International Journal of Biological Macromolecules 72 (2015) 290–298

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Gene cloning and characterization of a thermostable organic-tolerant

␣-amylase from Bacillus subtilis DR8806
Shamsi Emtenani a , Ahmad Asoodeh a,b,∗ , Shirin Emtenani a
a
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Vakil-Abad Blv., Mashhad, Razavi Khorasan 9177948974, Iran
b
Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The gene encoding an extracellular ␣-amylase from Bacillus subtilis DR8806 was cloned into pET28a(+)
Received 7 June 2014 vector and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme with molecular mass of
Received in revised form 18 August 2014 76 kDa exhibited optimal activity at pH 5.0 and 70 ◦ C with high stability in pH and temperature ranges
Accepted 19 August 2014
of 4.0–9.0 and 45–75 ◦ C. The enzyme showed a half-life of 125 min at 70 ◦ C. The ␣-amylase activity
Available online 26 August 2014
enhanced in the presence of Na+ , K+ , and Ca2+ ions, while Zn2+ , Pb2+ , and Hg2+ ions inhibited the activity.
The recombinant ␣-amylase exhibited high stability towards ioninc detergents sodium dodecyl sulfate
Keywords:
(SDS) and cetyl trimethylammonium bromide (CTAB). Organic solvents in reaction media increased the
␣-Amylase
Bacillus subtilis DR8806
␣-amylase activity. TLC analysis showed that maltoriose and maltose were the major end products of
Cloning enzymatic starch hydrolysis. Presenting various properties of recombinant ␣-amylase makes it well
Organic solvent suited as a potential candidate for industrial usages.
Maltotriose, Thermotolerant © 2014 Elsevier B.V. All rights reserved.

1. Introduction liquefaction step is restricted to function at pH around 6.50 that

Carbohydrate-converting enzymes produced by a broad spec-
trum of living organisms from plants to microorganisms are of
significant importance from industrial and biotechnological points
of view [1]. Different micoorganisms such as Bacillus species secrete
extracellular starch-hydrolyzing enzymes including ␣-amylase, ␤-
amylase, and ␣-glucosidase to utilize starch as a main energy
source [2]. Belonging to the family 13 (GH-13 or GH-H) of glyosyl
hydrolases, ␣-amylases catalyze the hydrolysis of ␣-1,4 glyco-
sidic linkages in an endo-acting manner, releasing a mixture
of oligosaccharides. ␣-Amylases fit a wide range of applications
in starch processing, brewing, baking, and detergent industries
by which they rank the first in terms of commercial utilization
[3]. ␣-Amylases with desirable features such as thermal stabil-
ity, substrate specificity, a broad pH profile, improved activity,
and proper resistance against denaturing agents and heavy metals
extensively draw the attention in industry [4]. Starch processing
generally employs thermostable, acidophilic ␣-amylases in lique-
faction step. As the activity and stability of most industrial amylases
currently utilized in starch processing decline at low pH, starch
∗ Corresponding author at: Department of Chemistry, Faculty of Sciences,
Ferdowsi University of Mashhad, Vakil-Abad Blv., Mashhad, Razavi Khorasan
9177948974, Iran. Tel.: +98 51 38795457; fax: +98 51 38795457.
E-mail address: asoodeh@um.ac.ir (A. Asoodeh).
is higher than the pH of starch slurry. Thus, the identification
and exploitation of microbial thermostable, acidophilic ␣-amylases
(active at pH less than 6.50) are of great demand [5,6]. Moreover,
hydrolyzing enzymes including proteases and ␣-amylases capa-
ble of removing protein/starch-based stains have been dominantly
offered on thriving detergent market. Today, various commercial
laundry/dishwashing detergents contain cocktails of enzymes that
improve washing performance and the environmental compatibil-
ity of detergents, indicating the increasing demand for exploiting
well-suited hydrolyzing enzymes in detergent formulations [7,8].
Organic solvents and ionic liquids (ILs) have recently garnered
widespread attention as attractive solvents in terms of chemical
and biotechnological applications. Operating organic solvents as
non-aqueous media for enzymatic reactions has numerous advan-
tages such as the improved thermal stability, the inhibition of
undesirable water-dependent side reactions, and also the elimina-
tion of microbial contamination. However, there are few reports on
thermoacidophilic amylases with orgaic solvent tolerance [9,10].
Hence, the isolation of organic solvent-tolerant enzymes seem to
be biotechnologically advantageous. On the other hand, ILs, known
as environmental friendly green solvents, are now considered as
more desirable carbohydrate conversion media compared to con-
ventional organic solvents, particularly in non-aqueos enzymology.
Comprehending the activity and stability of starch-hydrolyzing
enzymes in ILs may lead to advances in carbohydrate processing
[11].

http://dx.doi.org/10.1016/j.ijbiomac.2014.08.023
0141-8130/© 2014 Elsevier B.V. All rights reserved.
 S. Emtenani et al. / International Journal of Biological Macromolecules 72 (2015) 290–298
Overall, the level of production of wild-type enzymes is gen-
erally low; therefore, recombinant DNA technology provides the
production of recombinant proteins for both basic research and
commercial purposes [1]. In the present study, we cloned and
expressed an ␣-amylase encoding gene from thermophilic strain
Bacillus subtilis DR8806, which was previously isolated from Dig
Rostam hot mineral spring in Iran, in mesophilic host, E. coli [12].
Furthermore, biochemical properties of recombinant ␣-amylase
were characterized and its stability towards organic solvents, ILs,
and commercial detergents as well as its catalytic mode of action
on starch were determined.
2. Materials and methods
2.1. Baterial strains and vectors
The strain B. subtilis DR8806 (acquisition number: IBRC-
M10742, Iran) was used as the source of ␣-amylase gene. E. coli
DH5␣ and E. coli BL21(DE3) were used as host strains for cloning and
expression, respectively. The plasmid pTZ57RT (Fermentas, Mary-
land, USA) containing an ampicilin-resistance gene was used as
cloning/sequencing vector. The vector pET28a(+) (Novagen, USA)
bearing kanamycin-resistance gene was exploited for heteroge-
neous protein expression. B. subtilis DR8806 and E. coli strains
were grown aerobically at 37 ◦ C in Lauria–Bertani (LB) medium
(1% w/v peptone, 0.5% w/v yeast extract, and 1% w/v NaCl, pH
7.2).
2.2. Molecular gene cloning
Chromosomal DNA of B. subtilis DR8806 was extracted
as described [13]. A set of two oligonucleotide primers was
designed according to multiple alignment of ␣-amylase gene
sequences from Bacillus species to isolate ␣-amylase gene.
The complete open reading frame (ORF) of ␣-amylase gene
(Amy8806) was amplified using forward primer 5 -CGCGGATCCGC-
GATGTTTGCAAAACGATTCAAAACC-3 and reverse primer 5 -
CCCAAGCTTGGGTCAATGGGGAAGAGAACCGC-3 containing BamHI
and HindIII restriction sites, respectively. Amplification was carried
out by Thermocycler (Bio-Rad, CA, USA) under following program:
denaturation at 94 ◦ C for 5 min followed by 35 cycles at 94 ◦ C for
45 s, 68 ◦ C for 45 s, and 72 ◦ C for 2 min and a 10 min-final extension
at 72 ◦ C. The isolated fragment was then inserted into pTZ57R/T
vector for cloning and nucleotide sequencing. Positive colonies of
E. coli DH5␣ carrying pTZ-Amy8806 plasmids were first confirmed
by colony PCR and double digestion. Afterwards, the target gene
was sequenced by automated DNA sequencing. Amy8806 fragment
was cloned into pET28a(+) expression vector and transferred into
E. coli BL21(DE3).
2.3. Expression and purification of recombinant ˛-amylase
E. coli cells carrying recombinant pET28a(+) plasmids were cul-
tivated overnight in LB medium at 37 ◦ C with stirring at 180 rpm.
An aliquot of overnight culture (1% v/v) was inoculated into fresh
LB medium and incubated at 37 ◦ C to reach OD600 0.5–0.7. To
induce the expression, 1 mM isopropyl-␤-d-thiogalactopyranoside
(IPTG) was added to growing culture followed by additional
8 h-incubation at 25 ◦ C. The induced cells were collected by cen-
trifugation (12,000 × g, 20 min, 4 ◦ C) and resuspended in buffer A
(50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole and 1 mM PMSF,
pH 8.0) followed by sonication (6 cycles of intermittent sonica-
tion at 25 W for 30 s). The cell lysate was centrifuged at 12,000 × g
for 20 min and the supernatant containing soluble recombinant
protein was collected. The clear supernatant was loaded onto a
291
pre-equilibrated Ni2+ -NTA affinity column (Qiagen, CA, USA). Fol-
lowing the consecutive washing of column with buffer B (50 mM
NaH2 PO4 , 300 mM NaCl, and 20 mM imidazole, pH 8.0), target pro-
teins were recovered by elution with buffer C (50 mM NaH2 PO4 ,
300 mM NaCl, and 250 mM imidazole, pH 8.0). All purification steps
were performed at 4 ◦ C.
2.4. Characterization of recombinant ˛-amylase
2.4.1. Protein quantification and enzyme assay
Protein concentration was determined according to the method
of Bradford [14] by reading the absorbance at 595 nm with bovine
serum albumin as standard. ␣-Amylase activity was assayed in
accordance with Bernfeld method [15] using dinitrosalicylic acid
(DNSA) reagent. A reaction mixture containing purified enzyme
and 1% (w/v) soluble potato starch (Merck, Germany) as substrate
in sodium acetate buffer (50 mM, pH 5.0) was incubated at 70 ◦ C
for 10 min. The enzyme activity was determined by measuring
reducing sugars released following starch hydrolysis at 540 nm.
One unit of enzyme activity was defined as the amount of protein
needed to liberate 1.0 ␮mol of reducing sugar/min under the assay
condition.
2.4.2. Electrophoresis and zymogram analysis
The purity and molecular mass of recombinant ␣-amylase were
assessed by sodium dodecyl sulphate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) as described [16]. Using molecular mass
standards (Vivantis, CA, USA), the molecular mass of recombi-
nant protein was estimated on a 12% gel. Amylolytic activity was
detected on 10% polyacrylamide gel supplemented with 0.5% starch
under non-reducing condition. Non-heated samples were mixed
with 6× loading buffer a ratio of 5:1 (v/v). After electrophoresis, a
washing solution containing 2.5% Triton X-100 was used to remove
SDS from acrylamide gel. Upon staining the gel with Lugol’s solu-
tion, purified enzyme was visualized as white area in a dark blue
background representing amylolytic activity.
2.4.3. pH and temperature studies
The effect of pH on Amy8806 activity was measured at pH range
of 2.0–10.0 using different 50 mM buffer systems (glycine–HCl
buffer pH 2.0–3.0, sodium acetate buffer pH 3.5–5.5, sodium
phosphate buffer pH 6.0–7.5, Tris–HCl buffer pH 8.0–9.5, and
Na2 HPO4 –NaOH buffer pH 10.0). ␣-Amylase pH profile was
obtained by incubating the enzyme in each buffer system for 10 min
at 70 ◦ C with 1% soluble starch as substrate, followed by measur-
ing the amylolytic activity. The enzyme pH stability was studied by
pre-incubating the ␣-amylase for 60 min in mentioned buffers and
subsequently measuring the activity at 70 ◦ C. The enzyme activity
at the beginning of the reaction was taken as 100%.
Optimum temperature assays were performed at various tem-
peratures ranging from 30 to 90 ◦ C. The reaction mixture containing
the purified enzyme and 1% starch was incubated at different
temperatures (pH 5.0). Afterwards, the amount of reducing sugar
liberated was determined. For the measurement of thermal stabil-
ity, the enzyme was pre-incubated at respective temperatures for
60 min. After cooling samples, the residual activity was measured
using 1% soluble starch. To evaluate enzyme half-life, ␣-amylase
was pre-incubated in sodium acetate buffer (pH 5.0) at varying tem-
peratures (60–80 ◦ C) for 150 min following withdrawn of aliquots
at 30 min-intervals. After cooling on ice, enzyme aliquots were
assessed for residual activity, as formerly described.
2.4.4. Effect of various metal ions, enzyme inhibitors, and
denaturing agents on ˛-amylase activity
The influence of different metal cations as well as a number
of inhibitors and additives on ␣-amylase activity was studied. An
292 S. Emtenani et al. / International Journal of Biological Macromolecules 72 (2015) 290–298

aliquot of enzyme solution was pre-incubated with each reagent 5.0) was incubated with recombinant enzyme and the measure-
for 30 min. Subsequently, ␣-amylase activity was measured as ment of ␣-amylase activity was carried out under assay conditions.
described previously. The enzyme activity assayed in the absence A value of 100% activity was considered for soluble starch
of additive was assumed as 100%. hydrolysis.

2.4.5. Influence of organic solvents and ILs on ˛-amylase activity

3. Results and discussion
The amylolytic activity was investigated in order to study the
effect of polar/non-polar organic solvents and imidazolium-based
3.1. Construction of plasmid and sequence analysis of ˛-amylase
ILs as reaction media for starch hydrolysis. Enzyme aliquots were
gene
pre-incubated in different organic solvents (10% and 20% v/v in
buffer) and ILs (2–10% v/v in buffer) at ambient temperature for
␣-Amylase gene was amplified using genomic DNA of B. subtilis
30 min under 150 rpm stirring. The remaining activity was sub-
DR8806 as template resulting in an about 2000 bp DNA fragment.
sequently calculated according to aforementioned procedure. The
The target gene was ligated into pTZ57RT cloning vector and trans-
activity of control sample (lack of any organic solvents or ILs) was
formed to E. coli DH5␣ for cloning and sequence determination.
assumed as 100%.
The Amy8806 sequence was submitted to the GenBank nucleotide
sequence database under the accession number KC262177. The
2.4.6. Enzyme performance in the presence of commercial
deduced primary structure of the protein encoded by Amy88
detergents
gene is shown in Fig. 1. Sequence analysis revealed an ORF of
The stability and compatibility of recombinant ␣-amylase
1980 nucleotides encoding a protein composed of 659 amino acid
with commercial solid and liquid detergents were assayed [7].
residues. The molecular mass of recombinant protein was predicted
To simulate washing conditions, solid detergents including Barf
to be 72.49 kDa with a theoretical pI of 5.80 using ExPASy server
(Paxan, Iran), Softlan (Pakshoo, Iran), Vash (Henkel, Germany), Per-
(http://web.expasy.org/protparam/). Detected by SignalP 4.0 soft-
sil washing powder and handwash powder (Henkel, Germany),
ware, putative signal peptide was proposed to comprise 33 amino
Pril (Henkel, Germany), Shoma (TolyPers, Iran), Finish (Reckitt
acid residues followed by a cleavage site between positions 33 (Ala)
Benckiser, Canada), and Darya (TolyPers, Iran) were dissolved in
and 34 (Glu).
tap water at final concentration of 5 mg/mL. Additionally, a num-
Using CLC Main Workbench program (ver. 6.6.1, Denmark),
ber of commercial liquid detergents such as Goli (Paxan, Iran), Persil
␣-amylase amino acid sequence of B. subtilis DR8806 was multi-
(Henkel, Germany), Ave (Pakshoo, Iran), and Ganj (RaminGostar,
ply aligned with other ␣-amylase sequences of various bacteria
Iran) were diluted 100-fold followed by a heat-inactivation of com-
belonging to genu Bacillus and Geobacillus (Fig. 2a) in which highly
mercial enzymes within detergents at 80 ◦ C for 30 min. An aliquot
conserved regions (regions I–VII), catalytic residues, and function-
of purified ␣-amylase was pre-incubated in detergent solutions for
ally essential residues are demonstrated. All members of ␣-amylase
60 min at 50 ◦ C and pH value of 7.5–8.0. Afterwards, the residual
superfamily possess three domains consisting of catalytic domain A
activity was determined in the presence of 1% starch and compared
folded in the form of a (␣/␤)8 -barrel plus domains B and C. Bacillus
with residual activity of Bacillus licheniformis ␣-amylase (BLA). The
␣-amylases show seven conserved segments in the primary struc-
enzyme activity of control sample (without detergent), incubated
ture which mostly includes 138 DAVINH143 (located at strand ␤3 of
under similar conditions, was taken as 100%.
catalytic barrel), 213 GFRYDAAKH221 (strand ␤4), 245 FQYGEILQ252
(strand ␤5), 305 WVESHD310 (strand ␤7), 185 LYDWNT190 (loop3),
2.4.7. Thin-layer chromatography (TLC) of enzyme end products 74 GYAAIQTSP82 (strand ␤2), and 340 STPLFFSRP348 (strand ␤8). A
The end products of starch hydrolysis by recombinant ␣-
catalytic triad comprising acidic residues of Glu (region II), Asp
amylase of B. subtilis DR8806 were analyzed using TLC according to
(region III), and Glu (region IV) is compeletly conserved within
the method of Zhang [17]. Reaction mixtures containing 1% soluble
␣-amylase superfamily [18,19]. Moreover, two functionally impor-
starch and pure enzyme were incubated for 3, 6, 18 and 24 h at 70 ◦ C.
tant histidine residues (His143 and His309) being critical for
Defined amounts of reaction mixtures were applied on pre-coated
transition-state stabilization are highly conserved. Although the
silica gel plate (Merck 60 HPTLC plate, Darmstadt, Germany) in a
␣-amylase family can be characterised by several well-conserved
solvent system composed of n-butanol/acetic acid/water (4:8:1,
sequence regions, only four amino acid residues are totally con-
v/v) at room temperature. Spots were visualized by immersing
served invariantly throughout GH-13 family which involve the
the air-dried plate in a developing system consisting of 37.5% HCl
three catalytic residues as well as the arginine (Arg215) in the
(1 ml), aniline (2 ml), 85% H3 PO4 (10 ml), ethyl acetate (100 ml),
position i-2 with respect to catalytic nucleophile Asp (region III).
and diphenylamine (2 g), followed by heating at 110 ◦ C in a hot-air
Moreover, the phylogenetic tree was constructed by the neighbor-
oven for 10 min. Glucose (G1), maltose (G2), maltotetraose (G4),
joining method using different protein sequences of B. subitilis
and maltohexose (G6) were used as standards. Furthermore, the
␣-amylases (Fig. 2b).
product specificity of recombinant ␣-amylase in the presence of
four imidazolium-based ILs (10% v/v) and some tested commer-
cial detergents was evaluated using TLC. Following the enzyme 3.2. Affinity purification of recombinant protein and zymogram
incubation with the mentioned agents for 30 min, the enzymatic analysis
hydrolysis of starch was carried out under assay conditions. Simi-
lar to starch hydrolysis assay, a certain volume of reaction mixture Recombinant pET28a(+)-Amy8806 vector was transformed to
was applied on TLC plate and finally the end products of starch E. coli BL21(DE3) as an expression host. The recombinant ␣-amylase
hydrolysis released by the treated enzyme were determined and was expressed under the control of IPTG-inducible T7 promoter.
compared to standards. ␣-Amylase gene was inserted in frame with an N-terminal region
including six histidine residues that allowed the purification of
2.4.8. Substrate specificity recombinant protein using Ni2+ -NTA affinity chromatogrphy. Sin-
Enzyme preference towards different polysaccharides includ- gle step purification of recombinant enzyme resulted in a specific
ing glycogen (Merck, Germany), ␣-cyclodextrin, ␤-cyclodextrin activity of 3272 U/mg and a yield of 60%. The extracellular ␣-
(Merck, Germany), wheat starch, and corn starch was studied. Sub- amylase activity in the culture broth was negligible, indicating
strate (1% w/v) prepared in sodium acetate buffer (50 mM, pH that most part of recombinant protein remained within the host
 S. Emtenani et al. / International Journal of Biological Macromolecules 72 (2015) 290–298
Fig. 1. The deduced amino acid sequence of ␣-amylase from Bacillus subtilis DR8806. The well-conserved regions (I–VII) are indicated within colored boxes. Three catalytic
residues Asp217 (region II, strand ␤3), Glu249 (region III, strand ␤5), and Asp310 (region IV, strand ␤7) are marked with asterisk. Residues involved in the primary and
secondary Ca2+ binding sites are indicated by diamond and triangle, respectively.
cell carrying Amy8806 gene. By comparison, a recombinant ␣-
amylase bearing an N-terminal His-tag from B. halodurans LBK 34
was purified with a 47% yield and specific activity of 1200 U/mg
[20].
Recombinant Amy8806 exhibited a single band with an appar-
ent molecular mass of 76 kDa on 12% SDS-PAGE gel that is slightly
larger than the predicted molecular mass (Fig. 3a), since an extra
sequence containing 6×His tag added 3.8 kDa to protein mass. Thus,
the correct expression of ␣-amylase coding sequence was con-
firmed. The molecular mass of most amylases are typically ranged
in 10–210 kDa [3,21]. ␣-Amylase activity staining of recombinant
enzyme indicated a clear zone following enzymatic hydrolysis of
starch (Fig. 3b).
3.3. Effect of pH and temperature on ˛-amylase activity and
stability
The optimum pH of recombinant a ␣-mylase was determined
over a pH range of 2.0–10.0. According to pH-activity profile shown
in Fig. 4a, maximum activity was observed at pH 5.0. The rela-
tive activities at pH 3.0 and 9.0 were 64% and 80%, respectively,
of that at pH 5.0, although the enzyme activity rapidly declined at
extremely basic pH. According to pH-stability profile depicted in
Fig. 4b, DR8806 ␣-amylase was highly stable in pH range of 4.0–9.0
with >80% residual activity. The recombinant ␣-amylase retained
100% of its amylolytic activity between pH 4.0 and 7.0 whereas at
pH 3.0 and 9.0, the ␣-amylase retained 77% and 81% of its original
activity, respectively.
The effect of various temperatures on the ␣-amylase activ-
ity (temperature-activity profile) is illustrated in Fig. 4c. The
293
recombinant Amy8806 showed the highest activity at 70 ◦ C. The
enzyme was active at 35–75 ◦ C, while its activity declined at higher
temperatures. At 65 ◦ C and 75 ◦ C, relative activities were 92% and
74%, respectively. After 1 h-preincubation at different tempera-
tures, ␣-amylase was found to be quite stable in a wide temperature
range from 30 to 85 ◦ C (Fig. 4d); the enzyme retained 100% of its
activity when kept at optimum pH and temperature, while retain-
ing >50% of its initial activity at 75–80 ◦ C. A few thermoacidophilic
amylases from Bacillus and Geobacillus species have been cloned
and characterized including Geobacillus thermoleovorans (pH 5.0 at
80 ◦ C) [19], Bacillus licheniformis NH1 (pH 6.5 at 90 ◦ C) [22], and
Bacillus acidicola (pH 4.0 at 60 ◦ C) [21]. Furthermore, according
to thermal inactivation experiments, Amy8806 showed a half-
life value of 125 min at 70 ◦ C. After 120 min, a rapid decline in
enzyme activity (<40% of original activity) was observed at tem-
peratures above 70 ◦ C and below 65 ◦ C (data not shown). At 70 ◦ C,
the half-life of Amy8806 was more than amylases from Bacillus
sp. KR-8104 (>10 min) [6] and Bacillus sp. Ferdowsicous (75 min)
[23]. The optimal activity and stability of ␣-amylase at low pH
values is of significant importance from industrial point of view.
␣-Amylase with enzymatic performance at acidic pH of natural
starch slurry (pH 4.5), as the alternative to currently-used lique-
fying amylases with optimal pH of 6.8, seem essential to fulfill
industrial demands. Additionally, by utilizing acid-stable amylases,
the costly and time-consuming step of pH adjustment to 4.5 for sac-
charification process would be omitted and by-product formation
(usually at higher operation pH) would reduce [24]. Consequently,
recombinant ␣-amylase with optimal activity in acidic and high
tempareture conditions would be a good candidate for enzymatic
starch processing in combination with a glucoamylase.
294 S. Emtenani et al. / International Journal of Biological Macromolecules 72 (2015) 290–298

Fig. 2. (a) The well-conserved regions (I–VII) of ␣-amylase sequences obtained by multiple alignment of amino acid sequences from B. subtilis DR8806 (KC262177), B. subtilis
(AFD33644.1), Bacillus KR-8104 (ACD93218.3), B. vallismortis (WP 010331366.1), B. tequilensis (WP 024715525.1), B. amyloliquefaciens (WP 014416850.1), B. methylotrophicus
(WP 025650188.1), B. atrophaeus (WP 010788973.1), B. acidicola (JN680873.1), B. licheniformis strain NH1 (EF125542.1), Geobacillus thermoleovorans strain NP54 (JQ409473.1),
and Geobacillus thermodenitrificans (ABX83871.1). The catalytic triad (Asp217, Glu249, and Asp310), invariant arginine (Arg215) and conserved histidine residues (His143
and His309) are marked by asterisk, circle, and rectangle, respectively. (b) The phylogenetic tree of ␣-amylase amino acid sequences of Bacillus subtilis strains. Protein
sequences of the corresponding ␣-amylases from B. subtilis strains obtained from GenBank were incorporated into the tree by using the neighbor-joining method. Each name
at the termini represents the species from which the ␣-amylase protein sequence originated. The accession numbers for each sequence are as follows: B. subtilis DR8806
(KC262177), B. subtilis (WP 003241207.1; 93% homology), B. subtilis (WP 019257405.1; 94%), B. subtilis (WP 029973920.1; 86%), B. subtilis (WP 014112574.1; 94%), and B.
subtilis (AFD33644.1; 86%). The sequence of Geobacillus thermodenitrificans (ABX83871.1) was used as the out-group.

3.4. Influence of metal ions and enzyme inhibitors on ˛-amylase
activity
The effect of numerous metal cations and inhibitors on recom-
binant ␣-amylase was assessed and the results are summarized in
Table 1. Among metal ions tested, ␣-amylase activity enhanced in
Fig. 3. (a) SDS-PAGE analysis of recombinant ␣-amylase expressed in E. coli. M:
marker, Lane 1: purified fraction of recombinant protein following affinity purifica-
tion, Lanes 2–5: cell fractions at 0 (lane 2), 4 (lane 3), 6 (lane 4), 8 (lane 5) hour-post
induction with 1 mM IPTG. (b) Starch-zymogram of recombinant enzyme with amy-
lolytic activity.
the presence of 5 mM Na+ , K+ , and Ca2+ ions with remaining activ-
ity of 120%, 113%, and 116%, respectively. However, the enzyme
activity was strongly inhibited by Zn2+ , Cu2+ , Pb2+ , Hg2+ , and Mn2+
ions and a moderate inhibition was observed by Co2+ and Mg2+ .
Thermostable ␣-amylases from Geobacillus thermoleovorans [19]
and B. subtilis JS-2004 [25] exhibited similar behavior towards
tested metal ions. Calcium ion is generally known to have positive
effect on the catalytic activity of most thermostable ␣-amylases and
also enhance their themostability, as observed for Amy8806. The
inhibition of enzyme activity by copper and mercury ions demon-
strated the role of thiol/carboxyl groups as well as the oxidation of
indole ring in side chain of aromatic tryptophan within the enzyme,
respectively. Worth mentioning, the role of tryptophan and other
residues in substrate binding and catalysis has been indicated [19].
Oxidizing agents including potassium iodide and ammonium
persulfate exhibited an inhibitory effect on enzyme function
(Table 1). Additionally, in the presence of reducing agents such
as ascorbic acid and ␤-mercaptoethanol the residual activity was
found to be 74% and 59%, respectively. Recombinant ␣-amylase
remained 98% of initial activity following incubation with denat-
urant urea (5 mM). Similar to our enzyme, an ␣-amylase from
Geobacillus sp. IIPTN was highly resistant to 5 mM urea [26].
Moreover, a moderate enzyme inhibition was observed by 10%
Triton-X100. Interestingly, Amy8806 almost retained its catalytic
efficiency in the presence of cationic surfactant CTAB and anionic
 S. Emtenani et al. / International Journal of Biological Macromolecules 72 (2015) 290–298 295

(a) (b)
120 120

Remaining activity (%)

100
Relative activity (%)

100

80 80

60 60

40 40

20 20

0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11
pH pH

(c) (d)
120 120

Remaining activity (%)

100 100
Relative activity (%)

80 80

60 60

40 40

20 20

0 0
25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Tempreture (°C) Tempreture (°C)

Fig. 4. Effect of pH and temperature on the activity and stability of recombinant ␣-amylase. Activity (a) and stability (b) as a function of pH; the purified enzyme exhibited
maximum activity at pH 5.0, retaining 100% of its initial activity after 1 hour-incubation under optimal conditions. Temperature-activity (c) and stability (d) profiles of
␣-amylase at pH 5.0. Maximum amylase activity and stability was observed at 70 ◦ C. Experiments were carried out in triplicate.

surfactant SDS (10% w/v). The enzyme stability towards SDS is of
great significance, particularly in detergent formulation, because
SDS-resistant amylases have been rarely reported [7]. The enzyme
retained 80% of its initial activity in the presence of 5 mM che-
lating agent EDTA, suggesting that metal ions including calcium
are essential for enzyme activity. The inhibitory effect of EDTA has
been reported for ␣-amylase from Geobacillus sp. LH8 [27]. It is
proposed that through sulfonating serine residues at the enzyme
catalytic site, phenylmethylsulphonyl fluoride (PMSF) inhibits the
enzymatic activity, as observed for our recombinant ␣-amylase and
B. acidicola ␣-amylase [21].
3.5. Effect of organic solvents and ILs on amylase activity
As shown in Table 2, organic solvents as hydrolysis media were
found to have a promoting effect on ␣-amylase activity compared
to control. The enzyme activity was stimulated by 105%, 144%, and
163% in the presence of 20% (v/v) methanol, ethanol, and butanol,
respectively. Moreover, ␣ -amylase activity slightly enhanced by
following water miscible solvents: hexane (112%), heptane (104%),
and toluene (139%). Similar results have been reported for an amy-
lase from B. agaradhaerens Mi-10-62 which considerably retained
its activity in 30% (v/v) organic solvents such as dodecane, heptane,
methanol, and propanol [28]. Furthermore, a halophilic ␣-amylase
from Nesterenkonia sp. strain F exhibited a proper stability towards
20% (v/v) chloroform and toluene [10]. The hydrophobicity of an
organic solvent is detrmined by a parameter termed “log Po/w ”
value. Typically, organic solvents having low log Po/w values (water-
miscible or polar solvents) exhibit more biological toxicity resulting
in more inhibitory effect on biocatalysts compared to high Po/w
solvents (water-immiscible or nonpolar solvents) [29]. In this
investigation, no clear relation was obtained between the log Po/w
value of tested organic solvents and the stability of recombinant
␣-amylase. Additionally, organic solvents with low log Po/w values
(−0.208 to 0.8) revealed a slightly destabilizing effect on Amy8806
activity, whereas nonpolar organic solvents with high Po/w values
(>1.9) stimulated ␣-amylase activity. In spite of the advanteges
of organic solvents, hydrocarbon-containing environments result
in denaturation of most enzymes and loss of their catalytic activ-
ity. Consequently, enzymes being intrinsically stable and active in
organic solvent solutions appear to be quite attractive for industrial
applications such as treatment of carbohydrate-polluted industrial
wastewater contaminated with organic solvents [9]. Considering
the remained catalytic activity of recombinant amylase, organic
solvent-based solutions could be employed as suitable reaction
media for carbohydrate hydrolysis.
Due to the impact of ILs as dispersion/modification media
on enzymatic transformation, the activity of recombinant
enzyme was measured towards four imidazolium-based ILs
including 1-ethyl-3-methylimidazolium bromide ([EMIm][Br]),
1-butyl-3-methylimidazolium bromide ([BMIm][Br]), 1-hexyl-
3-methylimidazolium bromide ([HMIm][Br]), and 1-butyl-3-
methylimidazolium chloride ([BMIm][Cl]) (Fig. 4). The results
demonstrated that as the IL concentration increased, the enzyme
activity decreased. At 10% (v/v) concentration of ILs, the highest
retaining activity (74.7%) was observed in reaction mixture con-
taining [BMIm][Cl], while [HMIm][Br] was found to cause the most
inhibitory effect amongst Br-containing ILs (60% retaining activity).
Generally, as the chain length of alkyl substituents on imidazolium
ring of cation and anion size decrease, the polarity as well as enzyme
296 S. Emtenani et al. / International Journal of Biological Macromolecules 72 (2015) 290–298

Table 1
Effect of various metal ions and chemical reagents on Amy8808 activity.a

Reagent Relative enzyme activity (%)

Metal ions 1 mM 5 mM
Hg2+ 36 14
Ba2+ 81 112
Co2+ 73 38
Cu2+ 100 0
Mg2+ 44 22
Pb2+ 0 0
Mn2+ 23 0
Ca2+ 120 116
Fe2+ 29 26
Zn2+ 102 0
Na+ 115 120
K+ 100 113
Oxidizing agents 1 mM 5 mM Fig. 5. Amylase activity in the presence of four imidazolium-based ILs (2–10% v/v).
Potassium iodide 39 23 Values presented are the means of triplicate analyses.
Ammonium persulfate 13 0
Reducing agents 1 mM 5 mM
Ascorbic acid 67 74
␤-mercaptoethanol 61 59
Chelating agents 1 mM 5 mM
EDTA 85 80
Sodium citrate 65 32
Detergents 10% 20%
SDS 85 70
Triton X-100 45 12
CTAB 113 93
Additives 1% 2%
Glycerol 55 48
PEG 4000 71 73
Urea 100 98
Inhibitors 1 mM 5 mM
DTNB 71 46
PMSF 33 23
Dithiothreitol 50 39
Dimethylfomamide 42 0
Phenanthroline 72 64
Tetrafluoroethylene 76 68
a
Enzyme preparation was pre-incubated with different concentrations of chemi-
cal agents and the residual activity was determined under assay conditions. Amylase
activity of control sample in the absence of any additives was taken as 100%. The
standard errors were less than 5% of the mean.
Fig. 6. The effect of commercial solid and liquid detergents on the activity of ␣-
amylase from B. subtilis DR8806 compared with B. licheniformis amylase. The enzyme
activity increase [11]. It was previously reported that thermophilic
was first treated with detergent solution and the residual activity was then mea-
␣-amylases exhibited more stability and activity in ILs-containing sured in the presence of substrate. The activity of control samples in the absence of
solutions compared to mesophilic amylases [30]. This finding detergents was taken as 100%. All determinations were performed in triplicate.
allows [BMIm][Cl] to be used as a dispersion/modification meduim
for biocatalysis in enzymatic applications (Fig. 5.).
our recombinant ␣-amylase was totally stable towards both solid
3.6. Influence of commercial detergents on recombinant and liquid detergents, retaining 82–100% of its initial activity. Inter-
˛-amylase activity estingly, Amy8806 exhibited good compatibility and activity in the
presence of detergents compared with BLA. The residual activity
In this study, the effect of commercial laundry/dishwashing of Amy8806 was 99%, 99%, and 97% following 1 h-preincubation
detergents on the activity of Amy8806 was assessed and com- with Ave (Pakshoo, Iran), Persil (Henkel, Germany), and Shoma
pared with B. licheniformis ␣-amylase (BLA). As shown in Fig. 6, (TolyPers, Iran) detergents, respectively, while BLA retained 96%,
71%, and 60% of its original activity towards aforementioned deter-
gents under the same conditions. Totally, both amylases exhibited
Table 2
the highest stability towards Ave detergent whereas the minmal
Effect of various organic solvents (10% and 20% v/v) on amylase activity.
stability for Amy8806 and BLA was observed following the enzyme
Organic solvents Relative amylase activity (%) incubation with Persil washing powder and Ganj. By comparison,
10% v/v 20% v/v thermostable ␣-amylase from B. licheniformis NH1 showed good
compatibility with various solid/liquid laundry detergents under
Acetone 116 85
Methanol 113 105 similar assay conditions [7]. Enzymes retaining active in the pres-
Ethanol 128 144 ence of potentially inhibitory ingredients, routinely formulated into
Butanol 68 163 commercial detergents, are more favored for detergent industry [8].
Isopropanol 77 105
According to the enzyme stability towards commercial detergents
Hexane 112 114
Heptane 104 129
and ionic surfactants like SDS and CTAB as well as its activity up to
Isoamylalcohol 117 64 pH 9.0, recombinant ␣-amylase could be potentially applied in the
Diethyl ether 99 77 formulation of liquid and powder laundry/dishwashing detergents
Toluene 139 98 to remove starch-containing stains.
 S. Emtenani et al. / International Journal of Biological Macromolecules 72 (2015) 290–298
Fig. 7. (a) TLC analysis of hydrolysis products of starch by B. subtilis DR8806
recombinant ␣-amylase after 3, 6, 18, and 24 h tretaments. Lane 1 (G): standard
oligosaccharides (G1, glucose; G2 maltose; G4, maltotetraose; and G6, maltohex-
ose). (b) The product specificity of DR8806 ␣-amylase in the presence of four
imidazolium-based ILs (10% v/v). (c) The product specificity of recombinant ␣-
amylase treated with commercial solid detergents including Barf, Pril, Persil washing
powder, and Finish as well as liquid detergents including Ganj and Ave.
3.7. End product profile of starch hydrolysis
The end products of starch hydrolysis by Amy8806 were
assessed on TLC chromatogram for 3-24 h (Fig. 7a). TLC analy-
sis indicated a range of short length maltooligosaccharides from
random degradation of potato starch, gradually converted to malt-
ose (after 24 h treatment) in which maltose (G2) and maltotriose
(G3) are the major end products. Based on TLC analysis of the
end-products, Amy8806 could be classified as an endo-acting
maltose/maltoteriose-forming ␣-amylase, making it suitable to be
utilized for the production of high maltose-content maltodextrins
from starch.
In addition to enzyme staibility assay, the product specificity
of Amy8806 was determined in the reaction solutions containing
imidazolium-based ILs and a number of commercial detergents.
Totally, the results show that the recombinant ␣-amylase not
only was stable stable in the presence of tested ILs and com-
mercial detergents, but also could hydrolyze starch into different
297
maltooligosaccharides, particlurly maltotriose. In the case of ILs,
maltoteriose was the major end product of starch hydrolysis by
the treated enzyme (Fig. 7b). Interestingly, as shown in the chro-
matogram the highest maltoteriose production was observed for
sample containing B[MIm]Cl ionic liquid in which the enzyme
showed the highest stability. Moreover, recombinant Amy8806
was stable toward the tested detergents and could catalyze the
hydrolysis of starch into short lenght maltooligosaccharids during
the incubation time (Fig. 7c).
3.8. Substrate specificity analysis
Recombinant ␣-amylase from B. subtilis DR8806 was investi-
gated for its ability to hydrolyze different carbohydrates. Among
the substrates tested, soluble potato starch was most efficiently
hydrolyzed by Amy8806. The recombinant enzyme hydrolyzed
some substrates (1% w/v) including soluble potato starch, glyco-
gen, wheat starch, and corn starch with degradation rates of
100%, 65%, 62%, and 43% without any noticeable activity on ˛-
and ˇ-cyclodextrins. Thus, Amy8806 can hydrolyze ␣-glycosidic
bonds on both linear and branched carbohydrate polymers. Sim-
ilar hydrolyzing tendency was also obtained by Sharma et al.
[21], where B. acidicola ␣-amylase hydrolyzed soluble potato
starch (100%) and corn starch (68%) without any affinity towards
cyclodextrines.
To conclude, recombinant ␣-amylase (Amy8806) is a ther-
mostable, acidophilic, and organic solvent-resistant enzyme with
high compatibility towards detergents, which suggests its poten-
tial applications in different biotechnological fields such as starch
processing and detergent formulation.
Acknowledgment
Authors gratefully appreciate the Institute of Biotechnology and
research council of Ferdowsi University of Mashhad - Iran for their
financial support (grant numbers: 3/27142; 23-2-1392 and 4065;
06-02-1389).
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