ISSN 00262617, Microbiology, 2015, Vol. 84, No. 2, pp. 210218. Pleiades Publishing, Ltd., 2015.

EXPERIMENTAL
ARTICLES

Insight on Biochemical Characteristics of Thermotolerant Amylase

Isolated from Extremophile Bacteria
Bacillus vallismortis TD6 (HQ992818)1
Chandrasekaran Suganthi, Anbazhagan Mageswari,
Sivashanmugam Karthikeyan, and Kodiveri Muthukaliannan Gothandam2
School of Bio Sciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India
AbstractHalotolerant bacterium Bacillus vallismortis (HQ992818) was isolated from saltern sediments in
India, and produced significantly high levels extracellular amylase. A detailed investigation on the culture
conditions including period of incubation, media pH, and inoculum size in addition to different sources of
carbon and nitrogen, metal ions, NaCl, and amino acids was carried out for optimized production. Maxi
mum amylase production (62 U/mL) was attained after 26 h of incubation. The optimized conditions for
maximal production of amylase were found to be 1% NaCl, pH 8, temp 37C, 1% starch, 1% sodium nitrate,
phenyl alanine (0.01%) and calcium chloride (10 mM). The biochemical characteristics of the extracellular
amylase were studied with respect to change in temperature, pH and metal ions. The enzyme was found to be
optimally active in the temperature range of 4070C and pH 8. Activation of the enzyme by Ca2+ (135%),
Fe2+ (113%) and Mg2+ (109%) occurred at 5 mM concentration and strongly inhibited by Hg2+, Zn2+ and
Mn2+ occurred at 10 mM. Significant compatibility of the enzyme with the commercial laundry detergents
and the results of washing performance test confirmed its effectiveness. Available data on the optimized cul
ture conditions enables for easily adaptable setup of large scale production of the enzyme for use in detergent
formulations.
Keywords: amylase, Bacillus vallismortis, halotolerant, optimization, thermotolerant
DOI: 10.1134/S0026261715020162
1

Amylases are a major class of hydrolases involved in

starch catalysis. They cleave starch molecules to give
distinct products including dextrin and progressively
smaller polymers composed of glucose units. Based on
their mode of action, amylase are classified into;
endoamylases, exoamylases and debranching amy
lases. Amylase occupies 2530% of the world enzyme
market and it has great significance for biotechnologi
cal applications [1]. Amylase has a wide spectrum of
biotechnological applications in industrial sectors
such as textile, paper, sugar, brewing and baking. It is
also used in the preparation of cakes, fruit juices,
starch syrups and used as digestive aid in pharmaceu
tical industries [2]. Amylases obtained from meso
philic organisms possess extensive applications in
industries. However their industrial application is
often limited by factors such as salt solutions, high
temperature, extremes of pH, organic solvents and
heavy metals which inhibit the enzyme activity [3]. In
the field of biotechnology, bacteria are considered as a
source of diverse enzymes possessing features worthy
of industrial utility, in particular enzymes from extre
mophilic bacteria are of greater important due to the

1 The article is published in the original.

2 Corresponding author; email: gothandam@yahoo.com

production of stable and valuable enzymes [4]. Among

the extremophiles, halophiles are microbes which can
survive and multiply in hypersaline environments.
They are found to inhabit salt marshes, hypersaline
seas, salt evaporation pools, salt mines and salted
meats [5]. Halophiles can be classified into three dif
ferent groups based on the requirement of NaCl.
(A) Slight halophiles (25%), (B) moderate halo
philes (520%) and (C) extreme halophiles (2030%)
[6]. Amylases from halophilic bacteria would have the
advantage of the enzymes having optimal activities at
higher salt concentrations with prospective use in
industries [7]. Very few genera of halophilic or halotol
erant bacteria which include Halobacillus sp. strain
MA2 [8], Bacillus sp. strain TSCVKK [2], Chromo
halabacter sp. TVSP101 [9], Bacillus barbaricus [3]
have been reported to producer as amylase. The
increasing urge from biotechnological industries for
enzymes with specific properties necessitates the cur
rent investigations on microorganisms prevailing in
extreme environments [10]. Hence, screening
for microbes inhabiting the extreme environments for
amylase activities could facilitate the sighting of
novel amylases fitting industrial applications. Due to
the fact that bacteria are greatly influenced by media
components such as carbon and nitrogen sources,

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THERMOTOLERANT AMYLASE FROM EXTREMOPHILIC Bacillus

minerals and physical factors such as pH and inocu

lum density [11]. Media optimization for enhancing
amylase production from the selected organism was
performed in the present study. Hence this work aims
to identify a halotolerant bacterium capable with high
amylase activity, characterize the enzyme properties
and optimize culture parameters for sustained indus
trial production.
MATERIALS AND METHODS
Strains and culture conditions. Sample collected
from Tuticorin (Tamil Nadu, India) saltern sediments
at 34 cm depth. Collected samples were serially
diluted in sterile saline water and the dilutions were
plated in nutrient agar medium with 5% NaCl. Plates
were incubated at 37C for 48 h. After incubation,
morphologically varying colonies were picked and
purified on the same medium. For further studies pure
isolated colonies were maintained as glycerol stocks
and stored at 80C.
Screening for amylase activity and quantification.
All the isolates were screened for amylase production
in nutrient agar media supplemented with 1% starch
and 5% NaCl. The plates were incubated at 37C for
24 h. After incubation plates were flooded with iodine
solution. Amylase producing strains were chosen
based on the zone of clearance and were subjected for
quantification. Amylase produced by isolated strains
was quantitatively determined using the method of
Miller [12]. One unit of enzyme activity was defined as
the amount of enzyme releasing 1 mol glucose per
minute with glucose as standard.
Identification of bacteria. Morphological charac
teristics and Grams staining were studied for the iso
lated bacteria as per the standard protocols. Genomic
DNA of the microbes was isolated using the method of
Sambrook et al. [13]. The genomic DNA isolated from
the bacteria was amplified using the following univer
sal 16S rRNA primers: forward primer 5' GAG TTT
GAT CCT GGC TCA G 3' (E. coli positions 827)
and reverse primer 5' ACG GCT ACC TTG TTA CGA
CTT 3' (E. coli positions14941513). The amplified
PCR product was carried out for 16S rRNA gene
sequencing in Macrogen (Seoul, Korea). The
obtained sequence was compared with NCBI database
using BLAST search tool and the phylogenetic tree
was constructed with the MEGA v. 5.04 using neigh
bour joining method with a bootstrap value of 1000.
The 16S rRNA gene sequences of Bacillus vallismortis
TD6 was submitted to GenBank database.
Effect of salt concentration on amylase production.
To find out the optimal salt concentration for amylase
production in the medium by varying 15% of NaCl
concentration. Inoculated medium was incubated at
37C for 24 h. After incubation enzyme activity was
quantified.
Growth kinetics for amylase production. To deter
mine the growth and incubation period for extracellu
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lar amylase production, the culture were inoculated in

the nutrient broth medium with 1% NaCl and 1%
starch. Inoculated medium was incubated for a period
of 48 h at 37C and 130 rpm. At every six hours interval
culture was taken and amylase assay was quantified for
cell free extracts. For growth kinetics cells were mea
sured at 600 nm.
Effect of pH and inoculum. In order to investigate
the influence of pH on amylase production, the strain
was grown in 1% salt medium and 1% starch at differ
ent pH (3.011.0) and constant temperature at 37C.
Amylase production was studied for varying inoculum
(15%) level. After 26 h incubation amylase activity
was quantified.
Effect of various carbon and nitrogen sources. Vari
ous simple and complex carbon sources including glu
cose, fructose, galactose, maltose, lactose, sucrose,
starch and pectin were used as a sole source of carbon
(1%). Organic nitrogen sources peptone and yeast
extract, inorganic nitrogen sources including ammo
nium chloride, ammonium nitrate and sodium nitrate
(1%) were used. The pH was adjusted to 8. The
enzyme activity was monitored after 26 h incubation at
37C under shaking conditions of 130 rpm.
Effect of different amino acids and metal ions. The
effect of amino acids on the medium was studied with
(0.01%) of Lalanine, Lmethionine, Lphenylala
nine, Llysine and glycine. The pH was adjusted to 8.
The medium was incubated at 37C at 130 rpm. After
a period of 26 h the amylase activity was quantified.
Similarly the effect of metals ions also was investigated
with different metal ions (10 mM) such as calcium
chloride, manganese chloride, cobalt chloride, ferric
chloride, magnesium sulphate and sodium chloride.
Characterization of amylase enzyme. The extra cel
lular culture supernatant obtained from the optimized
medium was used for determining the biochemical
characteristics of the enzyme with respect to different
temperature, pH and metal ions.
Effect of temperature and pH on amylase activity.
The effect of pH on the activity of amylase was mea
sured in the 3.010.0 range, using the appropriate
buffers at a concentration of 50 mM (3, 4, citrate
buffer; 5, acetate buffer; 68, phosphate buffer; 910,
glycineNaOH) under standard assay conditions. The
effect of temperature on the activity of amylase
enzymes was measured in the range of 20100C.
Effect of metal ions. Enzyme assays were performed
in presence of different metal ions, at 5 and 10 mM
final concentration. The chloride salts of Zn2+, Ca2+,
K+, Mn2+, Hg2+, Cu2+, Na+, Ba2+, Fe2+, Co2+ and
Mg2+ were used.
Compatibility of amylase with commercial deter
gents. In order to validate the potential of Bacillus val
lismortis TD6 crude amylase as a detergent composi
tion and its compatibility towards commercial laundry
detergent such as Rin, Surf excel (Hindustan Uni
lever), Ariel, Tide Naturals (Procter and Gamble),

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CHANDRASEKARAN SUGANTHI et al.

Table 1. Morphological and biochemical characterization

of strain TD6
Characteristics
Gram staining
Shape
Motility
Growth in NaCl concentration
3%
5%
8%
10%
Catalase
Oxidase
Sugar fermentation
Glucose
Xylose
Mannitol
Sorbitol
Sucrose
Nitrate reduced to nitrite
Starch hydrolysis

Result
G + ve
Ellipsoidal
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+, positive reaction.

Ujala (Jyothi Laboratories), Wheel, Arasan, Pon

vandu (Hindustan Unilever), Mr. White and
Henko (Henkel India Ltd.) were used for detergent
compatibility studies. The solid detergents (7 mg/mL
was dissolved in tap water) were heated at 100C for 90
minutes to inactivate the indigenous enzymes present
in the commercially available detergents. Crude
enzyme containing 50 U/mL was mixed with the
detergent in the ratio of 1 : 1 (v/v) and incubated at
55C for 1 h, followed by the relative activity was cal
culated. The enzyme activity of control sample was
mixed with tap water and crude enzyme (1 : 1 v/v)
without detergent was taken as control 100%.
Evaluation of wash performance of halotolerant
amylase with detergent on different stained clothes
was done. Clean cotton fabrics (7 cm 7 cm) were
soiled with 100 L of five different stains which
include: Ketchup, chocolate, blood, tea and coffee
were applied on the cotton fabrics without any pre
treatment and dried. All the stained cotton fabrics
were subjected for washing performance in individual
100 mL conical flask containing 7 mg/mL of detergent
and 7 mg/mL detergent with 50 U/mL of TD6 crude
amylase. Then each flask was incubated for 1 h at
45C. After 1 h incubation stained cotton fabrics were
taken out, rinsed with tap water and dried. Stain
removal was confirmed by visual examination.

RESULTS AND DISCUSSION

Isolation and screening of amylase producing bacte
rium. In the present study, we have isolated fifteen hal
otolerant bacteria from saltern sediments of Tuticorin.
All the strains were screened for amylase production.
Among them seven isolates capable of producing zone
of clearance was considered as amylase positive organ
isms. Based on the level of amylase production isolate
TD6 was found to produce maximum units of enzyme
(20.32 U/mL) after 24 h of incubation period at 37C.
Therefore, TD6 was selected for further studies.
Identification of bacterium. The isolated strain TD6
was observed to be gram positive rods; smooth, circu
lar, spore forming, motile and rod like structure under
microscopic observation. The morphological and bio
chemical characteristics of the isolated strain TD6 are
shown in (Table 1). The strain was capable of growing
up to 10% NaCl and found to be catalase, oxidase and
nitrate reductase positive. It was capable of fermenting
glucose, xylose, mannitol, sorbitol and sucrose. On
the basis of 16S rRNA gene sequence analysis, the
strain was found to have 99% homology with Bacillus
vallismortis (DV1F3) isolated from desert soil in
Death Valley, California [14]. Hence the isolate TD6
was identified as type strain of Bacillus vallismortis
(Fig. 1). The 16S rRNA sequence of Bacillus vallis
mortis TD6 was submitted to GenBank (accession
number HQ992818).
Effect of salt concentration on amylase production.
Amylase production was initially observed for different
concentration of NaCl from 15%. Maximum
enzyme secretion was observed (Fig. 1b) at 1% NaCl
(40.67 U/mL). With increase the salt concentration,
enzyme production and growth was decreased. But
organism was found to grow up to 10% NaCl concen
tration. Due to high salt concentration in the medium
growth of bacteria was found to decrease which was
found to directly affect the production of enzyme.
Similarly were seen in Bacillus sp. [15]. Vijayabaskar
et al. reported that the maximum amylase production
was attained at 3% NaCl from moderately halophilic
bacterium Bacillus cereus [16].
Kinetics of bacterial growth and amylase produc
tion. The bacterial growth and enzyme production was
studied in the nutrient broth supplemented with 1%
NaCl. The effect of incubation period on bacterial
growth showed that the strain had a lag phase up to 2 h
after which exponential phase was observed up to 12 h
followed by stationary phase. The enzyme production
was observed from log phase and extended up to the
stationary phase (Fig. 1c). Maximum amylase produc
tion was achieved during stationary phase at 26 h
(38.72 U/mL) of incubation period, after which both
the enzyme production and growth was decreased as
the culture entered into death phase and also due to
depletion of nutrients in the medium [17]. In case of
Bacillus sp. ANT6 maximum amylase production was
attained after 24 h of incubation [18]. Most of the
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213

(a)
90 Bacillus licheniformis
75
Brevibacterium halotolerans
60

Bacillus subtilis
TD6
99 Bacillus vallismortis
Bacillus amyloliquefaciens
Bacillus aerius

Bacillus atrophaeus
99

Bacillus pumilus
Bacillus stratosphericus
96 Bacillus altitudinis
Bacillus aerophilus

100
0.002
(b)
Amylase activity, U/mL

OD600 nm
3

40
2

30

Bacillus safensis

(c)
Amylase activity, U/mL
50
40
30

20
1
10

10
0

20

10

20

30
Time, h

0
1

5
NaCl, %

Fig. 1. (a) Phylogenetic position of strain TD6 and other closely related Bacillus species based on 16S rRNA gene sequencing
analysis. The phylogenetic tree was constructed by neighbour joining method, strain TD6 showing the position of among phylo
genetic neighbours. Bootstrap (n = 1000) values below 50 are not shown. (b) Effect of varying NaCl concentration on amylase
production by Bacillus vallismortis TD6. (c) Effect of incubation period and growth on amylase production by Bacillus vallismortis
TD6 OD at 600 nm (circle) and amylase activity (triangle). Results represent the means of three separate experiments, and bars
indicate standard deviation. Absence of bars indicates that errors were smaller than symbols.

halophilic bacteria produce amylase during stationary

phase [2, 9].
Effect of pH and inoculum on amylase production.
Amongst the physical parameters, the pH of the
growth medium plays vital role by inducing morpho
logical change in the organism and in enzyme produc
tion. The pH also serves as a valuable indicator of the
initiation and end of enzyme synthesis. Most of the
Bacillus sp. produce amylases by SmF (Submerged
Fermentation) have most favourable pH ranging
between 6.0 and 9.0 for growth and enzyme produc
tion [19]. In the present study, there was moderate
growth and amylase production was seen in acidic pH
but maximum amylase production was observed at
alkaline pH 8 (38 U/mL) (Fig. 2a). Similar findings
have been reported on Bacillus barbaricus [3]. Bacillus
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subtilis JS2004 produced maximum amylase at pH 7

[20]. Inoculum percentage in the medium was investi
gated for amylase production; maximum amylase pro
duction was obtained in the presence of 2% inoculum
(Fig. 2b). Further increase in inoculum size was found
to decrease the enzyme production. Low level of inoc
ulum results in a less number of cells in the production
medium, which requires a longer time to grow an opti
mum number to use the substrate and form the desired
product. High level of inoculum in the medium may
secrete high amount of enzymes which rapidly
depletes the nutrients required for growth and product
synthesis. In the present study 2% inoculum
(45 U/mL) was found to be produce maximum amy
lase production. An inoculum size of 5% enhanced
amylase production by Bacillus cereus [21].

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CHANDRASEKARAN SUGANTHI et al.

Amylase activity, U/mL

(a)

Amylase activity, U/mL

(b)

60

50
40

40

30
20

20

10
0

0
4

Amylase activity, U/mL

10
pH

(c)

Amylase activity, U/mL

50

50

40

40

30

30

20

20

10

10

5
Inoculum, %

(d)

0
Mal Suc Fru Sta Lac Glu Pec Gal
Carbon sources

Pep

YE

AN
SN
Nitrogen sources

Fig. 2. (a) Effect of pH on amylase prodution in Bacillus vallismortis TD6. (b) Effect of inoculum level on amylase production by
Bacillus allismortis TD6. (c) Influence of various carbon sources on amylase production by Bacillus vallismortis TD6. Malmal
tose, Sucsucrose, Frufructose, Stastarch, Laclactose, Gluglucose, Pecpectin, Galglactose. (d) Influence of
various sources of nitrogen on amylase production by Bacillus vallismortis TD6. Peppeptone, YEyeast extract, ANammo
nium nitrate, SNsodium nitrate.

Effect of carbon and nitrogen sources on amylase

production. The addition of carbon source in the form
of either monosaccharide or polysaccharides may
influence the production of amylase enzyme. Among
the different carbon sources tested (Fig. 2c), starch (40
U/mL), lactose (29 U/mL) and galactose (29 U/mL)
were found to support maximum amylase production.
In general the synthesis of carbohydrate degrading
enzymes in most of the Bacillus sp. is subjected to cat
abolic repression by readily metabolizable substrates
such as glucose and fructose [22]. Similar result was
reported from B. subtilis strain ASS01 [17] maximum
amylase production was found when starch was used as
carbon source. Use of dextrin showed maximum amy
lase production from Halobacillus sp. strain MA2 [8].
The nitrogen sources also play significant function in
the growth of the organism and enzyme production. In

the present investigation, various nitrogen sources

were tested (Fig. 2d), amylase production was
highest when sodium nitrate was used (38 U/mL).
Ammonium chloride was found to have a significant
negative effect of both growth and enzyme production.
Bacillus sp. HPE 10 [23] and Bacillus sp. [24] reported
maximum amylase production on use of yeast extract.
Effect of amino acids and metal ions on amylase
production. Different amino acids, metal ions
were tested for amylase production (Figs. 3a and 3b).
Moderate growth and amylase production was seen in
Lalanine and glycine. LMethionine was found to be
inhibiting the growth and enzyme production.
The addition of Lphenyl alanine resulted in maxi
mum (58 U/mL) amylase production. LCysteine has
been reported to stimulate amylase production [25].
Metal ions and trace elements are often essential for
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Amylase activity, U/mL

(a)

(a)

Relative activity, %

70

215

160

60
120

50
40

80

30
20

40

10
0

0
LAla

LPhe

Amylase activity, U/mL

LLys
Glyc
Amino acids (0.01%)
(b)

Relative activity, %

50

140

40

105

30

10 Control
pH

(b)

70

20
35
10
0
0

40
FeCl2

MgSO4

CoCl2

MnCl2
CaCl2
Metal ions (10 mM)

Fig. 3. (a) Effect of amino acid sources on the production

of Bacillus vallismortis TD6 amylase. LAlaalanine,
LPhephenyl alanine, LLyslysine, Glyglycine.
(b) Effect of different metal ions on amylase production
from Baciilus vallismortis TD6.

enzyme production. The presence of calcium chloride

in the medium showed a remarkable enhancement in
amylase production (41 U/mL). Similar observations
were observed in Bacillus cereus [16] and Bacillus sp.
TSCVKK [2] where amylase production was
enhanced by addition of calcium chloride in the
medium.
Effect of pH and temperature on amylase activity.
The influence of pH and temperature on the enzyme
activity is shown in (Figs. 4a and 4b). The enzyme pro
duced from strain TD6 was found to be active in a
broad pH range of 68. The optimum pH was
observed at pH 8 in alkaline condition and it also
showed activity at pH 9 and 10. Similar results were
reported on Bacillus subtilis [20]. Enzyme activity
declined at a greater pace when the pH of the medium
turned acidic compared to change in pH to alkaline
conditions. These results are rather divergent from
most of the bacterial amylase which are active at
slight acidic to neutral pH [26]. The supernatant amy
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45

50

55

60

65
70 Control
Temperature, C

Fig. 4. (a) Effect of pH on amylase activity of Bacillus val

lismortis TD6. (b) Effect of temperature on amylase activ
ity of Bacillus vallismortis TD6.

lolytic activities were assayed at different temperatures

ranging from 20 to 100C. The enzyme was found to
be optimally active at 55C attained 127% of relative
activity and it was found to be active in the range of 40
to 70C confirming that the enzyme is thermotolerant.
A reduction in enzyme activity was observed at tem
perature above 70C. The optimum pH and tempera
ture was reported for amylase activity as 7.58.5 and
50C, respectively [8].
Effect of metal ions on amylase activity. The activity
of various amylases is influenced by the presence of
metal ions. Certain metal ions are required as cofac
tors and are thus called metalloproteins. The amylase
activity was determined in the presence of different
metal salts. Ca2+, were found to stimulate amylase
activity by 135% (Table 2). Other divalent metal ions
such as Fe2+ (113%) and Mg2+ (109%) were also found
to potentiate the amylase activity but not to the extent
of calcium ion. Ca2+ was found to be involved in the
salting out of hydrophobic residues in the enzyme,
thereby causing adoption of compact structure [27].
The enzyme activity of amylase from strain TD6 was
significantly enhanced in the presence of Fe2+ which is

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CHANDRASEKARAN SUGANTHI et al.

Table 2. Effect of metal ions on activity of amylase pro

duced by Bacilllus vallismortis TD6
Relative activity, %
Metal ions
5 mM

10 mM

100

100

Ca

135 1

106 1

Fe2+

113 5

105 9

Mg2+

109 5

81 6

K+

96 2

62 1

Mn2+

45 6

34 8

Hg2+

43 9

18 3

Cu2+

83 1

51 7

Na+

88 4

79 9

Ba2+

68 2

57 8

Zn2+

51 1

26 3

Co2+

57 7

51 5

Control
2+

Values are mean SD of three determinations.

Table 3. Compatibility of crude amylase from Bacillus val

lismortis TD6 against commercial laundry detergents pre
incubated at 35 and 45C
Relative activity, %
Detergents
35C

45C

100

100

102 1

112 1

Surf Excel

60 5

64 9

Ariel

47 5

50 6

Tide naturals

60 2

65 1

Ujala

98 6

97 8

Wheel

87 9

93 3

Arasan

85 1

92 7

Ponvandu

97 4

100 9

Mr. White

73 2

79 8

Henko

65 1

75 3

Control
Rin

Values are mean SD of three determinations.

a rare characteristic of amylase. Majority of amylases

are inhibited in the presence of Fe2+ where as only one
report from thermostable amylase of Bacillus sp. I 3
reported by Goyal et al., was found to be enhanced in
the presence of Fe2+ [16]. Enzyme activity was
strongly inhibited by Hg2+, Zn2+ and Mn2+ at 10 mM.
However, metal ions such as Na+, K+ and Cu2+ did not
have any significant effect on the enzyme activity at
5 mM concentration. The inhibition by Hg2+ may
specify the significance of indole amino acid residues
in enzyme function and has been reported for micro
bial amylases [28]. Amoozegar et al., has reported
that amylase from Nesterenkonia sp. was stimulated by
Ca2+ and strongly inhibited by Fe3+, Cu2+, Zn2+ and
Al3+ [30]. The amylase from Bacillus firmus was also
strongly inhibited by heavy metals such as Ni2+, Cd2+,
Zn2+ and Hg2+ [29]. The amylase from Bacillus fir
mus was also strongly inhibited by heavy metals such as
Ni2+, Cd2+, Zn2+ and Hg2+ [30].
Washing performance test. In order to assess the
compatibility of thermotolerant alkaline amylase as a
laundry detergent additive, the crude enzyme was pre
incubated with the local commercially available wash
ing detergents in the market for one at 35 and 45C.
Laundry detergents, which comprise the most impor
tant part of the detergent market and it was formulated
mostly function at a high alkaline pH. The thermotol
erant amylase from Bacillus vallismortis TD6 showed
(Table 3) a significant compatibility towards most of
the commercial laundry detergents at both tested tem
peratures. The crude amylase from TD6 was found to
be more compatible with laundry detergent Rin
retaining more than 102 and 112% followed by Pon
vandu retaining 98 and 100% at 35 and 45C of its
initial activity. But considerable loss of amylase activ
ity has been observed in the presence of Ujala,
Wheel, Arasan, Mr. White and Henko. Some of
the detergents such as Surf Excel and Ariel showed
partial loss of amylase activity which may be recog
nized as inhibitory effects of components such as
anionic surfactants, bleaching agents and water soft
ening builders etc. High stain removal was observed
(Fig. 5) when crude enzyme mixed with laundry deter
gent was added on the stained cotton fabric for wash
ing purpose. Therefore, the thermotolerant amylase
from Bacillus vallismortis TD6 is suitable laundry
detergent additive for washing performance.
In this present investigation, a halotolerant bacte
rium was isolated from saltern sediments from Tutico
rin and identified as B. vallismortis TD6. This study
was the first report for amylase production from B. val
lismortis. Culture conditions were optimized with
One at a time for amylase production. After optimi
zation one fold of amylase production was increased.
The strain TD6 was capable of amylase production in
alkaline condition and it was active at high tempera
ture and the crude amylase enzyme is compatible with
commercial laundry detergents and it can be used for
improving the wash performance. Based on the results
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(a)

(b)

(c)

(d)

217
(e)

Control

Detergent

Detergent
+
Enzyme

Fig. 5. Wash performance of the crude amylase from Bacillus vallismortis TD6 in the presence of the commercial detergent Rin.
(1a) Ketchup (2a) Chocolate (3a) Blood (4a) Coffee (5a) Tea (1b) Ketchup with detergent (7 mg/mL) (2b) Chocolate with deter
gent (7 mg/mL) (3b) Blood with detergent (7 mg/mL) (4b) Coffee with detergent (7 mg/mL) (5b) Tea with detergent (7 mg/mL)
(1c) Ketchup with detergent (7 mg/mL) and 50 U/mL of crude amylase enzyme (2c) Chocolate with detergent (7 mg/mL) and
50 U/mL of crude amylase enzyme (3c) Blood with detergent (7 mg/mL) and 50 U/mL of crude amylase enzyme (4c) Coffee
with detergent (7 mg/mL) and 50 U/mL of crude amylase enzyme (5c) Tea with detergent (7 mg/mL) and 50 U/mL of crude
amylase enzyme.

of present investigation amylase produced by strain,

TD6 could be a potential biological ingredient in laun
dry detergent formulation.
ACKNOWLEDGMENTS
The authors thank VIT University for providing the
necessary facility to carry out this work.
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