Volume 5 Number 2 (June 2013) 159-165
Isolation and characterization of a thermophilic Bacillus sp. with
protease activity isolated from hot spring of Tarabalo, Odisha, India
1
1
Mrunmaya Kumar Panda , Mahesh Kumar Sahu , KumanandaTayung
2*
Department of Bioinformatics, North Orissa University, Takatpur, Baripada-757003, Odisha, India.
2
Department of Botany, North Orissa University, Takatpur, Baripada-757003, Odisha, India.
1
Received: April 2012, Accepted: November 2012.
ABSTRACT
Background and Objectives: Thermophilic bacteria are less studied but important group of microorganisms due to their
ability to produce industrial enzymes.
Materials and Methods: In this study, thermophilic bacteria were isolated from hot spring of Tarabalo, India. A bacterium
that could tolerate high temperatures was characterized by morphology, biochemistry and sequencing of its 16S rRNA gene.
The isolate was screened for protease and amylase activity. Phylogenetic affiliations and G+C content of the isolate was
studied.
Results: The bacterium with the ability to tolerate high temperatures was identified as Bacillus sp. both by morphology,
biochemistry and sequencing of its 16S rRNA gene. BLAST search analysis of the sequence showed maximum identity with
Bacillus amyloliquefaciens (99% similarity). Strain exhibited considerable protease activity. Phylogenetic analysis of the
isolate revealed close affiliation with thermophilic Bacillus species. The G+C content was found to be 54.7%.
Conclusion: The study confirmed that the isolated Bacillus sp. to be a true thermophile and could be a source of thermostable
protease which can be exploited for pharmaceutical and industrials applications.
Keywords: Thermophilic Bacillus, Protease, Phylogenetic analysis, G+C content
INTRODUCTION
potential remains to explored from majority of the
thermal habitats. As a consequence of growth at high
temperatures and unique macromolecular properties,
thermophiles can possess high metabolism, physically and chemically stable enzymes and lower growth
but higher end product yields than similar mesophilic
species (2).
Thermophilic microorganisms have gained worldwide importance due to their tremendous potential
to produce thermostable enzymes that have wide applications in pharmaceuticals and industries (3). Proteases are such enzymes which account for nearly
60% of the total world-wide enzyme sales (4). Thermostable proteases are of greater advantage in applications because they not only do not usually denature
at high temperatures, but they also remain active at
such temperatures. Several workers have reported
thermophilic bacteria from diverse environmental
habitats such as geothermal sites and hot springs.
Bacteria are ubiquitous and highly diverse. They
can survive in all sorts of inhospitable environments.
Studies in the last two decades have revealed that 99%
of bacteria present in the environment are still unexplored or overlooked in laboratory cultivation and
hence remain obscure for their ecological functions
and unexploited for biotechnological applications (1).
Thermophilic bacteria are microbes that mostly inhabit hot springs, live and survive in temperatures
above 70°C. They have been less explored due to
difficulties in isolation and maintenance of pure culture. Therefore, their diversity and biotechnological
* Corresponding author: KumanandaTayung Ph.D.
Address: Department of Botany, North Orissa University,
Takatpur, Baripada-757003, Odisha, India.
E-mail: [email protected]
159
http://ijm.tums.ac.ir
Kumar Paramasivam ET AL .
The state of Odisha, situated in eastern region of India, is also rich in hot springs. One such hot spring is
Tarabalo. The microbial diversity of this hot spring
has not yet been fully studied, but there are few sporadic reports by Rath et al. (5). Therefore, the aims of
this study were to isolate thermophilic bacteria from
muddy soil samples of Tarabalo hot springs, determine the thermostability of the isolates, screen for
industrial enzymes and study the phylogenetic affiliation of the thermophilic bacterium in comparison
with other bacterial isolates occurring as mesophiles,
thermophiles and hyperthermophiles.
MATERIALS AND METHODS
Study site & collection of samples. The hot
spring Tarabalo is situated at the district of Nayagarh,
Odisha, India. It is the second largest hot spring of
India, as per the report of Tourism Development Corporation India. Tarabalo hot spring is situated at 20°
14’ 49.38” E Longitude and 85° 19’ 11.04”N Latitude.
Before exploration the place was a paddy field. Due
to its remoteness it is less influence by human interferences and believed to have rich microbial wealth.
The temperature of the hot spring during the sampling
period was about 60°C. The pH was recorded to be
in the range of 8-9 indicating alkaline environment.
Samples (muddy soil) were collected from different
sites of the hot spring in sterile poly bags and immediately brought into the laboratory.
Isolation of bacteria. Bacteria were isolated in
Nutrient Agar (NA) medium (Himedia, Mumbai)
India following serial dilution technique. The procedure adopted was as follows: 10 gram of soil sample
was diluted in 90 ml of sterile distilled water in 250
ml conical flask and kept it a orbital shaker at 150 rpm
to get a homogenized soil suspension. Serial dilution
was made and dilution of 10-7 was inoculated into
NA plates and incubated at 37°C for 24 h. Isolated
colonies growing on each diluted plates were transferred into freshly prepared nutrient agar slants. The
bacterial strains isolated on NA slants were kept at in
refrigerator for further study.
Determination for thermo-tolerance. Pure cultures of the bacterial isolates were determined for
their thermophilic characteristics. Each bacterial
isolates were inoculated into 5 ml of nutrient broth
medium in test tubes. The tubes were incubated at
160
IRAN. J. MICROBIOL. Vol. 5, No. 2 (June 2013), 159-165
initial temperature of 50°C for 12 h. After specified
incubation period each broth culture of bacteria were
streaked onto freshly prepared Nutrient Agar medium.
Bacterial isolates growing in the plates were selected
and again tested for their thermo-tolerance at higher
temperature. Finally a bacterium that could tolerate
temperature of 90°C was selected for further study.
Identification and characterization of the isolate.
The selected strain was observed morphologically
and growth characteristics were studied. The isolate was characterized by Gram staining technique.
Based on Gram’s staining the strain was found to be
Gram-positive and microscopic observation revealed
rod shaped bacterium arranged in chain. Various biochemical tests like endospore formation, motility, anaerobic; catalase and oxidase tests were performed.
Morphological, microscopic observation and biochemical test indicated the bacterium to be Bacillus sp.
Enzyme assay. The strain was determined for production of enzymatic activity if any. Two tests were
conducted i.e. test for protease and amylase activity.
For protease activity, Skimmed Milk Agar (SMA)
medium was prepared and the nutrient broth culture
of bacterium after 24 h of incubation was spot inoculated following agar well method. After inoculation
the SMA plate was incubated at 37°C for 24-48 h.
The plate was observed for halo zone around the well.
Observation of halo zone indicated positive protease
activity. Similarly, amylase activity was determined
in starch agar (SA) medium. Starch agar plates were
prepared and spot inoculated with the bacterial strain.
After incubating the plates at 37°C for 24-48 h, the
plates were observed for clear zone near inoculated
colony after treating with Iodine vapour.
PCR amplification and 16Sr DNA Sequencing.
Genomic DNA was extracted and purified according
to Sambrook and Russell (6) and its purity was spectrophotometrically assessed by the A260/A280 ratio.
Sequencing of 16S rDNA of the isolate and amplification of the target gene was done using Big Dye Chemistry, and performed as per the manufacturer’s protocols (Applied Biosystems, USA). Universal bacterial
primer 1492R (5’- TAC GGY TAC CTT GTT ACG
ACT T-3’) and the domain bacteria-specific primer
27F (5’- AGA GTT TGA TCM TGG CTC AG-3’)
were used for 16S rDNA amplification (7). The PCR
product was purified using QIA quick PCR purifica-
http://ijm.tums.ac.ir
protease from a thermophilic bacillus sp.
KUMAR
KUMAR PANDA
PANDA ET
ET AL
AL ..
Table
Table 1.
1. Biochemical
Biochemical characteristics
characteristics of
of thermophilic
thermophilic
Bacillus
Bacillus sp.
sp.
CHARACTERSTICS
CHARACTERSTICS
Rod
Rod shaped
shaped in
in young
young cultures
cultures
Diameter
Diameter over
over 2.5
2.5 mM
mM
Filaments
Filaments
Rods
Rods or
or filaments
filaments curved
curved
Cocci
Cocci in
in tetrads
tetrads or
or packets
packets
Endospores
Produced
Endospores Produced
Motile
Motile
Stain
Stain Gram
Gram positive
positive at
at least
least in
in young
young cultures
cultures
Strict
Strict aerobes
aerobes
Facultative
anaerobes
or
microaerophiles
Facultative anaerobes or microaerophiles
Strict anaerobes
anaerobes
Strict
Product
Product of
of carbohydrate
carbohydrate fermentation
fermentation
Sulfate
Sulfate actively
actively reduced
reduced to
to sulfide
sulfide
Catalase
Catalase
Oxidase
Oxidase
Marked
Marked acidity
acidity from
from glucose
glucose
Nitrate
Nitrate reduced
reduced to
to nitrite
nitrite
Requires
3-12%
NaCl
Requires 3-12% NaCl for
for growth
growth
Ability to
to grow
grow at
at >700
>700 C
C
Ability
Fig.
Fig. 1.
1. Protease
Protease activity.
activity.
Observation
Observation
+
+
__
__
__
__
+
+
+
+
+
+
ND
ND
ND
ND
__
ND
ND
__
+
+
ND
ND
+
+
ND
ND
ND
ND
+
+
+
+ indicates
indicates positive
positive and
and --- indicates
indicates negative
negative test;
test; NDND- not
not determined
determined
protocols
(Applied
Biosystems,
USA).
Universal
tion
kit (Qiagen).
Purified
16S rDNA
was sequenced
protocols
(Applied
Biosystems,
USA).
Universal
bacterial
primer
1492R
(5’TAC
GGY
TAC
CTT
partially
using
ABI
PRISM
big
dye
terminator
cycle
bacterial primer 1492R (5’- TAC GGY TAC CTT
GTT
ACT
T-3’)
domain
bacteriawas
studied
in
16
isolates
belonging
to
sequence
reading
kit the
(Applied
Biosystems)
and
program
and result
was obtained.
GTT ACG
ACG
ACT reaction
T-3’) and
and
the
domain
bacteriawas the
studied
in was
16 run
isolates
belonging
to three
three
specific
primer
27F
(5’AGA
GTT
TGA
TCM
TGG
bacterial
groups
(i.e.
mesophiles,
thermophiles
under
the
following
conditions:
initial
denaturation
specific primer 27F (5’- AGA GTT TGA TCM TGG
bacterial groups (i.e. mesophiles, thermophiles and
and
CTC
AG-3’)
were
used
for 16S
16S
rDNA
amplification
hyperthermophiles).
at
94°C
for 5were
min;used
30 cycles
of rDNA
denaturation
at 94°
RESULTS
CTC
AG-3’)
for
amplification
hyperthermophiles). It
It was
was calculated
calculated using
using the
the
(7).
The
PCR
product
was
purified
using
QIA
quick
software
ACUA
(9).
The
sequences
of
all
bacterial
for
45
sec,
annealing
at
48°C
for
45
sec,
extension
at
(7). The PCR product was purified using QIA quick
software ACUA (9). The sequences of all bacterial
1:
Protease
activity
PCR
kit
Purified
isolates
were
selected
including
sequence
of
72°C
for Fig.
90 sec,
a(Qiagen).
final extension
at 16S
72°CrDNA
for 5
Altogether
bacterial
isolates
were obtained
Fig.
1:and
Protease
activity
PCR purification
purification
kit
(Qiagen).
Purified
16S
rDNA
isolates
were 22
selected
including
sequence
of own
own
was
sequenced
partially
using
ABI
PRISM
big
isolate
and
uploaded
in
input
files.
The
GC%
min.
The
purified
sequencing
reaction
mixtures
were
from
muddy
soil
samples
collected
from
hot
spring
was sequenced partially using ABI PRISM big
isolate and uploaded in input files. The GC% option
option
dye terminator
terminator using
cycle an
sequence
reading
reaction
kit
was
selected
and
the
program
run
result
was
electophoresed
Appliedreading
Biosystems
model
of
Tarabalo
Thewas
bacterial
isolates
dye
cycle
sequence
reaction
kit
was
selectedOdisha,
and
theIndia.
program
was
run and
and
resultwere
was
bacterium
positive
for
motility,
catalase
activity,
endospore
production
bacterium tested
tested
positive
for
motility,
catalase
activity,
endospore
production
(Applied
Biosystems)
under
the
following
conditions:
obtained.
310
automatic
DNA
sequencer
(Perkin
Elmer,
Masscreened
for
their
thermo-tolerance
property
in
dif(Applied Biosystems) under the following conditions:
obtained.
ee test
and
sulphate
reduction.
The
biochemical
characteristic
test from
from anaerobic
anaerobic
and
sulphate
reduction.
The
biochemical
characteristic
initial
denaturation
at
94°C
for
5
min;
30
cycles
of
sachusetts
USA).
The
sequence
was
annotated
and
ferent
temperatures
starting
from
50°C
to
90°C.
Out
initial denaturation at 94°C for 5 min; 30 cycles of
rium
in
1.
and
biochemical
characterization
denaturation
at
for
at
RESULTS
submitted
to GenBank.
of
the total
isolates only one strain could sustain temrium is
is presented
presented
in Table
Table
1. Morphological
Morphological
and
biochemical
characterization
denaturation
at 94°C
94°C
for 45
45 sec,
sec, annealing
annealing
at 48°C
48°C
RESULTS
for
45
sec,
extensionsp.
at 72°C
72°C
for 90
90level
sec, and
and
final
peratures
of 90°C.
The was
bacterium was characterized
te
to
be
Bacillus
Species
confirmation
of
isolate
extension
at
for
sec,
aa final
te indicated
indicated it
itfor
to45
besec,
Bacillus
sp.
Species
level
confirmation
of the
the
isolate
was
extension
at
72°C
for
5
min.
The
purified
sequencing
Altogether
22
bacterial
were
Homology
search
and
phylogenetic
analysis.
by
morphological
observation
and biochemical
tests.
S
rDNA
sequencing.
Based
on
BLAST
search
analysis
of
the
sequence,
the
extension atBased
72°C foron
5 min.
The purified
sequencing
Altogether
22 bacterial
isolates
were obtained
obtained
S rDNA sequencing.
BLAST
search
analysis of
the sequence,
the isolates
reaction
mixtures
were
electophoresed
using
an
from
muddy
soil
samples
collected
from
hot
spring
The
16S
rDNA
sequence
of
the
isolate
was
been
Gram
staining,
microscopic
and
biochemical
characreaction
mixtureswith
were Bacillus
electophoresed
using an
from muddy
soil samples
collected from hot spring
wed
identity
amyloliquefaciens
(99%)
having
wed maximum
maximum
identity
with
Bacillus
amyloliquefaciens
(99%)
having
Applied
Biosystems
model
310
automatic
DNA
of
Tarabalo
Odisha,
The
bacterial
isolates
compared
with the non
redundant
databases in
all
terization
revealed
it toIndia.
be a Gram
Applied
Biosystems
model
310
automatic
DNA
of
Tarabalo
Odisha,
India.
The positive
bacterialrod.
isolates
number
The
sequence
of
the
isolate
has
been
deposited
in
number CP002627.1.
CP002627.1.
The
sequence
of
the
isolate
has
been
deposited
in
sequencer (Perkin
(Perkin Elmer,
Elmer, Massachusetts
Massachusetts
USA).
The
were
screened
for
theirpositive
thermo-tolerance
property
GenBank+EMBL+DDBJ+PDB
sequences
(but The
no
The screened
bacteriumfor
tested
for motility, property
catalase
sequencer
USA).
were
their
thermo-tolerance
with
number
JQ962977.
The
isolate
was
for
both
protease
with accession
accessionEST,
number
JQ962977.
isolate
was
screened
forendospore
both
protease
sequence
annotated
and
submitted
to
GenBank.
in
temperatures
starting
from
50°C
to
STS,was
GSS,
environmental
samples
phase screened
0,
activity,
production
and negative
sequence
was
annotated
andThe
submitted
toor
GenBank.
in different
different
temperatures
starting
from test
50°Cfrom
to
ee activity.
showed
protease
activity
(Fig.
1)
but
90°C.
Out
of
the
total
isolates
only
one
strain
could
1 orbacterium
2 HTGS sequences)
usingconsiderable
the program BLASTN
and
sulphate
reduction.
The
biochemical
activity. The
The
bacterium
showed
considerable
proteaseanaerobic
activity
(Fig.
1)
but
90°C. Out of the total isolates only one strain could
activity
Homology
search
and
phylogenetic
analysis.
sustain
of
bacterium
was
2.2.25
by selecting
optimization
parameter
to
characteristic
of the bacterium
presented
in Table
activity was
was observed.
observed.
Homology
searchthe
and
phylogenetic
analysis.
sustain temperatures
temperatures
of 90°C.
90°C. isThe
The
bacterium
was
The 16S
16S
rDNAsequences
sequence(Megablast).
of the
the isolate
isolate
was been
been
characterized
by and
morphological
observation
and
highly
similar
Phylogenetic
1.
Morphological
biochemicalobservation
characterization
The
rDNA
sequence
of
was
characterized
by
morphological
and
compared
with
the
non
redundant
databases
in
all
biochemical
tests.
Gram
staining,
microscopic
study
was
conducted
by
considering
16S
rDNA
seof
the
isolate
indicated
it
to
be
Bacillus
sp.
Species
compared with the non redundant databases in all
biochemical tests. Gram staining, microscopic and
and
GenBank+EMBL+DDBJ+PDB
sequences
(but
no
biochemical
characterization
revealed
to
aa gram
quences
of
30
bacterial
isolates
belonging
to
mesolevel
confirmation
of the isolate
wasit
16S
GenBank+EMBL+DDBJ+PDB sequences (but no
biochemical
characterization
revealed
itdone
to be
beby
gram
EST,
GSS,
samples
0,
positive
rod.
philes,
thermophiles
and hyperthermophiles.
The tree
rDNA
Based on BLAST search analysis
EST, STS,
STS,
GSS, environmental
environmental
samples or
or phase
phase
0,
positivesequencing.
rod.
or 22generated
HTGS sequences)
sequences)
using
the program
program
BLASTN
The
bacterium
tested
positive
for motility,
motility,
catalase
was
by Minimum
Evolution
(ME)
method
of The
the bacterium
sequence, the
isolate
showed
maximum
iden11 or
HTGS
using
the
BLASTN
tested
positive
for
catalase
Biochemical
of
thermophilic
Bacillus
sp.
Biochemical characteristics
characteristics
of
thermophilic
Bacillus
sp.
2.2.25
by
selecting
the
optimization
parameter
to
activity,
endospore
production
and
negative
test
using
MEGA
4.0
(8).
tity
with
Bacillus
amyloliquefaciens
(99%)
having
2.2.25 by selecting the optimization parameter to
activity, endospore production and negative test from
from
highly
similar
sequences
(Megablast).
Phylogenetic
anaerobic
and
reduction.
biochemical
accession
CP002627.1.
TheThe
sequence
of the
highly similar sequences (Megablast). Phylogenetic
anaerobic number
and sulphate
sulphate
reduction.
The
biochemical
study
was
conducted
by
considering
rDNA
characteristic
of
bacterium
is
Table
GC%
Guanine
cytosine 16S
percentage
isolate
has been
deposited
in GenBank
within
study
wasstudy.
conducted
by plus
considering
16S
rDNA
characteristic
of the
the
bacterium
is presented
presented
inaccesTable
TICS
Observation
sequences
ofin 16
30 isolates
bacterialbelonging
isolates Observation
belonging
to
1. Morphological
Morphological
and biochemical
biochemical
characterization
of
was
studiedof
tobelonging
three bactesion
number JQ962977.
The isolatecharacterization
was screened for
TICS
sequences
30
bacterial
isolates
to
1.
and
of
mesophiles,
thermophiles
and
hyperthermophiles.
the
isolate
indicated
it
to
be
Bacillus
sp.
Species
level
rial
groups
(i.e.
mesophiles,
thermophiles
and
hyperboth
protease
and
amylase
activity.
The
bacterium
ung
cultures
+
mesophiles, thermophiles and hyperthermophiles.
the isolate indicated it to be Bacillus sp. Species level
ung cultures
+
The
tree
was
generated
by
Minimum
Evolution
(ME)
confirmation
of
was
done
by
rDNA
thermophiles).
It was calculated
using
the_ software
showed
considerable
protease
activity
1) but
no
The
tree
was
generated
by
Minimum
Evolution
(ME)
confirmation
of the
the isolate
isolate
was
done (Fig.
by 16S
16S
rDNA
55 mM
mM
_
method
using
MEGA
4.0
(8).
sequencing.
Based
on
BLAST
search
analysis
of
the
ACUA
(9).
The
sequences
of
all
bacterial
isolates
amylase
activity
was
observed.
method using MEGA 4.0 (8).
sequencing. Based on BLAST search analysis of the
_
_
sequence,
thephylogeny
isolate showed
showed
maximum
identity
with
were selected including sequence of own isolate
and
Molecular
of the
isolate was
analysed
sequence,
the
isolate
maximum
identity
with
curved
_
curved
_ selected
GC%
study.
Guanine
plus
cytosine
percentage
Bacillus
amyloliquefaciens
(99%)
having
accession
uploaded
in
input
files.
The
GC%
option
was
considering
30
bacterial
taxa
occurring
as
mesoGC% study. Guanine plus cytosine percentage
Bacillus amyloliquefaciens (99%) having accession
rr packets
packets
uced
uced
_
_
+
+
44
IRAN.
http://ijm.tums.ac.ir
44
IRAN. J.
J. MICROBIOL.
MICROBIOL. Vol.
Vol. 5,
5, No.
No. 22 (June
(June 2013),
2013), ?-?
?-?
6
6
IRAN. J. MICROBIOL. Vol. 5, No. 2 (June 2013), 159-165
161
Kumar Paramasivam ET AL .
Table 2. List of bacterial isolates with their isolation source, environment, country and accession numbers considered for
the present study.
Bacteria
Accession no
Country
source
Environment
Bacillus sp.
FJ169970.1
China
Sea sediment
Mesophilic
Bacillus sp.
JQ074049.1
India
plant
Mesophilic
Bacillus sp.
EU706318.1
Kuwait
Coast of Kuwait
Mesophilic
Bacillus sp.
JF937435.1
China
Soil
Mesophilic
NR_025030.1
--
Hydrothermal
Mesophilic
JQ249031.1
India
Soil
Mesophilic
Halothiobacillus sp.
Bacillus sp.
Bacillus sp.
JQ340870.1
Iran
oil contaminated soil
Mesophilic
Bacillus sp.
HE617181.1
India
soda lake sediment
Mesophilic
Bacillus sp.
JQ659884.1
Singapore
plant tissue
Mesophilic
Bacillus sp.
JQ782986.1
China
marine sediment
Mesophilic
Geobacillus sp.
FJ823106.2
Korea
lakeshore duff
Thermophilic
Bacillus sp.
AB668060.1
Japan
hot spring
Thermophilic
Bacillus sp.
EU740977.1
China
hot spring
Thermophilic
Bacillus sp.
AB437939.1
Thiland
hot spring
Thermophilic
Bacillus sp.
FJ808719.1
Turkey
hot spring
Thermophilic
Paenibacillus sp.
AB618502.1
Japan
Fermented food
Thermophilic
Geobacillus sp.
HM989019.1
China
hot spring
Thermophilic
Bacillus sp.
FJ748503.1
Uganda
Landfill
Thermophilic
Bacillus sp.
HQ615926.1
China
soil
Thermophilic
Bacillus alveayuensis
NR_043013.1
--
deep-sea sediments
Thermophilic
Bacillus sp.
AF228764.1
Indonesia
LB media
Hyperthermophilic
Bacillus sp.
AF228765.1
Indonesia
LB media
Hyperthermophilic
Brevibacillus sp.
AF228763
Indonesia
oil wells
Hyperthermophilic
Thermocrinis ruber
AJ005640.1
USA
Yellow stone park
Hyperthermophilic
Thermotoga sp.
JF339227.1
Thailand
hot spring
Hyperthermophilic
Thermotoga thermarum
AB039769.1
Japan
oil reservoir
Hyperthermophilic
Thermoshipo africanus
DQ647058
--
hot water
Hyperthermophilic
Fervidobacterium sp.
HM004613.1
China
hot spring
Hyperthermophilic
Fervidobacterium sp.
HM004612.1
China
hot spring
Hyperthermophilic
M83548.2
--
--
Hyperthermophilic
Aquifex pyrophilus
-- Not known
philes, thermophiles and hyperthermophiles (Table
2). The generated tree revealed that the isolate shared
close affinity with thermophilic Bacillus spp. having accession numbers AB437939.1 and FJ808719.1
respectively with well supported bootstrap value of
100% (Fig. 2). The tree also showed that hyperthermopiles forms a separate group with distinct clade
supported by bootstrap of 100%. However, some of
the hyperthermophiles also shared the same clade
with thermophiles and mesophiles. The isolate was
also studied for its GC content by comparing with
15 bacterial isolates (selected randomly) occurring
162
IRAN. J. MICROBIOL. Vol. 5, No. 2 (June 2013), 159-165
as mesophiles, thermophiles and hyperthermophiles.
The result indicated that the GC content of the isolate
was 54.7% which is slightly higher than mesophiles
but lower than the hyperthermophiles (Fig. 3).
DISCUSSION
Thermophilic microorganisms have the adaptability to survive in high environmental conditions.
Many researchers believed that such capability may
be due to their molecular modifications at cellular
and subcellular level. In our present study muddy
http://ijm.tums.ac.ir
protease from a thermophilic bacillus sp.
x
Mesophiles
ƒ
Thermophiles
14
41
100
™ Hyperthermophiles
45
44
34
56
26
38
100
96
96
98
79
99
100
100
85
x
Bacillus sp (JQ340870.1)
ƒ
Bacillus sp (AB668060.1)
x
Bacillus sp (JQ074049.1)
x
Bacillus sp (EU706318.1)
x
Bacillus sp (JQ659884.1)
x
Bacillus sp (JF937435.1)
x
Bacillus sp (JQ782986.1)
ƒ
Bacillus alveayuensis (NR 043013.1)
ƒ
Tb P15 (JQ962977)
ƒ
Bacillus sp (AB437939.1)
ƒ
Bacillus sp (FJ808719.1)
x
Bacillus sp (FJ169970.1)
ƒ
Bacillus sp (FJ748503.1)
x
Bacillus sp (HE617181.1)
ƒ
Geobacillus sp (HM989019.1)
ƒ
Bacillus sp (HQ615926.1)
ƒ
Bacillus sp (EU740977.1)
ƒ
Geobacillus sp (FJ823106.2)
™ Bacillus sp (AF228764.1)
47
70
74
52
™ Bacillus sp (AF228765.1)
x
Bacillus sp (JQ249031.1)
ƒ
Paenibacillus sp (AB618502.1)
™ Brevibacillus sp (AF228763)
x
100
Halothiobacillus (NR 025030.1)
™ Thermocrinis ruber (AJ005640.1)
™ Aquifex pyrophilus (M83548.2)
99
100
™ Fervidobacterium sp (HM004613.1)
™ Fervidobacterium sp (HM004612.1)
™ Thermosipho africanus (DQ647058.1)
100
™ Thermotoga sp (JF339227.1)
66
95
™ Thermotoga thermarum (AB039769.1)
Fig. 2. Phylogenetic tree generated by Minimum Evolution method of 31 taxa representing mesophilic, thermophilic and
hyperthermophilic bacteria. The optimal tree with the sum of branch length = 0.85161164 is shown. The percentage of
fig. replicate
2: Phylogenetic
tree
by clustered
Minimum
Evolution
methodtestofare
31shown
taxa next
representing
mesophilic,
trees in which
thegenerated
associated taxa
together
in the bootstrap
to the branches.
The evothermophilic
and hyperthermophilic
bacteria.
optimal
tree with
sum
of units
branch
lutionary distances
were computed using the
MaximumThe
Composite
Likelihood
methodthe
and are
in the
of the length
number of=
base substitutions
per site.
P15 is own of
isolate.
The values
parenthesis
indicate
accession
no. of
the isolates.
0.85161164
is shown.
TheTb
percentage
replicate
treesinin
which the
associated
taxa
clustered
together in
the bootstrap test are shown next to the branches. The evolutionary distances were computed using the
Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.
Tb P15 is own isolate. The values in parenthesis indicate accession no. of the isolates.
IRAN. J. MICROBIOL. Vol. 5, No. 2 (June 2013), 159-165
163
http://ijm.tums.ac.ir
Kumar
KUMAR Paramasivam
PANDA ET AL . ET AL .
Fig.3.
3.GC
GCpercentage
percentageof
ofthe
theBacillus
Bacillusisolates
isolatesalong
alongwith
withother
otherbacterial
bacterialisolates
isolatesoccurring
occurringas
asmesophiles
mesophiles(M),
(M),thermophiles
thermophiles
Fig.
(T)and
andhyperthermophiles
hyperthermophiles(HT).
(HT).The
Thered
redbar
barindicates
indicatesGC
GCcontent
contentof
ofown
ownisolate.
isolate.
(T)
Fig. 3. GC percentage of the Bacillus isolates along with other bacterial isolates
occurring
as mesophiles
thermophiles
andnohyperthermophiles
TheRath
red
baral.
soil
samples believed
collected
fromsuch
hot(M),
spring
of Tarabalo,
Several
workers
have
reported
protease
researchers
that
capability
may be (T)characterized.
but
amylase
activity
was(HT).
observed.
et
indicates
content
of own
isolate.
Odisha,
was
investigated
for thermophilic
bacactivity
Bacillus
species (13,
14).
due
to India
theirGC
molecular
modifications
at cellular
(5) havefrom
also thermophilic
reported various
extracellular
enzymes
teria.
Out of the total
isolate
was muddy
found
Proteases
arethermophilic
essential constituents
all forms
of life
and subcellular
level.isolates,
In our one
present
study
from some
bacteria of
isolated
from
the
to
sustain
temperatures
to 90°C.
The ability
of the
on
earth
prokaryotes,
fungi,
plants were
and anisoil
samples
collected up
from
hot spring
of Tarabalo,
same
hotincluding
spring. However,
those
bacteria
not
bacterium
to tolerate
temperature
to be
mals.
ProteasesSeveral
are highly
exploited
in food,
Odisha,
India
was this
investigated
forindicated
thermophilic
characterized.
workers
haveenzymes
reported protease
DISCUSSION
thermophile.
al. (10)one
have
also characterleather,
pharmaceutical,
waste
bacteria. OutNarayan
the total et
isolates,
isolate
was found
activitydetergent,
from thermophilic
Bacillusdiagnostics,
species (13,
14).
ized
bacterial
isolates
as
thermophiles
from
Savusavu
management
and
silver
recovery
(15).
Bacillus
protoThermophilic
sustain
temperatures
up
to
90°C
.
The
ability
of
Proteases
are
essential
constituents
of
all
forms
microorganisms have the adaptability to survive in high environmentalof
hotspring
in
Fiji
by
determining
their growth
at 90°C.
tease
is earth
of special
importance
ofmolecular
itsplants
wide apthe
bacterium
to
tolerate
this temperature
indicated
lifecapability
on
including
fungi,
and
conditions. Many researchers
believed
that such
may
beprokaryotes,
due because
to their
The
bacterium
was at
identified
as
Bacillus
sp.
both
by
plications
in
various
industries
like
pharmaceutical,
tomodifications
be
thermophile.
Narayan
et
al.
(10)
have
also
animals.
Proteases
are
highly
exploited
enzymes
cellular and subcellular level. In our present study muddy soil samplesin
morphological
and molecular
characterization.
Simileather,
food
waste pharmaceutical,
processing
industries
(16).
characterized
bacterial
as of
thermophiles
from
food,
leather,
detergent,
diagnostics,
collected from
hotisolates
spring
Tarabalo,
Odisha,
India
wasand
investigated
for thermophilic
lar
work
carried
out
by
Rath
(11)
has
also
reported
Phylogenetic
study
of
the
isolates
with
other
bacteSavusavu
hotspring
in
Fiji
by
determining
their
waste
management
and
silver
recovery
(15).
Bacillus
bacteria. Out the total isolates, one isolate was found to sustain temperatures up to 90 0C.
thermophilic
Bacillus
andbacterium
Pseudomonas
from
isolates
as mesophiles,
thermophiles
growth
at 90°C
. The
wastospecies
identified
asthis rial
protease
is ofoccurring
special
importance
of its wide
The ability
of
the
bacterium
tolerate
temperature
indicated
to bebecause
thermophile.
three
hot springs
Odisha,
India. Thermophilic
and
and
hyperthermophiles
showed close
affiliation with
Bacillus
sp. bothofby
morphological
and molecular
applications
in various industries
like pharmaceutical,
Narayan et al. (10) have also characterized bacterial isolates as thermophiles from
hyperthermophilic
microorganisms
thebyability
thermophilic
species
and are grouped
characterization. Similar
work carried
Rath
leather, foodBacillus
waste
industrieswithin
(16).
0C.processing
The
bacterium
was
Savusavu hotspring
in Fiji have
by out
determining
their
growth and
at 90
to
produce
widereported
variety thermophilic
of thermostable
enzymes.
the
clade of thermophiles.
This validates
thebacterial
isolate
(11)
has also
Bacillus
and
Phylogenetic
study of the isolates
with other
identified as Bacillus sp. both by morphological and molecular characterization. Similar
Thermozymes
of great
interest
industrial
to
be a thermophilic
species.
The tree also
Pseudomonas are
species
from
threeforhot
springsapof
isolates
occurring asBacillus
mesophiles,
thermophiles
and
work carried out by Rath (11) has also reported thermophilic Bacillus and Pseudomonas
plications
(12). Thermophilic
Considering this
the thermorevealed
some hyperthermophiles
same clade
Odisha, India.
and facts,
hyperthermophilic
hyperthermophiles
showed closeshared
affiliation
with
species
fromhave
three
hot
springs
of Odisha,
India.
Thermophilic
and
hyperthermophilic
philic
Bacillus isolate
was
forproduce
proteasewide
and
with
thermophiles
andspecies
mesophiles.
ambigumicroorganisms
the screened
ability to
thermophilic
Bacillus
and are Such
grouped
within
microorganisms
haveenzymes.
the
ability
to produceity
wide
variety
of thermostable
enzymes.
amylase
Considerable
proteaseThermozymes
activity was
biased in submission
of sequences
variety activity.
of thermostable
the may
cladebeofdue
thermophiles.
This validates
the isolate
Thermozymes
are
greatwas
interest
for Rath
industrial
applications
(12).
Considering
thiswas
facts,
detected
but no
amylase
activity
observed.
in
The GC
content
ofspecies.
the isolate
also
are
of great
interest
for of
industrial
applications
(12).
to Genbank.
be a thermophilic
Bacillus
The
tree
also
et
al. (5) have this
also facts,
reported
extracellular
enfound
to be
relatively
high. This might
be same
the reason
Considering
thevarious
thermophilic
Bacillus
revealed
some
hyperthermophiles
shared
clade
zymes
thermophilic
bacteria isolated
from
for
its thermophiles
ability to survive
high temperature.
However,
isolatefrom
wassome
screened
for protease
and amylase
with
andinmesophiles.
Such ambiguity
the
same hot
spring. However,
bacteria
not 9 recent
report
Wu et
(17) suggests
that higher
activity.
Considerable
proteasethose
activity
waswere
detected
may be
due of
biased
in al.
submission
of sequences
in
164
46
IRAN.J.J.MICROBIOL.
MICROBIOL.Vol.
Vol.5,5,No.
No.22(June
(June2013),
2013),?-?
159-165
IRAN.
http://ijm.tums.ac.ir
protease from a thermophilic bacillus sp.
GC content could not only be the sole reason for an
organism to survive in extreme temperature. There
were of the opinion that organisms isolated from soil
preferably have higher GC content than aquatic isolates. Similarly, Hurst and Merchant (18) reported that
high GC content is not an adaptation to high temperature among prokaryotes. Thus GC content cannot be
taken into account to explain the adaptability of thermophiles in high temperature. Although this is not the
first report of thermophilic Bacillus from hot spring
of Tarabalo, but the organism is unique in the sense
that it could survive temperatures of 90°C and could
be a potential source of thermostable enzyme. Efforts
are on to optimize the protease production in different
temperature and pH and to purify the enzyme.
ACKNOWLEDGEMENTS
We acknowledge Department of Biotechnology,
Govt. of India for providing Bioinformatics Infrastructural Facility (BIF) at North Orissa University,
Odisha, India and Head of the Department Botany,
North Orissa University for necessary laboratory
facility.
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