradzadegan et al.
excellent candidates for filtration, drug delivery carrier, tissue
engineering, wound dressing, nano-sensors, and enzyme
immobilization [7–9]. Immobilization of enzymes on both
natural and synthetic polymeric supports has been widely
reported [10–17]. PVA can be used for enzyme immobilization
by electrospinning since it has good properties in forming
membranes and fibers. Besides, it is a biologically compatible,
nontoxic, hydrophilic, and readily available low-cost polymer
[18–22]. Cellulase loaded at 10% in 200 nm diameter PVA
fibers exhibited 65% of original activity [23]. Immobilization
of lipase in 100–150 nm diameter PVA nanofibrous, enzyme
loading in these biocomposite fibers reached as high as 50%
[24]. Glucose oxidase loaded at 8% in 70–250 nm diameter
PVA fibers and chronoamperometric measurmens demonstrated that electrospun fibrous enzymatic electrodes exhibited
a rapid response (1 s) and a higher response current (mA level)
to glucose in the normal and diabetic level [25] Acetylcholinesterase (AChE) (E.C 3.1.1.7) is a serine hydrolase,
which catalyzes the hydrolysis of the neurotransmitter acetylcholine [26]. Carbamates and organophosphates are potent
inhibitors of AChE, which causes the blocking of the nerve
signal transference into the postsynaptic membrane [27].
Because of the uniform mechanism of signal transduction in
animals, these inhibitors have been used as agricultural pesticides as well as chemical warfare agents (nerve agents) [28].
Immobilized AChE can be used for construction of biosensor
for pesticide determination [29, 30].
Two main methods have been employed to make an
assemblage of enzymes and polymeric fibers via electrospinning: (i) immobilization of the enzyme on the outer surface of
the nanofibers and (ii) mixing enzyme with the polymer
solution and subsequently spinning. In the first approach,
polymeric materials can be used, which are soluble in organic
media. However, enzyme is immobilized only on the outer
surface of the fibers while the interior of the fibers remains
inactive. In contrast to the first approach, water-soluble
polymers such as PVA or dextran can be used as a solid
support in the second approach. However, dissolution of the
fibers in an aqueous environment and subsequent enzyme
leaching would be a problem. To overcome this problem, it was
suggested to use vapor glutaraldehyde (GTA) [24] so that the
nanofibers can be cross-linked to prevent support dissolution.
In the present study, AChE was immobilized using mixed
spinning approach of the enzyme, PVA, and BSA biocomposite
nanofibers with the use of vapor GTA. Kinetic parameters of
both free and immobilized enzymes were determined. Also
storage stability of the immobilized system was compared with
free enzyme and reusable stability of immobilized enzyme was
assayed.
2
Materials and methods
2.1
Materials
AChE (E.C 3.1.1.7) was expressed with the baculovirus system
[31]. GTA 25% was purchased from TAAB Laboratories (England,
UK). BSA, acetylthiocholineiodide (ATChI) and 5,50 -dithiobis(2-nitrobenzoic acid) were obtained from Sigma-Aldrich (MO,
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
USA). PVA 72000 and 498% of degree of hydrolysis and all other
agents used in the experiments were of analytical grade and were
from Merck (Darmstadt, Germany). Aqueous solutions were
prepared in doubly distilled deionized water.
2.2
2.2.1
Methods
Preparation of nanofibrous membranes
PVA aqueous solution (6% w/w) was prepared by dissolving
PVA powder in deionized water at 801C with gentle stirring for
4 h to form homogenous solution after removing air bubbles.
After the solution was cooled to the room temperature,
prepared solutions in different concentrations were placed in a
plastic syringe (1 ml) bearing a metal capillary (0.8 mm
diameter) connected to a high-voltage power supply; the
grounded counter electrode connected to the aluminum foil
collector. Electrospinning was performed at 10–12 kV voltage,
15 cm distance between the needle tip and the collector. The
flow rate of the solution was controlled by a syringe pump to
maintain at 26 mL/h from the needle outlet. It usually takes
45 min to obtain a sufficiently thick membrane that can be
detached from the aluminum foil collector.
PVA/BSA solution was prepared by gently mixing PVA and
BSA solution to yield the spinning solution with final
concentrations of 6% w/w and 10 mg/mL for PVA and BSA in
spinning solution, respectively. The same procedure was
used to prepare PVA/BSA/AChE solution by adding an
aqueous solution of AChE to 25 mM phosphate buffer, pH 7.4,
different ratios of BSA and AChE to PVA were used. An aliquot
of 1 mL of each solution under the mentioned conditions
was electrospun to form the biocomposite nanofibrous
membranes. The electrospinning was performed at room
temperature and the resulting PVA/BSA and PVA/BSA/AChE
biocomposite fibers were collected on the surface of aluminum
foil collector.
Covalent attachment of BSA in nanofibrous PVA
membranes was carried out by cross-linking the electrospun
PVA/BSA membranes at room temperature by GTA vapor for
12 h. PVA/BSA membrane (20 mg) was immersed in glass vials
containing deionized water at room temperature and was
agitated at 300 rpm for 120 min to detach all un-bounded
protein from the membrane. The amount of released protein
was determined by measuring the residual protein present in
the supernatants by Bradford method using coomassie brilliant
blue G-250 and BSA as the standard protein [32]. The amount
of immobilized protein was estimated by subtracting the
amount of protein in the supernatants from the total amount
of protein used in the immobilization procedure. The protein
loading efficiency was determined by dividing the amount of
immobilized BSA (mg) in the membrane to the whole support
membrane mass (g).
In the case of AChE, enzyme immobilization was carried
out by cross-linking the electrospun PVA/AChE membranes at
41C by GTA vapor for 12 h. AChE immobilization was also
studied in presence of BSA to yield the PVA/BSA/AChE
nanofibrous membranes using GTA vapor at different enzyme/
BSA ratio. Finally, a solution of 6% w/w PVA, 10 mg/mL BSA
http://www.els-journal.com
Eng. Life Sci. 2010, No. 1, 57–64
and 3 mg/mL AChE was electrospun into biocomposite nanofibrous membranes and used for determination of kinetic
parameters, pH profile, and stability experiments. The
morphology of this nanofibrous PVA/BSA/AChE membrane
was characterized by a Philips XL-30 scanning electronic
microscope after being sputtered with gold.
2.2.2
Catalytic activity determination
The AChE activity was determined according to the ellman
method [33, 34] using ATChI as substrate and 5,50 -dithiobis(2-nitrobenzoic acid) as chromogen and increasing absorbance
at 412 nm was measured for 1 min. For the immobilized
enzyme assay, the electrospun nanofibrous membrane was
removed from the collector and cut into 1 cm 1 cm pieces,
3 mg of nanofibers containing immobilized enzymes were
added to each mixture reaction (1 mL) in vials, vortexed for
1 min, and then centrifuged rapidly at 16 000 g for 1 min.
The product concentration that was proportional to the
hydrolyzed ATChI, therefore, to the intensity of the yellow
solution in the resulting supernatant was determined spectrophotometrically at 412 nm. One unit of enzymatic activity
was defined as the amount of enzyme that catalyzes 1 mmol of
substrate to product per minute. Specific activity was expressed
as units per milligram of protein in the assay medium (1 mL)
and the relative activity was defined as the ratio between the
specific activity of a bound enzyme and free enzyme. All
reactions and measurements were carried out at room
temperature. In order to determine kinetic parameters (Km
and Vmax) for free and immobilized enzyme, several concentrations of ATChI were used ranging from 0.01 to 1 mM. The
activity of free and immobilized enzyme was determined
within the pH range of 4 to 8.5.
The amount of protein in the different samples was estimated
according to the Bradford method using BSA as standard [34].
2.2.3
Immobilization of acetylcholinesterase
59
tion can be electrospun into nanofibers with diameter around
190 nm as shown in Fig. 1A. With the addition of BSA and
AChE, electrospun fibers became irregular but the diameter of
the fibers did not change (Fig.1B). As a protein, it is impossible
for BSA or AChE to be electrospun alone into nanofibers due
to its complicated three-dimentional structures as well
as strong inter- and intra-molecular forces [12]. The blending
of BSA and AChE with PVA can interrupt its complex structure. PVA has a capacity for secondary binding; because of
this, the mixture of PVA and the enzyme can be electrospun
and form nanofibers by electrospinning. On the other
hand, the interaction of PVA and protein resulted in the
reduction of stability of PVA solution and induced the
appearance of irregular [24].
3.2
Covalent immobilization of BSA in PVA
membrane
After co-spinning of different concentration of BSA with PVA,
the nanofibers were incubated with vaporized GTA for 12 h.
The amount of released BSA was measured (Fig. 2). A significant decrease was observed after increasing the concentration
of BSA from 3 to 18 mg/mL. The optimum BSA concentration
for immobilization of AChE in nanofibers was found to be
10 mg/mL. GTA acts as a linker and links amine groups of
lysine residues of BSA and due to the conglutination between
BSA molecules, creates a protein networks onto PVA
membranes. This may be the reason for more immobilization
when high concentration of BSA was used.
Storage stability and reusability assay
Activities of the free and immobilized AChE were determined
after storage in 25 mM phosphate buffer solution (pH 7.4) at 4
and 301C. The measurements were performed at intervals of
2 wk within a period of 100 days. The reusability of bound
AChE was examined by conducting the activity measurement
of bound AChE at time intervals of 15 min. After each activity
measurement, the bound AChE was washed three times with
25 mM phosphate buffer. The supports were then centrifuged,
the supernatant was decanted, and the recycled supports
subjected to the activity assay for the second cycle and so on.
3
Results and discussion
3.1
Morphology of PVA/BSA/AChE electrospinning
composite membrane
The physical morphology of the electrospun samples was
examined using the scanning electron microscope. PVA solu-
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Scanning electron micrographs of the electrospun PVA
(A) and the electrospun PVA/BSA/AChE fibers (B).
http://www.els-journal.com
60
Figure 2. Immobilization of several concentration of BSA onto
PVA membranes. For more details please see Section 2.
3.3
Eng. Life Sci. 2010, 10, No. 1, 57–64
A. Moradzadegan et al.
Enzyme immobilization
AChE was covalently immobilized in modified membranes
using vapor GTA. Because of low concentration of AChE with
respect to BSA, no activity was detected in the supernatant at
the end of immobilization procedure. According to the
Drosophila AChE crystal structure [35] there are more than 20
lysine residues available on the AChE surface for reaction with
GTA.
GTA is highly reactive toward both the amine and hydroxyl
groups and acts as a linker [36]. These mechanisms result in
covalent attachment of AChE in PVA/BSA membranes. The
activity of immobilized AChE with different enzyme loading
(0.2–3.5 mg/mL) was determined (Fig. 3). The relative immobilized AChE activity (activity recovery) was more than 40%.
Decrease in activity is usually observed after enzyme immobilization. Enzyme immobilization via covalent binding will
change enzyme conformation or reduce its flexibility and may
cause reduction in enzyme mobility to induce fit to a substrate
[37] that may lead to a reduction of AChE activities. Moreover,
GTA as a cross-linking agent reacts with hydroxyl groups from
PVA chains and causes the PVA membranes to pack more
densely, thus decreasing surface area of the support as well as
mass transfer through it [23]. Besides, cross-linking can reduce
membrane porosity, which in turn causes limited accessibility
of substrates to active site.
In addition, different concentrations of BSA (0, 3, 6, 10, 14,
and 18 mg/mL) were electrospun into biocomposite
membranes containing 6% w/w PVA and 3 mg/mL AChE. All
samples were subjected to cross-linking with vapor GTA as
mentioned in Section 2. The fibers were washed three times
thoroughly with phosphate buffer solution (25 mM, pH 7.4) to
extract out un-bonded enzymes. Buffer solutions were found
to have no detectable enzyme activity, indicating that there was
negligible enzyme leakage during washing procedure. Therefore, the yield of AChE immobilization was found to be about
100%. The activity of the resulting immobilized AChE was
compared with the free enzyme to determine enzyme activity
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. Effect of several concentrations of enzyme on the
remaining activity of the immobilized enzyme. All experiments
were carried out at pH 7.4 (25 mM phosphate buffer) and room
temperature.
retention after immobilization. Results showed that the
presence of BSA had a significant effect on AChE activity
retention. In comparison to PVA, a significant increase in the
retaining activity of immobilized enzyme (23–40%) was
observed using PVA and BSA (10 mg/mL), simultaneously.
Several speculate could be used to explain this phenomenon.
First, the BSA-modified fibers could create a biocompatible
microenvironment for the immobilized AChE, which usually
leads to high activity retention of the immobilized enzyme
[10]. Second, decreased number of covalent bonds with the
support (number of hydroxyl groups of PVA is very high
compared with NH2 groups of BSA) may cause improvement
in the catalytic efficiency of the immobilized enzyme. Finally,
using BSA as a linker between AChE and the support would be
in favor of enzyme flexibility.
Although the presence of BSA led to a significant increase in
the activity retention of immobilized AChE, increasing loaded
protein from 14 to 18% resulted in decreasing activity retention from 40 to 33%. This may be due to the presence of large
numbers of amino groups on the surface of BSA molecules,
which are potential reaction sites for covalent coupling with
enzyme. Thus, it becomes easier to immobilize one enzyme
molecule through multipoint covalent attachments on the
BSA-modified membrane with higher concentrations of BSA,
which negatively affect the enzymatic activity. Based on
observed results, it has been suggested that enzyme immobilization using 3 mg/mL AChE into biocomposite membrane
loaded with 10% BSA would be the condition to achieve
maximum activity retention.
3.4
pH-activity profile
The pH is one of the important parameters capable of altering
enzymatic activities in aqueous solution. The effect of pH on
http://www.els-journal.com
Eng. Life Sci. 2010, No. 1, 57–64
the activity of the free and immobilized AChE was investigated
within range of pH 4.5–8.5 at room temperature. Relative
activity as a function of pH is depicted in Fig. 4. The optimum
pH of immobilized AChE was found to be about 7.5. So, the
three dimensional structure of enzyme’s active site may not be
affected from immobilization procedure and optimum pH of
immobilized enzyme can be observed as free enzyme [38]. The
immobilized enzyme was less sensitive to pH changes at acidic
pHs than alkaline pHs compared with that of free AChE. These
data indicate that the pH stability of AChE could be enhanced
by the immobilization process at low pH values [39].
PVA/BSA/AChE biocomposite contained about 15% w/w
protein and 85% polymer. The isoelectric point of BSA is 4.8
and is positively charged at lower pH values [40]. The presence
of protonated BSA amino groups on the surface of the
membrane might repel protons from the region in the vicinity
of the surface and create a higher pH at the boundary layer
between the support and the bulk solution. This microenvironment with a pH slightly higher than bulk solution may
lead to an increase in pH stability of the immobilized enzyme.
3.5
Immobilization of acetylcholinesterase
61
enzyme, respectively. There was approximately a 1.7-fold
increase in Km value for immobilized enzyme. This alteration
was either due to the conformational changes of the enzyme
resulting in a lower possibility of forming a substrate–enzyme
complex or the lower accessibility of the substrate to the active
sites of the immobilized enzyme caused by the increased
diffusion limitation.
3.6
Storage and reuse stability
In order to investigate the industrial practicability of an
immobilized enzyme, the loss of enzyme activity, known
as storage stability, is an important parameter to be taken
into account. The immobilized AChE was stored in phosphate buffer solution at 4 and 301C separately and activities
were measured periodically over duration of 100 days.
Upon 100 days of storage, the catalytic activity of immobilized
enzyme was retained 490% at 41C and 434% at 301C
(Fig. 6A).
Kinetic parameters
The kinetic parameters of the free and immobilized AChE were
determined using acetylthiocholine iodide as substrate at
constant temperature and pH. The Km and kcat of the immobilized enzyme were calculated from Lineweaver–Burk plot
and the results were compared with those obtained for the free
AChE (Fig. 5). In comparison with the free AChE, the kcat
obtained for immobilized AChE (3500 sec 1) was approximately four times lower as shown in Table 1. The Km values
were estimated as 0.5 and 0.3 mM for immobilized and free
Figure 5. The Michaelis–Menten diagram of soluble and
immobilized form of AChE. Soluble and immobilized enzymes.
Insets: Lineweaver-Burk plots of soluble and immobilized. A
solution of 6% w/w PVA, 10 mg/mL BSA, and 3 mg/mL AChE was
electrospun into biocomposite nanofibrous membranes and
used as immobilized enzyme. For more details see Section 2.
Table 1. Activity recovery and kinetic parameters of soluble and
immobilized AChE
Enzyme
Figure 4. Effect of pH on the activity of free and immobilized
AChE. The reactions were carried out at room temperature under
the variety of pH. A solution of 6% w/w PVA, 10 mg/mL BSA,
and 3 mg/mL AChE was electrospun into biocomposite nanofibrous membranes and used as immobilized enzyme. For more
details see Section 2.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Soluble AChE
Immobilized
AChEb)
Km (mM) Vmax (mmol/min mL) kcat (s 1)
0.3
0.5
0.37
0.27
15 000
3500
R (%)a)
–
40%
a) R is the remaining activity of the immobilized enzyme.
b) A solution of 6% w/w PVA, 10 mg/mL BSA and 3 mg/mL AChE was
electrospun into biocomposite nanofibrous membranes (please see
Section 2).
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62
Eng. Life Sci. 2010, 10, No. 1, 57–64
A. Moradzadegan et al.
got smaller after several arrays due to the hydrophilicity of PVA
[23].
4
Concluding remarks
PVA/BSA/AChE biocomposite nanofibrous membrane could
be fabricated by electrospinning and the enzyme molecules
could be covalently bound to the nanofiber with GTA. With
the huge specific surface area provided by the nanofiber,
immobilized AChE retains 40% of its initial activity. After
immobilization, there was a 1.7-fold increase in Km value for
immobilized enzyme and in addition, immobilized AChE can
retain most of the activity at lower pH values when compared
with the free enzyme. Immobilized system had good storage
stability and it was shown to have better reusable stability than
previous studies. This immobilized system may have industrial
application for pesticide determination.
Acknowledgements
The authors express their gratitude to Research Center of Basic
Science, Shahed University for the financial support during the
course of this project.
Conflict of interest statement
The authors have declared no conflict of interest.
References
Figure 6. (A) Storage stability of immobilized AChE in 4 and
301C. All experiments were carried out in phosphate buffer (pH
7.4). (B) The influence of the number of reuse on the activity of
immobilized AChE with repeated cycles. All cycles were carried
out at room temperature. A solution of 6% w/w PVA, 10 mg/mL
BSA, and 3 mg/ml AChE was electrospun into biocomposite
nanofibrous membranes and used as immobilized enzyme. For
more details see Section 2.
In addition, reusability of immobilized enzymes that was
important for their practical application was carried out by
measuring the activity of the immobilized enzyme successive
times. As shown in Fig. 6B, the immobilized enzyme maintained more than 70% of its original activity after ten reuses,
which is better than previous study that AChE was immobilized on PANCHI-B membranes, and the enzyme activity loss
was less than 50% after ten times [41]. The loss of catalytic
activity may be explained by the following two reasons. First,
there may be some AChE molecules, which were not chemically bonded on PVA fibers but only physically embedded,
were lost during the process of measurement. Second, the
diameter of PVA fibers may become larger and the surface area
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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