Enhanced Stability of α-Amylase via
Immobilization onto Chitosan-TiO2
V.U. Bindu1 and P.V. Mohanan2*
1,2Department of Applied Chemistry, Cochin University of Science and Technology, Kochi, Kerala, India
P.V. Mohanan, Assisant Professor, Department of Applied Chemistry, Cochin University of Science and Technology, Kochi 22, Kerala, India, Tel.: 9447184874; E-mail:
Received: May 05, 2017; Accepted: June 15, 2017; Published: July 10, 2017
P.V. Mohanan, V.U. Bindu (2017) Enhanced Stability of α-Amylase via Immobilization onto Chitosan-TiO2
Nanocomposite. Nanosci Technol. 4(2): 1-9. DOI: 10.15226/2374-8141/4/2/00146
The purpose of the present study is to enhance the stability
properties of α-amylase by immobilization onto chitosan-TiO2
nanocomposite. The immobilization of enzyme onto the nanocomposite
was done by adsorption method. α-amylase is an industrial enzyme
which is widely used in food, detergent, paper and textile industry.
The significance of the study is to allow the enzyme for industrial
usage by improving the stability properties. In order to determine
the optimum conditions of immobilization, various parameters such
as pH, incubation time and enzyme concentration were investigated.
The immobilized enzyme exhibited better immobilization yield of
80%. Compared to free enzyme, the immobilized enzyme showed
broader pH tolerance and temperature stability. Thermal stability
study showed that there is considerable enhancement in stability
of immobilized enzyme compared to free enzyme. The kinetic
parameters, Km and Vmax were determined from Line weaver-Burk
plot. The Km value for starch hydrolysis is found to be higher for
immobilized enzyme than that of free enzyme and the Vmax value
of free enzyme decreased moderately after immobilization. Storage
stability study demonstrated that the immobilized enzyme preserved
about 64.5% of its initial activity after six months of incubation. The
frequent use experiment exhibited that the immobilized enzyme
retained about 63% of its initial activity even after 10 cycles of reuses.
Keywords: Nanocomposite; Chitosan; TiO2; Adsorption
Immobilization; Maltose; Catalytic Activity;
Enzymes are very efficient catalysts for biochemical
reactions by providing an alternative reaction pathway of lower
activation energy. Enzymes have worthwhile industrial and
medical applications. The fermenting of wine, curdling of cheese,
leavening of bread and brewing of beer have been practiced from
earliest times, but not known these reactions to be as a result of
catalytic activity of enzymes until the 19th century. Since then,
enzymes have assumed an increasing importance in industrial
processes that involve organic chemical reactions. Because of the
low stability, high cost and high selectivity towards the reaction
conditions, they are limited to industrial applications [1-3]. In
order to facilitate their usage in industrial applications several
developments have been done in the field of enzyme technology
during the last two decades . Immobilization of enzymes
on solid supports has become most familiar technique after
Nelson and Griffin have done the immobilization of invertase
onto charcoal hydrolyses sucrose via adsorption in 1961 .
Immobilization provides many advantages, such as continuous
operations in enzyme reactor, enhanced stability, reusability and
easy separation from reaction mixture . The introduction of
immobilized catalysts has greatly improved both the technical
performance of the industrial processes and their economy.
The methods used in immobilization are adsorption,
covalent binding, and entrapment within a porous matrix,
microencapsulation and aggregation [7,8]. Among these
adsorption is the most important, easily operable method under
moderate conditions and so it is the most economical way for
enzyme immobilization [9,10]. In adsorption immobilization
there is physical interactions generated between the carrier and
enzyme that include van der Waals forces, ionic interactions and
hydrogen bonding . As the binding forces are too weak, it does
not change the native structure of the enzyme and so prevents
the distortion of active sites of the enzyme. Hence as a result of
adsorption immobilization enzyme will retain its activity .
Another factor considered to be for enzyme immobilization
is the selection of carrier [13,14]. They are classified as inorganic
supports, synthetic polymers and natural macromolecules .
Polymeric carriers have the advantageous of good mechanical
properties, ease of preparation, and applicability to introduce
bio-friendly components for improving biocompatibility [16,17].
In the recent past, nanosized materials have been widely
employed for enzyme immobilization. Due to the large surface
area, they provide superior loading capacity and low mass
transfer resistance [18,19]. The usage of metal oxides for enzyme
immobilization is now attracting too much attention. There is a
report on successful enzyme immobilization on titanium (IV),
iron (III), zirconium (IV), vanadium (III) and tin (IV) oxides .
Among this titanium (IV) oxide has been attracting too much
attention because of the properties such as semiconductivity,
biocompatibility, cost-effectivity and stability [21,22].
Chitosan, poly [β-(1→4)-2-amino-2-deoxy-D-glucose], is
the biopolymer which contains amino and hydroxyl functional
groups. It has many important chemical and biological properties
such as biocompatibility, biodegradability bioactivity, nontoxicity
and antibacterial properties [23,24]. The thermal, mechanical,
chemical and physical properties of the polymer matrixes can be
improved by addition of certain amount of inorganic oxides into
polymer matrix . In our study we have selected TiO2 to form
the nanocomposite with chitosan matrix so that the properties of
the polymer can be improved. This nanocomposite is employed
as the carrier for enzyme immobilization and seems to improve
the performance of immobilized enzyme system.
The enzyme, α-amylase was applied as a model enzyme for
immobilization. α-amylase is one of the largest selling enzymes
for a wide variety of industrial applications. They are endo acting
glycoside hydrolases which hydrolyzes α-1,4-glycosidic bond
present in polysaccharides to produce glucose, maltose and
maltotriose units . These low molecular weight products are
widely applied in the food, paper, textile, baking and brewing
industries . There are various types of supports have been
previously investigated for the effective immobilization of
The current work deals with the immobilization of α-amylase
onto chitosan- TiO2 nanocomposite. The nanocomposite was
synthesized by sol-gel method and characterized by IR, TGA, XRD
and SEM analysis. We have done the immobilization of α-amylase
onto the nanocomposite by adsorption. The immobilization
parameters such as pH, contact time and amount of enzyme
required for immobilization were optimized so as to acquire
maximum immobilization yield and efficiency. The properties
of the immobilized enzyme systems such as pH stability,
temperature stability, thermal stability, reusability and kinetic
parameters were compared with soluble enzyme. A novel
biochemical method based on the detection of maltose using 3,
5-Dinitrosalicylic acid (DNS) method was employed to check the
activity of the immobilized α-amylase. The results revealed that
chitosan-magnetite nanocomposite is the promising carrier for
Diastase α-amylase (1, 4 α-D-glucanglucanohydrolase, EC
220.127.116.11) was purchased from Himedia Laboratories Pvt Ltd,
Mumbai. Soluble Starch (potato) was acquired from S.D. Fine-
Chem. Ltd, Mumbai. Chitosan was purchased from Meron marine
chemicals, Cochin. All other chemicals were used analytical grade.
Amylase activity assay
Starch was selected as the substrate for determination
of amylase activity. The reaction mixture consists of 1 mL of
free α-amylase (0.5 mg/mL) and 1mL of 1% starch solution in
desired buffer. The reaction system was incubated in a water
bath with constant shaking at 30°C for exactly 15 min. The
reaction was stopped by adding 1 mL 3, 5-dinitrosalicylic acid
reagent. Incubation was performed in a boiling water bath for
5 min and cooling the reaction tubes to room temperature. The
amount of reduced sugar (maltose) produced was determined
spectrophotometrically at 540 nm . An enzyme activity unit
(EU) was defined as the amount of enzyme liberating 1 μmol
maltose per minute under the assay conditions.
Total protein assay
The amount of immobilized α-amylase was measured by
Lowry’s method . After the immobilization procedure the
supernatant and washings were subjected to protein estimation
using Folin Ciocaltaue’s reagent by measuring the absorption
at 660nm in a Thermo Scientific Evolution 201 UV-Visible
Synthesis of nanocomposite
Synthesis of TiO2 Nanoparticles
TiO2 nanoparticles were synthesized via sol-gel method on
the basis of already reported work . In the present study we
have used methanol as solvent for synthesis and H2SO4 as the
catalyst. Titanium tetraisopropoxide was dissolved in methanol
keeping the molar ratio, TTIP: MeOH=1:3. Then distilled water
was added drop wise into the solution in terms of a molar ratio
of TTIP: H2O = 1:4. H2SO4 was added to maintain acidic pH for
restrain the hydrolysis process of the solution. The solution
is then subjected to magnetic stirring for 40 minutes at room
temperature. After that the gel formed is dried under 50°C for 1.5
hour to evaporate water and organic material to the maximum
extent. Then it is undergone calcinations at 400°C for 2 h to obtain
desired TiO2 nanoparticles.
Synthesis of chitosan- TiO2 (CST) nanocomposite
About 1 g nano TiO2 powder was dissolved in 1% chitosan
solution in acetic acid (1% v/v) by sonication. The solution
stirred continuously until clear sol was obtained and after that
1M NaOH solution was added drop wise till the pH of solution
became 10. The precipitate formed was heated at 80°C for 5 hour.
Then the precipitate was filtered, washed with excess of water
and dried in a vacuum oven at 60°C overnight .
Preparation of immobilized enzyme system
Immobilized enzyme was prepared by stirring 1g
nanocomposite in enzyme solution in buffer at room temperature.
The immobilization parameters optimized were pH (5-9), time
(30-150 min) and the enzyme concentration (2 to 20 mg enzyme
support). The biocatalyst then filtered and washed with the
same buffer solution. The supernatant and washings were
subjected to protein estimation and the prepared immobilized
enzyme was kept in a refrigerator at 4°C for further studies.
Immobilization yield (IY) was calculated by Eq. (1)
is the concentration of protein introduced for
immobilization and C2
is concentration of protein present in the
supernatant after immobilization.
The activity yield (AY) was determined by Eq. (2)
Immobilization efficiency was calculated using Eq. (3).
Characterization of CST nanocomposite
The nanocomposite was characterized by using wellknown
physico-chemical methods. Surface groups and chemical
structure were determined by FT-IR spectrometry using JASCO
FT/IR-4100. Surface morphology of the nanocomposite was
investigated by using the JEOL Model JSM –6390LV scanning
electron microscope. Bruker AXS D8 Advance is used for XRD
analysis. Thermal behavior of the nanocomposite was evaluated
by using Perkin Elmer, Diamond TG/DTA.
Characterization of immobilized enzyme system
Optimum Temperature and Optimum pH
The optimum pH for maximum activity of free and immobilized
enzyme was assayed by incubating the enzyme with starch over a
pH range 5- 9 at 30°C. The temperature for maximum activity was
also assayed by varying the temperature from 30- 60°C.
Thermal stability of free and immobilized enzyme was carried
out by subjecting them to various temperatures ranging from 30-
70°C for 1hour in a water bath. After 1hour of pre-incubation
both free and immobilized enzyme in buffer cooled to optimum
temperature and the enzymatic reaction was performed for a fixed
time interval by adding definite amount of 1% starch solution to
each reaction medium. Thermal inactivation curves with respect
to incubation time for both the free and immobilized enzyme
were obtained by pre-incubating them at their optimum pH and
temperature. After definite time intervals, the enzymatic reaction
was carried out and then the enzyme activity was calculated.
The reusability of the immobilized enzyme was determined
by repeated batch experiments maintaining a couple hours in
each cycle. The residual activity of immobilized enzymes at its
optimum conditions was measured at fixed time intervals. At the
end of each cycle the immobilized enzyme was removed, washed
with buffer solution and the reaction medium was changed with
fresh substrate solution. The assay was carried out repeatedly 10
cycles under standard assay conditions.
The storage stability of immobilized enzyme was studied by
evaluating its activity after being stored at 4°C in buffer solution
for 6 months. The assay was conducted at regular intervals of
time. The activity was compared with initial activity and was
represented as percentage relative activity.
To determine the kinetic parameters, Michaelis constant (Km)
and maximum rate (Vmax) for free and immobilized enzyme,
the enzymatic assays were carried out by varying the substrate
concentrations ranging from 0.2 to 1.0 mg/mL at optimum
temperature and pH. The parameters were calculated from the
Lineweaver - Burk plot.
Results and Discussion
The IR spectra of chitosan nanoparticle, TiO2 nanoparticle
and chitosan-TiO2 (CST) nanocomposite were shown in
the supplementary data (Appendix A). In case of chitosan
nanoparticle and CST nanocomposite, the absorption band in the
region of 3750-3000 cm−1 was attributed to the combined peaks
of amino (-NH2) and hydroxyl (-OH) groups stretching vibration.
This band become much broaden in case of CST nanocomposite
which may arise from the hydroxyl group that belongs to linear
polymeric chain of TiO(OH)2. The IR spectra of TiO2 nanoparticle
showed the peaks at 3400 and 1620 cm−1due to the stretching
and bending vibration of the -OH group. Also the band at 520 cm-1
showed stretching vibration of Ti-O and at 1450 cm-1 showed
stretching vibrations of Ti-O-Ti. The Ti–O band in the range of
400–700cm-1 can be seen in case of CST nanocomposite which
was ascribed to the composite formation of TiO2 with chitosan.
The peaks related to CH2 asymmetric vibration could be seen at
2925, 2870, 1430, 1320 and 1250 cm−1 . The absorption band
at 1074 cm−1 represented the C-O stretching vibration of primary
alcoholic group in chitosan. The peak observed at 1585 cm−1
corresponding to amide II and the two peaks at 1655 and 1320
cm−1 representing the amides I and III . Increased intensity
of amide II peak in the spectrum of CST nanocomposite was
observed which may be resulted from the interaction of Ti+4 with
the amide group of chitosan.
SEM analysis was performed in order to observe surface
topography and morphology. From the SEM micrographs of TiO2
nanoparticle and CST nanocomposite (supplementary data-
Appendix B), it is clear that the nanocomposite has aggregated
particle structure. The image reveals the surface structure of
nanocomposite with small-flake like surface presented separately
in the exterior morphology. This aggregated and agglomerated
morphology of the CST nanocomposite confirms the dispersion
of TiO2 nanoparticles into the chitosan matrix and also the
The TGA analysis has done under nitrogen atmosphere at a
heating rate of 10°C/min in the temperature range of 30–730°C.
The TG curves of chitosan nanoparticles and CST nanocomposite
(supplementary data- Appendix C) exhibit almost similar
thermal degradation trend. Two stages of weight loss can be seen
for both the samples. The first stage of weight loss, from room
temperature to 200°C could be as a result of the evaporation of
adsorbed water. Nearly 10% weight loss was observed in the first
step. The major weight loss was observed in second step between
250 and 500°C. It was reported that this weight loss was caused
by decomposition of the polysaccharide and loss of (O–H) groups
of TiO2 dispersed in polymer matrices . After 500°C there is
no significant weight loss was observed. At the end of the analysis,
about 60% of weight loss for chitosan nanoparticle and 50% of
weight loss for CST nanocomposite were observed at 700°C.
XRD diffraction patterns of TiO2 nanoparticle and CST
nanocomposite are shown (supplementary data- Appendix D).
Diffraction peaks in XRD pattern of nanoTiO2 are sharper and
stronger at 26.9°, 36.5°, 39°, 44°, 54° and 55.5°. All the diffraction
peaks are also seen in case of CST nanocomposite. This revealed
that the CST nanocomposite is successfully formed. The TiO2 has
the crystal structure assigned to anatase-type titanium (JCPDS
21-1272). Anatase structure of the TiO2 crystals was preserved
even after the formation of nanocomposite. The peaks at 2θ= 5°
and 25° were characteristic peaks corresponding to chitosan.
The average crystalline size can be quantitatively evaluated
from the XRD data using the Debye–Scherrer equation which
gives a relationship between peak broadening in XRD and particle
size: d = (kλ/βcos θ).
Where d is the particles size, k is the Debye–Scherrer
constant (0.89), λ is the X-ray wavelength (0.15406 nm) and β
is the full width at half maximum, θ is the Bragg angle. The most
intense diffraction peak at 26.9° attributed to anatase phase of
TiO2 can be seen for TiO2 nanoparticle and CST nanocomposite.
According to the Debye-Scherrer equation, the crystalline size of
the TiO2 nanoparticle is 26.75nm and that of CST nanocomposite
is 28.32nm. This data indicates that the synthesis of CST
nanocomposite did not much affect the size of TiO2 nanoparticle
Optimization of immobilization parameters
The parameters affecting immobilization of enzyme such
as pH of the immobilization medium, incubation time and the
amount of enzyme were optimized. The retained activity of the
immobilized enzyme was designated in terms of relative activity.
Figure 1a exhibited the effect of pH on immobilization. The
optimum pH for the immobilized enzyme, CSTE was at pH 6. The
isoelectric point of α-amylase is around 4.6 and the amino group
in chitosan has a pKa value of about 6.5. At the optimum pH 6
there is significant electrostatic interaction between the support
and the enzyme since chitosan having net positive charge and the
enzyme molecule having net negative charge. The decrease in
activity above and below this pH may be due to lower adsorption
which occurred as a result of unfavorable charge distribution on
enzyme and the support.
Figure 1a: Effect of pH of immobilization medium on the relative activity
of immobilized α-amylase.
Figure 1b showed the effect of adsorption time on the
relative activity of the immobilized enzyme. The adsorption
time was varied in the range between 30 and 150 min. and the
other parameters kept constant. The enzyme activity increased
up to 90 min. and after this point, a decrease in the activity of
immobilized enzyme was observed. The decrease in activity
could be as a result of multilayer adsorption of enzyme on the
surface of support which deformed the active site of the enzyme.
Figure 1b: Effect of contact time on immobilized enzyme activity.
The amount of enzyme adsorbed and the activity of
immobilized enzyme were determined. As the enzyme
concentration increased, the amount of enzyme adsorbed on the
support increased and then reached a saturation point. It was
depicted in the figure 1c. The effect of enzyme concentration
on activity also exhibited in the figure 1d and the decrease in
the activity after the saturation point is due to conformational
changes of the enzyme or the steric hindrance caused by the
support at the active site . The immobilization yield, activity
yield and immobilization efficiency were calculated and the
results are given in the table1.
Figure 1c: Effect of initial protein amount on protein loading.
Figure 1d: Effect of initial protein concentration on immobilized enzyme
Table 1: Immobilization efficiency of CST nanocomposite
Initial protein (mg)
Immobilized protein mg/g
Initial activity (EU)
Immobilized enzyme activity (EU)
Parameters affecting enzyme activity
Effect of pH on enzyme activity
We have investigated the effect of pH on the activities of
free and immobilized α-amylase and the results are shown in
the figure 2a. The relative activity of immobilized enzyme, CSTE
was higher than that of free enzyme. Chitosan did not show any
acidic shift due to its solubility in lower pH range. In the present
study, interaction of Ti+4 with the –NH2 group of chitosan caused
the decrease of solubility of chitosan matrix in acidic media.
The positively charged TiO2 surface was keeping away the H+
ions from the microenvironment of enzyme and the activity of
immobilized enzyme, CSTE could be protected in lower pH range
. It was found that the free enzyme showed its maximum
activity in the range of pH 5–5.5. The optimum pH of immobilized
enzyme, CSTE was at pH 6, representing 0.5 units increase when
compared to free enzyme. This result agreed to Egwim et al. 
reported that the optimum pH of free lipase was 6.5, while that of
immobilized lipase was at pH 7 (a 0.5 unit increase). The shift of
optimum pH of the enzyme to alkaline region could be explained
by poly cationic nature of chitosan. They attract more OH¯ ions
making the microenvironment of enzyme more alkaline than the
bulk solution and leads to a shift in pH towards basic region.
Figure 2a: Effect of pH on the activity of free enzyme and CSTE.
Effect of temperature on the enzyme activity
The temperature effect on activity of free and immobilized
enzyme was shown in the figure 2b. It was found that the
relative activity of immobilized enzyme was higher than that of
free enzyme. This result indicate that adsorption of enzyme on
nanocomposite limited the movement of enzyme and increased
the conformational integrity. The addition of TiO2 nanoparticles
into the chitosan matrix increased the temperature characteristics
and decreased the diffusional restriction of product and substrate
at higher temperature. This led to the increased activity of the
immobilized enzyme system. The free α- amylase exhibited the
optimum temperature of 50°C and this value shifted to 35°C for
the immobilized enzyme, CSTE. The decrease of the optimum
temperature may arise from the change of the conformational
integrity of the enzyme structure through the interactions with
the nanocomposite which resulted in an alteration of enzyme
substrate affinity. The decrease of optimum temperature of α-
amylase was reported when immobilized on silica gel .
Figure 2b: Effect of temperature on activity of free enzyme and CSTE.
The immobilized enzyme with higher thermal stability is
beneficial for different industrial applications. This property of
free and immobilized enzyme was investigated in the range of 30–
70⁰C to assess the potential advantage and the results are shown
in figure 3a. After 1 hr pre-incubation at 50°C, the free enzyme
lost almost 60% of its initial activity and that of immobilized
enzyme was 15%. At 70°C the free enzyme retained 10% of its
initial activity, whereas immobilized enzyme retained above 70%.
The immobilized enzyme, CSTE exhibited better thermal stability
since its activity decreased slower rate as the temperature
increased to higher region. It preserves the tertiary structure
of the enzyme and protects the enzyme from conformational
changes of the active center caused at higher temperatures .
Figure 3a: Thermal stability of free enzyme and CSTE pre-incubated for
1h at different temperatures.
The thermal stability of free and immobilized α-amylase
with respect to pre-incubation time was studied and the result
was depicted in figure 3b. The figure showed that immobilized
α-amylase retained more than 90% activity even after 120 min of
pre-incubation at optimum temperature. This indicates that the
relative activity of the immobilized enzyme was decreased slowly
compared to that of free enzyme. The slow decrease in activity
of immobilized enzyme was due to its restricted mobility which
preserved the three dimensional conformation of enzyme from
denaturation. The increase in thermal stability of α-amylase was
observed when immobilized on nano ZnO .
Figure 3b: Effect of pre-incubation time on activity of free enzyme and
CSTE at their optimum temperature.
The kinetic parameters, Michaelis–Menten constant (Km)
and maximum reaction velocity (Vmax), for free and immobilized
α-amylase were determined using Lineweaver–Burk plot. These
parameters were estimated using starch as the substrate. Vmax
indicates the intrinsic characteristics of the enzyme which gives
the maximum reaction velocity and Km is a measure of the
substrate’s affinity for the enzyme. Km can change due to the
conformational changes of the enzyme molecule. Higher values
of Km indicate lower substrate affinity for the enzyme  as the
shown in the table 2, the Km value for the immobilized enzyme
(0.58 mg/ mL) was higher than that of the free enzyme (0.45mg/
Table 2: Kinetic parameters of the free and immobilized α-amylase.
0.58 ± 0.03
Vmax(μmol mg-1 min-1)
The increase in Km value for immobilized enzyme was due
to its conformational changes, resulting in a lower affinity for
the substrate compared to the free enzyme. The increase in Km
value for immobilized α-amylase was also reported by M.A. Abdel
Naby et al. . Vmax values were found 34.48 and 24.39 μmol
mg-1 min-1 for free and immobilized enzyme respectively. The
decrease in Vmax value represents that the action of the enzyme
immobilization reduced the enzyme activity . This reduction
may be caused due to the lower accessibility of the substrate to
the active site of immobilized enzyme. The structural changes in
the enzyme as a result of immobilization may also lead to change
in affinity to its substrate .
Effect of frequent use on activity of immobilized
The frequent use of immobilized enzyme is the key factor
for its cost effective employability in industrial applications.
We evaluated the reusability of the immobilized enzyme, CSTE
keeping a couple hours in each cycle. CSTE can be reused either
by centrifugation or filtration and it retained nearly 63% of initial
activity after ten reuses (Figure 4a). The subsequent decrease in
activity after every cycle could be due to desorption, denaturation
or conformational changes of the enzyme molecule upon uses.
The reusability of immobilized α-amylase was investigated in
several reports [47-49].
Figure 4a: Reusability of CSTE
Effect of storage time on activity of immobilized
The free enzyme in solution is not stable during storage
and the activity gradually reduced. The storage stability of
immobilized enzyme is the important factor in order to ensure
its long shelf life. In our study, immobilized enzyme was stored
in buffer at 4°C and the activities were measured after definite
periods of storage. The immobilized enzyme retained 64.5%
of its activity after 6 months and the results are depicted in the
figure 4b. The study showed that immobilized enzyme gained
more stable character than the free one. The ionic interactions
generated between enzyme and the carrier imparted a higher
conformational stability to the immobilized enzyme. Many
reports are there showing the storage stability of immobilized
Figure 4b: Storage stability of CSTE
In this work, α-amylase was successfully immobilized onto
chitosan-TiO2 nanocomposite by adsorption. The method used for
immobilization was simple and economical. We have optimized
the immobilization parameters such as pH, incubation time and
enzyme concentration. We improved the stability properties of
α-amylase by using the immobilized enzyme system. Enzyme
assays demonstrated that substantial improvement in thermal,
pH and storage stability. The immobilized enzyme showed
excellent reuse potential maintaining high levels of activity for
repeated use without much affecting the integrity of the catalytic
sites of the enzyme. The kinetic study showed that the substrate
affinity for the immobilized enzyme in enzymatic reaction is less
and exhibits higher Km and lower Vmax compared to the free
enzyme, indicate decreased activity. The results represented the
potential applicability of the immobilized enzyme for industrial
applications, which could be used for the efficient starch
The author, V.U. Bindu is thankful to UGC for providing the
senior research fellowship.
- K. Hernandez, R. Fernandez-Lafuente. Control of protein immobilization: Coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance. Enzyme and Microbial Technology. 2011;48(2):107-122. doi: 10.1016/j.enzmictec.2010.10.003
- L. Cao. Carrier-bound immobilized enzymes: principles, applications and design. Wiley-VCH, Weinheim. 2005. DOI: 10.1002/3527607668
- G. Pandey, D.M. Munguambe, M. Tharmavaram, D. Rawtani, Y.K. Agrawal. Halloysite nanotubes - An efficient ‘nano-support’ for the immobilization of α-amylase. Applied Clay Science. 2017;136:184-191.
- I.A. Veesar, I.B. Solangi, S. Memon. Immobilization of α-amylase onto a calixarene derivative: Evaluation of its enzymatic activity. Bioorganic Chemistry. 2015;60:58-63. doi: 10.1016/j.bioorg.2015.04.007
- J.M. Nelson, E.G. Griffin. ADSORPTION OF INVERTASE. Journal of the American Chemical Society. 1916;38(5):1109-1115. DOI: 10.1021/ja02262a018
- E. Cappannella, I. Benucci, C. Lombardelli, K. Liburdi, T. Bavaro, M. Esti. Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: Bench-scale fluidized-bed reactor study. Food Chemistry. 2016;210:49-55. doi: 10.1016/j.foodchem.2016.04.089
- Y.I. Doğaç, İ. Deveci, M. Teke, B. Mercimek. TiO 2 beads and TiO 2-chitosan beads for urease immobilization. Mater Sci Eng C Mater Biol Appl. 2014;42:429-435. doi: 10.1016/j.msec.2014.05.058
- E. Biró, Á.S. Németh, C. Sisak, T. Feczkó, J. Gyenis. Preparation of chitosan particles suitable for enzyme immobilization. Journal of Biochemical and Biophysical Methods. 2008;70(6):1240-1246.
- I. Deveci, Y.I. Doğaç, M. Teke, B. Mercimek, Synthesis and characterization of chitosan/TiO2 composite beads for improving stability of porcine pancreatic lipase. Applied Biochemistry and Biotechnology. 2015;175(2):1052-1068. doi: 10.1007/s12010-014-1321-4
- C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology. 2007;40(6):1451-1463.
- S. Datta, L.R. Christena, Y.R.S. Rajaram, Enzyme immobilization: an overview on techniques and support materials. 3Biotech. 2013;3(1):1-9.
- T. Jesionowski, J. Zdarta, B. Krajewska. Enzyme immobilization by adsorption: a review. Adsorption. 2014;20(5):801-821.
- B. Krajewska. Application of chitin- and chitosan-based materials for enzyme immobilizations: a review. Enzyme and Microbial Technology. 2004;35(2–3):126-139.
- R.A. Sheldon. Enzyme immobilization: the quest for optimum performance. Advanced Synthesis & Catalysis. 2007;349(8‐9):1289-1307.
- P. Ye, Z.-K. Xu, A.-F. Che, J. Wu, P. Seta. Chitosan-tethered poly(acrylonitrile-co-maleic acid) hollow fiber membrane for lipase immobilization. Biomaterials. 2005;26(32):6394-6403.
- O. Türünç, M.V. Kahraman, Z.S. Akdemir, N. Kayaman-Apohan, A. Güngör. Immobilization of α-amylase onto cyclic carbonate bearing hybrid material. Food Chemistry. 2009;112(4):992-997.
- M.G. Sankalia, R.C. Mashru, J.M. Sankalia, V.B. Sutariya. Reversed chitosan–alginate polyelectrolyte complex for stability improvement of alpha-amylase: optimization and physicochemical characterization. European journal of pharmaceutics and biopharmaceutics. 2006;65(2):215-232.
- S.-W. Choi, H.-Y. Kwon, W.-S. Kim, J.-H. Kim, Thermodynamic parameters on poly(d,l-lactide-co-glycolide) particle size in emulsification–diffusion process. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2002;201(1–3):283-289.
- Y. Jiang, C. Guo, H. Xia, I. Mahmood, C. Liu, H. Liu. Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification. Journal of Molecular Catalysis B: Enzymatic. 2009 58(1):103-109.
- J.F. Kennedy, J.M. Cabral, M.R. Kosseva, M. Paterson. Transition metal methods for immobilization of enzymes and cells. Immobilization of enzymes and cells. 1997;1:345-359.
- Y. Kurokawa, T. Sano, H. Ohta, Y. Nakagawa. Immobilization of enzyme onto cellulose–titanium oxide composite fiber. Biotechnology and Bioengineering. 1993;42(3):394-397.
- K.S. Huang, A.M. Grumezescu, C.Y. Chang, C.H. Yang, C.Y. Wang. Immobilization and stabilization of TiO2 nanoparticles in alkaline-solidificated chitosan spheres without cross linking agent. International Journal of Latest Research in Science and Technology. 2014;3(2):174-178.
- S. Ahmed, S. Ikram. Chitosan & its derivatives: a review in recent innovations. International Journal of Pharmaceutical Sciences and Research. 2015;6(1):14-30.
- M. Periayah, A. Halim, A. Saad. Chitosan: A Promising Marine Polysaccharide for Biomedical Research. Pharmacognosy reviews. 2016;10(19):39-42.
- Tulden Kalburcu, Nalan Tuzmen, Sinan Akgöl, Adil Denizli. Immobilized metal ion affinity nanospheres for\ alpha-amylase immobilization. Turkish Journal of Chemistry. 2014;38(1):28-40.
- B. Lonsane, M. Ramesh. Production of bacterial thermostable α-amylase by solid-state fermentation: a potential tool for achieving economy in enzyme production and starch hydrolysis. Advances in applied microbiology. 1990;35:1-56.
- P. Tripathi, A. Kumari, P. Rath, A.M. Kayastha, Immobilization of α-amylase from mung beans (Vigna radiata) on Amberlite MB 150 and chitosan beads: A comparative study. Journal of Molecular Catalysis B: Enzymatic. 2007 ;49(1–4) 69-74.
- M.V. Kahraman, G. Bayramoğlu, N. Kayaman-Apohan, A. Güngör. α-Amylase immobilization on functionalized glass beads by covalent attachment. Food Chemistry. 2007;104(4):1385-1392.
- M.G. Bellino, A.E. Regazzoni, G.J.A.A. Soler-Illia. Amylase-Functionalized Mesoporous Silica Thin Films as Robust Biocatalyst Platforms. ACS Applied Materials & Interfaces. 2010;2(2):360-365. doi: 10.1021/am900645b.
- N. Jaiswal, O. Prakash, M. Talat, S.H. Hasan, R.K. Pandey. α-Amylase immobilization on gelatin: Optimization of process variables. Journal of Genetic Engineering and Biotechnology. 2012;10(1):161-167.
- R.S.S. Kumar, K.S. Vishwanath, S.A. Singh, A.G.A. Rao. Entrapment of α-amylase in alginate beads: Single step protocol for purification and thermal stabilization. Process Biochemistry. 2006;41(11):2282-2288.
- A.M. Pascoal, S. Mitidieri, K.F. Fernandes. Immobilisation of α-amylase from <em>Aspergillus niger</em> onto polyaniline. Food and Bioproducts Processing. 2011;89(4):300-306.
- O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall. Protein measurement with the Folin phenol reagent. J biol Chem 1951;193(1):265-275.
- K. Thangavelu, R. Annamalai, D. Arulnandhi, preparation and characterization of nanosized TiO2 powder by sol-gel precipitation route. International Journal of Emerging Technology and Advanced Engineering. 2013;3(1).
- Y. Haldorai, J.J. Shim. Novel chitosan‐TiO2 nanohybrid: Preparation, characterization, antibacterial, and photocatalytic properties. Polymer Composites. 2014;35(2):327-333.
- P. Norranattrakul, K. Siralertmukul, R. Nuisin. Fabrication of chitosan/titanium dioxide composites film for the photocatalytic degradation of dye. Journal of Metals. Materials and Minerals. (2013);23(2).
- Y. Haldorai, J.J. Shim. Novel chitosan‐TiO2 nanohybrid: Preparation, characterization, antibacterial, and photocatalytic properties. Polymer Composites. 2014;35(2):327-333.
- P.C. Ashly, M.J. Joseph, P.V. Mohanan. Activity of diastase α-amylase immobilized on polyanilines (PANIs). Food Chemistry. 2011;127(4):1808-1813.
- R. Jayakumar, R. Ramachandran, V.V. Divyarani, K.P. Chennazhi, H. Tamura, S.V. Nair. Fabrication of chitin–chitosan/nano TiO2-composite scaffolds for tissue engineering applications. International Journal of Biological Macromolecules. 2011;48(2):336-344. doi: 10.1016/j.ijbiomac.2010.12.010
- Evans Egwim, A.A. Adesina, Oluwafemi Oyewole, I.N. Okoliegbe. Optimization of Lipase Immobilized on Chitosan Beads for Biodiesel Production. Global Research Journal of Microbiology. 2012;2(2):103–112.
- M.D.T.V.G. PREDA. Influence of immobilization on biocatalytic activity of a microbial (Bacillus amyloliquefaciens) alpha-amylase. Romanian Biotechnological Letters. 2012;17(3):7253.
- S.B. Navya Antony, P.V. Mohanan. Immobilization of diastase a-amylase on nano zinc oxide. Food Chemistry. 2016;211:624-630. doi: 10.1016/j.foodchem.2016.05.049
- M.K. Mohammad Kalantari, Fatemeh Tabandeh and Ayyoob Arpanaei, Lipase immobilisation on magnetic silica nanocomposite particles: effects of the silica structure on properties of the immobilised enzyme. Journal of Materials Chemistry. 2012;22:8385-8393.
- M.A. Abdel-Naby, A.M. Hashem, M.A. Esawy, A.F. Abdel-Fattah. Immobilization of Bacillus s ubtilis of its enzymatic properties. Microbiol. Res. 1998;153:1-000.
- M.S.K. Seyyedeh Leila Hosseinipour, R.S. Hamed Hamishehkar. Enhanced stability and catalytic activity of immobilized a-amylase on modified Fe3O4 nanoparticles for potential application in food industries. Journal of Nanoparticle Research. 2015;17:382.
- R.-S.J. Min-Yun Chang. Activities, stabilities, and reaction kinetics of three free and chitosan–clay composite immobilized enzymes. Enzyme and Microbial Technology. 2005;36:75–82.
- V. Swarnalatha, R.A. Esther, R. Dhamodharan. Immobilization of α-amylase on gum acacia stabilized magnetite nanoparticles, an easily recoverable and reusable support. Journal of Molecular Catalysis B: Enzymatic. 2013;96:6-13.
- N. Hasirci, S. Aksoy, H. Tumturk. Activation of poly (dimer acid-co-alkyl polyamine) particles for covalent immobilization of α-amylase. Reactive and Functional Polymers. 2006;66(12):1546-1551.
- P. Ashly, M. Joseph, P. Mohanan. Activity of diastase α-amylase immobilized on polyanilines (PANIs). Food Chemistry. 2011;127(4):1808-1813.
- M. Talebi, S. Vaezifar, F. Jafary, M. Fazilati, S. Motamedi. Stability Improvement of Immobilized a-amylase using Nano Pore Zeolite. Iranian Journal of Biotechnology. 2016;14(1):33-38.
- S. Aksoy, H. Tumturk, N. Hasirci. Stability of α-amylase immobilized on poly (methyl methacrylate-acrylic acid) microspheres. Journal of Biotechnology. 1998;60(1-2):37-46.
- E. Çakmakçı, A.B. Çiğil, Ö. Danış, S. Demir, M.V. Kahraman. Immobilization of alpha‐amylase on aminated polyimide membrane: Preparation, characterization, and properties. Starch‐Stärke. 2014;66(3-4):274-280.