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Bioresource Technology xxx (2007) xxxxxx

Highly thermostable, thermophilic, alkaline, SDS and chelator

resistant amylase from a thermophilic Bacillus sp. isolate A3-15
Burhan Arikan

Art and Science Faculty, Department of Biology Molecular and Microbiology Laboratory, University of Cukurova, 01330 Adana, Turkey
Received 29 March 2006; received in revised form 1 June 2007; accepted 1 June 2007

Abstract
A thermostable alkaline a-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in
amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8 mm zone diameter in agar medium) on starch agar
medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDSPAGE electrophoresis,
which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 C with
an optimum of 60 C on petri dishes. The partial purication enzyme showed optimum activity at pH 11.0 and 70 C. The enzyme was
highly active (95%) in alkaline range of pH (10.011.5), and it was almost completely active up to 100 C with 96% of the original activity
remaining after heat treatment at 100 C for 30 min. Enzyme activity was enhanced in the presence of 5 mM CaCl2 (130%) and inhibition
with 5 mM by ZnCl2, NaCl, Nasulphide, EDTA, PMSF (3 mM), Urea (8 M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%,
80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.011.0 and 60 C for 24 h. So our result showed that the
enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.
2007 Elsevier Ltd. All rights reserved.
Keywords: Thermophile Bacillus sp.; a-Amylase; Highly thermostable; Alkaliphilic; Chelator resistant

1. Introduction
Thermophilic microorganisms are adapted to thrive at
temperatures above 60 C. They are a source of interesting
enzymes that are both thermoactive and thermostable
(Niehaus et al., 1999). The enzymes that have been isolated
recently from these eotic microorganisms show unique features, are extremely thermostable and usually resistant
against chemical denaturants such as detergents, chaotrophic agents, organic solvents and extremes of pH (Jorgensen et al., 1977).
Amylases are among the most important enzymes and
are of great signicance in present-day biotechnology.

Tel.: +90 322 3386084x2571; fax: +90 322 3386070.

E-mail addresses: barikanus@yahoo.com, arikan@cu.edu.tr

Although they can be derived from several sources, such

as plants, animals and microorganisms; enzymes from
microbial sources generally meet industrial demands. The
spectrum of amylase application has widened in many
other elds, such as clinical, medical and analytical chemistries, as well as their widespread application in starch saccharication and in the textile, food, brewing and distilling
industries (Pandey et al., 2000).
Because of the industrial importance of amylases, there
is ongoing interest in the isolation of new bacterial strains
producing amylases suitable to new industrial applications,
such as alkaline amylase for the detergent industry (McTigue et al., 1995).
Amylases constitute a class of industrial enzymes having
approximately 25% of the enzyme market (Rao et al.,
1998). It is desirable that a-amylases should be active at
the high temperatures of gelatinization (100110 C) and

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doi:10.1016/j.biortech.2007.06.019

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liquefaction (8090 C) to economize processes; therefore,

there has been a need and continual search for more thermophile and thermostable a-amylases (Sidhu et al., 1997).
Several Bacillus sp. and thermostable Actinomycetes
including Thermomonospora and Thermoactinomyces are
versatile producers of the enzymes (Ben et al., 1999). The
genus Bacillus produces a large variety of extracellular
enzymes of which amylases and proteases are of signicant
industrial importance. An highly thermostable and alkaline
a-amylases is available from the mesophile Bacillus sp. PN5
(Saxena et al., 2007). The thermophilic bacterium B. stearothermophylus oers an alternative for commercial production of thermostable a-amylases. The advantages of using
thermostable amylases in industrial processes include the
decreased risk of contamination and cost of external cooling, a better solubility of substrates, a lower viscosity
allowing accelerated mixing and pumping (Lin et al.,
1998). The development of saccharifying amylolytic
enzymes that are active at high temperatures (90 C) would
directly benet the starch-processing industries. However,
running a-amylase production processes at higher temperatures will require new process design and improved
knowledge of thermophilic bacteria (Leveque et al.,
2000). Processes using thermophiles still lack the maturity
of classical processes with mesophilic bacteria and yeasts
(Coolbear et al., 1992). Alkaliphilic Bacillus strains often
produce enzymes active at alkaline pH, including alkaline
a-amylase, protease and carboxymethylcellulase (Horikoshi, 1996).
This article deals with the partial purication and some
properties of the amylase produced by thermophile and
alkaliphilic Bacillus sp. isolate A3-15 and their applications
such as, new detergent formulation, textile, starch liquefaction and other industrial area.
2. Methods

2.2. Enzyme production

The organisms were propagated at 60 C for 2 days in
100 ml of an M9 minimal medium, containing 1% soluble
starch (Merck), placed in 1000 ml asks, with shaking on
a shaker (250 rpm/min). The initial pH of the medium
was about 9.0. After removal of cells by centrifugation
(10,000 rpm, 20 min) at 4 C, the supernatant was used
for partial purication (McTigue et al., 1995).
2.3. Partial purication of amylase
Bacillus sp. A3-15 strain was grown 2 days. Cells were
removed by centrifugation at 8000 rpm for 20 min at
4 C. The clear supernatant was concentrated (250 ml),
with ethanol previously chilled to 20 C was added drop
wise at 4 C with continuous stirring of 75%, and solution
was left at 20 C for 24 h. The precipitate was recovered
by centrifugation at 13,000 g for 20 min at 4 C. It was then
resuspended in a minimum of phosphate (100 mM) at pH
7.0 (McTigue et al., 1995; Burhan et al., 2003).
2.4. Enzyme assay
The relative amylase activity was assayed by adding
0.5 ml of enzyme to 0.5 ml soluble starch (1% v/v) in
100 mM BoraxNaOH buer pH 11.0, and incubating at
60 C for 60 min. The reaction was stopped by the addition
of 2 ml of 3,5-dinitrosalicylic acid reagents and A550nm was
measured in a Cecil 5500 spectrophotometer. One unit of
amylase activity was dened as the amount of enzyme that
released one micromole of reducing sugar equivalent to
glucose per minute under the assay condition (Bernfeld,
1955).
2.5. Eects of pH and temperature on activity
and stability

2.1. Microorganism and culture conditions

Bacillus sp. A3-15 was isolated from poultry manure
compost samples collected in Adana, Turkey. To select of
the gram-positive spore-forming bacteria Bacillus sp., compost was pasteurised at 80 C for 10 min (Hamilton et al.,
1999). A3-15 was maintained and grown as described by
McTigue (McTigue et al., 1995). The identication of isolated bacteria was various morphological and biochemical
tests (Sodhi et al., 2005). This organism was found to produce an amylase on agar plates, a media composed with
Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g, MgSO4 0.24 g,
CaCl2 0.01 g, peptone 3 g, 1% (wt/vol) soluble starch
(Merck), and Agar 15 g (Burhan et al., 2003). The initial
pH was adjusted with NaOH to pH 9.0 after autoclaving
(Milner et al., 1997). The organism was propagated at different temperatures (2065 C) and pHs (6.012.0). Amylase production was detected after ooding the plates
with iodine solution (Burhan et al., 2003; Saxena et al.,
2007).

The optimal temperature for activity was determined by

assaying activity between 60 and 100 C for 60 min (Egas
et al., 1998; Lo et al., 2001). Enzyme of pH optima, pH
and heat stability were assayed under standard conditions
at optimum enzyme temperature. The eect of pH on amylase activity was performed at 70 C in 100 mM Naphosphate buer (pH 6.58.0), GlycineNaOH buer (pH 8.5
10.5) and BoraxNaOH buer (pH 11.013.0) for 60 min,
respectively (McTigue et al., 1995).
For the measurement of pH stability, the enzyme was
pre-incubated at 60 C for 24 h at pH 9.0, 10.0 and 11.0
in buer solutions. The residual activity was determined
under the standard condition at optimum temperature
for 60 min.
Thermostability of the amylase was performed by
enzyme samples at optimum pH for 30 min pre-incubating
at temperatures between 60 and 100 C. The remaining
activity was determined by incubating enzymes at optimum
temperature for 60 min (Egas et al., 1998).

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2.6. SDSPAGE zymogram

For determination of molecular weight, enzyme preparations and known molecular weight markers were subjected to electrophoresis with the use of homogenized
10% acrylamide gel. 0.2% soluble starch was incorporated
into the separating gel prior to the addition of ammonium
persulphate and polymerisation. After the electrophoresis,
the gel was stained for 1 h with Comassie Blue R 250 dye
in methanolacetic acidwater solution (4:1:5, by volume)
and destained in the same solution without dye (Maniatis
et al., 1982; Bollag et al., 1996).
For zymogram of amylase activity, SDS was removed
by washing the gel at room temperature in solutions containing 50 mM Na2HP04, 50 mM NaH2P04 (pH 7.2), isopropanol 40% for 1 h and 50 mM Na2HP04, 50 mM
Na2HP04 (pH 7.2) for 1 h, respectively. Renaturation of
enzyme proteins was carried out by keeping the gel overnight in a solution containing 50 mM Na2HP04, 50 mM
Na2HP04 (pH 7.2), 5 mM b-mercaptoethanol and 1 mM
EDTA at 4 C. Gel was stained in a solution of iodine
(Iodine 5 g/l, KI 50 g/l), for 30 min, clear bands indicate
the presence of amylase activity (Lee et al., 1994; Lin
et al., 1998). The molecular mass of the enzyme was nally
estimated from the position of standard proteins (Sigma
M3788, 36,000, 45,000, 55,000, 66,000, 84,000, 97,000,
116,000 and 205,000 Da).
2.7. Eect of metal ions and chelating agent
The eect of metal ions on amylolytic activity was determined by adding of dierent concentrations of each ion to
the standard assay. All metals were used in the chloride
such as EDTA (5 mM), CaCl (5 mM), NaCl (5 mM),
Nasulphite (5 mM), ZnCl2 (5 mM), PMSF (3 mM), Urea
(8 M) and SDS (1%). The activity of the enzyme alone in
BoraxNaOH buer (pH 11.0) was taken to be 100%.
The eect of metal ions, surfactants and chelating agent
on amylolytic activity was determined by pre-incubating
the enzyme in the presence of inhibitor for 30 min at
60 C, and then performing the assay in the presence of
the same inhibitor concentration at optimum temperature
for 60 min (Egas et al., 1998; Lo et al., 2001).

production and growth of bacteria were obtained at 60 C

and 2065 C, respectively. According to the paper chromatography results, the enzyme was determined as aamylase.
3.1. Molecular weight
Molecular weight was determined by SDSPAGE electrophoresis as described in Section 2. Analyses of the
enzyme by SDSPAGE revealed two bands that show amylolytic activity in starch gel. The molecular weight of these
bands was estimated as 86 and 60.5 kDa (Fig. 1).
3.2. Properties of the partially puried a-amylase
For estimation of the optimum temperature of the
enzyme, the activity was determined at dierent temperatures (60100 C). The enzyme has a broad temperature
range between 60 and 100 C and the optimum temperature was observed to be around 70 C. The activity of
enzyme at the temperatures 60 C, 70 C, 80 C, 90 C
and 100 C was as 99%, 100%, 91%, 87.5% and 60%,
respectively (Fig. 2). The enzyme was highly active over
94% between the temperatures 6090 C. Experiments were
repeated three times and mean values used.
The pH optima were determined in three buer systems.
The enzyme showed good activity at alkali pH. The optimum activity pH of the enzyme was 11.0. The optimal
activity was around 89% between 9.5 and 12.5 pH ranges
and especially pH 10.012.0 with an average 94% activity
(Fig. 3). The pH stability of enzyme was determined by
pre-incubating at 60 C for 24 h at three dierent pHs
(pH 9.0, 10.0 and 11.0) and the relative activity was measured by the standard assay method. The enzyme showed
about 75% (at pH 9.0), 67% (at pH 10.0) and 65% (at

3. Results
The isolated Bacillus sp. A3-15 from poultry manure
was gram positive, rod shaped, aerobic, catalase positive
and spore forming. According to the basis of various morphological and biochemical characteristic, it was identied
as Bacillus sp. Enzyme synthesis of Bacillus sp. A3-15
occurred at temperatures between 25 and 65 C with an
optimum of 60 C, while A3-15 Bacillus sp. grew well at
between 20 and 65 C on starch agar medium. There was
a slight variation in amylase synthesis within the pH range
6.0 and 11.0 with an optimum pH 8.5 on starch agar medium in petri dishes. The optimum temperatures for amylase

Fig. 1. Analysis of A3-15 a-amylase by 10% homogenized SDSPAGE.

The samples were subjected to SDSPAGE and the gel was stained with
iodine. Lanes (1) 15 ll, (2) 20 ll, (3) 25 ll, (4) 25 ll of A3-15 a-amylase
and (5) Marker: (Sigma 3788, 36,000, 45,000, 55,000, 66,000, 84,000,
97,000, 116,000 and 205,000 Da).

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105

Retain relative enzyme activity (%)

101

Relative enzyme activity (%)

100
95
90
85
80
75
70
65
60
55

100
99
98
97
96
95
94
Cont

60

50
60

70

80
T e mperature C

90

70
80
90
Pre-incubated temperatures C

100

100

Fig. 4. Eect of temperature on thermal stability of Bacillus sp. A3-15 aamylase.

Relative enzyme activity (%)

Fig. 2. Eect of temperature on the activity and stability of Bacillus sp.

A3-15 a-amylase. The enzyme displayed maximal activity at 70 C.

110

Table 1
Eect of dierent chemical sources with dierent concentrations on the
activity of a-amylase from Bacillus sp. A3-15

100

Eectors

Concentration

Remaining enzyme activity (%)

Control
EDTA
CaCl2
ZnCl2
NaCl
Nasulphite
SDS
Urea
PMSF

None
5 mM
5 mM
5 mM
5 mM
5 mM
1%
8M
3 mM

100
95.0
130
82.0
80.0
64.0
82.0
20.0
90.0

90
80
70
60
50
40
30
6.5

7.5

8.5

9.5

10 10.5

11 11.5 12

12.5

pH

Fig. 3. Eect of pH on the activity of Bacillus sp. A3-15 a-amylase. The

maximal enzyme activity was obtained at pH 11.0.

pH 11.0) remaining activity after treatment under standard

enzyme assay condition.
For thermal stability estimations, after pre-incubation
of the enzyme for 30 min at temperatures, of 60 C,
70 C, 80 C, 90 C and 100 C, the original activity
retained was 100%, 99%, 98%, 98%, and 96%, respectively.
The enzyme was completely active up to 100 C, with 96%
of the original activity remaining after heat treatment at
100 C for 30 min (Fig. 4).
3.3. Eect of dierent metal ions, chelator, surfactants and
inhibitors
The eect of dierent metal ions and inhibitors were
investigated on partially puried enzyme pre-incubated at
60 C for 30 min. The residual activity was measured at
optimum pH and temperature. The activity of the enzyme
alone in BoraxNaOH (pH 11.0) buer was taken to be
100%. Among the tested eectors, urea (8 M) inhibited
activity up to 80%. The denaturation of the original

enzyme activity with urea (80%) supports to that the

enzyme consists of hydrophobic amino acid composition.
When the A3-15 amylase enzyme was incubated with
EDTA (5 mM), NaCl (5 mM), Nasulphite (5 mM), ZnCl2
(5 mM), PMSF (3 mM) and SDS (1%), the enzyme activity
was retained at 95%, 80%, 64%, 82%, 90% and 82% of the
original activity. EDTA generally shows non-competitive
inhibition of amylase activity and a slight inhibition
showed us it is a metallo enzyme. On the other hand, a
stimulated activity was observed in the presence of Ca2+
(5 mM) around 130% from the original activity (Table 1).
The highly increase activity in the presence of Ca2+ also
showed that the enzyme has a rigid structure.
4. Discussion
In industry, bacterial a-amylases are produced mainly
from cultures of Bacillus subtilis var. amyloliquefaciens.
(Uhlig, 1998; Goyal et al., 2005). Bacillus stearotermophilus
and Bacillus licheniformis a-amylases are well characterized
and are heavily used in the starch-processing industry.
Since, thermostability is an important feature for use of
amylolytic enzymes in starch-processing, amylases from
thermophilic and hyperthermophilic bacteria are of special
interest as a source of novel thermostable enzymes (Leveque et al., 2000; Saxena et al., 2007).

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The optimal temperature for amylase production and

growth of the Bacillus sp. A3-15 strain were found to be
dierent as the organism has growth and production
optima at 60 C on petri dishes. Similar observations were
also reported for Bacillus sp. PN5 (Saxena et al., 2007).
According to the zymogram analyses of A3-15 a-amylase
enzyme was determined that consisting of two subunits as
86 kDa and 60.5 kDa. While bigger subunit, 86 kDa,
showed higher activity, small unit, 60.5 kDa, showed lesser
activity (Fig. 1). On pH optimum analyses, A3-15 a-amylase
produced two peaks at pH 8.0 and 11.0. However, the optimum enzyme activity was also detected at pH 11.0. These
results were supported that, 86 kDa subunit was showed
an optimum activity at pH 11.0 and the subunit 60.5 kDa
was showed an optimum activity at pH 8.0. Multiple amylase production has also been reported from many Bacillus
strains (1989; Hagihara et al., 2001). In literatures were
reported that the two enzyme referring 76 and 53 kDa from
thermophilic Bacillus, while two independent band with 150
and 42 kDa from thermophilic and alkaliphilic Bacillus sp.
TS-23 (Lin et al., 1998; Mamo and Gessesse, 1999).
It has been reported that the alkaline amylase of Bacillus
sp. Ant-6 was alkaline, thermophile and thermostable
(Burhan et al., 2003). Bacillus sp. A3-15 amylase enzyme
results showed that the optimum temperature and pH were
70 C and 11.0, respectively (Figs. 2 and 3). The enzyme
was highly active at pH range of 10.011.5 (95%). The
enzyme was stable between pH ranges 10.011.0 for 24 h
(70%). These ndings were highly similar with the literature
results (Mamo and Gessesse, 1999; Horikoshi, 1999; Goyal
et al., 2005). These results was showed that, A3-15 amylase
enzyme highly alkaline, pH stable and thermophile.
While the optimal temperature and pH for activity 70 C
and 9.0, respectively from amylase of Bacillus sp. TS-23
(Lin et al., 1998), The temperature prole our enzyme
showed that the enzyme had an optimum of 70 C with
60% activity retained 100 C at pH 11.0. Mamo and Gessesse (1999) also reported optimal temperature of 75
80 C for amylase from Bacillus sp. WN11. While Horikoshi (1999) observed temperature optimum for the activity
of a-amylase was at 70 C.
Our results showed that A3-15 a-amylase enzyme had
nearly 100% activity temperature 60100 C for 30 min. It
was also retaining 96% original activity at 100 C, pH
11.0. Similarly 100% activity at 90 C for 1 h for amylase
from Bacillus sp. has been reported by Teodoro and Martins (2000). A novel strain of Bacillus stearothermophilus
was isolated from the samples of a potato processing industry, which has a highly thermostable amylase. In fact,
a-amylases from Bacillus genus is heat stable and may be
used starch hydrolysis up to 90 C. Most other thermophile
Bacillus amylases reported to so far, two amylases exhibited higher temperature optimum for activity and showed
good thermal stability (Brawn and Kelly, 1993; Dong
et al., 1997; Horikoshi, 1999). These are the properties considered to be very important for industrial starch
liquefaction.

Although most a-amylases are inhibited by metal ions,

alkaline amylases vary in their response to the chelator,
EDTA with some being unaected (McTigue et al., 1995;
Jana et al., 1997).
In the presence of 1 mM EDTA, Amy-K38 retained full
activity ion in the presence of as high as 100 mM (Hagihara
et al., 2001), while Egas et al. (1998) reported 88% activity
with 10 mM EDTA. A3-15 a-amylase has also slight inhibition by 5% with 5 mM EDTA. A3-15 a-amylase enzyme
was strongly inactivated by 8 M Urea (80%). It was
reported that amylases from alkaliphilic Bacillus strains
were not inhibited by 10 mM EDTA at 30 C but was completely inactivated by 8 M urea (Horikoshi, 1999). These
results show that the A3-15 a-amylase is an alkaliphilic
enzyme.
The eect of Zn2+ also varied between amylases. For
instance, it had a potent inhibitory eect on the amylases
from Schwanniomyces alluvius and Bacillus cereus NY 14,
whereas it could have no eect at all on the enzyme of
Aspergillus kowachii (Lin et al., 1998). As the thermostable
a-amylases from a thermophilic Bacillus 46% (Lin et al.,
1998) and 13% inhibition were reported suggesting that
the inhibition with Zn2+ determines the thermostability
of enzyme (Mamo and Gessesse, 1999). The inhibition of
A3-15 (by 18%) in the presence of Zn2+ ion indicates that
is thermostable enzyme. Similar results were found thermostable a-amylase from a thermophilic Bacillus for AmyII
inhibition of 13% with zinc (Mamo and Gessesse, 1999).
The thermophilic and alkaliphilic Bacillus sp. TS-23
enzyme activity remained up to 115% (Lin et al., 1998),
while thermostable amylase activity from Bacillus stearothermophilus was retained up to 122% with 1 mM CaCl2
(Srivastava, 1987). Similarly A3-15 a-amylase enzyme
showed an enzymatic activity around 130% in the presence
of 5 mM CaCl2. The positive eects of Ca2+ ion presence
on the thermostability enzymes have been shown including
the amylases from B. Licheniformis, Pyrococcus juriosus
and Thermococcus litoralis (Brawn and Kelly, 1993; Dong
et al., 1997).
A3-15 a-amylase has also slight inhibition by 5% with
3 mM PMSF and Lin et.al were also reported similar result
(97% with 10 mM) (Lin et al., 1998).
In order to have applications in detergent industries,
amylase must be stable to various detergent ingredients,
such as surfactants and chelators. The amylase from Bacillus sp. A3-15 exhibited more than 82% activity when preincubated with 1% SDS at 60 C for 30 min. Saxena et al.
(2007) reported that, a highly thermostable and alkaline
amylase enzyme, there was 86.36% stability after 1 h incubation with SDS. Our results was indicated that, A3-15
amylase enzyme highly stable with 1% SDS (82%).
It was stated that LAMY was resistant to incubation at
40 C for 1 h with 0.1% SDS (Igarashi et al., 1998), additionally Lo et al., reported that SDS had no marked eect
on enzyme activity ion as concentration 8% (Lo et al.,
2001). This resistance, which is essential requirements, suggest that the enzyme may be used as an eective additive in

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detergents. Our results are conrmed with the results in the

literature in terms of CaCl2, ZnCl2 and SDS (Lo et al.,
2001).
In conclusion, the ability of Bacillus sp. A3-15 a-amylase enzyme to withstand a temperature up to 100 C for
30 min, and its high alkaline pH and temperature optimum
for activity and stability, and highly resistant for SDS,
could suggest that the enzyme has a potential in starch liquefaction and detergent industry.
Acknowledgement
This research supported by the Cukurova University research found (FEF 2004BAP8).
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Please cite this article in press as: Arikan, B., Highly thermostable, thermophilic, alkaline, SDS and chelator ..., Bioresour. Technol.
(2007), doi:10.1016/j.biortech.2007.06.019