International Journal of Biological Macromolecules 111 (2018) 917–922

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Agar-agar immobilization: An alternative approach for the entrapment of

protease to improve the catalytic efficiency, thermal stability and
recycling efficiency
Hafsa Sattar a, Afsheen Aman 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: The catalytic performance of an immobilized enzyme could be enhanced by using entrapment technique. In this
Received 4 November 2017 contemporary study agar-agar, a natural polysaccharide, is subjected to entrap serine-protease produced by As-
Received in revised form 5 January 2018 pergillus niger KIBGE-IB36. The results revealed that maximum enzymatic activity was attained when 3.0% agar-
Accepted 16 January 2018
agar was used. It was observed that in case of both free and entrapped forms the enzyme was stable at pH-5.0.
Available online 19 February 2018
While, an increment in reaction temperature and time was noticed from 50 to 55 °C and 15.0 to 20.0 min, respec-
Keywords:
tively. Km value increased from 1.883 mM to 2.399 mM and Vmax value decreased from 1753 U mg−1 to 1372 U
Protease mg−1 after agar-agar entrapment of protease as compared to soluble enzyme. Additionally, entrapped protease
Entrapment within the polymer exhibited significant increase in the thermal stability at various temperatures and retained
Agar-agar approximately 68.0% of its residual activity at 60 °C. However, at this extreme temperature the soluble protease
Stability lost its catalytic performance. Storage stability considerably improved as entrapped protease revealed enzymatic
Reutilization efficiency activity up to 30 days as compared to soluble enzyme. Recycling efficiency was calculated up to eight cycles
which is an exceptional characteristic for economic feasibility and continuous reusability of protease.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction However, these issues can be resolved by selecting appropriate carrier

With the advancement in technology, the application of biocatalysts
have been extended in many fields due to its specific and nontoxic prop-
erties [1]. However, still biocatalysts have to face various problems for
commercial utilization that include harsh industrial environment, less
stability under hostile condition and inability to recycle again [2].
These problems associated with biocatalyst could be overcome by utiliz-
ing enzyme immobilization technology [1]. Nowadays, this technology
has been successfully applied in many biological fields for immobilizing
proteins, peptides, DNA and plant extracts [3]. Conventionally, there are
four different methods of immobilization that include adsorption, en-
trapment, and encapsulation and cross linking [4]. Among them entrap-
ment method, is the physical confinement of an enzyme within the
polymer network which insignificantly disturb the active site of an en-
zyme [5]. The cost of a suitable carrier is one of the major limitation of
immobilization method however, this limitation can be minimized by
using non-synthetic or natural carriers for enzyme entrapment [6,7].
Along with this, there are some drawbacks of the entrapment technique
as well. Proper diffusion of substrate and weak interaction between the
enzyme and the carrier are some of the limitations for this technique.
⁎ Corresponding author.
E-mail address: saqader@uok.edu.pk (S.A.U. Qader).
for different enzymes.
Agar-agar is a natural polysaccharide obtained from cell wall of
Rhodophyta (red algae). Agar-agar is considered as a biocompatible,
non-toxic and cost effective inert biopolymer. Structurally, agar-agar is
a polymer of agarobiose that consist of repeating units of D-galactose
and 3,6-anhydro-L-galactopyranose. The robust gelling ability of agar
agar exhibits no reactivity with other bio-molecules which favors it
commercial application in pharmaceutical, food and biotechnological
industries [8,9]. Moreover, the agar agar matrix exhibits stability be-
tween 80 to 85 °C, even when gelatinized at low concentration of 0.1%
[10]. Considering this fact, thermosensitive enzymes are encapsulated
within the polymeric network of polysaccharides carrier to shield the
3D structure of an enzyme molecule. Among various microbial sources,
Aspergillus species are generally regarded as safe (GRAS) for fermenta-
tion and are mesophilic organisms in nature therefore, the enzyme pro-
duced by fungal species are temperature sensitive. Among different
fungal enzymes, protease contributes approximately 60% of the global
enzyme market have been extensively utilized in detergent formulation
and for production of non-aqueous peptide synthesis [11]. At extreme
environmental conditions (high temperature), the non-covalent inter-
action formed within protease structures are disturbed and may cause
exposure of the buried hydrophobic residues within the enzyme struc-
ture [12]. The deactivation of covalent mechanisms can also occur, such

https://doi.org/10.1016/j.ijbiomac.2018.01.105
0141-8130/© 2018 Elsevier B.V. All rights reserved.
918 H. Sattar et al. / International Journal of Biological Macromolecules 111 (2018) 917–922
as deamidation of the Asn (asparagine) and Gln (glutamine) residues,
beta-elimination of disulfide bonds and oxidation of amine groups par-
ticularly, Cys (cysteine) and Met (methionine) residues [13,14]. Under
these conditions there is a need of a novel procedure which could shield
these functional sites present within the biocatalyst. Immobilization is
among one of the procedures that could protect the catalytic activity
of enzymes.
Previously, immobilization of protease produced by Aspergillus niger
KIBGE-IB36 has been studied using polyacrylamide gel as a support car-
rier [15]. However, data is very scarce for protease entrapment on agar
agar as a carrier. Most of the literature available regarding protease im-
mobilization is on different polymeric supports [16–19]. This is the first
study in which entrapment technique was utilized to preserve the cat-
alytic behavior of protease produced by Aspergillus niger KIBGE-IB36
on agar agar. Various concentrations of agar-agar were investigated to
achieve the maximum entrapment yield. The enzymatic activity of sol-
uble and entrapped protease was also studied to observe the impact of
agar agar on the catalytic efficiency and stability under different envi-
ronmental conditions. Operational and storage stability was also ex-
plored to study the long-term usage of entrapped protease.
2. Material and methods
2.1. Protease production and purification
In the current study, Aspergillus niger KIBGE-IB36 [Genbank:
KF905651] was previously isolated from soil samples [20]. The strain
(KIBGE-IB36) was selected based on the enhanced production of prote-
ase (data not shown) as compared to other Aspergillus species [15].
The selected strain was incubated at 30 °C for 5 days in fermentation
medium containing (g L−1): Casein, 2.5; Peptone, 20; Yeast extract,
0.50; Glucose, 2.50; Dipotassium hydrogen phosphate, 1.0; Magnesium
chloride, 0.10 and Calcium chloride, 0.10 at pH: 6.0. After fermentation,
the spores were harvested by using filter paper and then the fermented
broth was centrifuged at 40,000 ×g for 15.0 min at 4 °C. The spore free
broth of extracellular enzyme was precipitated with 40% ammonium
sulphate salt. The precipitates were solubilized in tris-HCl buffer (pH:
5.0, 50 mM). The solubilized precipitates were desalted and then prote-
ase activity and total protein of the samples were calculated. The enzy-
matic activity of protease from Aspergillus niger KIBGE-IB36 used in the
current study was 860 U mg−1 [21].
2.2. Immobilization process for entrapment of protease
The entrapment of protease within agar-agar carrier was initiated by
mixing an equal volume of purified enzyme with agar-agar solution in a
ratio of 1:1. First of all, 2.0% of agar-agar solution was prepared in Tris-
HCl buffer (pH: 5.0, 50 mM) and dissolved it by continuous shaking at
100 °C. The dissolved solution was allowed to cool down between 30
to 40 °C. Then the purified enzyme was mixed thoroughly in the dis-
solved solution of agar-agar. The prepared solution of enzyme and
agar was poured immediately on petri plates (60 × 15 mm) before so-
lidification of the gel at room temperature. The solidified gel of agar-
agar with entrapped enzyme was cut into equal sizes with the help of
metallic borer of 3.0 mm and washed with double deionized water to
remove unentrapped enzyme from the surface of the gel beads. These
prepared beads were weight and stored at 4 °C in the same buffer and
utilized for further enzyme characterization.
For the calculation of entrapped protease, the entrapped protease
beads (1.0 g) were initially suspended in tris-HCl buffer of pH 5.0
(10.0 ml) containing 6.0 mg ml−1 protein (protease: 860 U mg−1) and
it was placed at 25 °C for 30 min for equilibration. After the entrapment
of enzyme within the agar agar polymer, the left over unbound enzyme
solution was collected and examined for the estimation of residual en-
zymatic activity and protein concentration. The amount of immobilized
protease was calculated after immobilization [22,23]. The amount of
total protein was calculated by subtracting the amount of protein (Vf)
remaining in the washing solution at the end of entrapment process
and in the multiple washing solution (Cf) from the amount of protein
(Vi) which was initially used for the immobilization purpose (Ci):
CiVi−CfVf
Protein loading efficiency ð%Þ ¼ X 100 ð1Þ
CiVi
Similarity, the entrapment yield of protease can be calculated ac-
cording to a following equation:
Entrapment yield ð%Þ
Catalytic Activity of Entrapped Protease
¼ X 100 ð2Þ
Catalytic Activity of Soluble Protease
2.3. Analysis of protease activity
The catalytic activity of soluble and entrapped protease was esti-
mated by modified method of Anson [24] in which casein as a substrate
and L-tyrosine amino acid was detected as a standard. Partially purified
protease (0.5 ml) mixed with 1.0 ml sodium-caseinate (substrate)
while for entrapped protease 0.5 g of beads were mixed with same
amount of casein solution which was previously prepared in tris-HCl
buffer (pH-5.0, 50 mM) and keeping them at 50 °C for 15.0 min. Then,
the entrapped protease beads were removed carefully, and the reaction
was stopped by adding 5.0 ml trichloroacetic acid solution (10% TCA)
and after that the tubes were incubated for 30.0 min at 37 °C. The re-
maining casein molecules which were undigested, removed by
Whatman® filter paper. Then, the filtrate (2.0 ml) was withdrawn
from each reactant tubes. The reaction mixture was then mixed with
5.0 ml of sodium carbonate (500 mM) and 1.0 ml of Folin-Ciocalteu's
phenol reagent (1:4) and keeping them at 37 °C for 15.0 min in dark.
The casein which is hydrolyzed by partially purified protease released
tyrosine amino acid. These tyrosine residues react with Folin's reagent
and generate a measurable color at 660 nm [25]. One unit of protease
is defined as “the amount of enzyme required to release 1.0 μmol of tyro-
sine per minute under standard assay conditions.”
2.4. Agar-agar concentration
Agar-agar concentration was varied from (1.0 to 5.0%) to evaluate
the effect of entrapped protease on different concentration of agar-
agar carrier.
2.5. Kinetics studies of soluble and agar entrapped protease
The reaction time of enzyme kinetics is the time at which enzyme-
substrate reaction form a product at a specific time. In the current
study, reaction time of soluble and agar-agar entrapped protease was
studied from 5.0 min to 30.0 min under optimized assay conditions.
Along with reaction time, the effect of reaction pH was also studied by
performing the enzyme-substrate reaction at various pH values (3.0 to
9.0). For this purpose, different buffers were prepared including acetate
buffer (pH-3.0 and 4.0), tris-HCl buffer (pH-5.0 and 6.0), potassium
phosphate buffer (pH-7.0 and 8.0) and glycine-NaOH buffer (pH-9.0).
The substrate was prepared in the abovementioned buffers and the en-
zyme assay was conducted to analyze the enzymatic activity. For mea-
suring the temperature of the enzyme reaction, soluble and agar-agar
entrapped protease was kept at a wide range of temperatures (40 °C
to 65 °C) under optimized assay conditions. The enzymatic activity
was performed for both soluble and agar-agar entrapped protease.
The control was also treated in the similar conditions as the test reac-
tion. The influence of substrate concentration was also studied by
performing the protease assay at various concentration of casein (1.0
to 40.0 mM). The kinetic parameters including (Vmax) maximum rate
 H. Sattar et al. / International Journal of Biological Macromolecules 111 (2018) 917–922
of reaction and (Km) Michaelis-Menten constant for both the soluble
and carrier entrapped protease were calculated using GraphPad
Prism® 7.0 software which was utilized for the estimation of non-linear
regression analysis.
2.6. Surface morphology of agar agar beads with and without protease
The surface morphology of agar-agar beads with and without
entrapped protease were examined through scanning electron micros-
copy (SEM). Initially, the samples were dried at 40 °C for 24 h and
then coated with 300°A gold particles. The sample was then loaded on
the holder by using auto coater (Jeol Japan, Model JFC-1500). The sur-
face morphology of coated beads was then observed under different
resolution (Jeol Japan, Model JSM 6380).
2.7. Stability of soluble and entrapped protease
Thermal stability of soluble and agar-agar entrapped protease beads
were evaluated by keeping them at various temperature (30 °C to 60 °C)
for different time intervals (zero minute to 120.0 min). The enzymatic
activity was tested after the interval of each 20.0 min. Storage stability
of soluble and agar-agar entrapped protease was evaluated at 4 °C for
a period of 30 days. The enzymatic activity was conducted after every
five days of the storage period.
2.8. Reutilization of agar agar entrapped protease beads
The operational stability of agar-agar entrapped protease was deter-
mined by reusing the agar-agar entrapped protease beads for several
cycles. After completion of each reaction cycle (enzyme-substrate reac-
tion), the agar-agar entrapped protease beads were washed with dou-
ble deionized water to remove excessive substrate and the reaction
residues from it. The agar-agar entrapped protease beads were then
used for following next cycle. The substrate was prepared freshly each
time. The initial enzymatic activity of agar-agar entrapped protease
beads was considered as 100%.
3. Results and discussion
The selected fungal species is capable of producing serine-protease
(data not shown) was subjected to immobilization to enhance its cata-
lytic performance [21]. The immobilization approach used for this pur-
pose was entrapment technique. Effect of various concentration of
agar-agar were examined on the enzymatic activity of entrapped prote-
ase. Maximum immobilization yield was observed when 3.0% of agar-
agar was utilized for entrapment of protease (Fig. 1). Agar-agar concen-
tration beyond this concentration displayed a decline in immobilization
Fig. 1. Effect of agar-agar concentration on immobilization yield of protease. (n = 3;
Standard deviation ± SD: 2%; p-value b .005).
919
yield. This gradual decline might be due to the intermolecular complex-
ation of galactose residues present within the agar polymer and this in-
termolecular complexation basically alters the 3,6-anhydro-bridge
structure within the agar molecule [26]. A solidified matrix is generated
as soon as it was poured into the petri plate thus, a tough and rigid gel
was generated at higher concentrations of agar. Along with this any
lower relative activity detected during entrapment could be due to the
two different reasons. The first reason could be either due to the steric
hindrance of the enzyme and secondly it might be due to the mass
transfer limitations of the substrate molecule. Higher concentration of
enzyme molecules causes steric hindrance by forming multilayers of
protein complexes on the charged surface particle within the carrier
polymer [27]. The second possible reason is the mass transfer limitation
which may happen with enzyme immobilization. The mass transfer lim-
itation can be either internal or external. In case of external mass trans-
fer limitation, the diffusion of the substrate molecule into the carrier
support might be restricted due to the polymeric network present in
the agar agar. However, in case of the internal mass transfer limitation
the secretion of product molecule outside the macroenviroment of the
gel is restricted [28–30].
The enzyme-substrate reaction time for soluble protease was com-
pared with that of agar-agar entrapped protease. Entrapped protease
attained maximum relative activity at 20.0 min; later, a sharp declined
in activity was observed whereas, soluble protease exhibited enzymatic
activity at 15.0 min of the reaction time (Fig. 2A). This increment of re-
action time is case of entrapped protease might be due to the excess
times which is required for the penetration of substrate molecules
within the microenvironment of agar-agar matrix. Excessive time re-
quirement might be prerequisite for the substrate to reach at the active
site of the enzyme. Later sharp declined in the relative activity was de-
tected after 20.0 min of reaction time which could be due to the exces-
sive formation of product and by-products at the competitive site of
enzyme that inhibit the enzyme-substrate reaction [23].
The reaction pH is the important parameter for determining the op-
timum enzymatic activity. In the current study, enzymatic activities of
soluble and agar-agar entrapped protease were assayed at various pH
values. For soluble and agar-agar entrapped protease, maximum rela-
tive activity was displayed at pH-5.0 (Fig. 2B). No shift in the pH was ob-
served when the enzyme was entrapped. However, at a wide range of
pH, the entrapped protease revealed much better enzymatic activity
as compared to the soluble enzyme. Soluble protease retained 29% of
the relative activity whereas, agar-agar entrapped protease exhibited
approximately 59% of the activity at the same pH value (pH-9.0). Similar
results were obtained in previously reported studies [31].
The reaction temperature is also vital parameter along with reaction
time and pH. Extreme temperature is the targeted element for commer-
cial application of enzyme, generally enzymes are considered as
thermosensitive. Beyond optimum reaction temperature, the enzymatic
activity remained inactive, whereas above optimal reaction tempera-
ture the denaturation of enzyme occurred. Considering its importance,
the agar entrapped protease showed significant relative activity at
high temperature with respect to free enzyme (Fig. 2C). Maximum ac-
tivity was attained at 55 °C in case of agar entrapped protease, whereas
at 50 °C of reaction temperature, the free enzyme exhibited its maxi-
mum activity. The decreased in activity in case of free protease might
be due to the denaturation of 3D structure of enzyme above 55 °C.
While, agar entrapped protease revealed around 50% of relative activity
at 65 °C. This may be due do the mechanical strength of agar polymer
that shield the structural conformation of biocatalyst from hostile envi-
ronmental conditions.
The kinetic behavior was studied by calculating kinetic constant
(Km) and maximum reaction rate (Vmax) of soluble and entrapped pro-
tease by measuring the enzyme activity at different casein concentra-
tions (substrate). The Km and Vmax of protease were affected by
immobilization. It was found that the Km value of soluble enzyme in-
creased from 1.883 mM to 2.399 mM with an R2 value of 0.986 and
920 H. Sattar et al. / International Journal of Biological Macromolecules 111 (2018) 917–922

hostile environmental conditions. In the current study, thermal stability

of free and entrapped protease was detected at a wide range of temper-
ature (30 to 60 °C) for a time interval of 120.0 min. After every 20.0 min,
the residual activity was measured. Free protease was much more stable
at 30 °C and 40 °C as compared to 50 °C. Sharp declined in residual activ-
ity was noticed at 60 °C within 20.0 min (Fig. 3A). On the other side, im-
provement in thermal stability was detected for agar-agar entrapped
protease at all above-mentioned temperatures (Fig. 3B). It was manifest
from the findings that agar-agar carrier maintained the stability of pro-
tease by providing the mechanical strength to the native structure of an
enzyme under punitive conditions. This fact could be due the functional
property of agar-agar because agar agar had shown high mechanical
strength between 80 and 85 °C [10]. The same phenomenon could
also be factual in case of agar-agar entrapped protease.
Shelf life determines the strength and viability of an enzyme that
how long it can retain its functional property in the bioprocess. Gener-
ally, active site of enzymes deteriorate when stored in solution for lon-
ger time period [36]. The storage stability of soluble and agar entrapped
protease was examined at 4 °C for 30 days. Agar entrapped protease
showed 79% residual activity while, insignificant residual activity was
retained by free protease (Fig. 4A). This lose in residual activity of solu-
ble enzyme could be due to the fact of autolytic behavior of proteolytic
enzymes in the solution which can easily deteriorate the active site of an
enzyme. The agar-agar entrapped protease displayed better residual ac-
tivity even after 30 days as compared to soluble protease. This might be
due to the fact of shielding effect provided by the agar agar carrier that
prevent the intermolecular interaction against proteolysis and interac-
tion with gas bubbles from an entrapped enzyme [37–39].
The operational stability (recycling efficiency) of an enzyme is a cen-
tral characteristic that has a significant impact on the commercial utili-
zation of the immobilization process developed. For the continuous

Fig. 2. Kinetics behavior of soluble and immobilized protease. (A) Effect of enzyme-
substrate reaction time on soluble and entrapped protease; (B) Effect of reaction pH on
soluble and entrapped protease; (C) Effect of reaction temperature on soluble and
entrapped protease. (n = 3; Standard deviation ± SD: 2%; p-value b .005).

Vmax value decreased from 1753 U mg−1to 1372 U mg−1 with an R2

value of 0.9721 respectively after agar-agar entrapment of protease.
This increased in Km value in agar-agar entrapped protease may be
due to the lower affinity of an enzyme for its substrate molecule within
the agar-agar carrier. This fact is could be due to the formation of
“Nernst layer” surrounds an immobilized enzyme due to which higher
concentration of substrate is required as compared to the soluble en-
zyme which resulted in the increment in Km value [32]. However, a de-
crease in Vmax value might be due to the confinement of an enzyme
within the carrier support which restricts the diffusion of substrate
and product molecules and thus limits the rate of reaction to some ex-
tent. Similar kinetic behavior has been demonstrated by other
immobilized enzyme. Immobilized protease, pectinase and
amyloglucosidase on similar and different support showed change in ki-
netic behavior after entrapment as compared to soluble form of enzyme
[33–35].
In general, exposure of enzyme under extreme conditions (high
Fig. 3. Stabilization of soluble and immobilized protease. (A) Thermal stability of soluble
temperature) change the structural conformation of the active site of protease at various temperature with different time intervals (B) Thermal stability of
an enzyme. Therefore, different immobilization techniques have been entrapped protease at various temperature with different time interval (n = 3; Standard
employed to shield the functionality of active site of an enzyme under deviation ± SD: 2%; p-value b .005).
 H. Sattar et al. / International Journal of Biological Macromolecules 111 (2018) 917–922 921

biosynthesis of a specific bioproduct within a bioreactor, appropriate

carrier is essential to preserve enzyme from the reaction mixture. In
the current study, the residual activity of agar entrapped protease
beads was examined for several cycles. Agar entrapped protease re-
vealed significant reusability and retained approximately 50% of resid-
ual activity till eight cycle (Fig. 4B). The gradual decreased in residual
activity of agar entrapped protease might be due the several washing
steps of the agar entrapped beads as this could be due to the leakage
of the entrapped enzyme from the carrier. Zaak et al. [31] reported
five cycles of casein hydrolysis when ficin (protease extracted from
fig) was entrapped within glyoxyl agarose gel. Similarly, protease
from Aeromonas caviae displayed recycling efficiency for up to three
cycle when entrapped within alginate carrier [39]. The agar-agar
entrapped manganese peroxidase was reused for up to ten cycles [37].
From aforementioned findings, it could be suggested that protease
from Aspergillus niger KIBGE-IB36 entrapped within the microenviron-
ment of agar-agar is one of the suitable carrier which retained protease
activity for several consecutive batches.
The topological surface of microenvironment of agar-agar with and
without entrapped protease was determined by scanning electron mi-
croscopy (SEM) (Fig. 5). The surface morphology of agar beads with and
without entrapped protease noticeably displayed significant difference
in their structure. The SEM images revealed that agar-agar without
entrapped protease exhibited uniform even surface with several covered
layers (Fig. 5A) whereas, entrapped protease within agar-agar displayed
heterogeneous globular aggregates on the surface (Fig. 5B). The surface
morphological studies provided the evidence of significant change in sur-
face before and after entrapment of protease. Haneef et al. [33] also re-
ports parallel findings according to the surface morphologies of same
carrier (agar agar) with and without immobilized enzyme using SEM.

4. Conclusions
Fig. 4. Storage stability of soluble and immobilized protease at 4 °C for 30 days (A); Recycling
efficiency of immobilized protease within agar agar beads (B). The values are shown as mean The current study validates the stability of the entrapped protease
of three independent experiments (n = 3; Standard deviation ± SD: 2%; p-value b .005). within inert microenvironment of agar-agar. Maximum entrapment

Fig. 5. Scanning electron microscopy of agar agar with and without protease entrapment. (A) and (C): SEM micrographs of agar agar 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.
922 H. Sattar et al. / International Journal of Biological Macromolecules 111 (2018) 917–922
yield was achieved by using 3% of agar-agar carrier. The kinetic param-
eters of soluble and entrapped protease beads were studied to investi-
gate the difference in their catalytic performance. The optimum pH
and temperature for soluble enzyme was 5.0 and 50 °C respectively,
whereas for entrapped enzyme the pH remained same but the temper-
ature was shifted to 55 °C. It was noted that the thermal stability of sol-
uble protease was much improved after entrapment. The entrapped
protease within agar-agar showed catalytic performance even at 60 °C
whereas, in case of soluble enzyme the activity was completely lost.
The reutilization of entrapped protease displayed significant retained
residual activity (50.0%) at eight cycle. These results suggested that
the fabrication of agar-agar entrapment method considerably improved
the catalytic performance and operational stability of an enzyme.
Acknowledgement
This research was funded and supported by The Karachi Institute of
Biotechnology and Genetic Engineering (KIBGE), University of Karachi,
Pakistan.
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