Urease-carrying electrospun polyacrylonitrile mat for urea hydrolysis

Article abstract of DOI:10.1016/j.reactfunctpolym.2014.12.004

Electrospinning was used to fabricate beadless microfibrous polyacrylonitrile (ePAN) mats with an average fiber diameter of 1448 ± 380 nm from a 10 wt.% PAN in dimethylformamide (DMF) dope solution at applied voltage of 18 kV and 20 cm fiber collection distance. Urease (EC 3.5.1.5) was then covalently immobilized on dispersed microfibrous ePAN mats following the chemical treatment of fibers with ethylenediamine (EDA) and glutaraldehyde (GA). The optimal concentration of GA for immobilization was 5%. The amount of loaded urease reached 157 μg/mg mat, exhibiting 54% of the free urease activity. The surface chemistry of as-spun and chemically treated fibers was examined with Fourier transform infrared (FTIR) spectroscopy. Field emission scanning electron microscopy (FESEM) was used to study the morphology and diameter of the pristine, chemically treated, and urease-immobilized microfibrous mats. Immobilized urease showed increased temperature for maximum activity (from 37 to 50 °C for free and immobilized urease, respectively) and improved storage stability (20 days). The immobilized urease was also less sensitive to the changes in pH, especially in acid conditions. In addition, nearly 70% of initial activity of the immobilized urease was retained after 15 cycles of reuse, which proved the applicability of the electrospun fibers as successful enzyme carriers.

Full text of DOI:10.1016/j.reactfunctpolym.2014.12.004

Reactive & Functional Polymers 87 (2015) 37–45
Contents lists available at ScienceDirect
Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
Urease-carrying electrospun polyacrylonitrile mat for urea hydrolysis
Aref Daneshfar a,b, , Takeshi Matsuura a,c, Daryoush Emadzadeh a, Zahra Pahlevani d,
⇑
Ahmad Fauzi Ismail a,
⇑
a Advanced Membrane Technology Research Centre (AMTEC), Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
b Faculty of Chemical Engineering, Department of Polymer Technology, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
c Industrial Membrane Research Laboratory, Department of Chemical and Biological Engineering, University of Ottawa, 161 Louis Pasteur St, Ottawa, ON K1N 6N5, Canada
d School of Engineering, Department of Materials Science and Engineering, Shiraz University, Shiraz, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2014
Received in revised form 22 November 2014
Accepted 22 December 2014
Available online 6 January 2015
Electrospinning was used to fabricate beadless microfibrous polyacrylonitrile (ePAN) mats with an aver-
age fiber diameter of 1448 380 nm from a 10 wt.% PAN in dimethylformamide (DMF) dope solution at
applied voltage of 18 kV and 20 cm fiber collection distance. Urease (EC 3.5.1.5) was then covalently
immobilized on dispersed microfibrous ePAN mats following the chemical treatment of fibers with eth-
ylenediamine (EDA) and glutaraldehyde (GA). The optimal concentration of GA for immobilization was
5%. The amount of loaded urease reached 157 lg/mg mat, exhibiting 54% of the free urease activity.
Keywords:
The surface chemistry of as-spun and chemically treated fibers was examined with Fourier transform
infrared (FTIR) spectroscopy. Field emission scanning electron microscopy (FESEM) was used to study
the morphology and diameter of the pristine, chemically treated, and urease-immobilized microfibrous
mats. Immobilized urease showed increased temperature for maximum activity (from 37 to 50 °C for free
and immobilized urease, respectively) and improved storage stability (20 days). The immobilized urease
was also less sensitive to the changes in pH, especially in acid conditions. In addition, nearly 70% of initial
activity of the immobilized urease was retained after 15 cycles of reuse, which proved the applicability of
the electrospun fibers as successful enzyme carriers.
Electrospinning
Enzyme immobilization
Polyacrylonitrile
Urea hydrolysis
Urease
Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction
To overcome the intrinsic vulnerability of free enzymes to the
changes that intermittently happen in the reaction medium such
In quest of a reliable way for daily removal of blood urea from
patients suffering from permanent renal failure, artificial wearable
or portable kidney machines have recently been developed [1,2]. In
order to maintain the device within an acceptable working condi-
tion, the dialysate solution, which is mainly contaminated with
urea, should be regenerated continuously. Urea is a remarkably
stable molecule with degradation half-life of 3.6 years at 38 °C
when dissolved in water. Due to its non-ionic polar nature, it can
hardly be removed from water by ion-exchange media, activated
carbon granules, or reverse osmosis membranes [3]. Thus, it seems
that the most effective method for regeneration of the dialysate
solution in the wearable kidney machine is the utilization of urease
[4], a nickel-based enzyme that catalyzes urea hydrolysis to
ammonia and carbon dioxide 1014 times faster than uncatalyzed
urea hydrolysis.
as changes in pH and temperature, the immobilized form applica-
tion of enzymes is preferable. More importantly, the immobilized
enzyme provides a steady process, easy removal of the products,
and convenient recovery of the catalyst from the stream. Three
major methods are scrutinized for immobilization of different sorts
of enzymes, i.e. adsorption, covalent binding, and encapsulation
(entrapment) [5]. Among these techniques, covalent immobiliza-
tion is widely studied as a durable technique for localization of
enzymes on solid supports due to irreversibility of the bonds
formed between the bioactive compartments and the support
surface.
The choice of the support material and its conformation signif-
icantly affects the performance of the immobilized enzyme. Ide-
ally, it should be inert, fairly inexpensive, and possess acceptable
mechanical strength. In addition, its chemistry should allow easily
incorporating of functional groups essential for direct or indirect
covalent attachment of enzymes.
⇑
Corresponding authors at: Advanced Membrane Technology Research Centre
(AMTEC), Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia (A. Danesh-
far). Tel.: +60 75535592; fax: +60 75581463.
Polyacrylonitrile (PAN) is an important engineering polymer
with good film and fiber forming characteristics which has
widely been utilized for biomedical applications and enzyme
E-mail addresses: darf2@live.utm.my (A. Daneshfar), afauzi@utm.my (A.F.
Ismail).
http://dx.doi.org/10.1016/j.reactfunctpolym.2014.12.004
1381-5148/Ó 2015 Elsevier B.V. All rights reserved.

38
A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
immobilization. The presence of nitrile groups (C„N) in the
backbone of the polymer chains provides the possibility for
incorporation of new reactive functional groups, macromolecules,
or biofriendly species making PAN films or fibers hydrophilic and
biocompatible [6]. Different PAN-based membranes has long been
studied for direct or indirect (use of coupling agents) covalent
immobilization of different sorts of enzymes. Poly (acrylonitrile-
co-methylmethacrylate-co-sodium vinylsulfate) was activated
with NaOH and hexamethylenediamine (HMDA) or hydroxylamine.
Glucose oxidase (EC 1.1.3.4) was then covalently attached to the
surface of membrane using glutaraldehyde (GA) as the coupling
agent [7]. Similar procedure was also used for immobilization of
urease (EC 3.5.1.5) on PAN hollow fibrous membranes [8].
grade), ethylenediamine (EDA) (analytical grade), potassium dihy-
drogen phosphate (monobasic) (analytical grade), potassium
monohydrogen phosphate (dibasic) (analytical grade), and urea
(biology grade) were purchased from Merck (Germany). The activ-
ity of the native or immobilized urease was measured by Atlas
Medical Berthelot urea kit. All other chemicals were of analytical
grade. Deionized water (DI) (>18 M
X cm) was used for washing
or preparation of all aqueous solutions unless otherwise stated.
The urea and urease solutions were prepared in 22 mM phosphate
buffers (PBS) containing 1 mM ethylenediamine tetra acetic acid
(EDTA) as an ion chelating compartment of the PBS.
2.2. Preparation of NH2-ePAN mats
The support conformation should pose minimum diffusional
limitation and provide utmost surface area per unit mass for high
enzyme loading [9]. Electrospun polymeric fibers are promising
for enzyme immobilization [10–13] because the electrospun mats
are more easily produced, dispersed in and recovered from the reac-
tion media than other choices of nanoscaled geometries, e.g. nano-
particles and nanotubes [14]. Furthermore, electrospun fibers with
high specific surface area could be prepared from a wide choice of
polymers with disparate chemical characteristics, which render
possibilities for different modes of enzyme immobilization [15–17].
Recently, enzyme immobilization on PAN-based nanofibrous
membranes has become increasingly interesting because of the rel-
atively easy procedures for introducing necessary functional
groups on the fiber surface prior to immobilization and for fiber
production. Lipase (EC 3.1.1.3) and cellulase (EC 3.2.1.4) have been
successfully immobilized on electrospun PAN (ePAN) fibers
through an amidination activation of nitrile groups and reaction
with amine pendant groups of the enzymes [18–20]. Others have
used PAN derivatives and tethered the electrospun fibers prior to
immobilization with biomacromolecules such as collagen or pro-
tein hydrolysate from egg skin in order to circumvent the hydro-
phobic nature of PAN and to make it biocompatible [21,22].
Direct amine functionalization of nanofibrous PAN mats have
recently been studied for efficient heavy metal ion removal from
aquatic environments [23–25]. Amine containing reagents such
as ethanolamine, ethylenediamine [24,26], or hydroxylamine [25]
react with PAN pendant nitrile groups through nucleophilic addi-
tion [24]. After the amine functionalization, bioactive compart-
ments containing ANH2 groups can be immobilized on the
surface of aminated carrier by use of glutaraldehyde (GA) through
the formation of stable amino-aldehyde bonds [27]. However,
there are no references concerning the application of directly ami-
nated PAN membranes or fibers for immobilization of enzymes.
In this work, the surface aminated electrospun polyacrylonitrile
(NH2-ePAN) mats were prepared by electrospinning of PAN dope
solution followed by amination with ethylenediamine (EDA). Ure-
ase was then covalently immobilized on NH2-ePAN mats using glu-
taraldehyde (GA). Properties of the immobilized urease were
studied and compared to those of free enzyme. Results will possi-
bly lead to the use of urease-immobilized ePAN mats (Urs-ePAN) in
miniaturized wearable kidneys to regenerate the valuable dialy-
sate fluid by preserving the catalytic activity for increasing the
number of reuses.
The 10 wt.% PAN dope solution was prepared by slowly adding
of PAN powder to DMF followed by mechanical stirring for 24 h at
ambient temperature. The solution was then loaded to a 10 ml
glass syringe and was upheld for 2 h to remove the dispersed air
bubbles. A metal needle spinneret (D = 0.7 mm) was attached to
the syringe tip. Electrospinning was performed for 7 h to collect
a detachable thickness of the fibers. A syringe pump (Cole-ParmerÒ
100 Touch Screen) was used to deliver the polymer solution to the
tip of the needle at a constant flow rate of 1 ml/h. High voltage
power supply (ES30P-5W-Gamma High Voltage Research, Florida,
USA) with low current output (166 lA) was used as the electricity
power source. Positive charge (18 kV) was applied to the needle tip
and the produced electrospun fibers were collected on a grounded
aluminum sheet. The distance between the needle tip and the col-
lector was kept constant at 20 cm. Afterwards, the nonwoven
sheets were cut into 2 ꢀ 2 cm2 sheets which were dried overnight
in a vacuum oven and weighed carefully after drying. The sheets
were then soaked in 70% ethanol aqueous solution in small glass
bottles, followed by shaking on a rocking shaker at 250 rpm for
24 h to disentangle the fibers by increasing their wettability.
Further surface functionalization was carried out on the dispersed
spun fibrous mats. The dispersed fibers were washed successively
and kept immersed in 3 M aqueous ethylenediamine (EDA) solu-
tion at 99 1 °C for 4 h. Those pieces were further washed to
remove residual EDA. Aminated electrospun ePAN mats, called
NH2-ePAN, were stored in DI for later use.
2.3. Immobilization of the urease using glutaraldehyde
Aminated electrospun mats, NH2-ePAN, were soaked in aqueous
GA solutions at different concentrations, 0–10 wt.%, for 2 h at
ambient temperature, followed by washing with a copious amount
of water. GA treated mats (GA-ePAN) were further loaded in glass
bottles containing 5 ml of urease solution in PBS (1 mg/ml) at pH 7.
Then, the bottles were shaken for 1 h at ambient temperature.
Afterwards, the bottles were transferred to a refrigerator for fur-
ther urease covalent immobilization at 4 °C 1 °C for 24 h.
2.4. Characterization
The attenuated total reflection-FTIR (ATR-FTIR) (Thermo Nicolet
Instrument Corporation, Madison, WI) was used to characterize the
surface chemistry before and after the surface modification of the
pristine electrospun PAN mats (ePAN).
2. Experimental
2.1. Materials
The morphology and diameter of the pristine, the chemically
treated, and the urease immobilized ePAN mats were investigated
by field emission scanning electron microscope FESEM (JEOL JSM-
6701F, Japan). The samples were prepared by placing 0.5 ꢀ 0.5 cm2
pieces over FESEM specimen stabs, followed by sputtering a thin
Jack bean urease type III (EC 3.5.1.5), PAN (MW = 150,000), and
GA (25 wt.% aqueous solution) were purchased from Sigma (USA)
and used as received. Dimethyl formamide (DMF) (analytical

A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
39
layer of gold. Surface imaging was carried out using 10 kV poten-
2.8. Reuse cycles and storage durability
tials for 1000–25,000 times magnification.
The process of separating products of a typical enzymatic reac-
tion that mostly takes place in solution, even if the free enzyme
remains active at the end of the reaction, will inexorably lead to
deactivation of the enzyme. Enzyme immobilization on the surface
of the insoluble supports enables recovery of the active catalyst
from the reaction mixture and facilitating the separation of the
products from the reaction mixture. However, a big question
remains; is it viable to reuse the immobilized enzyme-support sys-
tem repeatedly?
In order to study the reusability of the immobilized urease, its
specific activity was measured at pH 7 and ambient temperature
15 times within 75 min. After each time of reuse, the urease-ePAN
mats were washed with PBS at pH 7 three times.
2.5. Measurement of the activity and immobilized protein
Urease catalyzes the hydrolysis of urea according to the follow-
ing reaction (1):
COðNH Þ þ 2H O urease 2NH þ CO
ƒƒ!
ð1Þ
2
2
3
2
2
The numbers of moles of ammonia liberated from the urease-
catalyzed hydrolysis of urea in different time sequences were mea-
sured and the specific catalytic activity of the enzyme was
expressed in
l lmol
mol NH3 minꢁ1 mgꢁ1 dissolved enzyme and
NH3 minꢁ1 mgꢁ1 immobilized enzyme, for free and immobilized
urease, respectively.
Free and immobilized ureases were stored for 20 days at 4 °C or
25 °C and the samples were taken from time to time for the activity
measurement. The activities relative to the first day activity is
reported as % maximum activity.
For free urease, 2 ml of 0.25 mg/ml of enzyme solution was
mixed with 2 ml of 10 g/l of urea solution. For immobilized urease,
the urease-immobilized electrospun mat was immersed in 2 ml of
urea solution (10 g/l) and 2 ml of PBS. These two solution mixtures
are called working solutions. The pH of the working solutions was
7 and the experiments were conducted at ambient temperature
unless otherwise stated. The concentration of liberated ammonia
was measured by the Berthelot method using Atlas Medical urea
3. Results and discussions
3.1. Preparation and characterization of chemically treated ePAN mats
kit reagents. As a function of time, 10 ll of the sample was with-
drawn and mixed with 1 ml of reagent R2 followed by addition of
1 ml of R1 of the kit. A blue–green indophenol color was developed
after 30 min as the result of the reaction between the liberated
ammonia, sodium hypochlorite (R2), and sodium salicylate in the
presence of sodium nitroprusside (R1). Absorbance of the colored
An as-spun ePAN sheet and several pieces of 2 ꢀ 2 cm2 square
mats are shown in Fig. 1(a). An efficient enzyme immobilization
requires the maximum surface area available per unit mass of
the support. This implies that a non-woven electrospun mat should
be disentangled to get access to inner fibers. In order to increase
the wettability of the fibers and the surface area available for
immobilization, the square mats were soaked either in DI or 70%
ethanol aqueous solution. The as-spun ePAN mat remained undi-
spersed even though it was immersed in DI and shaken for 24 h
(Fig. 1(b)). In contrast, the mat was almost completely disentan-
gled after immersion in 70% ethanol solution followed by washing
and vigorous shaking for 5–7 times with DI (Fig. 1(c)). Undispersed
and dispersed as-spun ePAN mats in the flask are shown in Fig. 1(d)
for comparison. As shown in Fig. 1(c), immersion the fiber mats in
aqueous alcohol solution liberated the small amount of air trapped
within the mat and enabled the complete wetting of the surfaces of
the inner fibers. This happens due to the surfactant-like action of
ethanol. Alkyl group of ethanol preferentially were absorbed to
the surface of ePAN fibers while its hydroxyl group interacted with
water. Thus, the hydrophobic interaction between neighboring
fibers would be reduced so that further washing and shaking
would weaken agglomeration of the fibers and make them dis-
persed in water well [28].
solution was measured at 580 nm and the concentration (lmol/
ml) of evolved ammonia was determined using the calibration
curve. The ammonia concentrations were plotted versus time and
the slope of the linear portion was used to calculate the specific
activity.
The protein content of the urease-immobilized ePAN mats was
determined by subtraction of the amount of unbound protein in fil-
trate and washing liquids from the content of the initial solution
using the Bradford method.
2.6. pH and thermal stability
The effect of pH on the activity of both free and immobilized
urease was determined through changing the pH of the working
solution in a range from 5.5 to 8.5 by adjusting the amount of
PBS. The relative enzyme activity at different pH was reported as
percent maximum activity.
To study the effect of temperature on the free and immobilized
urease, the temperature of the working solution (pH = 7) was chan-
ged in a range from 4 to 90 °C by keeping the working solution
either in a laboratory fridge or in an oven for 75 min. The activity
measurement was made at room temperature.
During amine activation in EDA solution, the color of fibers
changed gradually from white to mild yellow, as shown in Fig
2(b), and amine content of the fibers, determined through acid–
base titration, reached to 2.3 0.4 lmol/mg mat after 4 h.
Extended duration of amination resulted in only negligible change
in amine content of the fibers. Following the reaction with GA, the
color was changed to orange or mild pink (Fig. 2(c)). The changes in
the color of the fibers during the activation of the ePAN fibers prior
to immobilization proved the occurrence of stepwise chemical
reactions between functional groups after each step.
The FTIR spectra of the pristine, NH2-ePAN and GA-ePAN mats
are shown in Fig. 3. For pristine ePAN, the characteristic peaks
are 2939 cmꢁ1 (CAH stretching band related to the backbone of
PAN), 2245 cmꢁ1 (C„N stretching), and 1450 cmꢁ1 (CAH scissor-
ing of methylene groups). The peak at 3633 cmꢁ1 is related to
the OH stretching possibly due to the absorbed water on the sur-
face of the fibers [29]. The peaks at 1736 cmꢁ1, 1250 cmꢁ1, and
1173 cmꢁ1 are assigned to the stretching vibration of (C@O),
2.7. Kinetic study of free and immobilized urease
The Eq. (2) relates the rate of enzyme reaction, V, represented
here by specific activity of urease (l
mol NH3 minꢁ1 mg ureaseꢁ1),
and the substrate (urea) concentration S (mol/l), according to the
Michaelis–Menten kinetics. Vm is the maximum reaction rate and
Km is the Michaelis constant (mol/l).
1
V
1
Km
1
S
¼
þ
ꢀ
ð2Þ
Vm Vm
Thus, Eq. (2) allows calculating Vm and Km from the intercept
and the slope of (1/V) versus (1/S) plot, which is also called the
Lineweaver–Burk plot.

40
A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
Fig. 1. (a) As-spun PAN (ePAN) mat and 2 ꢀ 2 cm2 pieces, (b) undispersed fibrous mats after shaking in and washing with deionized water, (c) dispersed ePAN fibers after
immersion in 70% aqueous ethanol solution and washing with DI, and (d) front view of dispersed and undispersed fibers.
Fig. 2. The changes in color of ePAN mats following the stepwise chemical treatment (a) pristine ePAN mat (white), (b) NH2-ePAN mat (mild yellow), and (c) GA-ePAN mat
(pink to mild orange). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
CAC(@O)AC, and AOCH3 in the methyl methacrylate constituent
groups, respectively [30,31], suggesting that the precursor material
was the co-polymer of acrylonitrile and methyl methacrylate.
Amination with ethylenediamine (EDA) resulted in the forma-
tion of a new broad peak at 3371 cmꢁ1 (NH stretching) that indi-
cates the existence of primary amine groups in NH2-ePAN.
Characteristic bands related to the acetate groups of methyl meth-
acrylate co-monomer show a considerable decrease in their inten-
sity whereas the peak related to the nitrile group of the PAN does
not reveal a dramatic change. Of particular importance, this is
interpreted as the preferential reaction of EDA with acetate groups
forming amide II groups [32]. The strong peak of NH2-ePAN
observed at 1635 cmꢁ1 can be assigned to either amide II groups
[29] or the CN bending in C(@NH)ANHA (amidine group) [33].
However, because the nitrile groups remained almost intact, the
peak is largely contributed by the amide groups. The peak at
1566 cmꢁ1 is related to the bending vibration of NH in primary
amine.
Following the treatment of NH2-ePAN mat with 10 wt.% GA
solution, the peak at 1566 cmꢁ1 has nearly vanished. This might
be attributable to the condensation reaction between the aldehyde
groups of GA and the amine end groups, to form imine groups
(Schiff base imine) and intensification of the peak at 1635 cmꢁ1
[34]. In addition, the characteristic peak of aldehyde carbonyl
group at 1720 cmꢁ1 has overlapped with the peak of methacrylate
carbonyl at 1735 cmꢁ1 [35]. The two other characteristic peaks
related to ACH2A groups of the GA [36] are combined with those
of PAN and resulted in intensification of the peak at 2940 cmꢁ1
.
Scheme 1 shows formation of possible functional groups on the
surface of ePAN fibers following the reaction with EDA and GA.
The reaction between GA and primary amine end groups present
on the surface of the NH2-ePAN fibers proves the feasibility of

A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
41
100
98
cross-linking took place as a result of Schiff base reaction, as indi-
cated earlier, between free unreacted amines and aldehyde end
group of the neighboring fibers at their touching points. Schiffman
and Schauer [38] reported the similar observations when chitosan
nanofibers were cross-linked using GA.
(a) Pristine ePAN
(b) NH2-ePAN
(c) GA-ePAN
96
94 (c)
The FESEM images presented in Fig. 4(d) and (e) shows urease
aggregates immobilized on or within ePAN fibers when the con-
centration of GA changed from 5 wt.% to 10 wt.%. As shown in
Fig. 4(d), urease was mainly immobilized on individual separate
fibers, whereas, in Fig. 4(e) some enzyme aggregates were
entrapped between the cross-linked fibers.
92
90
88
86
84
82
80
78
1565.915
1635.34
(b)
3370.96
1172.508
1249.647
(a)
2869.559
1450.207
3.2. Effect of GA concentration on the amount of immobilized urease
and activity
2938.984
3500
2244.736
2500
1735.62
2000
4000
3000
1500
1000
It has been demonstrated that the ligand mobility is the govern-
ing factor affecting the activity of an immobilized enzyme [39].
Therefore, rather than the total number of immobilized biomole-
cules, the number of active immobilized enzymes and their freedom
of movement in the surrounding microenvironment determine the
activity retention. In Fig. 5, the specific activity and the amount of
immobilized urease are plotted versus GA concentrations. As it
can be seen, the amount of immobilized enzyme increased steadily
with the increase in GA concentration. However, the specific activity
shows a drastic decline when the GA concentration was >5 wt.%. It
was because GA reacts preferentially with ANH2 and ASH groups
[40] of urease molecule, which are essential for catalytic activity
[41].
Zhu and Sun [11] proved that upon increment in GA concentra-
tion, the number of aldehyde groups on the surface of polyvinyl
alcohol fibers and the overall content of immobilized lipase
increased. The decrease in activity, however, was ascribed to more
multi-point chemical bondings that reduced the ligand mobility.
Another explainable scenario was the limited mass diffusion of
the substrate (feed) to the enzyme active site as a result of the
intermolecular steric hindrance of crowded enzyme molecules
immobilized on the fibers.
wave number (cm-1)
Fig. 3. ATR-FTIR spectral bands related to the (a) pristine ePAN mats, (b) EDA
treated ePAN resulted in NH2-ePAN, and (c) GA treated NH2-ePAN resulted in GA-
ePAN.
the covalent bonding of enzymes via the reaction between the sur-
face amine groups of the enzyme and the unreacted end group of
the GA-ePAN fibers [37].
FESEM images of pristine and chemically treated ePAN fibers,
presented in Fig. 4(a)–(c), revealed changes occurred during the
stepwise chemical treatments. For each micrograph, the diameters
of 20 arbitrarily chosen fibers were measured and the average fiber
diameter was reported. Fig. 4(a) shows smooth nonwoven as-spun
ePAN ultrafine microfibers with an average fiber diameter of
1448 380 nm. During the amination, as can be seen in Fig. 4(b),
fiber surfaces were corroded and rugged due to EDA. However,
fibers preserved their integrity and no flaws were observed. The
average diameter of NH2-ePAN fibers slightly increased
(1503 300 nm). Fig. 4(c) depicts the morphology of cross-linked
NH2-ePAN mats when treated with 10 wt.% GA solution. Possibly,
Scheme 1. (a) Polyacrylonitrile-co-methyl methacrylate precursor of the fibers and reaction with EDA, (b) possible amidine and amide II groups formed during the amination
reaction leaving unreacted amine end groups, and (c) reaction of aldehyde groups with amine end groups.

42
A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
Fig. 4. The FESEM images of (a) as spun ePAN, (b) NH2-ePAN, (c) GA-ePAN when NH2-ePAN mat was treated by 10 wt.% GA and crosslinking of the neighboring fibers at their
touching points, (d) urease immobilized on 5 wt.% GA-ePAN, and (e) urease immobilized on 10 wt.% GA-ePAN.
partially or completely deactivate urease molecules or ban sub-
strate to reach the active site of enzyme, respectively.
350
300
250
200
150
100
50
3.0
2.5
2.0
1.5
1.0
0.5
0.0
With the specific activity of free urease at pH 7 and 25 °C equal
to 5.26 (
ePAN microfibers retained 54% of initial activity of free enzyme and
immobilized urease content reached 157 g/mg ePAN mat. This
lmol NH3/min mg urease), urease immobilized on 5% GA-
l
concentration was then used in the following studies of urease
activity.
3.3. Effect of pH and temperature on urease activity
The pH of the solution must be set in order to maintain the ionic
groups on the active sites in a suitable form to function. Rather
than changes in the ionic groups of the active site, variations in
pH alter the three-dimensional shape of the enzyme and, thus,
the enzyme activity and reaction rate. The pH-activity profiles for
both free and immobilized urease are shown in Fig. 6. The maxi-
mum activity slightly shifted from pH 7 for free urease to 6.5 for
immobilized urease. Also, immobilized urease retained relatively
high values in a broader range of acidic pH than free urease did.
Immobilization through multi-point attachment can preserve
the structure of enzyme molecule when pH changes. However,
0
0%
3%
5%
8%
10%
Glutaraldehyde Concentration
Fig. 5. Effect of GA concentration on the specific activity and the amount (
free urease immobilized on mg ePAN mat at 25 °C and pH 7.
lg) of
Based on FESEM micrographs (Fig. 4(d) and (e)), it was probable
that enzyme molecules developed multiple bonds with two or
more adjacent fibers or were entrapped between them. This could

A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
43
the charge of the surface affects the microenvironment of the
H
H
H
H
immobilized enzyme substantially. Generally, immobilization on
polycationic surfaces-such as chitosan or polyethyleneimine shifts
the optimum pH toward acidic pH while immobilization on poly-
anionic surfaces such as polylactic acid tends to shift it towards
more alkaline pH [42]. In this work, PAN microfibers were tethered
with amine groups, which changed the microenvironment around
the immobilized urease; i.e., the positively charged surface of
NH2-ePAN microfibers repulsed the H+ ions and preserved the
activity of the immobilized urease at low pH. Schematic represen-
tation of the effect is shown in Scheme 2.
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Effect of temperature on the activity of free and immobilized
urease is shown in Fig. 7. The maximum activity for free and immo-
bilized urease was found to be at 37 and 50 °C, respectively. In
addition, thermal stability of urease was enhanced in a broad range
of temperature by immobilization. This might be due to the
enzyme multi-point attachment to the surface of the fibers
through covalent bonding, which would lead to reduce enzyme
conformational changes at higher temperatures.
ePAN Fiber
Free enzyme
Immobilized enzyme on
polycationic surface
3.4. Kinetic studies
The changes in the rate of the enzymatic reaction as the function
of substrate concentration provide information about the kinetic
parameters i.e., Vm (maximum reaction rate) and Km (the Michaelis
constant). Km is an approximate representation of binding tightness
of the substrate to the enzyme. The lower the Km, the higher the
affinity between enzyme and substrates will be. Vm, on the other
hand, demonstrates the maximum velocity that a given enzymatic
reaction can theoretically reach. The Lineweaver–Burk plots for
both free and immobilized urease are shown in Fig. 8. It was found
that Km values for the free and immobilized urease were 40.35 and
57.6 mM, respectively. Whereas the Vm value for the immobilized
pH
Scheme 2. The effect of polycationic surface charge on microenvironment pH and
immobilized enzyme optimum activity.
urease (10.1
than that of free urease (17.54
l
mol NH3 minꢁ1 mgꢁ1 immobilized urease) was lower
l
mol NH3 minꢁ1 mgꢁ1 free urease).
100
80
The linear nature of these plots shows that both the free and immo-
bilized urease follows Michaelis–Menten kinetics.
Immobilization of urease on different support materials and
conformations has been widely studied and comprehensively
reviewed by Krajewska [43]. It was found that a similar trend for
an increase in Km and a decrease in Vm, more or less, almost always
happens regardless of the carrier material chemistry, its conforma-
tion, and covalent immobilization technique. The increase in Km is
the result of inefficient substrate access to the immobilized
enzyme active site which results in lower rate of the reaction
and Vm. Mass transfer limitations within the support structure,
conformational changes of enzyme during the immobilization
60
40
20
0
Free urease
Immob urease
0
10
20
30
40
50
60
70
80
90
Temperature
Fig. 7. Effect of temperature on the activity of free and immobilized urease.
100
80
and washing steps, restricted freedom of movement due to
multi-point attachment, and non biospecific orientation of the
enzyme active site through hydrophobic interaction of the enzyme
with support are the most important reasons that explain the dif-
ficulty of the substrate to reach the enzyme active site afterwards
the immobilization on a solid support [42]. However, in compari-
son with the results of other works, the urease immobilized on
the fibers of the present study showed much lower increase in
Km (57.6 (immobilized)/40.35 (free) = 1.42). This result demon-
strates that the affinity of the urease for the substrate (urea) has
not changed significantly and that the binding sites in the enzyme
is not significantly affected due to its immobilization on the ePAN
microfibers. One possible reason for the difference seen is that
there would be a reduced diffusion path for the substrate around
the dispersed fibers.
60
40
Free Urease
20
Immob Urease
7.5
0
5
5.5
6
6.5
7
pH
8
8.5
9
Fig. 6. Effect of pH on activity of free and immobilized urease.

44
A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
4
3
2
1
0
120
100
80
60
40
20
0
100
200
300
400
500
600
700
1/[S] (mol/L)-1
0
1
3
5
10
12
15
18
20
Storage Days
Fig. 8. Lineweaver–Burk plots for free and immobilized urease at pH 7 and 25 °C.
Fig. 10. Storage stability for free and immobilized urease at 4 °C and 25 °C.
3.5. Reusability and storage stability
4. Conclusion
The activity of immobilized urease was measured repeatedly
and, as shown in Fig. 9, the activity decreased only slowly. After
10 times of reuse cycles the enzyme retained over 85% of its initial
activity. Even after 15 cycles, this system preserved around 70% of
its initial activity. Generally, immobilization prevents enzyme to
lose its 3-dimensional conformation, its active site and its activity
during repeated number of reuse. Moreover, in this study the con-
siderable urease loading (157 lg/mg mat) implies strong enzyme
aggregates which prevents the enzyme from leaching into solution
and denaturing during reuse cycles [44].
This study revealed the successful covalent immobilization of
urease for the first time on electrospun polyacrylonitrile (ePAN)
microfibrous mats. At first, electrospinning of a 10 wt.% PAN
(MW = 1,500,000)/DMF dope solution at 18 kV and 20 cm resulted
in microfibrous ePAN mats with an average fiber diameter of
1448 380 nm. Then, urease was immobilized on disentangled
fibers following the treatment of ePAN fibers with EDA and GA
solutions. At 5% GA concentration urease loading reached 157 lg/
mg mat while preserving 54% of the free urease initial activity.
The FESEM images revealed enzyme aggregates on the surface of
the fibers and they confirmed the success of immobilization.
Immobilized urease tolerated the changes in pH (5.5–7) and tem-
perature (10–90 °C) significantly better than the free urease did.
Furthermore, it also showed improved storage stability (20 days)
and could retain nearly 70% of initial activity after 15 cycles of
reuse. This study provided an inexpensive, simple, efficient and
reliable technique for immobilization of urease on ePAN fibers
which is prognosticated to find application in dialysate liquid
regeneration system in artificial kidney machines.
Another important challenge that faces real application of
enzymes is their activity retention during the storage period. In
this work, we studied the effect of storage on the activity of both
free and immobilized urease by measuring the activity retention
for 20 days storage duration at 4 °C and 25 °C. The results are
shown in Fig. 10.
As can be seen in Fig. 10, immobilization has improved enzyme
storage stability. Immobilized urease stored at 4 °C has retained
almost 80% of its initial activity while the activity of free enzyme
was below 40% of the initial value. Also, immobilized enzyme
retained near 40% of its initial activity, while free enzyme lost
almost all activity at 25 °C. These results which demonstrate the
preservation of enzyme structure after immobilization suggest
the applicability of ePAN mats as successful biocatalyst carriers.
Acknowledgements
We acknowledge the Ministry of Higher Education (Malaysia)
and Universiti Teknologi Malaysia for the financial support through
Fundamental Research Grant Scheme Vot. R. J130000.7809.4F157.
100
90
80
70
60
50
References
[1] V. Gura, C. Ronco, F. Nalesso, A. Brendolan, M. Beizai, C. Ezon, A. Davenport, E.
Rambod, Kidney Int. 73 (2007) 497–502.
[2] V. Gura, A.S. Macy, M. Beizai, C. Ezon, T.A. Golper, Clin. J. Am. Soc. Nephro. 4
(2009) 1441–1448.
[3] J.P. Chen, S.H. Chiu, Enzyme Microb. Technol. 26 (2000) 359–367.
[4] D.H. Chen, J.C. Leu, T.C. Huang, J. Chem. Technol. Biotechnol. 61 (1994) 351–
357.
[5] R.A. Sheldon, Adv. Synth. Catal. 349 (2007) 1289–1307.
[6] Z.G. Wang, L.S. Wan, Z.K. Xu, J. Membr. Sci. 304 (2007) 8–23.
[7] T.S. Godjevargova, A.R. Dimov, N. Vasileva, J. Appl. Polym. Sci. 54 (1994) 355–
359.
[8] M.C. Yang, C.C. Lin, Biomaterials 22 (2001) 891–896.
[9] J. Kim, J.W. Grate, P. Wang, Chem. Eng. Sci. 61 (2006) 1017–1026.
[10] T. Srisook, T. Vongsetskul, J. Sucharitakul, P. Chaiyen, P. Tangboriboonrat,
React. Funct. Polym. (2014)
[11] J. Zhu, G. Sun, React. Funct. Polym. 72 (2012) 839–845.
[12] X.-J. Huang, D. Ge, Z.-K. Xu, Eur. Polym. J. 43 (2007) 3710–3718.
[13] Z.G. Wang, J.Q. Wang, Z.K. Xu, J. Mol. Catal. B-Enzym. 42 (2006) 45–51.
0
5
10
15
Number of Reuses
Fig. 9. Reusability of the urease immobilized-ePAN microfibrous mats.

A. Daneshfar et al. / Reactive & Functional Polymers 87 (2015) 37–45
45
[14] K. Rege, N.R. Raravikar, D.-Y. Kim, L.S. Schadler, P.M. Ajayan, J.S. Dordick, Nano
[28] N. Wu, J. Parris, Colloid Surf. A 167 (2000) 179–187.
[29] S. Deng, R. Bai, J.P. Chen, Langmuir 19 (2003) 5058–5064.
Lett. 3 (2003) 829–832.
[15] T.E. Herricks, S.H. Kim, J. Kim, D. Li, J.H. Kwak, J.W. Grate, S.H. Kim, Y. Xia, J.
Mater. Chem. 15 (2005) 3241–3245.
[30] A.M. Shoushtari, M. Zargaran, M. Abdouss, J. Appl. Polym. Sci. 101 (2006)
2202–2209.
[16] Z.G. Wang, L.S. Wan, Z.M. Liu, X.J. Huang, Z.K. Xu, J. Mol. Catal. B-Enzym. 56
(2009) 189–195.
[31] P. Tahaei, M. Abdouss, M. Edrissi, A. Shoushtari, M. Zargaran, Materialwiss.
Werkst. 39 (2008) 839–844.
[17] T. Vongsetskul, P. Kongjumnean, P. Sunintaboon, R. Rangkupan, P.
Tangboriboonrat, Polym. Bull. 69 (2012) 1115–1123.
[18] S.F. Li, J.P. Chen, W.T. Wu, J. Mol. Catal. B-Enzym. 47 (2007) 117–124.
[19] T.C. Hung, C.C. Fu, C.H. Su, J.Y. Chen, W.T. Wu, Y.S. Lin, Enzyme Microb.
Technol. 49 (2011) 30–37.
[32] C. Xu, J. Du, L. Ma, G. Li, M. Tao, W. Zhang, Tetrahedron (2013).
[33] Y.G. Ko, Y.J. Chun, C.H. Kim, U.S. Choi, J. Hazard. Mater. 194 (2011) 92–99.
[34] L. Marin, B. Simionescu, M. Barboiu, Chem. Commun. 48 (2012) 8778–8780.
[35] S. Jain, S. Chattopadhyay, R. Jackeray, H. Singh, Anal. Chim. Acta 654 (2009)
103–110.
[20] S.F. Li, W.T. Wu, Biochem. Eng. J. 45 (2009) 48–53.
[21] P. Ye, Z.K. Xu, J. Wu, C. Innocent, P. Seta, Biomaterials 27 (2006) 4169–4176.
[22] X.-J. Huang, A.-G. Yu, J. Jiang, C. Pan, J.-W. Qian, Z.-K. Xu, J. Mol. Catal. B-Enzym.
57 (2009) 250–256.
[23] B. Bagheri, M. Abdouss, M.M. Aslzadeh, A.M. Shoushtari, Iran. Polym. J. 19
(2010) 911–925.
[36] H.C. Wu, T.W. Wang, P.L. Kang, Y.H. Tsuang, J.S. Sun, F.H. Lin, Biomaterials 28
(2007) 1385–1392.
[37] S. Nair, J. Kim, B. Crawford, S.H. Kim, Biomacromolecules 8 (2007) 1266–1270.
[38] J.D. Schiffman, C.L. Schauer, Biomacromolecules 8 (2006) 594–601.
[39] V. Bulmußs, K. Kesenci, E. Pisßkin, React. Funct. Polym. 38 (1998) 1–9.
[40] S. Di Martino, H. El Sheriff, N. Diano, A. De Maio, V. Grano, S. Rossi, U.
Bencivenga, A. Mattei, D. Mita, Appl. Catal. B: Environ. 46 (2003) 613–629.
[41] K.H. Yeon, R.M. Lueptow, J. Chem. Technol. Biotechnol. 81 (2006) 940–950.
[42] J.N. Talbert, J.M. Goddard, Colloids Surf., B 93 (2012) 8–19.
[43] B. Krajewska, J. Mol. Catal. B-Enzym. 59 (2009) 22–40.
[44] B.C. Kim, S. Nair, J. Kim, J.H. Kwak, J.W. Grate, S.H. Kim, M.B. Gu,
Nanotechnology 16 (2005) S382.
[24] N. Arsalani, R. Rakh, E. Ghasemi, A.A. Entezami, Iran. Polym. J. 18 (2009) 623–
632.
[25] L.C. de Santa Maria, M.C.V. Amorim, M.R.M.P. Aguiar, P.I.C. Guimarães, M.A.S.
Costa, A.P. de Aguiar, P.R. Rezende, M.S. de Carvalho, F.G. Barbosa, J.M.
Andrade, R.C.C. Ribeiro, React. Funct. Polym. 49 (2001) 133–143.
[26] N. Arsalani, M. Hosseinzadeh, Iran. Polym. J. 14 (2004) 345–352.
[27] L. Betancor, F. López-Gallego, A. Hidalgo, N. Alonso-Morales, G.D.-O.C. Mateo,
R. Fernández-Lafuente, J.M. Guisán, Enzyme Microb. Technol. 39 (2006) 877–
882.