International Journal of Food Properties

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Stabilization of α-Amylase, the Key Enzyme in

Carbohydrates Properties Alterations, at Low pH

Jay Kant Yadav & V. Prakash

To cite this article: Jay Kant Yadav & V. Prakash (2011) Stabilization of α-Amylase, the Key
Enzyme in Carbohydrates Properties Alterations, at Low pH, International Journal of Food
Properties, 14:6, 1182-1196, DOI: 10.1080/10942911003592795

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International Journal of Food Properties, 14:1182–1196, 2011
Copyright © Central Food Technical Research Institute
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942911003592795

STABILIZATION OF α-AMYLASE, THE KEY ENZYME

IN CARBOHYDRATES PROPERTIES ALTERATIONS,
AT LOW pH

Jay Kant Yadav and V. Prakash

Department of Protein Chemistry and Technology, Central Food Technological
Research Institute (A constituent laboratory of CSIR), Mysore, India

The present study correlates the mechanism of α-amylase denaturation under acidic con-
dition and its structural stabilization in presence of selected cosolvents (sorbitol, sucrose,
trehalose, and glycerol). The objective of the present study was to minimize the enzyme
inactivation at lower pH by stabilizing the enzyme structure. The above cosolvents were
found to be an effective stabilizer of α-amylase against denaturation at extreme low pH.
The optimum activity of α-amylase was found to be in the pH range of 4.5 to 7. Decreasing
the pH of enzyme solution below this range results in a decrease in enzyme activity. The
intrinsic and ANS fluorescence indicated the gradual unfolding of protein below pH 4 and
resulted in exposure of hydrophobic cluster. The pH-induced unfolding also resulted in
protein aggregation. The intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) flu-
orescence, acrylamide quenching, near and far UV-CD spectra of α-amylase at lower pH
(pH 2.25) indicated the complete loss of tertiary structure and substantial loss of secondary
structure. Cosolvents were shown to prevent the acid induced unfolding of the enzyme to a
certain extent and help in retaining the secondary structure of enzyme equivalent to native
state. Among all the cosolvents used, sorbitol was proven to be the most efficient stabilizer.
At lower pH, in presence of cosolvents the enzyme was shown to retain significant amount
of secondary structure and poorly defined tertiary structure. This structure resembles the
molten globule of the protein, which has substantial amount of secondary structure but
poorly defined tertiary structure.

Keywords: Molten globule, Aggregation, Cosolvents, Secondary structure, Denaturation,

Fluorescence.

INTRODUCTION
Although the structural stability of protein largely depends on the primary struc-
ture of protein,[1] the pH of the protein solution has a very crucial role in determining
the overall stability. At extreme acidic or alkaline pH, away from isoelectric points, the
protein tends to unfold due to electrostatic repulsion between like charges formed due to
ionization of functional groups in response to a change in pH.[2,3] At extreme pH, pro-
teins gradually undergo denaturation and results in the formation of unfolded structure
containing substantial amounts of secondary structure and poorly defined tertiary struc-
ture, which resemble the molten globule state of protein in protein folding pathways.

Received 28 October 2009; accepted 2 January 2010.

Address correspondence to V. Prakash, Director, Central Food Technological Research Institutea, Mysore,
570020, India. E-mail: prakash@cftri.com

1182
 STABILIZATION OF α-AMYLASE AT LOW pH 1183

Characterization of intermediates of protein unfolding pathways is very crucial for study-

ing the structural stability and designing the protein molecule with novel characteristics.
The partially unfolded protein can be refolded in various ways depending upon the con-
ditions provided. Many proteins at low pH undergo partial denaturation, which loses the
tertiary structure and retains a certain degree of secondary structure, known as molten
globule. Molten globules are likely to form during early stages of protein folding after
translation.[4] In order to understand the cascade of folding-unfolding reaction, it is impor-
tant to characterize the forces, which are required to integrate the protein structure in the
native state. In protein folding-unfolding reactions, the intermediates are transiently stable.
These intermediates are largely fluctuating structures and their stability is largely deter-
mined by a delicate balance of interactions, such as electrostatic repulsion between charged
residues and hydrophobic interactions.[5] A large number of proteins are demonstrated to
undergo acid induced unfolding and formation of stable molten globule at low pH.[2,6–9]
The solvent perturbation has a great role in providing the structural stability of
protein molecule. The interaction of protein with solvent depends on the various physico-
chemical parameters of solvents. The compositions and conditions determine various types
of forces operating in protein molecule responsible for protein stability. The cosolvents are
known to stabilize several proteins against thermal denaturation, mainly by alteration of
preferential interaction parameters.[10] In this paper the terms cosolvents, additives, excip-
ients, polyols, and sugars have been used interchangeably.[10–12] In addition to stabilizing
the native state of protein, cosolvent can also stabilize the intermediate state of protein fold-
ing pathway, which may provide valuable information in understanding the nature of these
intermediates. Several studies indicated that cosolvents stabilize the protein by enhancing
the hydrophobic interaction.[13–17]
In the present investigation α-amylase (α-1,4-D-glucan glucanohydrolase, EC:
3.2.1.1), a well-studied enzyme with complete sequence available, was selected as a
model protein. It is a metalloenzyme; requires at least one calcium ion for its structure
and function.[18] It contains a characteristic (β/α)8 structure, called a triose-phosphate-
isomerase-barrel catalytic domain with highly symmetrical folds of eight inner parallel
β-strands surrounded by eight α-helices.[19] The whole structure of enzyme is constituted
by three domains named as domain A, B, and C. The domain B is inserted between the 3rd
β-strand and 3rd α-helix of (β/α)8 structure of (β/α)8 fold. Each enzyme in this family
contains one glutamic acid and two aspartic acids residues in the catalytic active site.[19,20]
The domain C is thought to stabilize the catalytic domain by shielding the hydrophobic
domain.[21] The stability of α-amylase has been studied extensively using cosolvents and
found that stability has significantly improved.[16] In the present investigation, efforts have
been made to correlate the structural alteration and stabilization of protein at low pH in
presence of selected cosolvents. Study has also been extended to correlate the formation
of stable molten globule state of α-amylase at low pH in presence of cosolvents. This may
be a new characteristic of cosolvents that not only stabilize the native protein but may also
reverse the denaturation reaction.

MATERIAL AND METHODS

Chemicals and Reagents
α-Amylase type II (A6380), trehalose, sorbitol, sucrose, glycerol, starch, dinitrosal-
isylic acid, 8-anilinonaphathalene-1-sulphonic acid (ANS), bovine serum albumin (BSA),
1184 YADAV AND PRAKASH

and CaCl2 were procured from Sigma Chemicals Company (St. Louis, MO, USA). The
2-Chloro-p-nitrophenyl-α-D-maltotrioside (CNPG3) was procured from Pointe Scientific,
Inc. (Canton, MI, USA). KCl, NaCl, and HCl were obtained from Qualigen Fine Chemicals
Pvt. Ltd. (India). Acrylamide, NaOH, glycine, and dibasic sodium phosphate were pro-
cured from E-Merck (Mumbai, India). All chemicals were of analytical grade. The
crystalline α-amylase was dissolved and dialyzed in 0.02 M citrate buffer (pH 5.9) to
remove the excipients, freeze-dried (−80◦ C) and desiccated at 0◦ C for future use. Quartz
triple distilled water was used for all the experiments. All the cosolvents were analyzed
for calcium ion contamination by using atomic absorption spectroscopy. The calcium ion
content was found to be approximately 0.1 mM in 50% aqueous solution of each cosolvent,
which was low enough to make any appreciable change in activity and structural stability
of enzyme.

Protein Estimation
Protein concentration was determined by measuring the absorbance of enzyme sam-
ple at 280 nm on Shimadzu UV-spectrophotometer model UV-1601 using an extinction
coefficient (E1% 1cm ) of α-amylase as 14.46.[16] Alternatively, the protein concentration was
determined by the Lowery method using BSA as standard protein.[22]

α-Amylase Activity Measurement

α-Amylase activity was measured by using the Bernfeld method[23] for estimation
of reducing sugar. The crystalline amylase was dissolved in 0.02 M citrate buffer, pH 5.9,
containing 1 mM of CaCl2 and final concentration of enzyme was adjusted to 1 μg/mL.
The reaction mixture containing 1 mL of 1% starch solution and 1 mL of enzyme solution
incubated for 5 min on a temperature-controlled circularly rotating water bath at 37◦ C. The
reaction was terminated by addition of 2 mL of 1% alkaline dinitrosalicylic acid solution to
the reaction mixture. The whole solution was then subjected to heat in a boiling water bath
for 10 min. After cooling, it was diluted five times using triple distilled water, mixed prop-
erly, and absorbance was recorded at 540 nm. The activity was calculated by using maltose
standard plot. The unit of enzyme activity is defined as the amount of enzyme required for
starch hydrolysis to produce 1 μmol of maltose equivalent under given conditions.
Alternatively, the α-amylase activity was estimated by using an artificial substrate
CNPG3. α-Amylase hydrolyzes the CNPG3 to release 2-Chloro-nitrophenol, maltotri-
side, and glucose.[24,25] The amount of 2-Chloro-nitrophenol released reflects the degree
of hydrolysis, which could be monitored by spectrophotometry. The 25 μL of enzyme
solution was added to 1 mL of CNPG3 solution and incubated at 37◦ C for 3 min, and
absorbance was recorded at 405 nm in Shimadzu UV-1601 UV-Visible spectrophotometer.
The enzyme activity was determined using the equation:

Abs/min × total assay volume

Unit of α-amylase = (1)
MMA × SV × l

where MMA is the milimolar absorptivity of CNPG3, SV is sample volume, and l is the
path length of light.
 STABILIZATION OF α-AMYLASE AT LOW pH 1185

pH Induced Inactivation of α-Amylase

A series of enzyme solutions were prepared in 20 mM solution of different buffers
having different pH ranging from 1.5 to 10.00 (KCl–HCl buffer, pH 1.5–2.5, citrate buffer
pH 3.0–6.0, citrate-phosphate buffer pH 6.0–7.5, and glycin-NaOH buffer pH 8–10). These
enzyme solutions were incubated for 12 h to equilibration at 4◦ C before the activity
measurement. The pH of enzyme solutions was measured before and after the experiment.

Estimation of Protein Aggregation

The pH induced aggregation of α-amylase at different pH was measured by moni-
toring the absorbance at 360 nm in the Shimadzu UV-1601 UV-Visible spectrophotometer
(Japan). The absorbance at 360 nm arise mainly due to scattering of light by particulate
matter in the solution, and at this wavelength scattering can be measured free from interfer-
ence caused by intrinsic fluorescence or light absorption of the enzyme.[26] The α-amylase
samples were incubated at different pH at 25◦ C before taking the absorbance.

Intrinsic and ANS Fluorescence Measurement

Fluorescence measurement of α-amylase in presence of cosolvents at different pH
values was performed on a Shimadzu spectroflurophotometer model RF-5000 equipped
with temperature-controlled device. Intrinsic fluorescence spectra of α-amylase were mea-
sured at 25◦ C. The pH of protein solution was maintained as 1.5 to 10.00 (KCl–HCl buffer,
pH 1.5–2.5, citrate buffer pH 3.0–6.0, citrate-phosphate buffer pH 6.0–7.5, and glycin-
NaOH buffer pH 8–10). The enzyme samples were prepared in different concentrations of
cosolvents at different pH and incubated for 12 h before recording the fluorescence spectra.
For intrinsic fluorescence excitation it was set at 280 nm and emission was recorded in the
range of 300–400 nm using slit width of 10 and 5 nm for excitation and emission, respec-
tively. For ANS fluorescence, the molar ratio of ANS and protein was 100:1, excitation
was set at 380 nm, and emission spectra were recorded in the range of 400–600 nm, using
slit width of 10 and 5 nm for excitation and emission, respectively. Protein concentration
for all the fluorescence experiments was 0.05 mg/mL.

Acrylamide Quenching
Aliquots of 10 μL of acryamide stock solution (2M) were added to 2 mL of α-
amylase solutions incubated at different pH, mixed by inverting or with magnetic stirrer
set with spectrofluorophotometer chamber. Excitation was set at 280 nm and emission
maxima were recorded at 340 nm after each addition of acrylamide stock solution. The
acrylamide fluorescence quenching of protein samples were analyzed by the Stern-Volmer
equation:[27]

F0 /F = 1 + Ksv [Q] (2)

where F 0 and F are the fluorescence intensity in absence and presence of quencher, respec-
tively. Ksv is Stern-Volmer constant, which can be obtained from the slope of the curve of
F 0 /F versus quencher concentrations.
1186 YADAV AND PRAKASH

Circular Dichroism (CD) Measurement

CD measurement was carried out on a Jasco spectropolarimeter model J-
810 equipped with computer. The instrument was calibrated with D-10 camphorsulphonic
acid. α-Amylase samples were equilibrated at pH 7 and 2.25 in presence of different con-
centrations of each cosolvent and CaCl2 for 12 h at 4◦ C before CD measurement. Far and
near UV-CD was measured using protein concentration of 0.268 and 1 mg/mL, respec-
tively. The path length of far and near UV-CD were 1 mm and 10 mm, respectively, with
the scan speed of 50 nm/min. The molar ellipticitiy was expressed as mean residue ellip-
ticity in deg–cm2 dmol−1 using 110 g/mol residual mass of α-amylase. Each spectrum was
an average of three independent scans. The secondary structure of α-amylase was analyzed
using the program (J-810) of Yang et al.[28] The statistical analysis of the data was carried
out using OriginPro 7 software.

RESULTS
pH Induced Inactivation of α-Amylase
α-Amylase activity was measured in the pH range of 3.2 to 10.00 and it showed
optimum activity in the range of pH 5.5–7.5 (Fig. 1). Enzyme samples were incubated at
different pH from 3.20 to 10 in appropriate buffer for 12 h followed by activity measure-
ment. Figure 1 indicates the activity of α-amylase in presence of 40% of each cosolvents,
viz. trehalose, sorbitol, sucrose, and glycerol. Decreasing the pH below 4.5 resulted in
drastic reduction in enzyme activity, and at pH 3.2 there was complete loss of activity.
Addition of cosolvents was shown to have protective effect on the enzyme activity under
condition of relatively low pH but at pH 3.2 no activity was observed even in presence of
cosolvents. The maximum activity at pH 4, 4.5, and 5.0 were found to be 1100, 1740, 2300,
and 2720 units, respectively, and in presence of 40% trehalose the activity was reported to

3.5

3.0
Enzyme activity (x103 units)

2.5

2.0

1.5

1.0

0.5

0.0
3 4 5 6 7 8 9 10
pH

Figure 1 Effect of cosolvents on α-amylase activity at different pH. The curves represent the α-amylase activity
of (∀) control, and in presence 40% (w/v) (8) glycerol, (X) sorbitol, (–) trehalose, and (M) sucrose.
 STABILIZATION OF α-AMYLASE AT LOW pH 1187

0.10

0.08

Absorbance at 360 nm 0.06

0.04

0.02

0.00

1 2 3 4 5 6 7
pH

Figure 2 pH induced aggregation of α-amylase. The protein aggregation was monitored by recording the
absorbance at 360 nm at each pH ranging from 1.5 to 7.

be 1500, 2120, 2660, and 3050 units, respectively. The relative activity of enzyme in the
presence of cosolvents at low pH was found in the order of trehalose > sucrose > glycerol
> sucrose. In alkaline region, the activity enzyme was not affected significantly until pH
10. The pH induced enzyme inactivation was found to be reversible in certain ranges of
pH (5–4). The loss of activity at pH below 4.0 was found to be irreversible. At pH close
to optimum activity range, enzyme may not undergo any major structural change and the
inhibition of enzyme in this range may be because of change in ionization potential of
amino acid residues in the catalytic sites.
At pH less than 4.5 enzymes show irreversible inactivation and accompanied
by pH-induced aggregation (Fig. 2). These protein aggregates were easily soluble in
aqueous solution of 0.5% SDS, which is an indication of hydrophobic nature of the
aggregates.[29,30] The pH-induced aggregation was prominently found in the range of
pH 2.5–4.4. On further, lowering the pH aggregation decreases due to the reburial
of hydrophobic residue in the protein interior. The pH-induced change structure indi-
cates that the protein-folding pathway may primarily be governed by the hydrophobic
interactions.

Intrinsic and ANS Fluorescence

The pH-induced unfolding can be monitored by intrinsic and ANS fluorescence in
the ranges of pH 1.5 to 7.0. At pH above 7 (up to 10) there was no appreciable change in
the intrinsic and ANS fluorescence spectra. The intrinsic fluorescence intensity gradually
decreases the pH until 2.25 (Fig. 3). Although, no significant change was observed in the
fluorescence intensity of the enzyme on further lowering of pH, the fluorescence emis-
sion maxima (λmax ) was found to be altered significantly. The λmax of native enzyme (at
pH 7.0) was found to be 340 nm and with decreasing the pH a red shift was observed
in the λmax , and at pH 2.25 it was recorded to be 343 nm. Further lowering the pH
1188 YADAV AND PRAKASH

250

Intrinsic Fluorescence intensity (a.u.)

a
200 b
c
d
150

e
f
100
h
g

50

300 320 340 360 380 400

Wavelength (nm)

Figure 3 Intrinsic fluorescence spectra of α-amylase at different pH ranging from pH 1.5 to 7. The enzyme sam-
ples were incubated at different pH for 12 h before recording the spectra. The fluorescence spectra are represented
as: (a) pH 7, (b) pH 6, (c) pH 5, (d) pH 4, (e) pH 3, (f) pH 2.75, (g) pH 2.25, and (h) pH 1.5.

resulted in a blue shift in λmax , which is an indication of reorganization of unfolded enzyme

molecule, although this structure can not be compared with the native enzyme. This reflects
the changes in the micro environment of tryptophan and tyrosin residues in the protein.
A similar result was reported with glucose/xylose oxidase enzyme where extreme low
pH-induced an structuring effect.[31] The ANS fluorescence has been extensively used to
probe the conformational changes that occurred during protein denaturation.[32] Generally,
the protein denaturation leads to exposed hydrophobic clusters on the surface of enzyme
and, hence, it becomes accessible to solvents. ANS is a hydrophobic dye that preferen-
tially binds with the hydrophobic cluster on protein surface and fluoresces in the range
of 400–600 nm. As shown in Figs. 4a, 4b, and 4c ANS fluorescence intensity increases
with the lowering of pH with significant change in the λmax . This indicates the gradual
unfolding of α-amylase under acidic condition. The maximum ANS intensity was recorded
at pH 2.25, indicating the maximum exposure of hydrophobic clusters on the surface.
A decrease in intensity was observed by further lowering the pH. This could be due to
the reburial of hydrophobic core of protein molecules away from aqueous medium, which
creates variations in the structural compactness and flexibility in enzyme molecules at
different pHs.
The fluorescence quenching study was carried out in order to confirm the degree of
compactness of proteins at pH 7, 2.25, and 1.5. Acrylamide is a neutral molecule, and there-
fore, efficient quencher of intrinsic fluorescence has been used in this study.[27] Figure 5
shows the Stern-Volmer plot of acrylamide quenching of fluorescence by acrylamide in
native and acid denatured state. The protein at pH 2.25 shows very high quenching of
intrinsic fluorescence compared to native protein at pH 7.0. The degree to which an amino
acid side chains exposed to solvents depends upon the relative compactness of protein
molecule. It shows that solvent accessibility of acid-denatured state is comparatively higher
than the native enzyme (Fig. 5). The presence of additives, such as polyols-trehalose, sor-
bitol, sucrose, and glycerol, and salts-NaCl and CaCl2 has significantly reduced the ANS
 STABILIZATION OF α-AMYLASE AT LOW pH 1189

g
160 i

ANS fluorescence intensity (a.u.)

j
h

120
f

e
80
d
c
40
b
a
0
400 450 500 550 600
Wavelength (nm)

Figure 4a The 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence spectra of α-amylase at different pH

ranging from pH 1.5 to 7. The curves are represented as: (a) pH 7, 6.5 and 6.00, (b) pH 4.5, (c) pH 4.00, (d) pH
3.5, (e) pH 3.00, (f) pH 2.5, (g) pH 2.25, (h) pH 2.00, (i) pH 1.75, and (j) pH 1.5.

160
ANS fluorescence intensity at 470 nm

120

80

40

0
1 2 3 4 5 6 7
pH

Figure 4b Variation of 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence intensity of α-amylase with

change in pH at 25◦ C. After incubating the enzyme solution at different pH the aliquots ANS stock solution was
added, mixed and stand for 20 min in dark. The excitation wavelength was fixed at 380 nm and emission spectra
were collected between 400–600 nm.

fluorescence in acidic condition, as shown in Fig. 6. The decrease in ANS fluorescence

intensity in presence of cosolvents and additive indicates the stabilization of protein at
low pH. This result suggests that these additives prevent the acid-induced denaturation of
α-amylase.
1190 YADAV AND PRAKASH

510

ANS emission maxima (a.u.)

500

490

480

470

460

450
1 2 3 4 5 6 7
pH

Figure 4c Change in 8-anilinonaphathalene-1-sulphonic acid (ANS) emission maximum as a function of pH.

The emission spectra were collected between 400–600 nm and excitation wavelength was fixed at 380 nm.

3.0 b

c
2.5
a
F0/F

2.0

1.5

1.0

0.00 0.02 0.04 0.06 0.08 0.10

Acrylamide (M)

Figure 5 Stern-Volmer plot of acrylamide quenching of intrinsic fluorescence of enzyme at pH (a) 7.0, (b) 2.25,
and (c) 1.5. Aliquot of 2M stock solutions of acrylamide added sequentially and excitation wavelength was
fixed at 280 nm and emission maxima were recorded at 340 nm. The F0 and F are the fluorescence intensity
of the enzyme in absence and presence of acrylamide, respectively. The ratio of F0 and F indicates the relative
quenching of intrinsic fluorescence under given condition.

Near and Far UV-CD Spectra

The near and far UV-CD spectra of native and acid induced denatured state of α-
amylase were recorded after equilibration at each pH. Figure 7a shows the near UV-CD
spectra of amylase at pH 7.0 and 2.25. The spectrum in near UV-CD mainly contributed
by the conformation of aromatic amino acid residues. The near UV-CD spectra showed
 STABILIZATION OF α-AMYLASE AT LOW pH 1191

800
b

ANS fluorescence intensity (a.u.)

600

400
c
d
e
200
f
g
h
a
0

400 450 500 550 600

Wavelength (nm)

Figure 6 The 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence spectra of enzyme at native and acidic
pH in presence of cosolvents. Enzyme solutions were incubated at acidic pH in presence of cosolvents for 12 h
before recording the spectra. The curves (a) and (b) represent pH 7 and 2.25 in absence of cosolvents, respectively.
Curves (c), (d), (e), and (f) represent the spectra in presence of 30% of trehalose, sorbitol, sucrose, and glycerol
at pH 2.25, respectively. The curve (g) and represent 100 mM CaCl2 and 250 mM of NaCl, respectively.

(a) (b)

20 b 2000

a
[θ]MRE in deg–cm2 dmol−1

0
1
[θ]MRE in deg–cm2 dmol−1

0
−20

−40 −2000

b
−60
−4000 a

−80

−100 −6000
240 260 280 300 320 200 210 220 230 240 250 260
Wavelength (nm) Wavelength (nm)

Figure 7 (a) Near UV-circular dichroic spectra of enzyme at pH (1) 7.0 and (2) 2.25. The enzyme solutions were
incubated for 12 h before the circular dichroism measurement. The protein concentrations were maintained to be
1 mg/mL. The [θ]MRW is molar residual elipticity of enzyme. (b) Far UV-circular dichroic spectra of α-amylase at
pH (1) 7.0 and (2) 2.25. The enzyme solutions were incubated for 12 h before the CD measurement. The protein
concentrations were maintained to be 0.26 mg/mL. The [θ]MRW is molar residual ellipticity of enzyme.

the native orientation of aromatic amino acid residues. The appearance of major shoul-
ders at 265 and 295 nm reflect the alteration in the tertiary structure and more flexible
environment of aromatic amino acid residues. The far UV-CD spectra of amylase at pH
7.0 and 2.25 are showed in Fig. 7b. The curve of native α-amylase has two minima at
222 nm and 208 nm, which are the characteristics of the α-helical structure. The presence
of cosolvents did not show any effect on the secondary structure of the native enzyme (pH
1192 YADAV AND PRAKASH

7.0). In the acidic pH, cosolvents had significant protective effect on the secondary struc-
ture of enzyme. The α-helical content of α-amylase was found to be 16% and 7% at pH
7.0 and 2.25, respectively. Our purpose was to stabilize α-amylase against the acid-induced
denaturation using selected cosolvents and salts. Before recording the far UV-CD spectra
enzyme samples, they were equilibrated for 12 h in presence of different concentration of
each cosolvent and salt at pH 7.0 and 2.25. Table 1 summarizes the secondary structural
element of enzyme in acidic condition in presence of various cosolvents. The presence of
cosolvents helped in retaining the α-helical structure and the retention capacity of each
cosolvent was found to be depending on the concentration used. Out of all four cosolvents
used, sorbitol was proven to be the most efficient stabilizer at all concentrations against
acid-induced denaturation. At low concentrations (up to 20% w/v) of other cosolvents,
such as glycerol, trehalose, and sucrose, there is no significant change in the ellipticity,
but at higher concentrations (30–40% w/v) of trehalose and sucrose, more than 75% the
α-helical structure of native enzyme was protected. Glycerol, even at 40% concentrations,
was not able to make any significant change. Although it has been shown that glycerol
at 80% concentration retained up to 98% α-helical structure of the native human placen-
tal cystatin, whereas sorbitol at the same concentration has negligible effect.[33] Since the
acid-induced denaturation is a complete function of electrostatic repulsion, the neutraliza-
tion of charged groups may substantially reduce the electrostatic repulsion by counter ion
effect and render the protein stable. Therefore, investigation was also made in the presence
of different concentration of salts (i.e., NaCl and CaCl2 ). Our purpose was also to see the
effect of counter ions on the acid-induced denaturation α-amylase. It is well known that
calcium is essential for providing structural stability and activity of α-amylase; it may also
provide the suitable counter ion which can minimize the electrostatic repulsive forces. The
NaCl is neutral salt ionizes completely in the aqueous medium and form the Na+ and Cl−

Table 1 Comparison of secondary structural elements of α-amylase at pH 7.0 and 2.25 in

presence of cosolvents.

Percent secondary structure

Cosolvents Concentration (%w/v) α-helix β-structure Aperiodic

pH 7.00 16 ± 1 62 ± 3 22 ± 2
pH 2.25 7±1 65 ± 3 28 ± 2
Sorbitol 10 7±1 65 ± 3 28 ± 2
20 13 ± 1 52 ± 3 35 ± 2
30 14 ± 1 57 ± 2 29 ± 2
40 17 ± 1 52 ± 2 31 ± 2
Trehalose 10 7±1 69 ± 4 24 ± 2
20 7±1 68 ± 4 25 ± 2
30 8±1 74 ± 4 18 ± 1
40 9±1 73 ± 4 18 ± 1
Sucrose 10 7±1 64 ± 3 29 ± 2
20 8±1 70 ± 4 22 ± 2
30 9±1 67 ± 4 24 ± 2
40 10 ± 1 66 ± 3 24 ± 2
Glycerol 10 5±1 71 ± 4 24 ± 2
20 7±1 71 ± 3 22 ± 2
30 7±1 75 ± 4 18 ± 1
40 8±1 69 ± 4 23 ± 2
 STABILIZATION OF α-AMYLASE AT LOW pH 1193

Table 2 Comparison of secondary structural element of α-amylase at pH 2.25 in the presence

of CaCl2 and NaCl.

Percent secondary structure

Concentrations of salts (mM) α-helix β-structure Aperiodic

Control∗ 7±1 65 ± 3 28 ± 2
CaCl2
25 11 ± 1 56 ± 3 33 ± 2
50 15 ± 1 51 ± 2 34 ± 2
100 16 ± 1 53 ± 3 31 ± 2
NaCl
250 14 ± 1 47 ± 2 39 ± 2
∗ The enzyme in 0.02M KCl-HCl buffer at pH 2.25.

which interact with the counter ion on the protein surface. The secondary structural ele-
ments of enzyme in different concentration of each salt are summarized in Table 2. The
results showed that in the presence of 100 mM of CaCl2 more than 95% of α-helical con-
tent was preserved, whereas up to 85% of the native α-helical structure was retained in
presence of 25 mM of NaCl.

DISCUSSION
Under mild denaturation conditions, such as low pH, temperature, and low concen-
tration of denaturants, a number of protein exist in a stable conformation which neither
resemble with native state nor fully denatured state, and can be classified as molten glob-
ule state: the intermediate between native and unfolded state.[4,8,15,29] It has been shown
that in the presence of low concentration of urea, papain, and human serum albumin exist
as molten globule and fully denatured at higher denaturant’s concentrations.[34,35] A num-
ber of proteins are shown to undergo denaturation under the heavy acidic condition. It is
reported that the α-amylase exist as molten globule-like structure at pH 3.0 with sec-
ondary structure equivalent to native enzyme but altered tertiary structure.[36] Both, the
cosolvents and salts (CaCl2 and NaCl), have a protecting effect of α-amylase against acid-
induced denaturation. ANS binds preferentially to the hydrophobic clusters on the protein
surface.[32] Acid-induced (pH 2.25) denatured state of α-amylase has a maximum ANS flu-
orescence intensity, suggesting the exposure of hydrophobic patches on the protein surface.
Although the addition of cosolvent decreases the ANS fluorescence intensity significantly,
yet it was found to be more than the native enzyme. The decrease in intrinsic fluorescence
might be due to the relocation of tryptophan and tyrosin residues to polar environment
after acid denaturation, which reflects the alteration in the tertiary structure.[37,38] This
suggests that presence of cosolvents helped in retention of secondary structure equivalent
to native enzyme, but the acid denatured protein have not regained the native structure.
The reduction in fluorescence intensity in the presence of cosolvents varies to different
extent in presence of different cosolvents indicating the difference in the mechanism of
stabilization. In the presence of CaCl2 and NaCl, the ANS fluorescence was close to native
enzyme. Addition of additives did not affect the intrinsic fluorescence intensity and it was
similar to the acid denatured state. The secondary structure of enzyme in presence of 50%
of cosolvents found to be similar or close to the native enzyme. The mechanism of salt-
induced refolding can be explained on the basis of neutralization of protonated side chains
1194 YADAV AND PRAKASH

of amino acids by negative ions of salts, which decreases the internal repulsive force, which
favor unfolding.[9,15]
Cosolvents generally stabilized the protein by altering the solvent distribution in the
solution. In a previous report, it is shown that the presence of cosolvents in solution results
in preferential hydration of α-amylase.[39] It is also shown that molten globule state of
papain is more hydrated compared to the native one.[33] The preferential exclusion of cosol-
vents is directly proportional to surface area of the proteins and favors the protein to occupy
minimum surface area.[40] The preferential exclusion of cosolvents leads to increase com-
pactness of the protein molecule compared to acid denatured state of enzyme in absence of
cosolvents, which might provide the more rigid and stable structure compared to a native
one.[41–43] In the presence of cosolvents and salts, although the secondary structure was
comparable to native state, the enzyme remains inactive, and the ANS fluorescence remains
nearer to the denatured state. These results are indicative of the formation of molten globule
state of enzyme, which has substantial amount of secondary structure and poor in tertiary
structure.

CONCLUSION
Cosolvents were shown to have stabilizing effect on α-amylase at low pH. The sta-
bilizing effects of cosolvents were found to be proportional to their concentration used.
Although presence of cosolvent in the enzyme solution indicated the structural stabilizing
effect, still the enzyme did not achieve the native functional state. This might be proba-
bly due to the loss of stereochemistry of tertiary structure. In this situation, the structures
of the enzyme seems similar to the molten globule state of protein, which has signifi-
cant secondary structure and poorly defined tertiary structure. Addition of salts (NaCl and
CaCl2 ) also has significant effect on structural stabilization of α-amylase, although salts
and cosolvents follow different mechanism of structural stabilization of the enzyme.

ACKNOWLEDGMENTS
Jay Kant Yadav gratefully acknowledges the Council of Scientific and Industrial Research
(CSIR), New Delhi, India, for providing financial support in the form of Junior and Senior Research
Fellowship during the course of work.

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