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Polyacrylamide beads: Polymer entrapment increases the catalytic efficiency

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Article · February 2018

DOI: 10.1016/j.mcat.2017.12.022

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 Molecular Catalysis 446 (2018) 81–87

Contents lists available at ScienceDirect

Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat

Polyacrylamide beads: Polymer entrapment increases the catalytic

efficiency and thermal stability of protease
Hafsa Sattar a , Afsheen Aman a , Urooj Javed a , Shah Ali Ul Qader b,∗
a
The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi 75270, Pakistan
b
Department of Biochemistry, University of Karachi, Karachi 75270, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Immobilization is a unique method for the improvement of product yield. During solid phase catalysis,
Received 11 March 2017 the chemistry of the synthetic matrix plays an essential role in the performance of the biocatalyst. In the
Received in revised form current study, immobilized protease within polyacrylamide macrosphere beads exhibited 76.0% entrap-
14 December 2017
ment yield with a remarkable stability of enzyme (33.0%) after 30 days of storage period at 4 ◦ C. The
Accepted 16 December 2017
entrapment of free enzyme within polyacrylamide also increased the optimal reaction temperature by
55 ◦ C and provided a broad range of pH optima. Moreover, a significant enhancement in thermal stability
Keywords:
was also detected. Polyacrylamide entrapped protease revealed up to 30.93% activity after incubation
Entrapment
Immobilization
period of 30.0 min at 70 ◦ C whereas, the free enzyme was completely inactivated at this temperature.
Polyacrylamide Additionally, entrapped protease displayed an efficient recycling capacity and retained approximately
Thermal stability 24.0% of its initial activity after eight successive reaction cycles. After entrapment of protease, the anchor-
Recycling efficiency ing of substrate to the active site of the free protease exhibited change in Km and Vmax values. Therefore,
owing to economic feasibility, the polyacrylamide entrapped protease might be a promising candidate
for various applications in different industrial sectors.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction Besides vast commercial significance of protease, there are some

Casein is a complex milk protein and is an aggregate of micelle
structures consisting of ␣-, ␤-, and ␬-casein along with colloidal
phosphate calcium [1] Casein has been used for non-food applica-
tions predominantly, as binding materials for plastics, fibers and
for preparation of novel cotton fibers [2,3]. Apart from these appli-
cations, casein hydrolysate is also used to generates value added
products that has significance commercial applications such as
calcium binding peptides [4] and stabilizers to improve emulsion
stability of food products [5]. This complex structure of casein can
be hydrolyzed by a special mechanism in which proteolytic enzyme
cleave the peptide bonds within the polyamine chain and gener-
ate different peptides of varying length. Therefore, protease has
attractive characteristics that contributes to numerous industrial
applications such as tenderization of meat in meat industry, formu-
lation in pharmaceutical industries, dehairing in leather processing
industry [6] and bio-hydrolysis of nylon fibers in textile industry [7].
Additionally, specific proteases have been used in food biotechnol-
ogy industries [8].
∗ Corresponding author.
E-mail address: saqader@uok.edu.pk (S.A. Ul Qader).
major applicability difficulties with reference to free protease.
The limitation of using free protease includes extra cost of sub-
strate, media components and continuous maintenance of culture
for next inoculation batch [9]. Along with these issues, the cat-
alytic efficiency of soluble enzyme is also reduced which drastically
impact its commercial applications. For these reasons, novel pro-
cessing techniques like immobilization of enzymes, was developed.
Immobilization is a remarkable approach that not only confines or
localize an enzyme molecule within the matrix but it also preserves
the catalytic properties of the enzyme [10,11]. In addition, immo-
bilization approach increases the operational stability and makes
the enzyme feasible to be reused for continuous industrial process.
As a result, a reduction in process cost is noticed. There are vari-
ous methods of immobilization including adsorption, crosslinking
and entrapment of enzyme within polymeric matrix. Neverthe-
less, entrapment is mostly favored due to its simplicity and cost
effective approach. It also creates negligible impact on the confor-
mation of the enzyme in contrast to other immobilization methods
that causes structural and functional changes in enzyme molecules
[12,13]. Among various synthetic matrices that are currently being
used, the most suitable one is polyacrylamide. Polyacrylamide is
a water-insoluble polymer with pronounced application in agri-
cultural and industrial sectors [14,15]. The attractive properties of

https://doi.org/10.1016/j.mcat.2017.12.022
2468-8231/© 2017 Elsevier B.V. All rights reserved.
82 H. Sattar et al. / Molecular Catalysis 446 (2018) 81–87
Fig. 1. Optimization of entrapment conditions for maximum yield of immobilized protease. (a) Effect of acrylamide concentration on immobilization yield of protease;
(b) Effect of polyacrylamide bead size on entrapment of protease. The values are shown as mean of three independent experiments (n = 3; Standard deviation ± SD: 2%;
p-value < 0.005).
polyacrylamide have gained much attention in the field of biomed-
ical applications as well [16]. The unique feature of polyacrylamide
comprises of the negative charges that are created during the
gelling process of the polymer. The carboxylic group within the
polyacrylamide provides a suitable internal macroenvironment for
the enzyme [17]. Silva et al. [18] reported that the thermal stability
of polyacrylamide might be due to the presence of strong interac-
tion of the hydrogen bonds present within this matrix. This also
reduces the mobility of the polymer chain and therefore, increase
the thermal strength. It can be suggested that polyacrylamide net-
work improves the stability of an enzyme molecule under hostile
industrial conditions.
The current investigation aims to develop a suitable process
that provides mechanical strength to enzyme and maintains the
catalytic efficiency as well as the thermal stability under extreme
industrial conditions. Protease from Aspergillus niger KIBGE-IB36
was subjected to entrapment using polyacrylamide as a synthetic
matrix.
2. Material and methods
2.1. Production and purification of protease
For production of protease, Aspergillus niger KIBGE-IB36 [Gen-
bank: KF905651] was used which was previously isolated from
an indigenous source [19] The selected strain was subjected to
submerged fermentation at 30 ◦ C for 120 h in medium contain-
ing (g L−1 ): casein, 2.5; peptone, 20; yeast extract, 0.50; glucose,
2.5; dipotassium hydrogen phosphate, 1.0; magnesium chloride,
0.10 and calcium chloride, 0.10 and keeping the pH at 6.0. After
fermentation, the cells were harvested by using filter paper and fur-
ther mycelium spores were separated by centrifugation at 40,000g
for 15 min at 4 ◦ C. The clear supernatant containing extracellular
enzyme was precipitated by using 40% ammonium sulphate sat-
uration [20]. The precipitates were solubilize in Tris-HCL buffer
(pH: 5.0, 50 mM). The solubilized precipitates were desalted and
then protease activity and total protein of the samples were cal-
culated and expressed in term of specific activity (U mg−1 ). The
enzymatic activity of partially purified protease from Aspergillus
niger KIBGE-IB36 used in the current study was 860.0 U mg−1 [20].
2.2. Immobilization of protease within polyacrylamide gel
The immobilization of protease within polyacrylamide
was performed by polymerization of acrylamide and N,N -
methylenebisacrylamide. For this purpose, acrylamide (8.0%),
bisacrylamide (0.4%), of partially purified protease (5000 ␮l)
by the addition of ammonium persulphate (20%) and tetram-
ethylethylenediamine (TEMED) (0.015 ␮l). The prepared solution
was mix thoroughly for even distribution of enzyme and was
poured immediately into a petri plates (60 × 15 mm) for solidi-
fication. The prepared plates were kept at 4 ◦ C for up to 6 h. The
solidified gel with entrapped enzyme was cut into equal sizes
with the help of metallic borer (macrosphere diameter: 5.0 mm;
macrosphere width: 1.0 mm) and washed with Tris-HCl buffer
(pH: 5.0, 50 mM) to remove unbound enzyme from the surface
of the gel. The gel was also prepared for control studies in which
same buffer was used instead of enzyme.
2.3. Enzyme assay
The activity of free and entrapped protease was estimated by
Anson method [21] with slight modifications in which the casein
(0.2%) was used as a substrate. Tyrosine was used as a standard. For
the enzymatic assay of free protease, 0.5 ml of enzyme was mixed
with 1.0 ml of casein while, for entrapped protease, 0.5 g of macro-
sphere beads were mixed with same amount of casein solution
prepared in Tris-HCl buffer (pH:5.0, 50 mM) and incubated at 50 ◦ C
for 15 min. After 15.0 min, macrosphere were removed carefully
and the enzyme-substrate reaction was stopped by adding 5.0 ml
trichloroacetic acid solution (10% TCA). The tubes were incubated at
37 ◦ C for 30 min. The undigested casein precipitates were removed
®
by Whatman filter paper No. 1. After filtration, 2.0 ml of filtrate
was withdrawn from both test and control tubes. The reaction mix-
ture was incubated with 5.0 ml of sodium carbonate (500 mM) and
1.0 ml of Folin & Ciocalteu’s phenol reagent (1:4) and was further
incubated at 37 ◦ C for 15 min in dark. The digested casein released
tyrosine which reacts with Folin & Ciocalteu’s phenol reagent and
generates a measurable color that is visualized under spectropho-
tometer (660 nm) [22]. One unit of protease can be defined as the
amount of enzyme required to release 1.0 M of tyrosine per minute
under standard assay conditions.
2.4. Immobilization yield of protease
The immobilization yield of entrapped protease can be calcu-
lated according to a following equation:
Activity of Immobilized Protease
Immobilizationyield (%) = × 100
Activity of Free Protease
2.5. Optimization of immobilization conditions for maximum
yield of entrapped protease
The optimum concentration of acrylamide is essential to main-
tain the structure and porosity of the gel. Therefore, various
concentration of acrylamide (6.0%–10%) were investigated to form
a suitable matrix for protease entrapment. The beads size of
entrapped enzyme greatly effects on the yield of entrapped pro-
tease. Therefore, various sizes of beads (3 mm–10 mm) were also
prepared.
 H. Sattar et al. / Molecular Catalysis 446 (2018) 81–87
Fig. 2. Kinetics behavior of free and immobilized protease. (a) Effect of enzyme-
substrate reaction time on free and entrapped protease; (b) Effect of reaction
temperature on free and entrapped protease; (c) Effect of pH on free and entrapped
protease. (n = 3; Standard deviation ± SD: 2%; p-value < 0.005).
2.6. Kinetic studies of free and entrapped protease
Reaction time is a critical parameter in which enzyme inter-
act with substrate to form a product at specific time therefore,
reaction time of free and entrapped enzyme was estimated
(5.0 min–60.0 min) under optimized assay conditions. Reaction
temperature significantly influence the catalytic activity of free and
entrapped enzyme thus a wide range of temperature (40 ◦ C–65 ◦ C)
was also studied. The effect of pH was also determined by per-
forming the enzyme substrate reaction at various pH ranging from
3.0 to 9.0 under specified conditions. Different buffer including
acetate buffer (pH: 3.0 and pH: 4.0), tris-HCl buffer (pH: 5.0 and
pH: 6.0.), potassium phosphate buffer (pH: 7.0 and pH: 8.0) and
glycine-NaOH buffer (pH: 9.0) were used for this experiment. The
kinetic behavior including maximum rate of reaction (Vmax ) and
Michaelis-Menten (Km ) of free and entrapped protease was deter-
mined by measuring the rate of reaction using different substrate
(casein) concentrations (1.0 mM–40.0 mM). In the current study,
Michaelis-Menten equation was used to establish the values of Km
and Vmax for free and entrapped protease. Non-linear regression
®
(curve fit) was developed by using GraphPad Prism (version 7.0).
83
2.7. Thermal stability of free and entrapped protease
Thermal stability of free and entrapped protease was inves-
tigated in which both free and entrapped enzyme were
pre-incubated in Tris-HCl buffer (pH: 5.0, 50 mM) at different tem-
perature (30 ◦ C–70 ◦ C) for different time with a 30.0 min interval
(0.0 min–120 min). Enzyme activity was performed after each time
interval and percent residual activity was calculated with reference
to the control.
2.8. Storage stability of free and entrapped protease
Storage stability of free and entrapped protease was determined
after storing them at 4 ◦ C for up to 30 days. The enzymatic activity of
protease was estimated in term of residual activity by considering
the activity of control as 100.0%.
2.9. Recycling efficiency of entrapped protease
The recycling efficiency of immobilized protease within poly-
mer support was determined by reusing the immobilized beads for
several successive cycles. After each cycle the immobilized beads
were washed with Tris-HCl buffer (pH: 5.0, 50 mM) to remove
remaining substrate. Freshly prepared substrate was added for
every next reaction. The initial enzymatic activity of entrapped
protease within polyacrylamide beads were considered as 100.0%.
2.10 Topological analysis of polyacrylamide macrospheres
with and without entrapped protease
Surface topology of polyacrylamide beads with and without pro-
tease were analyzed through scanning electron microscopy (SEM).
All the samples were first dried at 40 ◦ C for 24 h and coated with
300 Å gold particles and then loaded on sample holder using auto
coater (Jeol Japan, Model JFC-1500). The surface morphology of the
samples was then examined under different magnification fields
(Jeol Japan, Model JSM 6380).
3. Results and discussion
3.1. Optimization of immobilization conditions
3.1.1. Optimization of acrylamide concentration
An appropriate ratio of acrylamide (monomer) and bisacry-
lamide (crosslinker) is essential to maintain the porosity and
rigidity of the matrix used. Considering its importance, different
concentration of acrylamide (6.0%–10.0%) was investigated for the
entrapment of protease when 0.4% of bisacrylamide was used.
The polyacrylamide macrospheres which were constructed using
8.0% of acrylamide showed maximum enzyme entrapment yield
(Fig. 1a). The macrospheres prepared at this concentration were
more smooth and rigid, as compared to the lower concentration
of acrylamide (6.0–7.5%) where they were thin as well as fragile
and showed a lower immobilization yield. After this concentra-
tion (8.0%), however the strength of the macrospheres increased
but the immobilization yield decreased, and this effect could be
due to the presences of compact crosslinking network which is
generated between microenvironment of the enzyme and support
matrix. This rigid crosslinking network also hamper the interaction
of entrapped enzyme with the substrate molecule. The differences
in structural morphology of polyacrylamide macrospheres could
also be due to the degree of swelling of polyacrylamide gel that
maintains the degree of crosslinking and the number of charged
molecules present in the gel [17]. The basic phenomenon is that
when any catalytic molecule is exposed to the strong electrostatic
field present in the synthetic matrix, it undergoes some changes in
84 H. Sattar et al. / Molecular Catalysis 446 (2018) 81–87
Fig. 3. Stabilization of free and immobilized protease. Thermal stability of free and immobilized protease at various temperature with different time interval (a) at 30 ◦ C; (b)
at 40 ◦ C; (c) at 50 ◦ C; (d) at 60 ◦ C; (e) at 70 ◦ C. (n = 3; Standard deviation ± SD: 2%; p-value < 0.005).
the three-dimensional network which distinctly affects it mode of
action and thus leads towards lower immobilization yield.
3.1.2. Optimization of polyacrylamide macrosphere bead size
A suitable bead size for macrosphere is essential as it provides
precise surface area for the maximum interaction between the
enzyme and substrate molecules [23]. After the selection of acry-
lamide concentration (8.0%), the size of the macrospheres were
varied. It was noticed that maximum relative activity of entrapped
protease within polyacrylamide was achieved with a macrosphere
size of 3.0 mm in diameter and 1.0 mm in height (Fig. 1b). The
macrosphere size greater than 3.0 mm exhibited lower relative
activity, and this could be due to the two different phenomena
that could be either due to the steric hindrance of the enzyme or
might be due to the mass transfer limitations of substrate. Larger
quantities of biocatalyst cause steric hindrance by forming multi-
layers of protein complexes on the charged surface particle of the
synthetic bead [24]. The second reason could be the mass transfer
limitation which may occurred with immobilization of enzyme. The
mass transfer limitation can be internal of external. In case of exter-
nal mass transfer limitation, the diffusion of the substrate molecule
into the microenvironment of enzyme may be hindered because of
the polymeric network present in the acrylamide gel. However, in
case of the internal mass transfer limitation the release of product
into the macroenviroment of gel is restricted [25–27].
3.2. Kinetic characterization of free and immobilized protease
3.2.1. Influence of enzyme-substrate reaction time on free and
entrapped protease
Enzyme-substrate reaction time was evaluated at different time
intervals to measure the effect of reaction time on the free and
entrapped protease. Free enzyme, exhibited maximum relative
activity within 15.0 min of the enzyme-substrate reaction and
afterwards a declined was noticed (Fig. 2a). The entrapped pro-
tease within polymer exhibited maximum activity around 30.0 min
of incubation period with the substrate molecule. This shift of
time from 15.0 min to 30.0 min with respect to entrapped protease
may be due to the sturdy confinement of enzyme within the poly-
meric network. Any additional time required for penetration of the
substrate molecule within the macroenvironment of macrosphere
could lead to the extended reaction time therefore, in the current
study casein molecules require additional 15 min to interact with
the entrapped protease entities. The decline in relative activity after
30.0 min exhibited by entrapped protease could be due to the for-
mation of excessive product formation (tyrosine) which might have
resulted in accumulation of the product within the macroenviron-
ment of the macrospheres. This accumulation most of the time
leads towards competitive inhibition of enzyme.
3.2.2. Influence of reaction temperature on free and entrapped
protease
Most of the enzymes produced by fungal species are consid-
ered as temperature sensitive because the strains are generally
mesophilic in nature, therefore their catalytic activity is temper-
ature sensitive. In the current study, enzyme-substrate reaction
of free and entrapped protease was also examined at broader
temperature range (40 ◦ C–65 ◦ C) under optimized assay conditions
(Fig. 2b). Maximum relative activity in case of free enzyme was dis-
played at 50 ◦ C whereas, entrapped protease within polyacrylamide
exhibited its maxima at 55 ◦ C. This minimal 5 ◦ C temperature
shift for enzyme-substrate reaction could be due to the phys-
ical limitation of the entrapped enzyme within this synthetic
matrix. Entrapped protease exhibited tolerance against this tem-
perature raise which might be due to the low susceptibility to
the temperature-mediated conformational changes within the cat-
alytic structure of the enzyme molecule [28]. From these findings,
it can be assumed that polyacrylamide network is supportive in
protecting the catalytic site of the entrapped enzyme at broader
temperature range.
3.2.3. Influence of reaction pH on free and entrapped protease
The effect of reaction pH is also vital along with the reaction time
and temperature for determining optimal operating conditions. The
enzymatic activities of free and entrapped protease were assayed
at various pH values. The optimum pH was determined and is pre-
sented in Fig. 2c. Both the free and entrapped protease displayed
optimum pH at 5.0. Interestingly, entrapped protease displayed rel-
atively improved relative activity as compared to the free enzyme
at all the pH values tested. In this respect, Gonzalez and Pizarro
[17] have suggested that it is not easy to quantify the degree of
protection which the immobilization matrix have exerted on the
free enzyme because for this purpose it is mandatory to know the
exact pH values for both the enzyme solution and the interior of
 H. Sattar et al. / Molecular Catalysis 446 (2018) 81–87 85

Fig. 4. Stability of entrapped protease. (a) Storage stability of free and immobilized protease at 4 ◦ C for 30 days; (b) Recycling efficiency of immobilized protease within
polyacrylamide macrosphere. The values are shown as mean of three independent experiments (n = 3; Standard deviation ± SD: 2%; p-value < 0.005).

Fig. 5. Scanning electron microscopy of polyacrylamide matrix with and without protease entrapment. A and C: SEM micrographs of polyacrylamide beads at 4000× and
5000× magnification, respectively before protease entrapment; B and D: SEM micrographs taken at 4000× and 5000× magnification, respectively after protease entrapment.

the support. However, the pH for the solution can be determined
directly through measurements but it is rather difficult to quantify
the pH of the internal macroenvironment of the polyacrylamide
matrix.
3.2.4. Influence of substrate concentration on free and entrapped
protease
The kinetic behavior of immobilized enzyme has been deter-
mined by effectiveness factor (EF) in which diffusion of substrate
and product formation is measured. In the current study, the Km
values for free and entrapped protease was1.8 mM and 2.6 mM,
respectively, with an R2 value of 0.9863. Whereas, the Vmax for free
and entrapped protease was around 1753 U mg−1 and 1359 U mg−1 ,
respectively, with an R2 value of 0.9751. The higher Km value for
the entrapped protease as compared to the free enzyme suggests
that the interaction of the enzyme with its specific substrate is
due to the lower binding affinity. The hindrance caused during the
anchoring of the specific substrate to enzyme molecule could be
due to the diffusion barrier generated by the macroenvironment
of the matrix. The Vmax value of entrapped protease (1359 U mg−1 )
shifted to 1753 U mg−1 which could be due to diffusion limitation
of the product formed inside the macrosphere and thus the product
is not released efficiently outside the matrix. Similar effect has also
been reported for different enzymes. Immobilized dextranase and
laccase on different matrices exhibited change in enzyme substrate
kinetic behavior but still exhibited better operational and thermal
stability as compared to the free enzyme [29,30].
3.3. Thermal stability of free and entrapped protease
Thermal stability of an enzyme at extreme environmental con-
dition is an important factor for its prolong utilization at industrial
scale. Immobilization of an enzyme within a suitable matrix can
improve operational stability and can also prevent the enzyme
conformational structure when exposed to harsh environmen-
tal conditions like extreme pH and temperature. In the current
study, free and entrapped protease were incubated at different
temperature (30 ◦ C–70 ◦ C) for up to 120 min to estimate the opera-
tional stability of protease (Fig. 3). The residual activity of free and
entrapped protease was measured after every 30.0 min. Entrapped
protease exhibited improved thermal stability at all the tempera-
tures as compared to the free enzyme. In case of thermal stability
at 30 ◦ C, both the free and entrapped protease displayed exactly
100.0% residual activity for up to 120.0 min (Fig. 3a) whereas, when
86 H. Sattar et al. / Molecular Catalysis 446 (2018) 81–87
the temperature was increased both the free and entrapped enzyme
showed gradual decline in residual activity. The residual activi-
ties at 40 ◦ C (Fig. 3b) and 50 ◦ C (Fig. 3c) was much more stable as
compared to the 60 ◦ C (Fig. 3d) and 70 ◦ C (Fig. 3e). There could be
different explanation for this kind of enzyme behavior under immo-
bilized conditions. The improved thermal stability as suggested by
Çetinus and Öztop could be due to the conformational flexibility
in the immobilized enzyme [31]. Therefore, the acrylamide macro-
spheres might have the property to preserve the tertiary structure
of the enzyme and thus protect the enzyme from conformational
changes caused by the macroenvironment of the beads. When the
biocatalyst is entrapped within a specific matrix, another plausible
explanation for this phenomenon could be due to the reduction in
the enzyme autolysis [32].
3.4. Storage stability of free and entrapped protease
Storage stability is a crucial parameter that determines the
shelf life and strength of an immobilized enzyme. Therefore, free
and entrapped protease were stored at 4 ◦ C for up to 30 days and
the residual activities were measured after every 5th day of stor-
age (Fig. 4a). Free protease exhibited lower residual activities as
compared to the entrapped enzyme during the 30 days of stor-
age period. The extensive range of storage stability of entrapped
protease within polyacrylamide macrosphere could be suitable for
solid-phase catalyst for multiple applications in commercial indus-
tries. The loss in the enzymatic activity of free enzyme could be
the fact of autolytic behavior of the protease [33]. In other scien-
tific reports of protease immobilization on different matrices, the
enzyme exhibited storage stability even after 60 days of storage
[34].
3.5. Recycling efficiency of entrapped protease
The production cost of an enzyme is the targeted parameter
during commercial production. The separation of enzyme from
the product is a costly procedure therefore, the entrapment of
enzymes within a suitable matrix have attracted attention where
the enzymes can be used for multiple processes. In current study,
the recycling efficiency of entrapped protease was examined by
utilizing polyacrylamide gel for up to several successive cycles
(Fig. 4b). Entrapped protease revealed significant reusability and
retained approximately 72.0% of its residual activity up to fourth
cycle and around 24.0% at eight cycles. The residual activity of
immobilized protease within polyacrylamide gradually decreased
with the increase in number of cycles. This decrease in the activ-
ity might be due to leaching out of entrapped enzyme from the
matrix during several washing steps which are performed prior to
each cycle. Similar findings are reported when alkaline protease
from Bacillus amyloliquefaciens SP1 was reused for up to only five
cycles [35]. When fungal protease from Aspergillus niger KIBGE-IB36
was immobilized on polyacrylamide gel it showed slightly higher
recycling efficiency than the previous findings [34,35].
3.6. Topological analysis of polyacrylamide macrospheres with
and without entrapped protease
The surface morphology and texture of polyacrylamide macro-
spheres with and without entrapped protease were determined
by SEM (Fig. 5). Surface topology displayed significant differ-
ence between their surfaces. The SEM images revealed that
the polyacrylamide macrospheres without entrapped protease
showed relatively smooth homogenous morphology as compared
to entrapped protease (Fig. 5). These results were further confirmed
at higher resolution (×5000) where, polyacrylamide macrospheres
without entrapped protease displayed rough and regular surface
(Fig. 5C). In case of entrapped protease, macrosphere heteroge-
neous particles were observed on the surface (×4000) (Fig. 5B). At
higher resolution (×5000) heterogeneous globular shaped particles
were observed on the matrix surface of protease entrapped poly-
mer macrosphere beads (Fig. 5D). This irregularity of entrapped
protease macrospheres might be due to the embedment of the
protease on to the porous surface of polyacrylamide macrospheres
beads.
4. Conclusion
The current study validates the biocompatibility of protease
with polyacrylamide matrix and has proven to be an effective
approach to enhance the stability against high temperature and
pH. Entrapped protease also displayed interesting parameter of
recycling efficiency which makes it more attractive candidate at
industrial level. The kinetic behavior of immobilized protease was
also evaluated with respect to free enzyme. Thus, it could be con-
cluded that this particular immobilization method is suitable for
entrapment of protease under harsh environmental conditions.
Conflict of interest
The authors declare that they have no conflict of interests.
Acknowledgement
This research was indigenously supported by The Karachi Insti-
tute of Biotechnology and Genetic Engineering (KIBGE), University
of Karachi, Pakistan.
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