Accepted Manuscript

Immobilization of α-amylase on chitosan-montmorillonite

nanocomposite beads

Tohid Mardani, Mahmood Sowti Khiabani, Reza Rezaei

Mokarram, Hamed Hamishehkar

PII: S0141-8130(18)31440-5
DOI: doi:10.1016/j.ijbiomac.2018.08.065
Reference: BIOMAC 10309
To appear in: International Journal of Biological Macromolecules
Received date: 27 March 2018
Revised date: 11 August 2018
Accepted date: 13 August 2018

Please cite this article as: Tohid Mardani, Mahmood Sowti Khiabani, Reza Rezaei
Mokarram, Hamed Hamishehkar , Immobilization of α-amylase on chitosan-
montmorillonite nanocomposite beads. Biomac (2018), doi:10.1016/
j.ijbiomac.2018.08.065

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 ACCEPTED MANUSCRIPT

Immobilization of α-amylase on chitosan-montmorillonite nanocomposite

beads
Tohid Mardaniab. Mahmood Sowti Khiabania. Reza rezaei Mokarrama. Hamed
Hamishehkarc.
a
Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
b
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
c
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

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ABSTRACT

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Enzyme immobilization is a way to increase efficiency of the enzyme and facilitate its recovery.
The aim of this study was to immobilize α-amylase on chitosan-montmorillonite nanocomposite

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beads. Nanocomposite beads were prepared as the carrier for the enzyme stabilization and their
surface was modified by Glutaraldehyde. Alpha-amylase was immobilized on nanocomposite
beads by covalent bonding. The results of scanning electron microscopy (SEM) showed that

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particle size range of montmorillonite was 10 - 30 nm. This study indicated that the enzyme
immobilization efficiency was 87٪. The activity of free and immobilized enzyme during 40 days
of storage at 4 °C decreased 95٪ and 36٪, respectively. The results showed that the immobilized
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enzyme activity after reusing five times decreased about 47٪. This study indicated that the
immobilized enzyme activity was higher than the free enzyme at different temperatures. Also the
immobilized enzyme was more stable than the free enzyme at lower pH. The results of kinetic
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parameters showed that Km values of the immobilized enzyme (9.12 μmol /ml) was higher than
free enzyme (6.80 μmol /ml). The Vmax values for the free and immobilized enzyme were 1.30
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and 0.629 μmol/mg.min, respectively.

Keywords: Immobilization. Enzyme activity. α-amylase. Montmorillonite. Chitosan. Nanocomposite
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1. Introduction
Enzyme immobilization is a technique that stabilizes the enzymes on carriers prior to reaction [2,
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3]. This technique has some advantages which are as follows: improving stability of the enzyme
against environmental conditions, facilitating enzyme isolation from the reaction medium,
reducing production costs and the possibility of reusing the enzyme in continuous processes [1,
4, 5]. Enzymes are biocatalysts that have been used in different industries because of their high
activity, selectivity, substrate specificity and easy production. Alpha-amylase (1,4-α-D-glucan-
glucanohydrolase, EC.3.2.1.1) are group of hydrolase enzymes which hydrolyze starch into
smaller molecules like glucose, maltose, and maltotriose for production of glucose syrups. Due
to the excessive application of enzymes in various processes, stability of the enzyme is a very
important factor that should be considered [1]. There are several techniques for enzyme
immobilization including entrapment, microencapsulation, physical adsorption and covalent
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bonds between the enzyme and carrier [1, 6]. The activity of immobilized enzyme depends on
factors such as the type of carrier and immobilization method [1] In the recent decade,
clay/polymer nanocomposites have been developed as carrier [7] The main feature of
nanocomposites is to apply natural materials such as chitosan in their composition [8]. Chitosan
is the second most abundant polysaccharide and one of the most common materials used as a
carrier in the process of enzyme immobilization [8, 9]. Chitosan has different advantages. It is
cheap and plentiful [10], non-toxic and harmless to the enzymes [9]. Chitosan has amine and
hydroxyl groups in its structure which facilitate its linkage with enzymes. It can be prepared in

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the form of beads [11] which have the ability to crosslink with glutaraldehyde. However, floating
in water due to its low density and soft tissue are the main drawbacks of chitosan beads. In order

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to improve density of chitosan beads, they can be combined with other compounds [9]. One of
the compounds that can be used for this purpose is montmorillonite nanoparticles.

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Montmorillonite is one of the most important clay minerals [12]. Clay particles have
characteristics that make them appropriate carrier for the immobilization of enzymes. Clay

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particles are inexpensive, chemically inert and resistant to high temperatures [1].

2. Materials and methods

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2.1. Materials
Alpha-amylase from Aspergillus Oryzae (Specific activity of α-amylase: 30 U (mg protein)-1, CAS
Number: 9001-19-8), Chitosan, soluble starch, 3,5-Dinitrosalicylic acid (DNS) and Glutaraldehyde
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(25% in H2O) were purchased from Sigma-Aldrich. Nanoparticles of montmorillonite were

purchased from Iranian nanomaterials Pioneers Co.
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2.2. Preparation of montmorillonite-chitosan nanocomposite beads

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First, the montmorillonite nanoparticles were activated with sulfuric acid. For this purpose, 5 g
of montmorillonite nanoparticles were refluxed by 25 ml sulfuric acid 1 M for 2 h. To prepare
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chitosan solution, 0.5 g of chitosan was dissolved in 2% glacial acetic acid and then 1 g of
activated montmorillonite was added to chitosan solution. Subsequently, montmorillonite-
chitosan mixture was poured into neutralization solution (1:1 (v/v) mix of 8% (w/v) NaOH and
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ethanol) and kept for 24 h [9, 10].

2.3. Surface modification of beads

To improve the surface of beads, 10 mg of nanocomposite beads and 1ml of glutaraldehyde 5%
were poured into a microtube and agitated at 200 rpm, 30 ° C for 1.5 h. Subsequently, the beads
were washed with distilled water and stored in phosphate buffer (0.05 M, pH= 6.5) [9].

2.4. Enzyme immobilization

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𝑚𝑔
Modified nanocomposite beads (10 mg) and enzyme solution (1 ⁄𝑚𝑙 ) were agitated at 20 rpm,
4°C for 24 h. The beads were strained. Finally, beads were washed with phosphate buffer (0.05
M, pH= 6.5) and stored at 4°C [13].

2.5. SEM study

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To determine size of the montmorillonite nanoparticles used in nanocomposite beads and to
investigate the morphological changes induced on surface of nanocomposite beads after enzyme

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immobilization, scanning electron microscopy was used. For this purpose, 1 mg of sample was

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dispersed in 1mg of distilled water and stirred by vortex. Then, for better dispersion the solution
was placed in an ultrasonic bath for 1 minute and a drop of the each sample was placed on
coverslip [17].

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2.6. FTIR spectroscopy
In order to study the bonding between components of the nanocomposite beads and the type of
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interaction between the enzyme and beads, Fourier-transformed infrared (FT-IR) spectrometry
was used. For this purpose, samples were dried at 37°C for 48 h and mixed with KBr and then
pressed to achieve transparent tablets. The tablets were then placed in a special cell and FTIR
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spectra was taken at a range of 500 – 3500 cm-1 [18].

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2.7. Determination of immobilization efficiency of the enzyme

The amount of immobilized enzyme was determined by Bradford method. In this method optical
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adsorption of the supernatant (free enzyme) was measured at 595 nm and the amount of enzyme
was calculated using bovine serum albumin as standard [14]. The enzyme immobilization
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efficiency was calculated by the following equation:

total amount of enzyme −amount of free enzyme
Enzyme immobilization efficiency = ×100 (1)
total amount of enzyme
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2.8. Determination of free and immobilized α-amylase activity

According to Miller method the amylolytic activity of free and immobilized α-amylase were
determined by formation of reducing sugars from starch solution (1% w/v) as the substrate in
phosphate buffer (0.05 M, pH 6.5) using dinitrosalicylic acid (DNS) at wavelength of 575 nm
[15]. One unit of α-amylase was defined to be the amount of enzyme that would produce
1.0 μmol of reducing sugar per minute. In this study, the time and temperature used for
hydrolysis of soluble starch were 10 minutes and 37℃, respectively. The initial activity was
considered 100%, and the remaining activity was expressed as a percentage of initial activity.
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µ mol of maltose released

Enzyme activity (IU/mg.min) = (2)
mg of a−amylase in minute

2.9. Enzyme stability at different temperatures and pH

In order to investigate the effect of pH on free and immobilized α-amylase activity, starch
solution (1% w/v) was prepared by phosphate buffer with different pH values (3.5, 5, 6.5 and 8),
then the starch solution with different pH was incubated with free and immobilized enzyme for
10 minutes at 37 ° C, and finally the enzyme activity was measured. Also, in order to investigate
the effect of temperature on free and immobilized enzyme activity, α-amylase was incubated

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with starch solution (1% w/v) prepared with phosphate buffer (0.05 M, pH 6.5) for 10 minutes

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at different temperatures (30, 45, 60 and 75 °C), and then activity of the enzyme was measured.

2.10. Stability of free and immobilized enzyme during storage

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To evaluate the stability of free and immobilized enzyme activities during storage, free and
immobilized enzyme were stored in phosphate buffer (0.05 M, pH=6.5) at 4℃ for 40 days. Also

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in order to investigate activity of enzyme, it was incubated with starch solution (1% w/v) every 10 days
[16].
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2.11. Effect of enzyme reuse on immobilized enzyme activity
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To investigate the effect of reuse on enzyme activity, the nanocomposite beads containing
immobilized enzyme were incubated with starch solution (1% w/v) at 37 ° C for 10 minutes and
the activity was measured. This work was repeated several times and the activity at each stage
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was compared with the initial activity of the enzyme [15].

2.12. Determination of Kinetic parameters (Km & Vmax)

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To determine the kinetic parameters (Km & Vmax), the activity of free and immobilized enzyme
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was measured at different concentrations of starch )2.5, 5, 7.5, 10 and 15 mg/ml) dissolved in
phosphate buffer (0.05 M & pH=6.5) and incubated at 37℃ for 10 min. Line weaver-Burk plots
were depicted and kinetic parameters (Km & Vmax) were calculated by the following equation:
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1 K  1   1  (3)
 m  S    V 
V0 Vmax  max 

Lineweaver- Burk plot of free and immobilized α-amylase are shown in figures 8 and 9,
respectively.
Results and discussion
3.1. Scanning electron microscopy
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The images of montmorillonite-chitosan nanocomposite beads before and after immobilization of

the enzyme at different magnifications are shown in Fig 1. According to scanning electron
microscopy (SEM) images, diameter size of the montmorillonite nanoparticles used in the
preparation of the beads was 10 - 30 nm. SEM of nanocomposite beads confirmed the presence
of nanoparticles. In addition, surface morphological changes of the nanocomposite beads after
enzyme immobilization were shown by SEM.

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3.2. FTIR study

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FTIR spectra of montmorillonite (a), chitosan (b), chitosan-montmorillonite nanocomposite bead

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(c), modified chitosan-montmorillonite nanocomposite bead with glutaraldehyde (d), α-amylase
enzyme (e) and nanocomposite beads with immobilized enzyme (f) are clearly shown in Fig. 2.
In Section (a) of Fig. 2, the peaks in 3438 cm-1 and 1037 cm-1 were related to bending vibrations

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of O-H and Si-O bonds in montmorillonite particles, respectively [8]. In Fig. 2(b), the peaks
attributed to bending vibrations of O-H and C-H bonds in chitosan can be seen at 3000-3750 cm-
1
and 2874 cm-1 ,respectively [8]. Analysis of FTIR spectra of chitosan- montmorillonite
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nanocomposite beads indicated peak of Si-O bonds at 1039 cm-1 which represents presence of
montmorillonite particles in the carrier Fig. 2(c). The peak at 1718 cm-1 related to bending
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vibrations of C=O of carbonyl groups showed surface modification of nanocomposite beads by

glutaraldehyde Fig. 2(d). FTIR spectra of α-amylase can be seen in Fig. 2(e). The peak at 1659
cm-1 is for the imide group, Fig. 2(f) demonstrates the successful immobilization of enzyme on
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beads.
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3.3. Immobilization effeciency

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In this study, immobilization effeciency of α-amylase on chitosan-montmorillonite

nanocomposite beads was 86%. In most studies, efficiency of α-amylase enzyme immobilization
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was reported to be 60% [19]. The reason for higher immobilization yield in this study was the
type of material selected as carrier for enzyme immobilization, namely chitosan. Chitosan was
able to make covalent bonds with amine groups of the enzyme due to its amine and hydroxyl
functional groups. Also modification of nanocomposite beads with glutaraldehyde could have
caused formation of aldehyde groups on surface of support and consequently, improves the
immobilization yield [20]., Mechanism of enzyme immobilization on nanocomposite beads is
shown in Scheme 1.

3.4. Effect of glutaraldehyde concentration on immobilized α-amylase activity

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In this study, the effect of different concentrations of glutaraldehyde on immobilized α-amylase

activity was investigated. The maximum activity of immobilized α-amylase was obtained at
concentration of 5٪ glutaraldehyde. The results showed that the enzyme activity decreased with
increasing concentrations of glutaraldehyde (Fig. 3). Glutaraldehyde is an active compound and
its excessive use can lead to enzyme denaturation and restructuring of the carrier [21, 22]. The
effect of concentrations lower than 5% of glutaraldehyde on α-amylase activity was investigated.
As results indicated that activity of enzyme was decreased in concentrations of less than 5%.
Therefore, these data were not presented.

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3.5. Effect of pH on free and immobilized α-amylase activity

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The results of pH assay on enzyme activity in free and immobilized forms showed that the highest
activity of free and immobilized α-amylase was at pH=6.5. The activity of immobilized α-amylase was
more than that of of free enzyme at various pHs. Immobilized enzyme activity at pH 3.5, 5 and 8 was 25

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%, 63% and 20% more than the activity of free enzyme, respectively (Fig. 4). Immobilization process
reduces the sensitivity of the enzyme to acidic and alkaline conditions [23]. Results of other research
showed that in the range of pH (3.5-5.8), immobilized enzyme activity was higher than that of the free
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enzyme activity. [24]. According to Fig. 4, optimum pH for free and immobilized enzyme activity was
6.5. Similar studies have demonstrated that the optimal pH for free and immobilized enzymes was the
same [1].
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3.6. Effect of Temperature on free and immobilized α-amylase activity

Fig. 5 shows that maximum activity of free and immobilized enzyme were at 30 and 45°C,
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respectively. The activity of immobilized enzyme at 60 and 75°C was more than the free enzyme
activity at the same temperatures (Fig. 5). In a study, catalytic activity of free and immobilized
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enzyme in a covalent system was compared at different temperatures and was reported that the
activity of immobilized enzyme was higher than free enzyme activity due to higher thermal
resistance [25, 26]. Reduced flexibility of enzyme configurations in the immobilization process
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has been reported to be the cause of stability of immobilized enzyme at higher temperatures [27,
28].
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3.7. Stability of free and immobilized enzyme during storage

Among the goals of enzyme stabilization is to increase the stability of enzyme during storage.
Generally, the activity of free enzyme decreases over time [29]. Comparison of free and
immobilized α-amylase activity during storage indicated that the reduction in the activity of
immobilized enzyme was lower than free enzyme (36% vs. 95%) (Fig. 6). In a study, the
decrease in activity rate of immobilized α-amylase stabilized on silica particles was reported to be
10% after 12 days in storage [30].
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3.8. Effect of enzyme reuse on immobilized enzyme activity

The other aim of enzyme immobiliztion is to reuse enzyme [13]. Acoording to Fig 7 after five
times of reusing, 47% of the immobilized enzyme activity was retained. Decrease in enzyme
activity may be due to separation of enzyme molecules from the carrier [3]. In another study, the
activity of immobilized glucose oxidase on cobalt was found to be 50% after five times of
reusing [31].

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3.9. Kinetic parameters

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Small amounts of Km indicates high affinity of enzyme to substrate [32]. Free and immobilized
α-amylase kinetic parameters (Km and Vmax) are reported in Table 1 which shows low tendency
immobilized enzyme to bind to the substrate after immobilization process probably due to

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structural changes in the enzyme as a result of enzyme immobilization. Reduction in tendency of
the enzymes to bind to substrate after immobilization process is probably due to steric hindrance
and structural changes in the enzyme which reduce the access of the enzyme to the substrate [9,
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33]. The results indicated that Vmax value of the immobilized enzyme was lower than that of the
free enzyme. Resistance in transfer of substrate mass to immobilized enzyme was reported due to
decrease of enzyme activity after immobilization process [34].
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Conclusion
In this work, chitosan- montmorillonite nanocomposite beads were prepared as carrier for
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enzyme immobilization. The results showed that the chitosan- montmorillonite nanocomposite
beads were suitable carrier for α-amylase immobilization. Surface modification of beads by
glutaraldehyde and immobilization of enzyme were proved by FTIR spectera and SEM.
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Immobilized enzymes were more resistant to environmental conditions than the free enzymes.
The immobilized enzyme activity was higher than that of free enzyme at high temperatures. In
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acidic conditions activity of immobilized enzyme was higher compared with that of the free
enzyme. Storage stability of the immobilized enzyme was more than the free form.
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Table 1 Kinetic parameters of free and immobilized α-amylase

Kinetic parameters Free enzyme Immobilized enzyme

Km (µmol / ml) 6.80 9.12
Vmax (µmol/mg.min) 1.30 0.629

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Figure Captions

Fig. 1. SEM of montmorillonite-chitosan nanocomposite beads before immobilization of enzyme at

different magnifications (a,b), SEM of montmorillonite-chitosan nanocomposite beads after
immobilization of enzyme (c,d) at different magnifications.
Fig. 2. FTIR spectra of montmorillonite (a), chitosan (b), chitosan-montmorillonite nanocomposite bead
(c), modified chitosan-montmorillonite nanocomposite bead with glutaraldehyde (d), α-amylase enzyme
(e) and nanocomposite beads with immobilized enzyme (f).

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Fig. 3. Effect of glutaraldehyde concentration on immobilized α-amylase activity

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Fig. 4. Effect of pH on free and immobilized α-amylase activity

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Fig. 5. Effect of temperature on free and immobilized α-amylase activity
Fig. 6. Storage stability of free and immobilized α-amylase

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Fig. 7. Effect of reuse on immobilized α-amylase activity

Fig. 8. Lineweaver- Burk plot of free α-amylase

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Fig. 9. Lineweaver- Burk plot of immobilized α-amylase

Scheme. 1. Mechanism of enzyme immobilization on nanocomposite bead

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a b

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c d
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Fig. 1. SEM of montmorillonite-chitosan nanocomposite beads before immobilization of enzyme at

different magnifications (a,b), SEM of montmorillonite-chitosan nanocomposite beads after
immobilization of enzyme (c,d) at different magnifications.
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c d
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e f
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Fig. 2. FTIR spectra of montmorillonite (a), chitosan (b), chitosan-montmorillonite nanocomposite bead
(c), modified chitosan-montmorillonite nanocomposite bead with glutaraldehyde (d), α-amylase enzyme
(e) and nanocomposite beads with immobilized enzyme (f).
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120
d
100 c b b
a

Enzyme activity (٪)

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60

40

20

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0

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5 10 15 20 25
Glutaraldehyde concentration (%)

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Fig. 3. Effect of glutaraldehyde concentration on immobilized α-amylase activity

120
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100 C
Enzyme activity (٪)

B
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60 c
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A
40 b
a
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3.5 5 6.5 8
pH
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Free enzyme Immobilized enzyme

CE

Fig. 4. Effect of pH on free and immobilized α-amylase activity; a, b, c, d, and A, B, C, D represent

the standard deviation of free and immobilized enzyme activity, respectively
AC
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120
b D
100 b
C

Enzyme activity (٪)

80

60 B
A

40
a
a
20

T
0

IP
30 45 60 75
Temperature (°C)

CR
Free enzyme Immobilized enzyme

Fig. 5. Effect of temperature on free and immobilized α-amylase activity; a, b, c, d, and A, B, C, D

US
represent the standard deviation of free and immobilized enzyme activity, respectively
AN
120

100 100
Enzyme activity (%)

82.33 77.66
80 74.66
67.83
ED

60
45.83
36.33
40 30.83

20
PT

5.83
0
0 5 10 15 20 25 30 35 40 45
CE

Time (day)

Free enzyme Immobilized enzyme

AC

Fig. 6. Storage stability of free and immobilized α-amylase

120

100
Enzyme activity (%)

80

60

40

20

0
1 2 3 4 5 6
Number of reuse
Fig. 7. Effect of reuse on immobilized α-amylase activity
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1.4

T
y = 5.2236x + 0.7674 1.45
R² = 0.9906 1.2

IP
(µmol/mg.min)-1

1.2
1 0.882 1.02
0.878

CR
0.8
1/V

0.7674
0.6
0.4

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0.2
0
0
-0.15 -0.1 -0.05 0 0.05 0.1 0.15
AN
1/S
(µmol/ml)-1
M

Fig. 8. Lineweaver- Burk plot of free α-amylase

ED
PT
CE

4
y = 14.493x + 1.5885 3.5 3.52
R² = 0.9942
(µmol/mg.min)-1

3
AC

2.5 2.69
1.94 2.283
1/V

2 1.93
1.5 1.5887

1
0.5
0 0
-0.15 -0.1 -0.05 0 0.05 0.1 0.15
1/S
(µmol/ml)-1

Fig. 9. Lineweaver- Burk plot of immobilized α-amylase

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T
IP
CR
US
AN
M
ED

Scheme. 1. Mechanism of enzyme immobilization on nanocomposite bead

PT
CE
AC
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Highlights:

 α-amylase enzyme was covalently immobilized on chitosan-montmorillonite nanocomposite beads.

 The size of montmorillonite nanoparticles used in the preparation of the beads was 10 - 30 nm.
 FTIR confirmed enzyme immobilization and connections between carrier components.
 Enzyme immobilization efficiency of 86% was obtained.

T
 Immobilized enzyme exhibited high thermal and pH stability.

IP
 Immobilized enzyme maintained 47% of initial activity after 5 times of reuse.
 Immobilized enzyme retained 64% of its initial activity after 40 days.

CR
 Km increased and Vmax decreased after enzyme immobilization.

US
AN
M
ED
PT
CE
AC