Application of Immobilized Ipomoea batata β Amylase in the Saccharification of Starch
Journal of Applied Biological Sciences
Application of Immobilized Ipomoea batata ? Amylase in the Saccharification of Starch
0 Mohammad Jahir KHAN Fahim Halim KHAN Qayyum HUSAIN
1 Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University , Aligarh (202002) , INDIA
Present study demonstrates the immobilization of acetone fractionated Ipomoea batata (sweet potato) ? amylase on an inorganic support, Celite-545 by simple adsorption mechanism. The adsorbed enzyme exhibited an activity yield of 244 U g-1 of the matrix with effectiveness factor '?' 0.83. Interaction between Celite-545 and enzyme was confirmed by fourier transform infrared spectroscopy and atomic force microscopy. The binding efficiency of enzyme to the support was analyzed by eluting it with 1.0 M NaCl. Both soluble and immobilized ? amylase exhibited same pH optima while temperature optimum of immobilized enzyme was shifted from 50oC to 60oC. The immobilized ? amylase preparation was superior to the free enzyme in hydrolyzing starch in a batch process: it hydrolyzed starch to 88% and 96% at 40oC and 50oC, respectively whereas soluble enzyme hydrolyzed only 83% and 80% of starch under similar experimental conditions. The immobilized ? amylase retained 84% of its original activity after 30 days storage at 4oC, while the soluble enzyme showed only 41% of the initial activity under identical conditions. Immobilized ? amylase retained 79% activity even after its 7th repeated use.
? Amylase (E.C. 22.214.171.124) is an exoamylase which is widely
distributed in higher plants and microorganisms. Among plant
sources sweet potato is thought to be a promising source
of ? amylase with fair thermostability . It catalyzes the
hydrolysis of ?-1, 4 glycosidic linkages at non reducing end
of starch and related carbohydrates . The biotechnological
application of ? amylase includes the production of maltose
and high maltose syrups. Maltose is widely applied in food and
pharmaceutical industries, since its properties are represented
by mild sweetness, good thermal stability and low viscosity in
An effective application of free enzymes is hindered due to
several drawbacks like thermal instability, rapid loss of catalytic
activity during operation and storage period and sensitivity to
numerous denaturing agents [
]. Such drawbacks can be
circumvented by using enzymes in their immobilized form
Several methods including entrapment, adsorption,
encapsulation in membranes, chemical cross linking by
using bifunctional or multifunctional reagents and bioaffinity
based procedures have been employed previously for enzyme
]. Among these methods, adsorption of
enzymes on particulate carriers is one of the simplest and cost
effective immobilization techniques .
Celite-545 has recently been used as an immobilization
matrix owing its inexpensive nature and other desirable physical
properties like large surface area with high enzyme loading and
enormous porosity which increase enzyme accessibility to the
substrate. Moreover, it shows higher thermal and chemical
stabilities with greater resistance to microbial degradation
In this study, I. batata ? amylase has been immobilized on
an inexpensive support, Celite-545. The immobilized enzyme
preparations were characterized by fourier transform infrared
spectroscopy (FT-IR) and atomic force microscopy (AFM) in
order to monitor the functional groups and surface topography,
respectively. The stability of soluble and immobilized ?
amylase has been investigated against several physical and
chemical denaturants. Hydrolysis of starch in batch processes
at varying temperatures by soluble and immobilized enzyme
was also evaluated.
MATERIALS AND METHODS
Celite-545 (20-45 ? mesh) was obtained from Serva
Labs (Heidelberg, Germany). Starch, maltose, glucose and
DNS were purchased from SRL Chemicals (Mumbai, India).
Acetone was obtained from Merck (Darmstadt, Germany). I.
batata was purchased from local market. Other chemicals and
reagents employed were of analytical grade and used without
any further purification.
M. J. Khan et al / JABS, 5 (2): 33-39, 2011
Extraction and partial purification of ? amylase from
Mature, healthy Ipomoea batata roots (200 g) were washed
thoroughly with distilled H2O, cut into small pieces and
homogenized in 100 ml of 0.1 M sodium phosphate buffer, pH
6.0. The suspension was filtered through 4 layers of cheesecloth.
Filtrate thus obtained was centrifuged at 10 000 x g for 30 min
at 4oC. The crude extract was initially fractioned by 50% (v/v)
chilled acetone saturation. This solution was continuously
stirred overnight at 4oC for complete precipitation of proteins.
After centrifugation at 10 000 x g for 30 min, precipitated
pellets were collected and resuspended in two-pellet volume of
cold buffer. The solution was dialyzed against 0.1 M phosphate
buffer of pH 6.0 for overnight. Undissolved particles were
removed by centrifugation and the clear solution was stored in
assay buffer at 4oC for further use.
Adsorption of ? amylase on Celite-545
Celite-545 (1.0 g) was suspended in 50 ml of 0.1 M
phosphate buffer and stirred for 1 h at room temperature.
The fine particles present in suspension were removed by
decantation and similar procedure was repeated thrice [
binding of ? amylase on support was carried out by incubating
1232 U of enzyme g-1 of Celite-545 and this mixture was stirred
overnight in 0.1 M sodium phosphate buffer, pH 6.0 at 4oC. The
enzyme bound on Celite-545 was collected by centrifugation at
3 000 x g for 15 min at 4oC. Unbound enzyme was removed by
washing thrice with buffer and immobilized enzyme was stored
in assay buffer at 4oC for further use.
FT-IR spectra of Celite-545 and Celite-545 adsorbed ?
FT-IR spectra of Celite-545 and Celite-545 adsorbed ?
amylase were recorded by the potassium bromide pellet method
on INTERSPEC 2020 (USA) in the range of 400-4000 cm-1.
The calibration was done by polystyrene film. The samples were
injected by Hamiet 100 ?l syringe in ATR box. The syringe was
first washed with acetone followed by distilled water. FT-IR
analysis was done to examine the functional groups present in
enzyme and support.
Tapping modeAFM experiments of Celit-545 and Celite-545
adsorbed ? amylase were performed using commercial etched
silicon tips as AFM probes with typical resonance frequency
of ca. 300 Hz (RTESP, Veeco). The samples were placed drop
wise on a mica wafer, air dried at room temperature for 12 h and
the images were recorded with a Veeco Innova nanoscope II
AFM. AFM scans were carried out on several surface positions
to check the surface uniformity.
Effect of NaCl on immobilized enzyme
The Celite-545 adsorbed ? amylase (1.0 U) was incubated
with 1.0 M NaCl in 0.1 M sodium phosphate buffer, pH 6.0
at 50 oC for varying times. Activity of untreated enzyme was
considered as control (100%) for the calculation of remaining
Effect of pH
Soluble and immobilized ? amylase (1.0 U) was assayed in
the buffers of different pH (pH 2.0-8.0). The buffers used were
glycine-HCl (pH 2.0), sodium acetate (pH 3.0, 4.0), sodium
phosphate (pH 5.0-7.0) and tris-HCl (pH 8.0). The molarity of
each buffer was 0.1 M. Maximum activity obtained at pH 6.0
was taken as control (100%) for the calculation of remaining
Effect of temperature
Effect of temperature on soluble and immobilized ?
amylase (1.0 U) was studied by measuring activity of enzyme
preparations at various temperatures (20-80oC) in 0.1 M sodium
phosphate buffer, pH 6.0.
In another set of experiment, soluble and immobilized ?
amylase (1.0 U) was independently incubated at 60?C in 0.1 M
sodium phosphate buffer, pH 6.0, for varying times. Aliquots of
each preparation were taken at indicated time intervals, chilled
quickly in ice for 5 min and activity was measured. The activity
obtained without incubation at 60?C was taken as control
(100%) for the calculation of remaining percent activity.
Soluble and the immobilized ? amylase were stored at 4oC
in 0.1 M sodium phosphate buffer, pH 6.0 for over 30 days. The
aliquots from each preparation (1.0 U) were taken in triplicates
at the gap of 5 days and were then analyzed for the remaining
enzyme activity. The activity determined on the first day was
taken as control (100%) for the calculation of remaining percent
Reusability of immobilized ? amylase
Immobilized enzyme was taken in triplicates for assaying
? amylase activity. After each assay the immobilized enzyme
preparation was taken out, washed, and stored overnight in 0.1
M sodium phosphate buffer, pH 6.0, at 4?C. The activity was
assayed for seven successive days. The activity determined
for the first day was considered as control (100%) for the
calculation of remaining percent activity after each use.
Starch hydrolysis in batch process
Starch solution (1% w/v) was independently incubated
with soluble and immobilized ? amylase (500 U) at 50oC and
60oC respectively under stirring condition for 6 h. Aliquots
were taken out at different time intervals and assayed for the
formation of maltose by DNS method [
Measurement of ? amylase activity
Activity of ? amylase was assayed by DNS method with
slight modifications [
]. 250 ?l of enzyme in buffer was
added to 250 ?l substrate (1% w/v) and the resulting mixture
was incubated for 30 min at 50oC. Reaction was stopped by
adding 1.5 ml of DNS solution and then heated in a boiling
water bath for 5 min. After cooling, reaction mixture was
diluted with distilled water. Absorbance was measured
spectrophotometrically at 540 nm with maltose as standard.
One unit (1.0 U) of ? amylase activity is defined as the
amount of enzyme that liberating 1 mg of maltose min-1 under
the standard assays conditions. A standard curve of absorbance
against amount of maltose was constructed to calculate the
amount of maltose released during assay.
Estimation of protein
Protein concentration was estimated according to the
procedure described by Lowry et al [
]. BSA was used as a
Each value represents the mean for three independent
experiments performed in duplicates, with average SDs, <5%.
Data were analyzed by one-way ANOVA. P-values <0.05 were
considered statistically significant.
RESULTS AND DISCUSSION
Immobilization efficiency of ? amylase on Celite-545
The present study involves direct immobilization of partially
purified ? amylase from I. batata on an inexpensive support,
Celite-545. Thus, the cost of enzyme purification is minimized.
Celite-545 is an inorganic mechanically stable, non-toxic and
non-biodegradable diatomaceous earth which has been used
widely to immobilize various enzymes and proteins [
The binding of ? amylase on support was significantly affected
by change in pH. Enzyme was maximally adsorbed at pH 6.0
and retained 244 U ? amylase activity g-1 of Celite-545 with 83%
preserved activities (Table 1). In the literature, for immobilized
? amylase, various values for binding capacities and preserved
activities are given. For example, when immobilization of sweet
potato ? amylase was achieved on chitosan beads and spheron
based support, preserved activities were reported as 59% and
]. Furthermore 49% immobilized
efficiency was observed on immobilizing barley ? amylase on
polyacrylamide polymer [
The FT-IR spectra of Celite-545 and Celite-545 adsorbed ?
amylase are used to investigate the interaction between enzyme
and support (Fig. 1). The premier intensity peak at 1079.63 cm-1
is due to asymmetric Si-O-Si stretching vibration of Celite-545.
The band at 791.57 cm-1 is attributed to the symmetric
stretching of the ring structure of (SiO)4 tetrahedra [
vibration of H2O caused by the hydrogen bonds of protein with
silanol groups is presented at 1639.48 cm-1 [
peak at 3362.70 cm-1 due to C-H stretching indicated strong
interactions of enzyme with Celite-545 [
]. Intensity peak of
Si-O-Si stretching vibration at 1079.63 cm-1 was decreased to
1074.20 cm-1 when enzyme was adsorbed to support surface.
Visualization of surface topography of support and enzyme
adsorbed on it with AFM revealed a significant amount of
enzyme molecules immobilized on the support (Fig. 2). The
functional groups existing on Celite-545 surface were also
verified by FT-IR spectroscopy. We used the peak-to-valley
distance in these images as an indicator of the surface roughness.
The support surface, before ? amylase immobilization, was
smooth (Fig. 2a), compared with the enzyme immobilized
surfaces. It is evident that as the immobilization progressed
the roughness of support surface increases, which could be
seen from the increase of peak-to-valley value (Fig. 2b). The
roughness of the sample surface is an important feature and
should play an important role in affecting the enzyme activity.
This observation is consistent with those in previous reports
Effect of 1.0 M NaCl on immobilized ? amylase
The activity of immobilized enzyme was evaluated after
incubating it with 1.0 M NaCl for 4 h at 50oC (Fig. 3). The
adsorbed enzyme exhibited retention of signifcant enzyme
activity even in presence of 1.0 M NaCl. Result showed that
binding of ? amylase with Celite-545 was quite strong and
such type of immobilized enzyme preparations could be easily
exploited for industrial applications. Ashraf and Husain, showed
similar results with radish peroxidase when immobilized on
activity under identical thermal exposure (Fig 5). Further
incubation of soluble enzyme at 60oC for 4 h resulted in a
loss of 64% activity whereas immobilized enzyme retained
significantly higher activity, 66%.
Noda et al in their studies observed higher temperature
optima for immobilized ? amylase in comparison to its soluble
countepart18. Similarly an increase in temperature-optima was
noticed when soybean ? amylase was immobilized on chitosan
beads. The shift in temperature optima of immobilized enzyme
might be due to conformational changes at higher temperatures
that might expose active sites more appropriate for substrate
interaction thereby increasing its apparent enzymatic activity
Reusability of immobilized ? amylase
Enzymes are quite expensive products. Immobilization as a
technique ensures the recycling and reusability of the enzyme.
The most important advantage of immobilization is its repeated
uses. Reusability pattern of immobilized ? amylase showed
about 79% of initial activity retention even after its 7th repeated
use (Fig. 7). The trivial activity loss upon reuse could be due
to frequent encountering of substrate into the same active site
which might distort it and this distortion could dwindle the
catalytic efficiency either partially or fully[
Effect of pH
A shift in enzyme activity upon immobilization towards
acidic or basic directions is natural since the microenvironment
of the free and immobilized enzyme is quite different. Charge
and structure of support material, along with nature of the
activators impart a significant effect on enzyme activity.
Therefore, comparing the activity of soluble and immobilized
enzyme as a function of pH forms an important part of the study
Fig. 4 demonstrates the pH-activity profiles for soluble
and immobilized ? amylase. Both soluble and immobilized
enzyme preparations showed the same pH-optima, pH 6.0.
However, immobilized ? amylase retained significantly higher
enzyme activity at alkaline sides as compared to soluble under
similar experimental conditions. Several earlier investigators
have previously reported the broadening in pH optima for the
immobilized amylases [
Effect of temperature
The inability to enhance the thermal stability of a native
enzyme is one of the most important limitations for their
application in continuous reactors. Studies showed an increase
in temperature-optimum from 50oC to 60oC for immobilized
enzyme (Fig. 5). At 70oC the activity retained by immobilized
enzyme was significantly higher (83%) as compared to soluble
enzyme (28%). Thermal inactivation studies showed 88% of
the initial activity retained by immobilized enzyme after 2 h
exposure at 60oC whereas the free enzyme exhibited 62%
120 150 180 210 240 270
Table 2 depicts the storage stability of soluble and
immobilized ? amylase. Immobilized enzyme retained 84%
of its initial activity after 30 days of storage while its soluble
counterpart exhibited only 43% of its original enzyme activity.
The greater stability offered by this inexpensive immobilized
? amylase preparation for longer duration and at higher
temperatures might bring about its use in the continuous
production of novel products of industrial importance at large
Starch hydrolysis in batch processes
Starch is a very important raw material for the production
of sweeteners, adhesives, thickening and binding agents. Table
3 illustrates the hydrolysis of starch in batch process by soluble
and immobilized ? amylase at 40oC and 50oC, respectively. It
was noticed that the hydrolysis of starch by soluble enzyme was
82% after 5 h at 40oC whereas immobilized enzyme exhibited
88% of the hydrolytic activity under identical conditions.
Hydrolysis of starch with free and immobilized ? amylase
at 50oC after 3 h incubation was 74% and 85% respectively.
It was observed that only 80% starch was hydrolyzed by the
free enzyme after 4 h at 50oC. However, the hydrolysis of
starch by soluble enzyme beyond this limit did not exhibit any
significant increase whereas the maximum starch hydrolysis
achieved by immobilized enzyme reached to 96% in 6 h (Table
3). The results showed that the rate of hydrolysis was more in
case of soluble enzyme for the first few hours as compared to
immobilized enzyme. It was due to the fact that soluble enzyme
was more accessible to starch for first few hours but after
prolonged incubation, the rate of hydrolysis decreased. It might
be due to enzyme unfolding or inhibition of enzyme activity
by its own product [
]. Satish et al have reported similar
results where starch was hydrolyzed by immobilized ?-amylase
on super porous CELBEADS .
Amylases are among the most important enzymes used
for industrial purposes, and now in the light of biotechnology
they are considered useful for biopharmaceutical applications.
Here, an attempt has been made to obtain a simple, inexpensive
and stable immobilized ? amylase. The proteins were directly
immobilized by adsorption from the crude homogenate, thus
avoiding the high cost of enzyme purification. The immobilized
? amylase exhibited better thermostability than the free enzyme
which resulted in several benefits including low viscosity of
substrates and products, minimized bacterial contaminations,
increased reaction rates and decrease of operation time.
Furthermore, immobilized enzyme significantly hydrolyzed
starch in batch processes at high temperatures. Thus the reactors
containing such types of inexpensive immobilized enzyme
preparations could be exploited for the continuous hydrolysis
of starch at large scale.
The authors are thankful to University Grants Commission,
New Delhi, India, for providing special grant DRS, SAP to the
department for developing infrastructural facility
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