ORIGINAL PAPER

Purification and biochemical characterization of an acidophilicamylase from a newly isolated Bacillus sp. DR90

Ahmad Asoodeh • Ashraf Alemi • Akbar Heydari •

Jafar Akbari

Received: 12 October 2012 / Accepted: 17 January 2013 / Published online: 21 February 2013

� Springer Japan 2013

Abstract An acidophilic and Ca2?-independent amylase

was purified from a newly isolated Bacillus sp. DR90 by

ion-exchange chromatography, and exhibited a molecular

weight of 68.9 kDa by SDS-PAGE. The optimum pH and

temperature of the enzyme were found to be 4.0 and 45 �C,

respectively. The enzyme activity was increased by Ba2?,

Fe2? and Mg2?, and decreased by Hg2? and Zn2?, while it

was not affected by Na?, K?, phenylmethylsulfonyl fluo-

ride and b-mercaptoethanol. Ca2? and EDTA did not have

significant effect on the enzyme activity and thermal sta-

bility. The values of Km and Vmax for starch as substrate

were 4.5 ± 0.13 mg/ml and 307 ± 12 lM/min/mg,

respectively. N,N-dialkylimidazolium-based ionic liquids

such as 1-hexyl-3-methylimidazolium bromide

[HMIM][Br] have inhibitory effect on the enzyme activity.

Thin layer chromatography analyses displayed that maltose

and glucose are the main products of the enzyme reaction

on starch. Regarding the features of the enzyme, it may be

utilized as a novel candidate for industrial applications.

Keywords Bacillus sp. DR90 � Acidophilic amylase �Starch industry � Characterization � Ionic liquids

Introduction

Amylases are enzymes that break down polysaccharides

such as starch or glycogen (Vidyalakshmi et al. 2009).

Amylases are one of the most important and widely used

industrial enzymes. They account for about 30 % of the

world’s enzyme production (Aqeel and Umar 2008).

Starch-degrading enzymes have potential applications in a

number of industrial processes such as food, textile, paper

and detergent industries, baking, the production of glucose

and fructose syrups, ethanol fuel, fruit juices, alcoholic

beverages, sweeteners, digestive aid and spot remover in

dry cleaning (Suman and Ramesh 2010).

Despite amylases can be derived from several sources

such as plants, animals and microbes (Valaparla 2010), the

microbial amylases meet industrial demands due to being a

substitute for chemical hydrolysis of starch (Vidyalakshmi

et al. 2009, Van Der Maarel et al. 2002). Although there are

many microbial sources available for producing amylases,

only few of them such as Bacillus subtilis, Bacillus li-

cheniformis and Bacillus amyloliquifaciens are recognized

as commercial producers (Alamri 2010). Because of the

complex structure of starch, a variety of enzymes are

required for its degradation (Niehaus et al. 1999, MacGr-

egor et al. 2001, Prakash and Jaiswal 2010). During the

starch processing, starch slurry is gelatinized by heating

followed by two enzymatic steps, liquefaction (by a-

amylase) and saccharification (by glucoamylase) steps (Bai

et al. 2012). The natural pH of starch slurry is around 4.5,

whereas the optimum pH for the most industrial a-amy-

lases is approximately 6–7, being unstable at low pH

Communicated by H. Atomi.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00792-013-0520-1) contains supplementarymaterial, which is available to authorized users.

A. Asoodeh (&) � A. Alemi

Department of Chemistry, Faculty of Sciences, Ferdowsi

University of Mashhad, Mashhad, Iran

e-mail: asoodeh@um.ac.ir

A. Asoodeh

Institute of Biotechnology, Ferdowsi University of Mashhad,

Mashhad, Iran

A. Heydari � J. Akbari

Department of Chemistry, Tarbiat Modares University, P.O. Box

14155, 4938 Tehran, Iran

123

Extremophiles (2013) 17:339–348

DOI 10.1007/s00792-013-0520-1

(Hassan et al. 2011). Because of the liquefaction step is

currently constrained to operate at pH 5.8–6.2, it is nec-

essary to improve the activity and stability of the enzymes

at low pH values to omit the pH adjustment step (Liu and

Xu 2008). In addition, most of the a-amylases of Bacillus

species have been found to be dependent on calcium ions

(Uyar et al. 2003) and destabilized and/or inhibited by

chelating agents such as EDTA (Sajedi et al. 2005).

In the present study, we report purification and bio-

chemical characterization of a novel amylase produced by

Bacillus sp. DR90 which was isolated from Dig Rostam hot

mineral spring.

Materials and methods

3,5-dinitrosalicylic acid (DNS), malto-oligosaccharides,

EDTA and PMSF were obtained from Sigma (USA).

Q-Sepharose was bought from GE Healthcare (Uppsala,

Sweden). TLC plate, sodium dodecyl sulfate (SDS), all

culture media and their supplements were purchased from

Merck KGaA Co. (Darmstadt, Hesse, Germany). PCR and

DNA extraction reagents were procured from Fermentas

(Thermo Fisher Scientific, USA).

Isolation and screening of amylase producing

microorganism

Water samples were collected from Dig Rostam hot min-

eral spring in the southeast of Iran. The spring possesses a

temperature of 55 �C and pH 7.2. Aliquots of water sam-

ples were used to inoculate a nutrient agar medium con-

taining 0.5 % (w/v) peptic digest of animal tissue, 0.5 %

(w/v) sodium chloride, 0.15 % (w/v) beef extract, and

0.15 % (w/v) yeast extract. The medium was incubated in a

rotary shaker incubator (180 rpm) at 37 �C for 48 h and

subsequently subcultured on the above medium, including

1 % (w/v) agar and 1 % (w/v) starch to isolate single

colonies.

Identification of microorganism and phylogenetic

analysis

The isolated strain was characterized according to 16S

ribosomal gene sequence analysis and some testes which

described in ‘‘Bergey’s Manual of Systematic Bacteriol-

ogy’’ (Bergey and Holt 1994). 16S rRNA gene of the

isolated strain was amplified using a universal upstream

primer 50-AGTTTGATCCTGGCTCAG-30 and a down-

stream primer P2: 50-GGCTTACCTTGTTACGACTT-30

(Asoodeh et al. 2010). PCR conditions were programmed

as follows: (1) denaturing temperature of 93 �C for 5 min;

(2) a run of 35 cycles with each cycle, including 45 s at

93 �C, 45 s at 50 �C and 30 min at 72 �C; and (3) a final

extension for 5 min at 72 �C. The amplified DNA seg-

ment was sequenced, submitted to GenBank, and evolu-

tionary analysis was carried out using CLC Main

Workbench version 6.8.1 software (CLC bio A/S, Den-

mark). To construct the phylogenetic tree, the neighbor-

joining method was used. Bootstrap analyses with 1000

replications were performed on the phylogenetic tree to

estimate the reproducibility of the tree topology (Asoodeh

et al. 2010).

Partial optimization of culture conditions

To choose a proper culture medium for growth of the

isolated strain and amylase production, four different

culture mediums were examined (Vahidi et al. 2005). A

nutrient broth composed of beef extract 0.1 % (w/v), yeast

extract 0.2 % (w/v), peptone 0.5 % (w/v) and sodium

chloride (NaCl) 0.5 % (w/v) was used. This culture

medium was enriched as follows: (1) 1 % (w/v) of starch;

(2) 1 % (w/v) of fructose, 1 % (w/v) of starch and 1 %

(w/v) of peptone; (3) 1 % (w/v) of peptone, 1 % (w/v) of

maltose and 1 % (w/v) of starch; and (4) 1 % (w/v) of

peptone, 1 % (w/v) of maltose, 1 % (w/v) of starch and

2 % (w/v) of yeast extract. In addition, the effect of three

temperatures (37, 42 and 50 �C) on the isolated strain

growth was studied. To produce enzyme, the isolated

strain was incubated on the selected culture medium

(200 ml) at the optimum temperature. After a 24-h incu-

bation, the culture medium was centrifuged at 4 �C,

8500g for 15 min, and the supernatant was used as the

enzyme source.

Enzyme purification

The cell-free supernatant (crude enzyme) was prepared as

mentioned above. Ammonium sulfate (85 % of saturation)

was added to the crude enzyme solution at 4 �C. After

36 h, the sediment was dissolved in minimum volume of

20 mM phosphate buffer (pH 7) and dialyzed overnight

against one liter of the same buffer at 4 �C. Dialyzed

solution was filtered through an ultrafiltration membrane

with a 10 kDa cut-off. Filtered solution was loaded on a

Q-Sepharose fast flow column (2 9 10 cm), which had

been previously equilibrated with 20 mM phosphate buffer

pH 7. The column was washed with the mentioned buffer

to remove unbound proteins. Subsequently, the column was

eluted with a linear gradient of sodium chloride

(0.1–1.0 M) in the same buffer. Fractions containing pro-

tein (absorbance 280 nm) were monitored for the amylase

activity. The positive fractions were dialyzed to equilibrate

with the above phosphate buffer. Ion-exchange chroma-

tography was carried out again at this buffer to obtain the

340 Extremophiles (2013) 17:339–348

123

pure enzyme. Fractions exhibiting amylase activity were

dialyzed against 50 mM sodium acetate buffer (pH 4),

lyophilized, and stored at -20 �C. Protein concentration

was calculated by Bradford’s method (1976) using bovine

serum albumin (BSA) as standard.

Amylase activity assay

Amylase activity was determined according to Bernfeld’s

method (1955) using starch dissolved in a 50 mM sodium

acetate buffer (pH 4) at 42 �C. To assess the activity, the

enzyme reaction was carried out in the final 1.0 ml volume

of a mixture containing 0.8 ml of starch (1.25 %,w/v),

0.1 ml buffer and 0.1 ml enzyme solution (0.1 mg/ml).

After incubation at 42 �C for 10 min, 1 ml of dinitrosali-

cylic acid (DNS) was added to the reaction, and then the

solution was heated for 15 min. After cooling on ice to

room temperature, 9 ml of water was added to the test, and

then the activity was measured by the absorbance at

540 nm. One unit (U) of activity was defined as the amount

of enzyme releasing 1 lmol of the reducing end groups per

minute at 42 �C. D-Maltose was used as standard of the

reaction.

Polyacrylamide gel electrophoresis

To evaluate the purity and the molecular weight of the

purified enzyme, denaturing SDS-PAGE was conducted

using a 5 % (w/v) stacking gel and 15 % (w/v) resolving

gel (Simpson 2004). The molecular weight was calcu-

lated according to semi-logarithmic plot of standard

molecular masses versus Rf value (relative mobility).

Protein bands were visualized by staining with Coo-

massie Brilliant blue R-250. Non-denaturing polyacryl-

amide gel electrophoresis was performed in a 10 %

resolving gel containing 1 % starch to detect the amylase

activity (Asoodeh and lagzian 2012). The sample was

mixed with b-mercaptoethanol-free 69 sample buffer in

a ratio of 5:1 (v/v), and the mixture was used without

heating. After electrophoresis, the acrylamide gel was

washed with 2.5 % (v/v) of Triton X-100 for 30 min to

eliminate SDS. Subsequently, Lugol staining solution

was used for visualization of the amylase activity in gel.

A clear zone in a dark-blue background exhibits the

amylase activity.

Effect of pH on the amylase activity and stability

The effect of pH on the activity of amylase was measured

in the pH range of 2.0–10.0 at 42 �C. Three different

buffers (50 mM) including sodium acetate buffer (pH 2–5),

sodium phosphate buffer (pH 5.5–7.5) and Tris–HCl buffer

(pH 8.0–10) were used. To determine pH stability, the

enzyme was incubated in different buffers for 1 h at 42 �C,

and the residual activity was calculated under enzyme

assay conditions. The enzyme activity at the beginning of

the reaction was assumed as 100 % activity. BLA (Bacillus

Licheniformis a-amylase) was used as standard in activity

and stability tests.

Temperature profile and stability

The effect of temperature on the enzyme activity was

determined from 30 to 95 �C at the optimum pH of

enzyme. The maximum activity of enzyme was considered

as 100 %, and the percentage of relative activity was

plotted against different temperatures.

To determine thermal stability, the purified enzyme

was incubated at different temperatures from 30 to 90 �C

for 45 min at the optimum pH of enzyme, in the pres-

ence and absence of 5 mM CaCl2. After cooling on ice,

the residual activity was calculated under assay

conditions.

Half-life of the enzyme was assayed at temperatures

ranging from 30 to 80 �C. The enzyme was incubated for

150 min at each temperature. Afterwards, samples were

collected at 1/2 h intervals and assayed for residual activ-

ity. Half-life (t1/2) was measured from the plot of log %

residual activity as a function of time (Asoodeh and

Ghanbari 2013). BLA (B. Licheniformis a-amylase) was

also used as standard in all experiments.

Effect of metal ions, enzyme inhibitors and denaturing

agents

The effect of metal ions at three concentrations (1, 5,

10 mM) on the enzyme activity was investigated in the

presence of CaCl2, ZnCl2, FeCl2, HgCl2, BaCl2, MgCl2,

NaCl and KCl. In addition, the effects of Trition X-100 (1,

5, 10 mM) and SDS (1, 5, 10 mM) as detergents on the

enzyme activity were studied. The inhibitory effect of

PMSF, b-mercaptoethanol and EDTA at three concentra-

tions (1, 5 and 10 mM) on the enzyme activity was

examined. The residual activity was assayed after incu-

bating the purified enzyme with these agents for 15 min at

optimum temperature and pH. The activity of the enzyme

in 50 mM sodium acetate buffer, pH 4.0 (in the absence of

additive) was taken as 100 %.

TLC analysis of hydrolytic products

To analyze the hydrolytic products of enzyme action on

starch, TLC was carried out according to Zhang’s method

(Zhang et al. 2007). Chromatography was performed on a

precoated silica gel plate using a mixture of formic acid/n-

butanol/water, 8:4:1 (v/v) as the mobile phase. The purified

Extremophiles (2013) 17:339–348 341

123

enzyme was incubated with 2 % starch for 24 h. The

hydrolysis products were removed at 30 min, 1, 2, 4, 6 and

24-h intervals. Spots were visualized by immersing the

plate in detection reagent (2 ml of aniline, 1 ml of 37.5 %

HCl, 100 ml of ethyl acetate, 10 ml of 85 % H3PO3, and

2 g of diphenylamine) after incubating the plate for 10 s at

150 �C.

Substrate specificity

Substrate specificity of the purified enzyme was assessed

for its ability to hydrolyze various carbohydrates. Three

different polymeric carbohydrates (1.0 % w/v), including

glycogen, a-cyclodextrin and b-cyclodextrin were used. A

1 % (w/v) substrate solution (900 ll) was mixed with

100 ll of the enzyme solution (0.1 mg/ml). Afterwards, the

enzyme assay was carried out under the standard condi-

tions. The amount of starch hydrolysis (1.0 %, w/v) was

proposed as 100 % activity.

Enzyme kinetics

To determine the kinetic parameters, 0.1 ml of enzyme

(0.1 mg/ml) was incubated in the presence of 0.9 ml starch

at different concentrations (0.1–1.2 % w/v). The assays

were accomplished at optimum pH and temperature of the

enzyme. The Michaelis constant (Km) and maximum

velocity of the reaction (Vmax) were calculated according to

Michaelis–Menten equation using SigmaPlot software 8.0

(Bangalore, India).

Effect of ionic liquids on the enzyme activity

Ionic liquids (ILs) constructed of relatively large asym-

metric organic cations and inorganic or organic anions.

They have been taken as a promising new class of solvents

for different reactions. The use of ionic liquids in enzyme

catalyze reactions has been widely investigated. As previ-

ously reported, ionic liquids can be extensively useful for

dispersing starch (Dabirmanesh et al. 2011). However,

whether starch hydrolyzing enzymes such as amylases are

active in ionic liquids is unknown. Finding the activity of

amylases in ionic liquids may lead to a more impressive

technology for hydrolyzing starch which is a significant,

application for many industrial processes. To investigate

the effect of ionic liquids on the enzyme activity, con-

centrations of 2–10 % of ionic liquids in 50 mM sodium

acetate buffer (pH 4) were prepared. Subsequently, the

enzyme (0.1 mg/ml) was added to various concentrations

of ionic liquids. After addition of 1 % (w/v) starch as

substrate, the residual enzyme activity was measured.

Enzyme activity in the absence of ionic liquids (100 %)

was considered as a control.

Results and discussion

Strain identification

Water samples were collected from Dig Rostam hot min-

eral spring. Three different colonies were detected and

screened for a-amylase activity on the agar plate. Finally,

one colony which produced a clear zone on the plate was

selected as amylase producer strain. According to the

methods described in ‘‘Bergey’s Manual of Systematic

Bacteriology’’, the isolated strain was a gram-positive, rod-

shaped, indole-positive, non-motile and hemolysis negative

bacterium (data not shown). Sequencing of PCR-amplified

16S rRNA was carried out, and the length of the sequenced

segment was observed to be 1351 bp.

To search homology of the sequence, BLAST program

was used and the results showed a high similarity to

Bacillus genus. The obtained sequence was submitted in

the NCBI GenBank as Bacillus sp. DR90 with accession

number JN713925. 16S rRNA sequences from Bacillus

species were achieved from National Center for Biotech-

nology Information (NCBI, http://www.ncbi.nlm.nih.gov/),

and multiple sequence alignments were performed with

Clustal W. The phylogenetic tree was constructed using

CLC Main Workbench ver.6.8.1 (CLC Bio, Denmark). The

result of the phylogenetic tree showed that the isolated

strain is most closely related to Bacillus as shown by this

tree (Supplementary Material). The strain was then

deposited in Iranian Biological Resource Center as the

acquisition number of IBRC-M 10741.

Fig. 1 Effect of temperature on the growth rate of bacterium. The

best growth was appeared at 42 �C (filled circle). Bacillus sp. DR90

did not grow at 50 �C (the standard errors were less than 5 % of the

means)

342 Extremophiles (2013) 17:339–348

123

Effect of temperature on bacterial growth

The effect of temperature on Bacillus sp. DR90 was studied

at 37, 42 and 50 �C. The bacterial growth (OD 600 nm)

was monitored during 72 h. The fastest growth was

observed at 42 �C among the three temperatures. Bacillus

sp. DR90 did not grow at 50 �C. The graph shows that the

highest cell density was reached at 42 �C. Also, the cells

have a faster rate of growth at 42 �C (Fig. 1).

Optimization of bacterial growth

The growth of Bacillus sp. DR90 and the production of

amylase depend on the type of culture medium. The

amylase activity of isolated strain was measured at various

intervals during growth in all media. Among the culture

mediums, the medium 4 (1 % of peptone, 1 % of maltose,

1 % of starch and 2 % of yeast extract) exhibited a con-

siderable impact on the microorganism growth and enzyme

production. Amylase production by Bacillus sp. DR90 was

started at early log phase and reached its maximum level at

the middle of exponential phase. In medium 2 (1 % of

fructose, 1 % of starch and 1 % of peptone), the production

of amylase enzyme is as same as medium 4, but the growth

of microorganism is lower than the medium 4. Further

results indicate that the mediums 1 and 3 show lower

values for amylase production and the growth of micro-

organism (data not shown).

Enzyme purification

Bacillus sp. DR90 amylase was purified by ammonium

sulfate precipitation, ultrafiltration and Q-Sepharose anion

exchange chromatography. The results of enzyme purifi-

cation are summarized in Table 1. In the first Q-Sepharose

anion exchange chromatography, the enzyme was sepa-

rated from the most of the proteins. Using the second

Q-Sepharose anion exchange chromatography, a 12.5-fold

purification with a yield of 4.2 % was obtained. The spe-

cific activity was achieved from 62.32 U/mg in the crude

extract to 780.79 U/mg in the final step. To determine the

molecular weight of purified enzyme, SDS-PAGE was

performed. Molecular weight of the purified enzyme was

evaluated to be 68.9 kDa (Fig. 2a). The native PAGE gel

showed a single band with amylolytic activity, which this

result is consistent with the SDS-PAGE (Fig. 2b). Molec-

ular masses of amylases from various microbial sources

vary from 10 to 210 kDa (Gupta et al. 2003). These results

were in agreement with the previous reports on the

molecular masses of amylases from Geobacillus

Table 1 The purification steps of amylase enzyme from Bacillus sp. DR9

Steps Volume (ml) Total protein (mg) Specific activity (U/mg) Total activity Fold purity Yield (%)

Crude extract 200.0 62.4 62.3 3887.0 1.0 100.0

Ultrafiltration 60.0 9.9 109.8 1090.0 1.8 28.0

First Q-Sepharose column 90.0 0.1 754.8 99.0 12.1 5.1

Second Q-Sepharose column 9.0 0.1 780.8 82.0 12.5 4.2

M1234 (b)(a)

62

130

42

29

22

14

175

95

10.5

51

70

2 1

Fig. 2 a Polyacrylamide gel electrophoresis analysis of the purified

amylase from Bacillus sp. DR90, from various purification steps.

SDS-PAGE: M marker, Lane 1 purified enzyme after second

Q-Sepharose column, Lane 2 amylase enzyme after first Q-Sepharose

column, Lane 3 sample after ultrafiltration, Lane 4 crude enzyme

extract. b Zymogram of purified enzyme. Lane 1 amylase activity

after first Q-Sepharose chromatography, Lane 2 the activity of

purified enzyme after second Q-Sepharose chromatography

Extremophiles (2013) 17:339–348 343

123

caldoxylosilyticus TK4 (Kolcuoglu et al. 2010) and

Bacillus sp. RM16 (Hassan et al. 2011).

Effect of pH on the enzyme activity and stability

pH profile of the amylase enzyme is shown in Fig. 3a. The

maximum activity was appeared at pH 4.0, and more than

50 % of the maximum activity was presented in the pH

range of 3.0–5.0. As depicted in Fig. 3a, BLA (Bacillus

licheniformis a-amylase) as control showed that the

activity of the enzyme is above 50 % in pH range between

4.0 and 9.0. The pH stability of isolated enzyme is

illustrated in Fig. 3b. The amylase was stable from pH

3.0–5.5, whereas most of the amylases are unstable at low

pH (Shaw et al. 1999). More than 80 % of its initial activity

retained after 60 min at pH 3.5, while at the same condi-

tions, BLA did not have activity at pH 3.5 after this time.

From the viewpoints of industrial applications, having a

high stability at pH 3.5 is very noticeable (Sajedi et al.

2005). In addition, the liquefaction step in the starch pro-

cess is requisite to perform at pH 5.8–6.2, which is in close

proximity to the optimum pH of the amylase in use

(Hashida and Bisgaard-Frantzen 2000). Employing an

amylase enzyme which is active and stable at low pH

Fig. 3 Characterization of purified amylase. Influence of pH on the

activity (a) and stability (b) of Bacillus sp. DR90 amylase (filledcircle). The optimum pH for the best activity was displayed at 4.0.

The behavior of BLA (filled square, Bacillus licheniformis a-

amylase) was assayed as control. Each data point depicts the mean

of three independent assays. c Influence of different temperatures (�C)

on the activity of the purified amylase. The activity of the enzyme at

45 �C was assumed as 100 %. d Thermostability of amylase enzyme

in the presence (filled triangle) and absence (filled circle) of Ca2?. As

shown in d, the thermal stability of this enzyme is not affected by the

addition of Ca2?

344 Extremophiles (2013) 17:339–348

123

values to omit the pH adjustment step is of a great sig-

nificant in the starch process. Moreover, the utilization of

amylase that performs at lower pH values declines

formation of some by-products, such as maltulose at higher

pH values (Goyal et al. 2005).

Effect of temperature on the enzyme activity

The influence of temperature on the enzyme activity is

displayed in Fig. 3c. The optimum temperature for the

amylase activity was 45 �C. The amylase activity reduced

sharply above 70 �C. The enzyme exhibited 68.9 and 60 %

of maximum activity at 30 and 60 �C, respectively. The

enzyme retained more than 70 % of its initial activity after

45 min of incubation below 60 �C. These findings are in

agreement with the reports for Bacillus sp. YX-1 (Liu, and

Xu 2008) and Bacillus cereus Ms6 (Al-ZaZaee et al. 2011)

which displayed an optimal temperature of 45 �C.

The activity of the purified amylase was also studied by

incubating the enzyme at temperature range from 30 to

95 �C in the presence of 5 mM CaCl2. As depicted in

Fig. 3d, the thermal stability of this enzyme is not affected

by the addition of Ca2?.

The thermal inactivation was examined at temperatures

ranging from 30 to 80 �C for 150 min. The enzyme

retained more than 50 % of its initial activity after 1 h of

incubation from 30 to 50 �C. The half-life estimated at the

optimum temperature (45 �C) was 138 min (data not

shown). The half-life of Bacillus sp.DR90 amylase at

60 �C was more than amylases enzyme from Lactobacillus

manihotivorans (Aguilar et al. 2000) Bacillus sp. KR-8104

(Sajedi et al. 2005) and Bacillus sp. YX-1 (Liu and Xu

2008) while less than from Bacillus aquimaris VITP4

(Anupama and Jayaraman 2011) and Bacillus subtilis

DR8806 (Asoodeh and Lagzian 2012).

Effect of metal ions, chemical reagents and detergents

The effect of metal ions (K?, Na?, Zn2?, Ba2?, Mg2?,

Ca2?, Fe2? and Hg2?), chemical reagents (EDTA, PMSF

and b-mercaptoethanol) and detergents (SDS and Triton

X-100) on the enzyme activity was studied at three dif-

ferent concentrations (1, 5 and 10 mM). As depicted in

Fig. 4a, among the examined metal ions, Mg2?, Fe2? and

Ba2? increased the amylase activity, while Hg2? and Zn2?

were established to inhibit enzyme activity. Zinc ions have

also inhibited the enzymatic activity of Bacillus amylo-

liquefaciens (Gangadharan et al. 2009) and Geobacillus sp.

LH8 (Mollania et al. 2010). It is recommended that this

inhibitory effect could be due to competition between the

protein-associated cations and the exogenous cations.

Furthermore, our results showed that K?, Na? and Ca2?

had no effect on the amylase activity. This finding is

consistent with the previous reports on the effect of K?,

Na? and Ca2? on amylase activity from Geobacillus sp.

LH8 (Mollania et al. 2010) and Bacillus sp.KR-8104

Control NaCl KCl CaCl2 ZnCl2 FeCl2 BaCl2 HgCl2 MgCl2

G1

G2

G3

G4

G5

G6

G7

s 4 h2 h 12 h 24 h6 h1 h0.5 h Starch

(a)

(b)

(c)

Fig. 4 a Effects of different mono- and divalent cations on the

activity of purified amylase enzyme. Each data point depicts the mean

of three independent assays (the standard errors were less than 5 % of

the means). b Effect of different chemical agents at three concen-

trations (1, 5 and 10 mM) on the activity of amylase from Bacillus sp.

DR90. c Thin-layer chromatography (TLC) analyses of the hydrolysis

products of the purified enzyme. From left to right, standard

oligosaccharides (S, Lane 1), hydrolysis products for 0.30 h (Lane2), 1 h (Lane 3), 2 h (Lane 4), 4 h (Lane 5), 6 h (Lane 6), 12 h (Lane7), 24 h (Lane 8) and starch sample that used as substrate (Lane 9).

Lane 1 exhibits the standard oligosaccharides (G7 maltoheptose, G6maltohexose, G5 maltopentaose, G4 maltotetraose, G3 maltotriose,

G2 maltose, G1 glucose)

Extremophiles (2013) 17:339–348 345

123

(Sajedi et al. 2005). As shown in Fig. 4b, PMSF and b-

mercaptoethanol had no effect on the amylase activity. To

utilize the amylase enzyme in detergent industries, it must

be resistant to various detergent ingredients, such as sur-

factants and cheleators (Arikan 2008). The amylase from

Bacillus sp. DR90 retained 60 and 90 % of its initial activity

during incubation with SDS and Triton X-100 (10 mM),

respectively. Therefore, this enzyme may be useful in

detergent industries. The enzyme was almost stable at high

concentrations of EDTA (10 mM) and preserved its activity

up to 94 %. The enzymatic activity in the presence of EDTA

was not inhibited in Bacillus sp. RM16 (Hassan et al. 2011)

and Alicyclobacillus sp. A4 (Bai et al. 2012).

Thin layer chromatography of enzyme hydrolysis

products

The hydrolysis products of starch were analyzed by TLC

technique (Fig. 4c). After 6 h of reaction, glucose, maltose

and maltotriose were observed as predominant products.

The value of glucose production enhanced with increasing

incubation time. Thus, after 24 h of incubation, glucose

and maltose were the main products; thus, the enzyme

acted specifically on a-1, 4 glucosidic linkages. These

results demonstrated that the amylase from Bacillus sp.

DR90 is an endo-type amylase (Metin et al. 2010) capable

of completing the hydrolysis of starch in 24 h.

Substrate specificity

The enzyme was studied for its ability to hydrolyze dif-

ferent carbohydrates, as shown in Fig. 5a. Among the

substrates, soluble starch was significantly hydrolyzed by

the enzyme. The activity of the enzyme towards glycogen,

a-cyclodextrins and b-cyclodextrins was 30, 5 and 5 %,

respectively. Similar results have been reported for Bacil-

lus sp. RM16 (Hassan et al. 2011).

Enzyme kinetics

The kinetic constants (Vmax and Km) for amylase from

Bacillus sp. DR90 were calculated by incubating enzyme

with various concentrations of starch as a substrate

(0.1–1.2 %). As estimated from Michaelis–Menten

[S] (mg/ml)

V (

µM/M

in/m

g)

(b)

0

50

100

150

200

250

0 2 4 6 8 10 12 14

(a)

(c)

Fig. 5 a Substrate specificity of

the purified amylase toward

various substrates. b Michaelis–

Menten plot for Bacillus sp.

DR90 amylase enzyme. c Effect

of ionic liquids on the activity

of amylase from Bacillus sp.

DR90

346 Extremophiles (2013) 17:339–348

123

equation (Fig. 5b), the Km and Vmax values were

4.5 ± 0.13 mg/ml and 307 ± 12 lM/min/mg, respec-

tively. The kinetic values of different amylases are difficult

to compare to each other’s because of their dependence on

different substrates and the reaction conditions. The Km

value of the purified amylase for starch was within range of

the majority of amylases (0.35–4.7 mg/ml) (Mollania et al.

2010). Similar finding was obtained for an amylase from

Lactobacillus manihotivorans (Aguilar et al. 2000).

Effect of ionic liquids on enzyme activity

The effects of four N,N-dialkylimidazolium-based ionic

liquids, including 1-ethyl-3-methylimidazolium bromide

([EMIM][Br]), 1-n-butyl-3-methylimidazolium bromide

([BMIM][Br]),1-hexyl-3-methylimidazoliumbromide([HMIM]

[Br]) and 1-butyl-3-methylimidazolium chloride ([BMIM]

[Cl]) on the activity of amylase were investigated. As

depicted in Fig. 5c, the results showed that the ionic liquids

had inhibitory effect on the amylase activity, among them,

[HMIM][Br] exhibited the highest inhibitory effect. Fur-

thermore, among Br-containing ionic liquids, enhancing

the alkyl group leads to more inhibitory effects on the

enzyme activity. Similar results have been reported for two

amylase enzymes from Bacillus amyloliquefaciens and

Bacillus lichiniformis (Dabirmanesh et al. 2011).

Conclusion

The present study describes the purification and biochem-

ical characterization of an acidophilic Ca2?-independent

amylase from Bacillus sp. DR90. The enzyme was purified

by ion-exchange chromatography with 12.5-fold purifica-

tion and a specific activity of 780.79 U/mg. It displayed a

molecular mass of 68.9 kDa by SDS-PAGE. Bacillus sp.

DR90 amylase was more active at acidic pH ranges with

the optimum pH of 4.0. These features of the enzyme are

more desirable in the starch industry than majority of the

commercial alpha-amylases (pH 6.0–6.5 and Ca2?-depen-

dent). Furthermore, the purified amylase showed stability

towards the non-ionic surfactants such as Triton X-100 and

anionic surfactants such as SDS. Considering its favorable

properties, it could be considered as a novel amylolytic

enzyme which would be an appropriate alternative in starch

industry. More studies would be required so as to under-

stand the mechanism of enzyme in low pH and its Ca2?-

independency feature. Ultimately, it is recommended that

the properties of this Ca2?-independent enzyme such as

thermostability and pH profile could be modified by protein

engineering to design a notable enzyme for industrial

applications.

Acknowledgments We gratefully thank the Institute of Biotech-

nology and research council of Ferdowsi University of Mashhad for

their financial support (Grant number: 3/17458; 06-02-1390 and 4065;

06-02-1389).

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