Enzyme and Microbial Technology 37 (2005) 723–734

A novel raw starch digesting thermostable ␣-amylase from Bacillus sp.

I-3 and its use in the direct hydrolysis of raw potato starch
Nidhi Goyal, J.K. Gupta, S.K. Soni ∗
Department of Microbiology, Panjab University, Chandigarh 160014, India

Received 19 September 2004; received in revised form 19 April 2005; accepted 28 April 2005

Abstract

A new strain of Bacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch.
Upon optimization of various environmental and cultural conditions, the yield of ␣-amylase reached 642 U/mL. The kinetic characterization
of partially purified enzyme exhibited the maximum activity at 70 ◦ C, pH 7.0 and revealed a high thermostability in the presence of 10 mM
CaCl2 ·2H2 O where it could retain more than 90% residual activity at 70 ◦ C after 3.5 h. At 80, 90 and 100 ◦ C, the enzyme retained 80, 59 and
26% of its maximum activity after 2.5, 0.5 and 0.5 h, respectively. The enzyme preparation had a strong affinity towards raw potato starch
granules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a short
period of time of 12 h.
© 2005 Elsevier Inc. All rights reserved.

Keywords: Raw starch hydrolysis; Thermostable ␣-amylase; Bacillus sp.; Raw potato starch; Raw starch adsorption; Direct hydrolysis

1. Introduction cost of starch-based products. In view of energy costs, effec-

Starch is the most abundant form of storage polysaccha-
rides in plants and constitutes an inexpensive source for pro-
duction of syrups containing glucose, fructose or maltose,
which are widely used in food industries [1]. In addition to
that, the sugars produced can be fermented to produce bio-
ethanol [2]. In starch granules, the molecules are densely
packed in a polycrystalline state with inter and intramolecular
bonds and are hence insoluble in cold water and often resistant
to chemicals and enzymes [3]. In the course of conventional
enzymatic saccharification, a slurry containing 15% starch is
gelatinized where it is heated up to a temperature of 105 ◦ C so
as to open the crystalline structure for the enzyme action [4,5].
This increases the viscosity of the slurry and poses prob-
lems with mixing and pumping [6]. The gelatinized starch is
then liquefied with high temperature ␣-amylase at the same
time followed by the saccharification with glucoamylase at a
much lower temperature of 50–60 ◦ C [5]. The whole process
requires a high-energy input, thus increasing the production
∗ Corresponding author. Tel.: +91 172 2534149; fax: +91 172 2541409.
E-mail address: sonisk@pu.ac.in (S.K. Soni).
tive utilization of natural resources and viscosity problems,
direct hydrolysis of starch below gelatinization temperature
is desirable [7]. In recent years, the importance of enzymatic
saccharification of raw starch without heating has become
well recognized, mainly from the viewpoints of energy sav-
ings and effective utilization of the biomass thereby reducing
the cost of starch processing [4,5,8]. This has generated a
world wide interest in the discovery of several raw starch di-
gesting amylases which does not require the gelatinization
and can directly hydrolyze the raw starch in a single step and
that too at moderate temperature much below the gelatiniza-
tion temperature [7,9].
It is more difficult for amylases to act on raw starch gran-
ules than on gelatinized starch. Previous studies indicated
that the saccharification of raw starch by amylolytic enzymes
might be related to the extent of adsorption of enzyme to the
starch granules. The microorganisms reported to be good pro-
ducers of amylase capable of digesting raw starch have mostly
been fungi, such as Aspergillus sp., Rhizopus sp. and Corti-
cium rolfsi [7–10], although there are reports of the possibility
of raw starch degradation by bacterial ␣- and ␤-amylases [4].
Several amylases from molds and bacteria have been reported

0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.04.017
724 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734
which digest the raw starch granules at ordinary temperatures
(30–50 ◦ C) involving mashes having 1% raw starch [11] in
which significant saccharification is achieved after extended
period of incubation. Different studies demonstrated that the
better hydrolysis of raw starch can be achieved by increasing
the incubation temperature approximately to 60 ◦ C [12,13].
Therefore, use of thermostable, raw starch digesting amy-
lases is expected to give better hydrolysis of raw starch at
temperatures ranging between 60 and 70 ◦ C without loosing
activity during prolonged incubation.
Most raw starch digesting enzymes reported to date hardly
digest potato starch [6,7,13–15]. On the other hand, next to
corn, potato is the most important source of starch [6,16].
Therefore, enzymes that are capable of digesting raw potato
starch are economically attractive for they can increase the
range of starch sources for direct hydrolysis.
The purpose of this research was to isolate a microor-
ganism capable of producing thermostable and efficient raw
potato starch digesting ␣-amylase for the direct hydrolysis
of raw starch in a considerably less duration of time, at a
concentration of starch normally used in the starch industries
(10–15%) at temperatures below the gelatinization tempera-
ture of starch.
2. Materials and methods
2.1. Microorganism
To screen the raw potato starch digesting microorganisms,
insoluble potato starch granules (SD Fine Chemicals, India)
sterilized by washing in ethanol, were added to sterile nutri-
ent agar at 50 ◦ C to a final concentration of 1%. The screening
medium was dispensed in petridishes, which were inoculated
with soil samples collected from the vicinity of various flour
mills of Chandigarh city and incubated for 48 h. Starch hy-
drolysis was assessed as clearing zones around the colonies.
The colonies showing starch hydrolysis in the plates were
further evaluated by their enzyme productivity levels in liquid
culture, after 48 h, employing nutrient broth, supplemented
with 1% raw (insoluble) potato starch granules and then by
checking the enzyme affinity towards raw potato starch gran-
ules by determining the adsorption rate and ability to hy-
drolyze 1% raw potato starch suspension at 60 ◦ C in 6 h.
2.1.1. Determination of raw potato starch adsorbability
Affinity of the enzyme preparations from various isolates
towards raw potato starch was studied by incubating 0.2 g
of raw potato granules with 1 mL of the enzyme at 37 ◦ C
for 15 min. After centrifugation, the ␣-amylase activity of
the supernatant was measured and the adsorption percentage
was calculated as follows:
A−B
Adsorption (%) = × 100
A stated (Table 1). The effect of carbon sources was studied
A is the original ␣-amylase activity and B is the ␣-amylase ac-
tivity in the supernatant after adsorption on raw potato starch
granules.
2.1.2. Determination of raw potato starch digestibility
The reaction mixtures containing 500 U of ␣-amylase
preparation from different isolates and 100 mg of raw potato
starch in a final volume of 10 mL dispensed in 100 mL Er-
lenmeyer flasks were incubated in rotary water bath shaker
at 60 ◦ C and 150 rpm. After a time interval of 6 h, the re-
ducing liberated in the reaction mixtures were determined by
dinitrosalicyclic acid method [18].
One strain, designated as I-3, giving the best adsorption
and hydolysis at 1% concentration of raw potato starch was
isolated and selected for further studies. It was identified on
the basis of various morphological, physicochemical and bio-
chemical characteristics following the criteria laid down by
Bergey’s Manual of Systematic Bacteriology [19].
2.2. Profile of growth and extracellular α-amylase
production by Bacillus sp. I-3 in submerged fermentation
The basal medium used for the submerged cultures was
composed of: peptone, 10.0 g/L; beef extract, 10.0 g/L; sol-
uble starch, 10.0 g/L and sodium chloride, 5.0 g/L; pH 7.0.
Twenty milliliters of medium in 100 mL Erlenmeyer flasks
was inoculated with 2 mL of the inoculum (A600 0.8; cell
count 2.5 × 108 /mL) obtained from 12 h old shake cultures
of Bacillus sp. I-3 in nutrient broth and incubated on new
Brunswick environmental shaker (150 rpm) for 72 h at 37 ◦ C.
The flasks were analyzed for growth (A600 ) and pH at regular
intervals of time. The culture was centrifuged at 10,000 × g
for 20 min at 4 ◦ C and the cell-free supernatant was used as
the enzyme source.
2.2.1. Enzyme assay
The activity of ␣-amylase was determined at 60 ◦ C by
mixing 0.25 mL of appropriately diluted enzyme source with
0.25 mL of 0.2% (w/v) soluble starch (E. Merck, India) dis-
solved in 0.1 M phosphate buffer, pH 7.0. The residual starch
was determined after 10 min [17]. One unit of ␣-amylase ac-
tivity was defined as the amount of enzyme that caused 10%
reduction in the starch–iodine color, under the assay condi-
tions. All values given are averages of three determinations.
2.3. Optimization of raw starch digesting α-amylase
production by Bacillus sp. I-3
The enzyme production was optimized by studying the
effect of various cultural and environmental variables, indi-
vidually, in terms of various carbon, nitrogen, metal ions,
vitamins and amino acids in the basal medium, whose com-
position is described in Section 2.2, after 48 h of incubation of
shake cultures (150 rpm) at 37 ◦ C, pH 7.0, unless, otherwise
 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734 725

Table 1 Table 1 (Continued )

Effect of different cultural and environmental factors on ␣-amylase produc-
Parameter Enzyme activity
tion by Bacillus sp. I-3 in submerged fermentation
(U/mL)
Parameter Enzyme activity
B-complex 185
(U/mL)
Amino acids (0.01%, w/v)
Carbon sources (1%, w/v)
Phenylalanine 176
Sugars
Threonine 165
Fructose 219
Glutamic acid 116
Galactose 324
Methionine 114
Glucose 51
Tryptophan 97
Glycerol 27
Leucine 93
Lactose 310
Lysine 72
Maltose 81
Valine 68
Mannitol 19
Cysteine 68
Sorbitol 53
Tyrosine 68
Sucrose 16
Arginine 67
Xylose 11
Glycine 59
Gelatinised starchy sources
Ornithine 59
Soluble starch 90
Histidine 54
Corn starch 81
Aspartic acid 33
Potato starch 181
Proline 31
Rice starch 51
Alanine 22
Wheat starch 218
Serine 6
Rice flour 15
Wheat bran 22 Inoculum size (%, v/v; cell count of 2.5 × 108 /mL)
Wheat flour 37 6 64
Raw starchy sources 7 73
Corn starch 35 8 78
Potato starch 172 9 82
Rice starch 53 10 90
Wheat starch 55 11 80
Rice flour 82
Incubation temperature (◦ C)
Wheat bran 51
30 67
Wheat flour 88
37 90
Nitrogen sources (1%, w/v) 42 84
Beef extract 383 50 80
Corn steep liquor 216 60 44
Malt extract 5
pH of the medium
Bacto peptone 87
4.0 10
Peptone M 28
4.5 31
Peptone type I 309
5.0 75
Proteose peptone 49
5.5 76
Soyabean meal 417
6.0 77
Soyapeptone 31
6.5 84
Tryptone 6
7.0 90
Yeast autolysate 33
7.5 81
Yeast extract 18
8.0 13
Metal salts (10 mM)
The basal medium having 1% soluble starch, 1% peptone, 1% beef extract
LiSO4 215
and 0.5% NaCl with pH 7.0 was used at 37 ◦ C as a control, which pro-
MgSO4 ·7H2 O 12
duced 90 U/mL. The C, N and mineral components of the basal medium
CaCl2 ·2H2 O 178
were replaced one after the other with other compounds keeping the other
FeCl3 30
ingredients same while the role of vitamins, amino acids, inoculum size,
MnSO4 ·H2 O 22
temperature and pH was evaluated using the basal medium.
CuSO4 ·5H2 O 12
HgCl2 9
ZnSO4 7
AgCl3 7 by replacing soluble starch with different sugars, gelatinized
Vitamins (0.01%, w/v) and raw natural crude starch sources (1%, w/v). Similarly,
Thiamine 100 the effect of nitrogen sources was studies by replacing pep-
Riboflavin 74 tone and beef extract with various nitrogen sources at a final
Pyridoxine 18 concentration of 1% (w/v) while the effect of salts was eval-
Ca pantothenate 104
uated by substituting NaCl with various metal salts (10 mM).
Nicotinic acid 212
The inductive effect of various vitamins and amino acids on
the enzyme production was studied by supplementing them
726 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734
separately at a level of 0.01% (w/v) in the basal medium.
The optimal levels of various nutrients were determined sep-
arately by varying their concentrations in the basal medium.
The inoculum size was standardized by evaluating the ef-
fect of varying inoculum size for seeding the basal medium.
The effect of incubation temperature and initial pH on en-
zyme production was studied by incubating the seeded basal
medium at different temperatures and initial pHs. The syner-
gistic effect of all the factors was also studied by employing
the optimal concentrations of various nutrients giving the best
results in the individual studies and an optimized medium was
worked out.
2.4. Partial purification of the α-amylase preparation
Since, the enzyme preparation was found to readily and
strongly bind to raw potato starch and digest it in signifi-
cantly short time, raw starch adsorption-hydrolysis process
was attempted for the purification of ␣-amylase from Bacil-
lus sp. I-3. The enzyme preparation was adsorbed onto the
raw potato starch granules and was then subjected to hy-
drolysis at 60 ◦ C. The hydrolyzed sample was centrifuged to
obtain the residual enzyme in the supernatant, which was di-
alyzed against 0.1 mM phosphate buffer, pH 7.0. The enzyme
was then precipitated with ammonium sulphate at a satura-
tion value of 60% and was again dialyzed against 0.1 mM
Tris–HCl buffer.
2.5. Characterization of partially purified α-amylase
preparation
The kinetics studies on the partially purified ␣-amylase
obtained was carried out in terms of the temperature and pH
versus activity and stability profiles, effect of various metal
ions on activity and by studying the affinity of it towards raw
potato starch granules.
2.5.1. Activity versus temperature and thermostability
profiles
The effect of temperature on amylase activity was stud-
ied by assaying the enzyme at different temperatures in
the range of 37–100 ◦ C at pH 7.0 using phosphate buffer
(0.1 M). The thermostability of the enzyme was tested by
pre-incubating the enzyme preparation, without and with
10 mM CaCl2 ·2H2 O at varying temperatures ranging from
50 to 100 ◦ C for 3.5 h and determining the residual activity
at regular intervals of 0.5 h.
2.5.2. Activity versus pH and stability profiles
pH activity and stability profiles were studied in a pH
range of 4.0–8.0 using different buffers at 0.1 M concen-
trations. For stability studies, one volume of enzyme was
mixed with respective buffers and incubated up to 60 min.
The residual activity was determined at normal assay condi-
tions.
2.5.3. Effect of metal salts on amylase activity
The effect of various metal ions was studied by supple-
menting these, separately, at a concentration of 1 mM in the
reaction mixture under normal assay.
2.6. Application of partially purified enzyme
preparation from Bacillus sp. I-3 in the hydrolysis of raw
potato starch
A reaction mixture containing 10 mL of 1% raw potato
starch was incubated with 10 U of enzyme preparation per
mg of starch at temperatures ranging from 40 to 90 ◦ C and
the degree of hydrolysis was determined after 3 and 5 h of
incubation. The starch granules recovered from the reaction
mixture were analyzed for SEM studies to confirm the extent
of degradation of raw potato starch granules. For SEM stud-
ies, the samples, both the untreated and ␣-amylase treated
raw potato starch granules, were centrifuged at 10,000 rpm
for 5 min and the pellet was washed twice with pure ethanol
then with t-butyl alcohol followed by centrifugation and dry-
ing. These were then attached to SEM stub with silver plate
[20]. The mounted samples were spatter coated with gold
using fine coat, JEOL ion sputter, model JFC-100 and then
scanned at an operating voltage of 10 kV.
Optimization of the raw starch hydrolysis by the enzyme
preparation was also attempted by varying the starch concen-
tration and the enzyme dose in the reaction mixture. Effect
of raw potato starch concentration was studied by varying
its concentration from 1 to 15% in the reaction mixture con-
taining 10 mL of 1000 U enzyme preparation at 70 ◦ C. To
determine the extent of starch hydrolysis, glucose estimation
was done after 12 h of incubation.
The end products of raw potato starch hydrolysis after 12 h
of incubation at 70 ◦ C were withdrawn and identified to ascer-
tain the extent of hydrolysis by ascending paper chromatog-
raphy with the solvent system of n-butanol–pyridine–water
(6:4:3) and a detection reagent comprising 100 mL of ace-
tone, 0.66 g of phthalic acid and 930 mL of acetone [21].
Effect of the enzyme concentration was studied was vary-
ing its concentration from 0.5 to 2 U/mg raw starch in the
reaction mixture containing 10 mL of 12.5% of raw potato
starch granules at 70 ◦ C. Similarly, the glucose estimation
was done after 24 h of incubation to determine the extent of
hydrolysis of starch.
3. Results and discussion
Although most of the reports indicate fungi belonging to
Aspergillus and Rhizopus to be the good producers of amy-
lases capable of digesting raw starches, few recent studies
suggest the possibility of raw starch degradation by bacterial
␣-amylases too [4,7]. Further, most of the reports published
so far indicate the digestion of raw starch granules at around
1% concentration by these enzymes at ordinary temperatures
(30–50 ◦ C) and at a very slow rate. As the starch processing
 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734
Fig. 1. Pattern of growth, pH and extracellular ␣-amylase production during
submerged fermentation with Bacillus sp. I-3 at 37 ◦ C, pH 7.0. The basal
medium used for enzyme production had 1% soluble starch, 1% peptone,
1% beef extract and 0.5% NaCl with pH 7.0.
industry employs the mashes containing around 15% starch
for the conventional conversions, there is, thus, an urgent need
to explore thermostable raw starch digesting amylases which
can be used effectively at around 70 ◦ C for the efficient hy-
drolysis of much higher concentrated mashes of raw starches
quickly. The bacterial strain of Bacillus sp. I-3 used in the
present study was isolated from local soil. It was a mesophile
having a growth temperature in the range of 30–60 ◦ C with
an optima at 37 ◦ C and pH 5.0–7.5 with an optima at pH
7.0. It produced 90 U/mL of thermostable ␣-amylase, which
showed very high affinity towards raw potato starch granules
(94% adsorption) and brought about 90% hydrolysis of 1%
solution of the starch, at 60 ◦ C after 6 h.
3.1. Pattern of growth and α-amylase production
The enzyme production by most of the organisms is
growth associated and is induced by the presence of the sub-
strate in the medium. Its levels generally increase during the
exponential phase and a peak is attained towards the end of
this phase or during the stationary phase. After the peak, the
enzyme levels tend to decline either due to the increase in
the glucose concentration resulting from the degradation of
starch, or due the rise of protease levels in the environment
or due to the drastic change in pH levels [22–24]. Bacillus
sp. I-3 grew lograthimatically from 2–24 h and then entered a
stationary phase, of a duration of nearly 30 h, before entering
the decline phase (Fig. 1). The amylase production pattern in
this organism also indicates that the induction of the amylase
took place during the log phase in the presence of starch and
the maximum yield is obtained, after 48 h of incubation, in
the mid stationary growth phase which is probably due the re-
lease of the all the intracellular fractions of the enzyme during
the stationary phase because of cell lysis. On further incuba-
tion, the enzyme yield declined gradually to give 40 U/mL at
the end of 72 h. The pH of the culture broth showed a decline
from 7.0 to 4.85 after 48 h.
727
3.2. Optimization of α-amylase production during SmF
Amylase production is known to be induced by a variety of
carbohydrates, nitrogen compounds and minerals [25]. In or-
der to achieve high enzyme yield, efforts are made to develop
a suitable medium and to work out the favorable environmen-
tal conditions for the proper growth and maximum secretion
of enzyme. The medium is generally selected on the basis
of economically feasible components. Development of such
medium requires the right selection of cheaper and readily
available components. The growth and enzyme production of
any organism are greatly influenced by both environmental
conditions and the nutrients available in the growth medium.
Some nutrients favor the growth while other induces better
yields of amylase.
Of the various carbon sources used in the present study,
galactose at the concentration of 1% proved to be the best
inducer for ␣-amylase production (324 U/mL) by Bacillus
sp. I-3 (Table 1; Fig. 2a) while soyabean meal at 3% concen-
tration proved to be the best nitrogen source for enzyme pro-
duction, revealing an activity of 471 U/mL (Table 1; Fig. 2b).
Strains of B. stearothermophilus and B. amylolyticus secrete
maximum in a medium supplemented with 1% peptone, 0.5%
yeast extract and 0.5% maltose under vigorous shaking con-
ditions [26]. Hamilton et al. [3] noted maximum raw starch
digesting ␣-amylase yield in Bacillus sp. in a medium con-
taining lactose as the carbon source and yeast extract as the
nitrogen source. Several earlier reports also indicate soyabean
meal as the best nitrogen source for amylase production in
Sacharomycopsis capsularis [23], Botryodiploidia theovro-
mae [27], Bacillus sp. SN-1 [25] and Bacillus sp. AS-1
[28].
The enzyme yield exhibited a gradual rise with increased
LiSO4 concentration up to 20 mM in the production medium
revealing 246 U/mL (Table 1; Fig. 2e). The requirement of
some vitamins has already been observed for the enzyme
production in different organisms. Nicotonic acid at 0.01 %
concentration proved to be the most favorable for the enzyme
production (Table 1; Fig. 2c). Also, among the various amino
acids supplemented in the production medium, phenylalanine
at a concentration of 0.02% (Fig. 2d) and inoculum size of
10% proved to be the best for the amylase production in the
present study (Table 1).
Role of temperature in the SmF growth is very impor-
tant and productions of enzymes or metabolites are usually
sensitive to the temperature. Enzyme synthesis occurred at
temperature between 30 and 60 ◦ C with an optimum of 37 ◦ C
(Table 1). pH is one another among the other most important
environmental factors for any fermentation process. Each mi-
croorganism has a pH range for its growth and activity with an
optimum pH range. Enzyme synthesis occurred at a wide pH
range from 4.0 to 8.0 with an optimum of 7.0 (Table 1). When
synergistic effect of the exogenous compounds required by
the organism grown at the optimum temperature and pH was
studied in the optimized media, the ␣-amylase yield touched
642 U/mL (Fig. 3).
728 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

Fig. 2. Effect of varying concentrations of different nutrients on ␣-amylase production by Bacillus sp. I-3. The basal medium used for optimization had 1%
soluble starch, 1% peptone, 1% beef extract and 0.5% NaCl with pH 7.0 and was incubated at 37 ◦ C for 48 h as shake cultures. (a) Soluble starch was replaced
with varying concentrations of galactose; (b) peptone and beef extract were replaced with varying concentrations of soyabean meal; (c) basal medium was
supplemented with varying concentrations of nicotinic acid; (d) basal medium was supplemented with varying concentrations of phenylalanine; and (e) NaCl
was replaced with varying concentrations of lithium sulphate.

3.3. Partial purification and characterization of
α-amylase preparation
As the raw starch digesting amylases have a lot of po-
tential in the bulk processing of starch at the industrial scale
where crude enzymes are generally exploited taking into con-
sideration the high cost of enzyme purification. However, it
is significant to obtain enzymes with higher specific activity
for their kinetic characterization. Traditionally, the purifica-
tion of amylases from fermentation media has been done in
several steps which include centrifugation of culture filtrate
after extraction from solid media, selective concentration of
the supernatant by ultrafiltration and selective precipitation
of the enzyme by ammonium sulphate or organic solvent such
as ethanol, in the cold, followed by affinity or ion-exchange
chromatography and gel filtration [29]. Several one-step
or two-step rapid purification methods including acetone
precipitation [30], ammonium sulphate precipitation [31],
hydrophobic interaction and ion-exchange chromatography
[32,33] have been developed for the partial purification of
amylases, but a different protocol has been used in the present
study to partially purify the enzyme preparation. Since, the
 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734
Fig. 3. ␣-Amylase yields by submerged cultures of Bacillus sp. I-3 on var-
ious media at 37 ◦ C, pH 7.0. Basal medium containing 1% soluble starch,
1% peptone, 1% beef extract and 0.5% NaCl with pH 7.0 was incubated at
37 ◦ C for 48 h as shake cultures. Optimized medium containing 1% galac-
tose, 3% soyabean meal, 0.02% phenylalanine, 0.01% nicotinic acid and
20 mM lithium sulphate with pH 7.0 was incubated at 37 ◦ C for 48 h as
shake cultures.
enzyme preparation was found to readily and strongly bind to
raw potato starch and digest it in significantly short time, raw
starch adsorption–desorption process was attempted for the
purification of ␣-amylase from Bacillus sp. I-3. The enzyme
was allowed to get adsorbed onto the raw potato granules.
Desorption of the adsorbed enzyme was tried by using vari-
ous elutants (0.1 M phosphate buffer pH 5.0, 5.5, 6.0, 6.5, 7.0
and 7.5 and sodium chloride 5, 10, 15 and 20 mM), but none
of these could remove the enzyme adsorbed on the raw potato
starch granules. In a similar study, Saha and Shen [34] tried
the elution of the adsorbed enzyme from raw starch by using
various buffers of different pH including raising temperature
and changing pH. These conditions, however, had no signifi-
cant effect on elution of the enzyme. On the other hand, Lin et
al. [35] purified a raw starch degrading amylase from culture
supernatant of Bacillus sp. TS-23 by adding raw corn starch
and the enzyme was eluted from the raw starch with an elu-
tion solution containing maltose in Tris–HCl buffer. In order
to elute the adsorbed enzyme, hydrolysis of the starch gran-
ules was carried out at 60 ◦ C and the mixture was dialyzed
to remove the glucose from it. It was then concentrated by
precipitation with ammonium sulphate and again dialyzed.
When defining the proposed unit of activity for any en-
zyme, the International Unit of Biochemistry stated that reac-
tion conditions should be specified and optimal. This implies
that enzymes activities are only valid within a range of physi-
cal properties. Therefore, optimum conditions for producing
maximum enzyme activities need to be determined. Activity
and stability data of enzyme is also useful in screening for
suitable new enzymes for a particular process, in the design
of multi-enzyme processes and in the characterization of en-
zyme. Every enzyme has an optimum temperature and pH
at which it has maximum activity. Metal ions, monovalent,
729
Fig. 4. Temperature and pH vs. activity profiles of ␣-amylase from Bacil-
lus sp. I-3. Optimized medium containing 1% galactose, 3% soyabean meal,
0.02% phenylalanine, 0.01% nicotinic acid and 20 mM lithium sulphate with
pH 7.0 was incubated at 37 ◦ C for 48 h as shake cultures for enzyme pro-
duction. For studying the effect of temperature on enzyme activity, the assay
pH was kept constant at 7.0 and for studying the effect of pH on enzyme
activity, the assay temperature was kept constant at 60 ◦ C.
divalent or polyvalent also exert a significant influence on the
activity of an enzyme.
As most of the industrial processes involving ␣-amylases
operate at high temperatures exceeding 50 ◦ C, due to higher
reaction rates at these temperatures, the thermostable raw
starch digesting amylases are, thus, of great importance
for starch hydrolysis. In this regard, the ␣-amylase from
Bacillus sp. I-3, which was optimally active at 70 ◦ C and
displayed 97, 93 and 41% of its peak activity at 80, 90 and
100 ◦ C (Fig. 4), could be a good candidate for the efficient
and quick hydrolysis of raw starches. The enzyme revealed
a high thermostability, retaining about 94% residual activity
at 70 ◦ C in presence of 10 mM CaCl2 ·2H2 O even after 3.5 h
(Fig. 5). The half lives at 80 and 90 ◦ C improved signifi-
cantly to 2.5 and 0.5 h, respectively, with supplementation of
10 mM CaCl2 ·2H2 O (Fig. 5). The stabilizing effect of Ca2+
on thermostability of the enzyme can be explained due to the
salting out of hydrophobic residues by Ca2+ in the protein,
thus causing the adoption of a compact structure [33] and
has also been reported in other organisms [32,36,37].
Many industrial processes involving enzymatic processes
do not have an adequate pH control and the pH usually fluc-
tuates around the required value. Thus, pH optima data deter-
mined under the controlled conditions of the laboratory need
to be applied carefully in processes. It is important to know
the pH activity curve in quantitative detail for the particular
730 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

Fig. 5. Thermostability profiles of ␣-amylase from Bacillus sp. I-3 at different temperatures. Optimized medium containing 1% galactose, 3% soyabean meal,
0.02% phenylalanine, 0.01% nicotinic acid and 20 mM lithium sulphate with pH 7.0 was incubated at 37 ◦ C for 48 h as shake cultures for enzyme production.
The enzyme assays were carried out at 60 ◦ C, pH 7.0.

enzyme substrate system under consideration, including the
other prevailing physical conditions under which the reaction
is to take place so that the maximum potential of the enzyme
can be attained. Further, in most of the cases, ␣-amylases
and amyloglucosidases are used together for the complete
hydrolysis of the starch as ␣-amylases generally do not bring
about the complete hydrolysis of starch, the same may be
true for raw starch digesting ␣-amylases. To exploit the op-
portunity of synergism, the temperature and pH optima of the
␣-amylases and AMGs should be compatible. Thermostable
␣-amylases currently in use are not stable at lower pH val-
ues where saccharification of liquefied starch is carried out
[38]. As a result, up to now, starch is liquefied at a pH around
7.0. The use of liquefying amylases that are active and sta-
ble around the saccharification pH is economically attractive
for it could avoid or reduce the use of acid to lower the pH
from liquefying to saccharifying range [38]. This again will
minimize the use of ion-exchange media used during down-
stream processing [39]. Moreover, the use of ␣-amylases that
operate at lower pH values reduce the formation of unwanted
products, such as maltulose, which is usually produced at
higher operation pH [40]. The ␣-amylase of Bacillus sp. I-
3 showed pH optima at pH 7.0 and displayed 96, 97, and
98% of its peak activity at pH 5.5, 6.0 and 6.5, respectively
(Fig. 4) where most of the amyloglucosidases are also active
[40]. The enzyme was also found to be almost 80% stable
at pH range of 5.0–5.5 and almost 90% stable at pH range
of 6.0–7.0 (Fig. 6) and, thus, can also be a potential candi-
 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734 731

Fig. 6. pH stability profiles of ␣-amylase from Bacillus sp. I-3. Optimized

Fig. 7. Effect of various metal salts and EDTA on the activity of ␣-amylase
medium containing 1% galactose, 3% soyabean meal, 0.02% phenylalanine,
preparation from Bacillus sp. I-3. Optimized medium containing 1% galac-
0.01% nicotinic acid and 20 mM lithium sulphate with pH 7.0 was incubated
tose, 3% soyabean meal, 0.02% phenylalanine, 0.01% nicotinic acid and
at 37 ◦ C for 48 h as shake cultures for enzyme production. The enzyme assays
20 mM lithium sulphate with pH 7.0 was incubated at 37 ◦ C for 48 h as
were carried out at 60 ◦ C, pH 7.0.
shake cultures for enzyme production. The enzyme assays were carried out
at 60 ◦ C, pH 7.0.

date for synergistic use along with amyloglucosidase for the

complete hydrolyisis of raw starchy mashes. 3.4. Applications of amylase preparation from Bacillus
The activity of various enzymes is influenced by the pres- sp. I-3 in the hydrolysis of raw potato starch
ence of metal ions. Some require certain metal ions as cofac-
tors and thus called metalloproteins while others are inhib- Raw potato starch granules are the most resistant to en-
ited by their presence in the reaction mixture. The enzyme zymatic hydrolysis because of the larger size of these gran-
activity of ␣-amylase from Bacillus sp. I-3 got significantly ules [26]. The potential application of raw starch digesting
promoted in the presence of FeSO4 , CuSO4 and MnSO4 sug- ␣-amylase preparation from Bacillus sp. I-3 was evaluated
gesting the enzyme to be a metalloprotein needing a cofactor
for its maximum activity (Fig. 7). This is also corroborated
by the inhibitory effect of EDTA on the enzyme activity.
Binding of the raw starch digesting amylases to the starch
granules is usually affected by a C-terminal domain within
the enzyme, which has been shown to be necessary for degra-
dation of granular starch by mould glucoamylases [26]. How-
ever, in case of bacterial ␣-amylases, binding to starch gran-
ules is not an obligatory requirement for the hydrolysis of
the same. Kelly et al. [7] found that raw starch digesting ␣-
amylase of Bacillus sp. IMD 370 showed no adsorbability to
any kind of raw starch, at any pH, therefore, showing that ad-
sorbability was apparently not necessary for raw starch diges-
tion by bacterial enzyme. However, Itkor et al. [4] observed
that the amylase preparation was more than 98% adsorbed
onto both raw corn and potato starches. Similar results were
also observed by Saha et al. [41] and Gautam and Gupta
[42] where the bacterial enzyme preparation could adsorb
onto raw corn starches. Study on the affinities of the enzyme
preparation from Bacillus sp. I-3 towards raw potato starch
Fig. 8. Pattern of 1% raw starch hydrolysis with ␣-amylase preparation from
granules revealed that the enzyme had a very strong affin-
Bacillus sp. I-3 after 3 and 5 h at different temperatures. Optimized medium
ity for the same. It showed 94% adsorption on to the raw containing 1% galactose, 3% soyabean meal, 0.02% phenylalanine, 0.01%
potato starch granules indicating the potential usefulness of nicotinic acid and 20 mM lithium sulphate with pH 7.0 was incubated at
the enzyme for the hydrolysis of raw potato starch granules. 37 ◦ C for 48 h as shake cultures for enzyme production.
732 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734

Plate 1. Scanning electron micrograph showing the smooth and intact sur-
face of untreated raw potato starch granules.

by studying the extent of hydrolysis of raw potato starch

granules. It was observed that the enzyme preparation could
substantially hydrolyze the raw starch granules in a short du-
ration of time in a temperature range of 60–90 ◦ C indicating
thermostable nature of the enzyme. Maximum hydrolysis of
the raw potato starch granules at 1% concentration occurred
at 70 ◦ C with 90 and 89% conversion to glucose was achieved
after 5 and 3 h, respectively, indicating that most of the hy-
drolysis occurred during the earlier 3 h (Fig. 8). This is in
contrast to the various published reports where it was seen
that the potato starch is least susceptible to hydrolysis even
after prolonged incubation of days [26].
The scanning electron microscopy of the treated starch
granules indicates that the granules were more or less com-
pletely damaged, thus revealing that the granules were almost
completely hydrolyzed by the enzyme preparation. Plate 1
shows that the surface of untreated starch granules in the raw Fig. 9. Hydrolysis of: (a) different concentrations of raw potato starch with
form was smooth, whereas Plate 2 shows that the pores of 100 U/mL of ␣-amylase preparation from Bacillus sp. I-3; and (b) 12.5% raw
the hydrolyzed raw starch potato granules were randomly potato starch with varying doses of enzyme preparation. Optimized medium
containing 1% galactose, 3% soyabean meal, 0.02% phenylalanine, 0.01%
distributed due to the breakdown of the granules by the en-
nicotinic acid and 20 mM lithium sulphate with pH 7.0 was incubated at
37 ◦ C for 48 h as shake cultures for enzyme production. Hydrolysis in both
the cases was carried out at 70 ◦ C.

zyme. This strongly supports the efficient action of amylase

Plate 2. Scanning electron micrograph showing the deformed and ruptured
raw potato starch granules after treatment with ␣-amylase preparation from
Bacillus sp. I-3.
preparation towards raw potato starch granules.
With a constant enzyme dose, as the concentration of
starch was increased, conversion efficiency to glucose
decreased. With an increase in starch concentration, the
hydrolysis remained almost constant (80–90%) up to 12.5%
starch concentration. At 15% concentration, the glucose
formation dropped to 64% after 12 h of incubation (Fig. 9a).
When the enzyme dose was varied from 0.5 to 2 U/mg
and incubated with 12.5% concentration of raw potato
starch, almost similar pattern of hydrolysis was observed
at all the enzyme doses. With an enzyme dose of 0.5, 1.0
and 2 U/mg, 80, 82 and 82% hydrolysis was observed,
respectively (Fig. 9b). This strongly suggests that at such a
low concentration of enzyme dose (0.5 U/mg) is sufficient
 N. Goyal et al. / Enzyme and Microbial Technology 37 (2005) 723–734
Plate 3. Two dimensional paper chromatogram of the end products of raw
potato starch after hydrolysis with ␣-amylase preparation from Bacillus sp.
I-3.
to bring about an appreciable hydrolysis of raw potato
starch at a reasonably high concentration of 12.5%. Paper
chromatography results revealed that the main end products
of hydrolysis carried out in the present study were a mixture
of glucose, maltose and maltotriose (Plate 3).
In conclusion, the present study has yielded a highly
thermostable bacterial raw starch digesting ␣-amylase by
submerged fermentation with a natural isolate of Bacillus sp.
I-3. The enzyme was active over a broad temperature range
of 50–100 ◦ C with optima at 70 ◦ C and a relative activity
of 93% at 90 ◦ C. This also displayed a reasonably good
thermostability profile with 94% residual activity after 3.5 h
of incubation at 60 or 70 ◦ C. The enzyme revealed a high
affinity towards raw potato starch and got almost completely
adsorbed onto it leading to good degree of hydrolysis at
various temperatures. Almost all raw starch digesting fungal
glucoamylases known to date adsorb to raw starch. On the
other hand, raw starch digesting bacterial ␣-amylases greatly
vary in their ability to bind to starch granules [12,21,43]. The
amylase from Bacillus sp. I-3 completely got adsorbed to
raw potato starch granules. This is a very interesting property
for it could offer the opportunity of developing an affinity
procedure for the isolation and concentration of the enzyme
directly from the culture broth. In view of these properties,
the ␣-amylase from Bacillus sp. I-3 appears to a good can-
didate for the direct hydrolysis of potato starches, omitting
an energy intensive gelatinization step, in a concentration
range of 1–15% using very low doses of the enzyme, i.e.
0.5 U/mg of starch for various biotechnological applications.
733
Since this study was purely on a laboratory scale at 100 mL
Erlenmeyer flask level, there is a need to scale-up the
production and application trials of the enzyme before any
conclusion can be drawn for the commercial utilization of
this enzyme. It is further pointed out that though the raw
potato starch digesting ␣-amylase from Bacillus sp. I-3 can
alone bring about significant hydrolysis of potato starch,
it may also be tried in synergism with some glucoamylase
preparation to bring about a complete hydrolysis.
Acknowledgement
The financial support provided by the Department of
Biotechnology (DBT), Government of India, is highly ac-
knowledged.
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