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Metal
oxide Nano composites, ZnO-Fe3O4 (S1) and ZnO-PANI (S2)
were synthesized and used as carriers for the immobilization of the enzyme,
α-amylase. The parameters such as pH, temperature, concentration and contact
time were optimized. The immobilized enzyme showed higher immobilization yield
compared to that of the free enzyme. The thermodynamic parameters such as free
energy change (ΔG°), entropy change (ΔS°) and enthalpy change (ΔH°) were
evaluated and found to be higher for both S1and S2. The kinetic parameters, Km
and Vmax were evaluated from Line weaver-Burk plots. The immobilized
enzymes also showed higher storage stability and reusability which ensure them
for various biochemical applications.
Keywords: ZnO-Fe3O4,
ZnO-PANI, α-amylase, Immobilization, Catalytic activity, Thermal deactivation
INTRODUCTION
In the current global world, bio-catalysis is
emerging as the most challenging field where there is increasing demand in
applications with the highest biological impact. They also play a major role in
the field of pharmaceutics, medical diagnostics [1,2], agrochemical products
[3], cosmetics [4] and in food industry [5]. Bio-catalysis refers to the use of
“enzymes” which reform them to process catalysts to execute under the reaction
conditions of industrial process [6]. Interest in the field of immobilization
of enzyme on solid supports and their applications have been emerging in the
last few decades [7]. Immobilization intensifies the catalytic properties and
makes them fit for many analyses [8].
Immobilization can be done by using various
methods such as adsorption, covalent binding and entrapment within a porous
matrix, microencapsulation and aggregation [9,10]. Adsorption is the simplest
and widely used method for immobilization process which is based on physical
adsorption or ionic binding [11,12]. The advantage of this method is that it
minimizes the disturbance inactive centers thereby retains the entire amount of
activity [13]. A wide variety of materials have been reported in literature
which is used as carriers for immobilization process [14-17]. Presence of more
reactive sites with good mechanical properties makes nanocomposite materials
more notable to possess several applications. Nanocomposites formed by
combination of conducting polymers and metal oxides nanoparticles, possess the
good properties of both the constituents and thus enhance the utility [18].
Zinc oxide structured metal oxides are more advantageous over other nano metal
oxides, makes it a favorable material for enzyme immobilization [19]. Since,
magnetite particles allow easy separation of the catalyst from the reaction and
with high surface area are more used in immobilization of enzymes in an
effective way [20,21]. Magnetite particles also have the capability to improve
the efficiency of the immobilized enzymes by weakening the diffusion barriers
in transporting the substrate and the reaction products [22]. Polyaniline
(PANI), one of the most versatile and environmentally stable polymers having
high stability to extreme pH and temperature makes suitable for enzyme
immobilization [23,24].
The enzyme α-amylase was chosen as the enzyme
for immobilization. It is one of the enzymes with great significance in
miscellaneous fields [25,26]. They are endo enzymes that
cleave α-1,4 glycosidic
bonds in poly-
In our study, we immobilized
α-amylase onto magnetite-ZnO (S1) and PANI-ZnO (S2) nanocomposites. The method
used is adsorption. The nanocomposites were characterized by IR,
TGA, XRD and SEM analysis. Effect of different immobilization parameters such
as pH, temperature, contact time and amount of enzyme required for
immobilization were evaluated to achieve maximum immobilization yield and
efficiency. The kinetic parameters Km and Vmax
were calculated from the Lineweaver-Burk plots. The thermal deactivation
studies were performed and thermodynamic parameters were evaluated. Thermal
stability, reusability and storage stability of the enzyme were studied under
free and immobilized conditions.
MATERIALS AND METHODS
Materials
Diastase α-amylase (1,4
α-D-gluconglucanohydrolse, E. C. 3.2.1.1) was purchased from HiMedia
Laboratories Pvt. Ltd, Mumbai. Soluble Starch (potato) and aniline were
procured from S. D. Fine Chem. Ltd. Mumbai. Iron (II) chloride. Tetrahydrate
(FeCl2.2H2O), Zinc acetate dehydrate (Zn(CH3COO)2,
Hydrochloric acid (HCl), Ammonia solution (NH3, 25%), Ammonium
peroxodisulphate and acetone were purchased from Merck Co.
Synthesis of ZnO-Fe3O4
nanocomposite (S1)
ZnO-Fe3O4
nanocomposite was prepared on the basis of already reported work [34].
Initially, magnetite nanoparticles were prepared by dissolving 1 g of FeCl2.4H2O
100ml of distilled water. 15 ml of NH3 solution (5 M) was added
slowly with continuous stirring. The acquired precipitate was black. It was
then centrifuged, cleansed several times with distilled water further dried and
weighed. About 1 g of the magnetite nanoparticles were dissolved in distilled
water and Zn (CH3COO)2.2H2O was added and
dissolved in the same solution. 5ml of ammonia (5 M) was slowly added to the
above solution. The precipitate obtained was then filtered, rinsed with
distilled water and dried at 200°C.
Synthesis
of ZnO-PANI nano composites (S2)
ZnO nanoparticles were first
prepared by adding NaOH containing 2 M ethanol was added drop wise to the
ethanolic solution of zinc nitrate drop by drop for about 2 h. The solution is
allowed to stand overnight. The precipitate settled may contain unwanted
contents which is removed by rinsing with distilled water for three times and
also with ethanol and dried at around 60°C [35]. ZnO-PANI nanocomposite was
prepared by dissolving 1 g of ZnO powder in 20 mL aqueous solution of 0.01 mol
aniline and 0.01 mol hydrochloric acid. 0.01 mol of APS was made to dissolve in
100 ml distilled water, further was added drop wise to the above mixture with
stirring in an ice bath for 5.5 h. The precipitate was obtained which is again
filtered, washed with distilled water and ethanol and dried at 500°C [36].
Characterization of
synthesised nanocomposites
The
synthesized nanocomposites were characterized by FT-IR spectrometry using JASCO
FT/IR-4100. Bruker AXS D8 Advance is used for XRD analysis. SEM images were
obtained by using the JEOL Model JSM-6390LV scanning electron microscope.
Thermal behavior of the nanocomposites was obtained by using Perkin Elmer,
Diamond TG/DTA.
Immobilization of enzyme, α-amylase
Immobilization of enzyme was
carried out by mixing the synthesized nanocomposites with equal volumes of
enzyme in buffer solution followed by shaking in a water bath shaker at room
temperature for two hours. The prepared biocatalyst was rinsed with the same
buffer to remove the unbound enzyme. The unbound enzyme in the supernatant and
washings were estimated by the spectrophotometric method, using
Folin-Ciocaltaue’s phenol reagent by measuring the absorption at 660 nm in
Thermo scientific evolution 201 UV-Visible Double Beam Spectrophotometer. The
freshly prepared immobilized enzyme was stored in a refrigerator at 4°C for
further studies [37].
Immobilization yield (IY) was
calculated by measuring concentration of protein in supernatants before and
after immobilization, according to Eqn 1,
IY%= C1 – C2/C1 * 100 (1)
Where C1 was the
concentration of protein taken for immobilization and C2 was the
concentration of protein present in supernatant after immobilization. And the
activity yield (AY) was determined by the Eqn 2,
AY% = Activity of immobilized enzyme / Activity of free
enzyme * 100 (2)
The immobilization efficiency
(IE) was calculated using Eqn 3,
IE = AY / IY (%) (3)
Activity determination of the free and
immobilized enzyme
The activity of immobilized α-
amylase was observed using starch as the substrate. The reagent used was
3,5-dinitrosalicylic acid (DNS). The test tubes containing the reaction mixture
(1 ml each of α-amylase and 1% starch) with desired buffer are shaken
thoroughly for 15 min at room temperature. 3,5-dinitrosalicylic acid (1 ml) was
then added to each of the test tubes to stop the reaction. Incubation was done
in a boiling water bath for 5 min and cooled until room temperature is
attained. The amount of sugar (maltose) produced was determined
spectrophotometrically at 540 nm [38].
Conditions
for optimization of free and immobilized enzymes
The optimization of the
immobilization parameters such as pH, temperature, concentration and contact
time was done. The maximum activity of optimum pH was assayed using starch in
enzyme and incubated over a pH range 4-9 at 30°C. The optimum temperature for
maximum activity was performed by varying the range of temperature from 30°C to
70°C. Thermal stabilities for free and immobilized enzyme were investigated by
measuring their residual activities at optimum conditions after being incubated
for 60 min in the temperature range of 30-60°C in a water bath and then cooled
to optimum temperature. Desired amount of 1% starch was added to each reaction
at definite time interval to carry out the reaction process.
Kinetic
studies of free and immobilized enzymes
Free and the immobilized enzymes
was incubated at different temperatures without adding starch to perform the
kinetic studies. The enzyme activity assay was done at definite time intervals
and the residual activity is expressed in terms of percent of initial activity.
The deactivation constant (kd) is obtained from the slope of the logarithmic
plot of percent residual activity versus time and the slope of Arrhenius plot
drawn between ln kd and reciprocal of temperature, 1/T (K) gives the
deactivation energy (Ed) using Eqn 4,
Thermodynamic parameters
Estimation of the values of
thermodynamic parameters such as free energy change (ΔG°), entropy change
(ΔS°), and enthalpy change (ΔH°) can be obtained using kd and Ed values.
Where R is the universal gas
constant, T is the absolute temperature (K), h is the Planck constant and kB
is the Boltzman constant.
Determination
of kinetic parameters
Michaelis constant (Km)
and maximum rate (Vmax) are the kinetic parameters that can be
evaluated from Lineweaver-Burk plot. Initial reaction rate were measured under
optimum conditions of pH and temperature by changing the concentration of
starch.
Reusability
and storage stability
The reusability study of the
immobilized enzyme was done using batch experiment by maintaining the fixed
hours for each cycle. The residual activity of the immobilized enzyme at
optimum pH and temperature was measured at fixed time intervals. At the end of
each cycle, the immobilized enzyme was removed and washed with buffer and then
added a substrate solution to begin a new cycle. The process is done for
several counts. The stability while storing immobilized enzyme in buffer
solution for a long time was measured by calculating their activities after
being stored at 4°C for several months. At regular intervals of time, assay was
performed. The activity obtained was compared with the initial one and
considers as the percentage relative activity.
RESULTS AND
DISCUSSION
Characterization studies of the synthesized
nanocomposites
The FT-IR spectrum of ZnO
exhibits broad absorption peaks between 3500 and 3600 cm-1,
corresponding to the stretching mode of O–H group of hydroxyl group and the
weak band near 1630 cm-1 is assigned to H–O–H bending vibration mode
due to the adsorption of moisture on the surface of nanoparticles. The band
3477 cm-1 corresponds to the stretching vibrations of the OH group
on the surface of ZnO nanoparticles. The peaks observed in the region 460-390
cm-1 corresponds to the Zn-O bond. The spectrum of Fe3O4,
the peak in the region 3600-3100 cm-1 is attributed to the
stretching vibrations of –OH, which is assigned to –OH (water molecules)
absorbed by Fe3O4 nanoparticles, the peak in the region
570-590 cm-1 is attributed to the Fe-O bond vibration of Fe3O4.
The spectrum of S1 (Supplementary Data – Appendix A) shows Fe–O bond around 565 cm-1.
In order to confirm existence of Zn–O bond in nanocomposite, ZnO nanoparticles
were prepared in absence of Fe3O4 by the same method and
its FT-IR spectrum is shown. It can be observed that the strong absorption band
at 435 cm-1 which is ascribed to phonon absorptions of the ZnO
lattice is present. The spectrum exhibits broad absorption peaks between 3500
and 3600 cm-1, corresponding to the stretching mode of O–H group of
hydroxyl group and the weak band near 1630 cm-1 is assigned to H–O–H
bending vibration mode due to the adsorption of moisture on the surface of
nanoparticles. The PANI show characteristic peaks at 503.1cm-1,
587.6cm-1, 798.7cm-1, 1121.8cm-1, 1296.7cm-1,
1472.4cm-1, 1559.8cm-1, 2369.3cm-1 and
3446.7cm-1 corresponds to C=N iminoquinone, C=C stretching modes of
quinoid rings, the C=C stretching mode of benzenoid rings, the stretching mode
C-N, C-H bending mode of aromatic rings. The S2 composite (Supplementary data – Appendix A) also show the same characteristic peaks. But some
of the peaks of PANI were shifted to higher values which may be due to the
hydrogen bonding between ZnO and NH2 group of PANI on the surface of
ZnO particles.
The XRD peaks of S1
and S2 are shown in Appendix B. The
strong and sharp peaks of S1 are found at 2θ=35.6°, 43.1°, 53.5°, 62.7°, corresponding to
(311), (400), (422) and (440) crystal planes of face centered cubic Fe3O4
respectively (JCPDS file no: 75-0033). FCC structure of Fe3O4
retained even after the formation of nanocomposite. The XRD peaks of ZnO are
found at 2θ=31.7°, 34.36°, 36.2°, 56.59°, 62.7° and 67.90° corresponding to (100), (002), (101), (110), (103) and
(112) planes assigned to the wurtzite pattern of ZnO (JCPDS card 36-1451). The
characteristic peak of polyaniline were at 2θ=26°. Using Debye Scherrer equation, the average particle size can be evaluated
quantitatively.
d = kλ / βcosθ
Where, d gives the particle size,
k is the Debye Scherrer constant (0.89), λ is the X- ray wavelength (0.15406
nm), β is the full width at half maximum and θ is the Bragg angle. According to
this equation, the particle size of S1 is 24.5 nm and of S2 is 21.8 nm.
The surface morphology was
observed from the SEM analysis. The SEM micrographs of S1 show that the
magnetite nanoparticles are well dispersed on ZnO and are found to be loosely
packed. The SEM image of S2 reveals that ZnO particles are surrounded by
polyaniline matrix and appears as aggregated particle structure (Appendix C).
The TG-DTG curve of the S1 and S2 are shown
in Appendix D. All of them underwent
two stages of weight loss. The first stage of weight loss from room temperature
to 200°C is ascribed to loss of water molecules. The reason behind the weight
loss between 250°C and 550°C may be due to the decomposition of the polymer
chain which occurs before 490°C in pure samples. The interaction with ZnO
nanoparticles reduces the decomposition and makes S1and S2 more thermally
stable compared to pure samples [39].
Conditions optimized
for immobilization
To find out the
optimum immobilization conditions for the enzyme, we have inquired the effect
of pH of the immobilization medium, effect of temperature of the immobilization
medium, contact time of carrier and amount of enzyme taken for immobilization.
The activity which is retained during optimization was represented in terms of
relative activity. Figure 1a shows
the effect of pH on immobilization process. The optimum pH for both S1 and S2
was obtained at pH 6. At pH 6, there is an effective surface interaction
between the carriers and the enzyme there by showing the highest activity.
Because of the lack of considerable interaction, there is a decrease in the
activity at higher pH.
By keeping other parameters constant, the
adsorption time was varied from 30-150 min. Figure 1b shows the effect of contact time after immobilization.
The relative activity of enzyme is high for S1 at 60 min and that of S2 is at
120 min. The contact time is more for S2 compared to that of S1. Enzyme
activity first increased with increase in contact time, longer an enzyme is
incubated with the carriers, greater the amount of product will be formed. As a
result, the rate of formation of product slows down as the incubation proceeds.
After that even if the contact time of enzyme increased, the activity was found
to be decreased which might be due to lower accessibility of substrate as a
result of multilayer adsorption of enzyme and also be due to the formation of a
disordered multilayer or formation of multiple bonds which deformed the active
site of the enzyme [40].
Figure 1c shows the effect
of enzyme loaded at optimum pH 6. The protein loaded and the enzyme activity (Figure 1d) increases as the
concentration increases and reaches the maximum and then decreases which shows
the formation of monolayer. After the saturation point the enzyme starts to desorb
from the surface of the support. The amount of adsorbed protein depends on the
strength of interaction between enzyme and the support and the method of
immobilization [41]. The immobilization yield, activity yield and
immobilization efficiency were calculated and the results are given in the Table 1.
Parameters
affecting the activity of enzymes
Effect of pH on the enzyme activity: We have examined the effect of pH on free and immobilized enzymes
at a range of pH 4-9 and the results shown in Figure 2a. The maximum activity of free enzyme was found to be at
pH 5.5. After immobilization, the optimum pH has been changed to pH 7. These
results also prove that the process of immobilization shields the enzyme from
alkaline and severe acidic medium [42]. After immobilization, the stability has
increased which may be due to low diffusional limitations.
Effect of
temperature on the enzyme activity: The effect of temperature on the activity of
free and immobilized enzymes (S1 and S2) was investigated at a temperature
range from 30°C to 70°C and shown in Figure
2b. The optimum temperature for starch hydrolysis was found at 50°C for
free enzyme. The immobilized enzymes S1 showed maximum activity at 50°C and S2
at 45°C. The decrease in optimum temperature might be due to the change in
conformational integrity of the enzyme structure by immobilization which
favored amylase activity below 50°C. The higher temperature may lead to the
denaturation of enzyme. The lowering of optimum temperature was reported when
polyaniline used for the immobilization of glucoamylase [43].
In order to estimate the activation energy,
Ea, an Arrhenius plot was drawn between log relative activity (%) and 1/T (K) (Figure 2c). Values obtained were
16.99, 24.02 and 27.07 for free enzyme, S1 and S2, respectively. The activation
energy was found to be decreased which might be due to the loss of
conformational integrity at the enzyme active site after immobilization.
Thermal
stability
Enzymes which are susceptible to industrial
applications should be thermally stable. In order to investigate this property
of the free and immobilized enzymes, it was incubated in buffer in solution for
60 min in a water bath over the temperature range of 30-70°C. The results (Figure 3) obtained reveals the high
heat resistance of S1 and S2 compared to free enzyme. Both free and immobilized
enzymes show high-rise in activity after 60 min of pre incubation at 30°C. The
stability was found to be decreased as the temperature rises. When the
temperature reached at 70°C, free enzyme lost almost 90% of the activity
whereas S1 and S2 lost 30-35%. With reference to adsorption, increase in
thermal stability was attributed to increase in enzyme rigidity due to strong
electrostatic interaction of carriers with free enzyme which retains the
tertiary structure of enzyme from conformational changes that might occur at
higher temperatures [44].
Thermal inactivation
and kinetics
The thermal inactivation study of free and
immobilized enzymes were conducted by pre incubation at various incubation
times at their respective optimum temperature and the corresponding relative
activity was determined. The log relative activity (%) versus time plot
illustrates the first law of thermodynamics [45]. The plot of ln kd and 1/T (Figure 4) whose slope of the linear
curve gives the deactivation energy (Ed) of both free and immobilized enzymes.
The values for Ed were 18.9, 21.6 and 27.4 for free enzyme, S1 and S2,
respectively. After immobilization, the Ed values were found to be enhanced
which makes them more stable towards denaturation and also reveals the
requirement of higher energy for thermal deactivation and makes them more
potent towards industrial applications [46,47].
Evaluation
of thermodynamic parameters
The feasibility and spontaneous nature of the
biosorption process was reflected by the thermodynamic parameters [48]. The
enthalpy change (ΔH°) at 60°C, for free enzyme was 15.52 KJ mol-1,
S1 was 18.83 KJ mol-1 and for S2, 24.63 KJ mol-1 (Table 2). The enhancement in
temperature decreases the value of ΔH° in all cases indicated that lower amount
of energy is needed for the denaturation of enzyme at high temperatures. As a
result of immobilization, greater amount of energy is required for the thermal
denaturation of enzymes at high ΔH° values [49]. The higher Gibbs free energy
change (ΔG°) of the immobilized enzymes describes that at high temperature, the
thermal unfolding is controlled by the immobilization process and also increase
in thermal stability provides resistance towards the thermal denaturation [50].
The ΔG° values at 60°C, for free enzyme, S1 and S2 were 108.27 KJ mol-1,
110.87 KJ mol-1 and 111.21 KJ mol-1, respectively and are
tabulated in Table 2. The negative
values of entropy of deactivation (ΔS°) showed the processes of ordered state
which enhances the stability of enzyme through strong intermolecular forces
[51]. Taking the magnitude values, S1 and S2 have lower values compared to free
enzyme that indicated their ordered state which were enhanced due to
immobilization.
Kinetic parameters
The two most important parameters to
characterize the kinetic properties of the enzyme are Km, the
Michelis constant and Vmax, the maximum reaction velocity from
Michelis-Menton kinetics. Km and Vmax for free enzyme, S1
and S2 were evaluated from Line weaver Burk plots (Figure 5). These parameters were studied by varying the
concentration of starch in the reaction medium. Km gives the measure
of affinity of enzyme active site for its substrate. High Km values
specify lower substrate affinity for the enzyme [52]. Km value for
the free enzyme is lower compared to S1 and S2 (Table 3). Vmax is the maximum catalytic potential of
the enzyme. There was a reduction in the value of Vmax which might
be due to immobilization. It changes the conformations of enzyme which leads to
decrease in affinity to the substrate [53,54].
Reusability
The major advantage of the immobilized
process is the repeated use of enzymes which makes them cost effective for
industrial applications. Reusability of the enzymes was evaluated in a repeated
batch process. S1 retained 55% and S2 to 54% activity after 5 cycles (Figure 6). In the 8th
cycle, the activity of S1 and S2 reached to 20% and cannot be reused further.
The decline in activity might be due to denaturation and desorption of the
enzyme molecules. Reusability studies of α-amylase have been outlined in literature
[55,56].
Storage stability
Efficiency in storage of an enzyme is an
important criterion for the applications in industry. The immobilized enzymes
cannot maintain their stability and activity for long term storage in its dried
form. So, it was then stored in buffer solution at 4°C to exhibit high activity
and stability. Immobilized enzyme S1 retained 76% and S2 retained 72% after 2
weeks. After 6 weeks, S1 reduced to 50% and S2 to 45%. The results (Figure 7) suggested that the
immobilized enzyme exhibit improved storage stability. This decrease in
activity among the immobilized enzymes is due to the time-dependent natural
loss in activity of enzymes.
CONCLUSION
ZnO-Fe3O4 (S1) and ZnO-PANI
(S2) composites were successfully prepared. The composites were confirmed by
FT-IR, XRD, TGA and SEM images. The α-amylase was successfully immobilized onto
S1 and S2 nanocomposites. The parameters of immobilization were optimized.
Immobilization brought about an increase in the Km value but a decline in the Vmax
and the changes correspond to immobilization induced conformational changes in
the enzyme. The immobilized enzymes S1 and S2, shows good thermal stability,
reusability and storage stability compared to the free enzyme. Thermodynamic
parameters ΔH°, ΔG°, ΔS° were determined whose values indicated that due to
immobilization the stability increased at high temperatures. These results
suggest that both S1 and S2 was a good support for immobilization of α-amylase
and suitable for various industrial applications.
ACKNOWLEDGEMENT
The author is
thankful to UGC for financial support.
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