scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Characterization Of A Novel Hydrolytic Enzyme Producing Thermophilic Bacterium Isolated From The Hot Spring Of Azad Kashmir-Pakistan

TL;DR: A thermophilic bacterium (TP-2) was isolated from the Tatta Pani hot spring in Azad Kashmir and was characterized using phenotypic and genotypic characters and Sequence analysis of its 16S rRNA gene showed that isolate TP-2 had 89% homology with Geobacillus debilis.
Abstract: A thermophilic bacterium (TP-2) was isolated from the Tatta Pani hot spring in Azad Kashmir and was characterized using phenotypic and genotypic characters. The strain developed cream colored, round, smooth, flat and slimy colonies while the cells were Gram positive rods that ranged in size from about 2.1-3.6 μm to 0.2-0.3 μm in width. Sequence analysis of its 16S rRNA gene showed that isolate TP-2 had 89% homology with Geobacillus debilis. It grew within pH range of 5.5 to 8.5 with optimum growth at pH 7.0. The isolate showed optimum growth at 65oC and gave positive results for gelatin hydrolysis (GEL), ortho nitrophenyl-β-D-galactopyranosidase (ONPG), and nitrate production and produced acid from sucrose, glucose and maltose. It utilized glucose, fructose, maltose, lactose, sucrose, xylan, starch, filter paper and carboxymethylcellulose as sole carbon source. Isolate TP-2 produced significant amount of industrially important enzymes i.e. extracellular α-amylase, CMCase, FPase, Xylanase, Protease and Lipase and intracellular CMCase and FPase.

Content maybe subject to copyright    Report

Braz. Arch. Biol. Technol. v.59: e16150622, Jan/Dec 2016
1
Vol.59: e16150662, January-December 2016
http://dx.doi.org/10.1590/1678-4324-2016150662
ISSN 1678-4324 Online Edition
BRAZILIAN ARCHIVES OF
BIOLOGY AND TECHNOLOGY
AN I N T E R N A T I O N A L J O U R N A L
Characterization Of A Novel Hydrolytic Enzyme Producing
Thermophilic Bacterium Isolated From The Hot Spring Of
Azad Kashmir-Pakistan
Sana Zahoor
1
*; Huhammad Mohsin Javed
2
; Masroor Ellahi Babarl
1
.
1
Virtual University of Pakistan Department of Molecular Biology, Lahore, Pakistan;
2
GC University, Lahore
Institute of Industrial Biotechnology, Lahore, Pakistan.
ABSTRACT
A thermophilic bacterium (TP-2) was isolated from the Tatta Pani hot spring in Azad Kashmir and was characterized
using phenotypic and genotypic characters. The strain developed cream colored, round, smooth, flat and slimy
colonies while the cells were Gram positive rods that ranged in size from about 2.1-3.6 μm to 0.2-0.3 μm in width.
Sequence analysis of its 16S rRNA gene showed that isolate TP-2 had 89% homology with Geobacillus debilis. It
grew within pH range of 5.5 to 8.5 with optimum growth at pH 7.0. The isolate showed optimum growth at 65ºC and
gave positive results for gelatin hydrolysis (GEL), ortho nitrophenyl-β-D-galactopyranosidase (ONPG), and nitrate
production and produced acid from sucrose, glucose and maltose. It utilized glucose, fructose, maltose, lactose,
sucrose, xylan, starch, filter paper and carboxymethylcellulose as sole carbon source. Isolate TP-2 produced
significant amount of industrially important enzymes i.e. extracellular α-amylase, CMCase, FPase, Xylanase, Protease
and Lipase and intracellular CMCase and FPase.
Keywords: Tatta Pani, characterization, enzyme production, Geobacillus
1
Authors for correspondence: azzamohamed99@yahoo.com
Human and Animal Health

Zahoor, S et al.
Braz. Arch. Biol. Technol. v.59: e16150622, Jan/Dec 2016
2
INTRODUCTION
Microorganisms occupy all possible sites in which
life survives, ranging from ideal environments for
growth and reproduction to those signifying
extreme environments. Extremophiles are adapted
to live and grow in conditions like extreme salinity,
temperature, pH and absence of oxygen.
Thermophilic bacteria grow optimally at
temperatures ranging from 45°C to 70°C, and
hence, it is likely to isolate them from numerous
habitats like deep-sea hydrothermal vents, deep
ocean-basin cores, petroleum reservoirs
and
artificially hot environments like fermented
compost (Rahman et al. 2004; Lebedinsky et al.
2007). One of the natural habitats of the
thermophilic bacteria is the hot spring. Hot springs
are springs that are geothermally heated. Hot
springs are located all over the earth, on all
continents and even beneath the seas and oceans
(Kauze et al. 2006).
Thermophiles have gained a great consideration
since they are not usually denatured at high
temperature, yet they are active at elevated
temperature (Lee et al. 1999; Beg et al. 2000).
Thermostable enzymes, isolated chiefly from
thermophilic microorganisms, are commercially
important because of their inherent stability that is
maintained under harsh industrial processes
conditions as well as benefits for accomplishing the
procedures at high temperature (Demirjian et al.
2001).
In recent times, molecular phylogenetic methods
utilizing the small-subunit rRNA gene have been
used to study the bacterial diversity of varied hot
springs (Reysenbach et al. 2000). Thermophilic
bacteria have been characterized by phenotypic and
genotypic means
from numerous geothermal
regions of the World, comprising
Italy (Maugeri et
al. 2001),
Turkey (Gul-Guven et al. 2008), Bulgaria
(Derekova et al. 2008), Iceland (Marteinsson et al.
2001), Yellowstone National Park (Boomer et al.
2009), China (Lau et al. 2009), India (Sharma et al.
2009) and Greece (Sievert et al. 2000).
Tatta Pani hot spring located in Azad Kashmir,
Pakistan has not been explored yet from the
microbiological aspects. The objective of this work
was to isolate and characterize
thermophilic
bacteria from Tatta Pani hot spring using genotypic
and phenotypic methods and to check the
biotechnological potential of the isolates.
MATERIALS AND METHODS
SAMPLE COLLECTION
Water samples were collected separately in sterile
thermal glass containers and in aseptic culture tubes
containing LB medium (1% NaCl, 0.5% yeast
extract, 1% tryptone, pH: 7.0). These tubes were
placed into the water in the same place for two
hours and taken to the lab for additional processing.
In-situ (temperature) and ex-situ (pH, EC, soluble
anions and cations) analysis of water samples was
carried out to characterize the hot springs (Khalil et
al. 1998).
ISOLATION OF BACTERIAL STRAINS
Thermophilic bacteria were isolated through serial
dilution method. Water sample (100 µL) was added
to the LB media and incubated at 65
o
C. Pure culture
was obtained by repeated streaking on LB agar
plates followed by incubation at 65
o
C (Khalil et al.
1998).
MORPHOLOGICAL CHARACTERIZATION
Colony morphology characteristics of isolates were
observed from 48 h culture on LB agar with respect
to color, shape, margin, internal structure, elevation
and configuration. Cell characteristics of isolate
were studied by Gram’s staining. Scanning electron
microscopy was done commercially from
University of Karachi.
PHYSIOLOGICAL CHARACTERIZATION
Optimum temperature for growth was determined
by incubating the isolate in LB medium at different
temperatures (30-90
o
C) with 5
o
C interval.
Optimum pH was determined by incubating the
isolate overnight at 65
o
1
o
C by altering the initial
pH of the LB medium from 4.0 to 10 with either 1N
NaOH or 1N H
3
PO
4
. The ability of the isolate to
grow at different NaCl concentration was examined
by incubating the isolate in LB medium (pH: 7.0)
containing different amount of NaCl ranging from
0-5% (w/v). Growth was checked by measuring
optical density (O.D.) at 600 nm using
spectrophotometer (Nair and Surendran 2004).
Study of utilization of different carbon sources by
the isolate was also checked. Mineral salts broth
(0.02% MgSO
4
·7H
2
O; 0.25% NaCl; 0.001%
FeSO
4
· 7H
2
O; 0.1% (NH
4
)
2
SO
4
; 0.15% KH
2
PO
4
;
pH:7.0) with 1% of different carbon sources, like
sucrose, maltose, lactose, fructose, glucose,
carboxymethylcellulose (CMC), xylan, starch,

Chracterization of a thermophilic bacterium
Braz. Arch. Biol. Technol. v.59: e16150622, Jan/Dec 2016
3
wheat bran extract and filter paper (0.05 g) was
added to test tubes. These were inoculated with
overnight grown culture (20 µL) of the isolate
followed by incubation at 65°C for 48 h. Growth of
the culture was determined by measuring the optical
density at 600 nm using spectrophotometer.
BIOCHEMICAL CHARACTERIZATION
Oxidase test, catalase test and QTS-20 tests were
performed following the method of Wiegel and
Ljungdahl (1981), Tarrand and Groschel (1982) and
Macfaddin (2000).
MOLECULAR CHARACTERIZATION
Genomic DNA was isolated after Kronstad et al.
(1983). 16S rRNA gene was amplified by PCR
using the following primers
“9F 5´-GAGTTTGATCCTGGCTCAG-
1510R 5´- GGTTACCTTGTTACGACTT-
PCR reaction was carried out in a 50 μl reaction
mixture, using DNA (1
μl; 50 ng ), primers (l µ l
e a c h ; 10 µM)
, dNTPs (5 µl; 2.5 mM), PCR buffer
(5 µl; IX), magnesium chloride (5 µl; 25 mM)
and
Taq polymerase (l µl; 2.5 U). Initial denaturation of
template was carried at 94
°C (5 min), followed by
35 cycles of 94°C (30 sec), 54°C (30 sec) and 72°C
(90 sec) and
one final cycle of extension at 72°C
(10 min). The PCR product was analyzed by
agarose gel electrophoresis (1%). The PCR product
was excised from the gel and purified using
Novagen spin prep gelmelt kit. The purified 16S
rRNA gene was sequenced using 3130xl Genetic
Analyzer. Sequence database similarity searches of
the 16S rRNA gene sequence were done using the
BLAST tool of GenBank. Alignment of 16S rRNA
gene sequences was done using software Clustal W
and observed by Jalview. Phylogenetic analysis was
done using neighbor-joining method using the
software MEGA 5.0 (Saitou and Nei 1987; Altschul
et al. 1990; Thompson et al. 1994; Waterhouse et
al. 2009). The stability of relationships was
evaluated by carrying out bootstrap analysis of the
neighbor-joining data based on 1000 resamplings.
NUCLEOTIDE SEQUENCE ACCESSION
NUMBER
The 16S rRNA gene sequence of isolate TP-2 has
been submitted in the GenBank with accession no.
JQ284017.
BIOTECHNOLOGICAL POTENTIAL
Production of intracellular and extracellular
enzymes was studied by incubating the isolate in
different media reported by Mandels and Reese
(1957), Srivastava and Baruah (1986), Chen et al.
(2004), Lee et al. (1999), Sharma et al. (2007) and
Kim et al. (1998) for the production of CMCase &
FPase, α-amylase, protease, lipase, xylanase and
phytase, respectively.
Extraction of Extracellular and Intracellular
Enzyme
Following incubation for particular time interval,
the fermented broth was centrifuged at 10,000 rpm
(4
o
C) for 15 min. The supernatant obtained was
utilized as extracellular enzyme. After
centrifugation of the fermented broth, the cells were
washed two times with 0.85% NaCl (5 ml). The wet
weight of the pellets was recorded and the pellets
were placed at 20°C before sonication. Frozen
cells were thawed in potassium phosphate (50
mM)-EDTA (5 mM); pH: 7.0 buffer and the final
cell concentration was kept at 20% wet
weight/volume and then cells were broken by
sonication on ice, using sonicator UP 400S at 70
amplitude for 30 cycles (30 sec on/50 sec off). The
mixture was centrifuged at 12,000 rpm for 10 min
at 4
o
C and the supernatant was utilized as
intracellular enzyme.
Enzyme Assay
CMCase, α-amylase, FPase, Lipase, protease,
xylanase and phytase were estimated by the
methods of Mandels et al. (1976), Rick and
Stegbauer (1974), Ghose (1987), Mustranta (1992),
McDonald and Chen (1965), Bailey et al. (1992)
and Harland and Harland (1980) and Heinonen and
Lahti (1981), respectively.
STATISTICAL ANALYSIS
Significance difference among replicates was
shown in the form of probability (p≤0.05) values
using the software Costat (Snedecor and Cochrane
1980).
RESULTS AND DISCUSSION
Tatta pani is located in Kotli, Azad Kashmir
(Pakistan) and Tatta Pani hot springs are positioned
on the right bank of the river Poonch and can be
approached from Kotli by covering a distance of 26
km. Heat produced due to the occurrence of sulphur
in Patala shales, friction alongside the Main
Boundary Thrust and deep circulation of meteoric
water underneath the surface is the possible cause
of heat giving rise to hot springs in Tatta Pani, Azad

Zahoor, S et al.
Braz. Arch. Biol. Technol. v.59: e16150622, Jan/Dec 2016
4
Kashmir. In-situ measurements of temperature
showed that the hot spring had temperature ranging
from 79-86
o
C. The hot spring had nearly neutral pH
of 6.93. Khan et al. (1999) noted pH and
temperature 1.70-1.86 and 56-60
o
C, respectively
for these hot springs. Ex-situ characterization of
water sample was done to determine the chemical
composition of the hot spring (Table 1). The key
cation in the Tatta Pani hot springs was found to be
Na
+
(208 mg/L). EC value and TDS were recorded
to be 462.6 μS/cm and 231.3 mg/L, respectively.
Khan et al. (1999) also stated Na
+
as the major ion
in Tatta Pani hot spring, having concentration of
110 to 120 ppm. Total dissolved solids (TDS) and
electrical conductivity (EC) were found to be 3911
to 4183 ppm and 7020 to 9560 μS/cm, respectively.
Khan et al. (1999) study displayed a little variation
in the water characterization results, they recorded
low pH and temperature and higher TDS and EC
values as compared to the current findings. This
variation might be due to the difference in time
period of study and due to the change in
concentration of various ions and amount of sulfur
that resulted in shift in pH from extremely acidic to
neutral. The change in temperature detected may be
because of different sampling points (distance from
the source) or this change might be because of the
earthquake that occurred in 2005.
Table 1: Composition of water in Tatta Pani
hot spring
Result
0.91
2.9
208
4.5
58.3
0.71
0.05
26.23
231.3
23.31
8.2
16.9
Temperature (
o
C)
79-86
462.6
6.93
A thermophilic bacterium (TP-2) was isolated from
Tatta Pani hot spring in Azad Kashmir. Single
colonies were identified and the pure culture was
isolated by repeated streaking on LB agar plates
incubated at 65
o
C. Colony morphology
characteristics were examined from over 48 h
culture on LB agar. Colonies were flat, cream
colored and slimy with smooth margins while the
cells were Gram positive rods (Fig. 1). Geobacillus
sp. strain GWE1 formed white, convex and circular
colonies while cells were gram positive rods
(Daniela et al. 2013). Geobacillus sp. NMS2
isolated from water and soil samples from a hot
spring in Sri Lanka formed yellow colored, round,
convex and smooth colonies while the cells were
rod shaped that stained positive with Grams
staining (Mathew and Rathnayake 2014).
Geobacillus thermoleorans strain Rekadwadsis
isolated from hot spring in Unkeshwar formed light
yellow, circular, convex and smooth colonies
whereas the cells were long Gram positive rods
(Rekadwad 2015).
Figure1: Gram’s staining of the isolate TP-2
Scanning electron microscopy results showed that
the cells have smooth surface and ranged in size
from 0.2-0.3 μm in width to 2.1-3.6 μm in length
(Fig. 2). The length and diameter of cells of G.
debilis, G. thermoglucosidasius and G.
stearothermophilus ranged in size from 1.0-14.2
μm to 0.5-1.0 μm, <3.0 μm to <0.9 μm and 2.0-3.5
μm to 0.5-0.9 μm, respectively (Banat et al. 2004;
Nazina et al. 2001; Sung et al. 2002). The length
and diameter of cells of Geobacillus sp. strain
GWE1, Geobacillus sp. NMS2, Geobacillus
thermoleorans strain Rekadwadsis ranged in size
from 8.0 to 0.8-1.0 μm, 8-10 to 1 μm and 6.0-1.0
μm, respectively (Daniela et al. 2013; Mathew and
Rathnayake 2014; Rekadwad 2015)

Chracterization of a thermophilic bacterium
Braz. Arch. Biol. Technol. v.59: e16150622, Jan/Dec 2016
5
Figure 2: Scanning Electron Micrograph of isolate TP-2
Physiological and biochemical characters are
significant as they give indications for selection of
efficient strains for additional studies principally of
applied value. Various range of pH (4.0-10) was
tested to record the pH optimal for the growth of the
isolate (Fig. 3). Maximum growth of isolate (TP-2)
was recorded at pH 7.0. The isolate grew at pH 5.5
to 9.0 while no growth was observed below pH 5.5
and above 9.0. The isolate was able to survive at
both slightly alkaline and acidic environments.
Though the isolate preferred a neutral pH range, it
adopted itself to survive at slightly basic and acidic
pH conditions in the environment. Sharma et al.
(2009) isolated Geobacillus spp. from the Soldhar
hot spring site (India) that can tolerate pH range of
4.0-11. Thermophilic bacteria that grew well at pH
ranging from 7.5-8.5 were isolated from the hot
springs of Saudi Arabia (Khalil 2011). Geobacillus
sp. SG 01 displayed optimum growth at pH 7.0
while no growth was observed at pH values higher
than 9.5 (Yang et al. 2013 ). Geobacillus pallidus
p26 isolated from Pasinler hot spring, Erzurum,
Turkey grew optimally at pH 7.5 (Celik et al. 2014).
Geobacillus sp. 12AMOR1 exhibited growth
within the pH range of 5.5-9.0 with optimal growth
within the wide pH of 6.5-8.0 (Wissuwa et al.
2016).
Figure3: Effect of pH on the growth of isolate TP-2. ±
indicates the standard deviation among the three parallel
replicates.
Minimum, optimum and maximum growth
temperature for the isolate was noted by incubating
the isolate at different temperature (30-90
o
C). The
isolate displayed good growth within temperature
range of 50-65
o
C with maximal growth observed at
65
o
C (Fig. 4). No growth was observed below 40
o
C
while the upper limit for growth with respect to
temperature was around 75ºC. The temperature
specificity of the isolate indicated that it is
thermophilic in nature since it required 65
o
C for
optimum growth and was unable to grow outside
the temperature range of 40-75ºC. Thermophilic
bacteria that showed good growth between 45-85
o
C
were isolated from hot springs of Fiji (Narayan et
al. 2008). G. thermoglucosidasius grew within the
temperature range of 37-68
o
C (Nazina et al. 2001).
Geobacillus toebii showed growth within the
temperature range of 45-70
o
C with optimum
growth at 60
o
C (Sung et al. 2002). Geobacillus sp.
strain GWE1 showed growth within the
temperature range of 60-80
o
C while optimum
growth was observed at 70
o
C (Daniela et al. 2013).
G. thermoleovorans DA2 isolated from Southern
Sinai, Egypt was able to grow within the
temperature range of 5080
o
C displaying optimal
growth at 65C (Fotouh et al. 2016). Geobacillus
thermoleorans strain Rekadwadsis also showed
optimal growth at 65
o
C (Rekadwad 2015).
0
0,25
0,5
0,75
1
1,25
1,5
5 5,5 6 6,5 7 7,5 8 8,5 9
Optical density at 600 nm
pH

Citations
More filters
Journal ArticleDOI
TL;DR: A new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time is revealed and the high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.
Abstract: Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 °C of temperature, pH 7 to 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.

20 citations


Cites background from "Characterization Of A Novel Hydroly..."

  • ...In this regard, most members of the genus are found in warm biotopes, such as most soil environments, heaps of compost, oil fields, and geothermal zones [12,15]....

    [...]

  • ...[15] reported the growth of isolate Geobacillus TP-5 in the range of 0% to 3....

    [...]

  • ...Studies based on molecular phylogenetic approaches using 16S rRNA to investigate different bacteria found in the hot spring have been undertaken lately [15]....

    [...]

  • ...They can be found in every part of the world, including underneath the oceans and seas [15]....

    [...]

  • ...Thermophilic bacteria have been characterized by phenotypic and genotypic means from numerous geothermal regions of the world [15]....

    [...]

Book ChapterDOI
01 Jan 2018
TL;DR: This chapter contains a review of studies of geobacilli and anoxybacilli from terrestrial geothermal springs worldwide with special emphasis on their distribution and diversity, ecological significance, adaptive mechanisms, enzymes, and biotechnological potential.
Abstract: A large number of thermophilic representatives of the Geobacillus and Anoxybacillus genera have been isolated from geographically distant and physicochemically different environments, including high-, moderate-, and low-temperature habitats However, terrestrial hot springs are the main habitats for Geobacillus and Anoxybacillus species The members of these genera possess a variety of thermo-adaptive features that enable them to thrive at elevated temperatures Due to their ability to withstand harsh environmental conditions, geobacilli and anoxybacilli are a valuable source for provision of thermostable enzymes, such as amylases, lipases, proteases, etc, and other components Thermostable enzymes obtained from thermophilic bacilli have found a plethora of commercial applications due to their sturdiness and toughness in withstanding the heat generated in various biotechnological and industrial processes This chapter contains a review of studies of geobacilli and anoxybacilli from terrestrial geothermal springs worldwide with special emphasis on their distribution and diversity, ecological significance, adaptive mechanisms, enzymes, and biotechnological potential

17 citations

Journal ArticleDOI
TL;DR: According to results obtained from this study, this new strain of Thermomonas haemolytica isolated from geothermal Nenehatun hot spring in Turkey is a promising candidate for industrial applications in production of detergent.
Abstract: In this study, it was aimed to determine the ability to produce protease enzyme of Thermomonas haemolytica isolated from geothermal Nenehatun hot spring in Turkey and utilization of this enzyme in the detergent industry to remove protein stains. The protease-producing strains were screened from hot springs, and a potential strain was identified as T. haemolytica according to morphological, physiological and biochemical characteristics and sequence of 16S rRNA gene. Maximum protease activity was observed at 55 °C and pH 9.0 at 72 h of incubation. Activity was very stable between 50 and 65 °C and pH 8.0–10.0, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA, EGTA, SDS, and urea. Some divalent metal ions such as Ca2+, Mg2+, and Mn2+ increased the enzyme activity, while Zn2+ and Cu2+ decreased. Michaelis–Menten constant (Km) and maximum velocity (Vmax) values were calculated by Lineweaver–Burk plot as 125 EU/ml and 1262 mg/ml, respectively. The biochemical characterization of the protease obtained from T. haemolytica was performed and applied on the blood and grass-stained fabrics with detergent to evaluate the stain removal performance of the enzyme. It was observed that the application of detergent with enzyme was more effective than the detergent without enzyme to clean up the stained fabrics. This is the first report of characterization of the protease of T. haemolytica. According to results obtained from this study, this new strain is a promising candidate for industrial applications in production of detergent.

14 citations

Dissertation
01 Jan 2019
TL;DR: 137 7.2.3 Oxidation of iron by Acidithiobacillus ferrooxidans IO-2C in the presence of different iron substrates 139 6.3 Discussion 150 Chapter 8: Culture independent microbial diversity of extremophiles 152 Abstract 152 8.2 results 8.3 discussion 168 Chapter 9: Discussion 171 Conclusion 187 References 188
Abstract: 137 7.1 Background 137 7.2 Results 138 7.2.1 Isolation of acidophilic bacteria 138 7.2.2 Molecular characterization 138 7.2.3 Oxidation of iron by Acidithiobacillus ferrooxidans IO-2C in the presence of different iron substrates 139 7.2.4 Draft genome of Acidithiobacillus ferrooxidans IO-2C 144 7.3 Discussion 150 Chapter 8: Culture independent microbial diversity of extremophiles 152 Abstract 152 8.1 Background 152 8.2 Results 153 8.2.1 Microbial diversity of hot spring 153 8.2.2 Microbial diversity of saline soil 157 8.2.3 Microbial diversity of acid seep 162 8.3 Discussion 168 Chapter 9: Discussion 171 Conclusion 187 References 188

3 citations


Cites background from "Characterization Of A Novel Hydroly..."

  • ...Zahoor et al. (2016) isolated hydrolytic Thermophiles Chapter 6 125 enzyme producing Geobacillus debilis from hot springs of Tatta Pani, Azad Kashmir-Pakistan....

    [...]

Journal ArticleDOI
Wargijono Utomo1
01 Jan 2023-Heliyon
TL;DR: In this article , the authors aimed at screening and characterizing thermophilic amylase-producing bacteria from water and sediment samples of unexplored hot spring of Tatta Pani Kotli Azad Kashmir.
Abstract: Present study was aimed at screening and characterizing thermostable amylase-producing bacteria from water and sediment samples of unexplored hot spring of Tatta Pani Kotli Azad Kashmir. Four thermophilic isolates were characterized on morphological, biochemical, physiological basis and were authenticated by molecular analysis. By 16S rDNA sequencing, isolates were identified as Anoxybacillus mongoliensis (MBT001), Anoxybacillus flavithermus (MBT002), Bacillus (MBT004). Among all identified strains, MBT003 showed maximum homology with both Anoxybacillus mongoliensis and Anoxybacillus flavithermus. Amylase activity was analyzed qualitatively in starch agar and quantitatively by DNS method. The optimal enzyme production was observed and authenticated by Response Surface Methodology at 7 pH, 70 °C, 1.25% substrate concentration, 300 μL of inocula volume after 48 h of incubation. Optimum amylase activity (4.4 U/mL) and stability (3.3 U/mL) was observed with 1.5% soluble starch at 70 °C. Maximum activity (3.7 U/mL) and stability (1.5 U/mL) was found at pH 8. Enzyme activity was increased in the presence of MgSO4 and CaCl2. Amylase was stable with surfactants and commercial detergents for 30 min. Supplementation of the enzyme with commercial detergent improved the washing ability of the detergent. This investigation has revealed that these thermostable bacteria are excellent source of amylase which can be used commercially for generating economic activity on sustainable basis.

3 citations

References
More filters
Journal ArticleDOI
TL;DR: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score.
Abstract: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straight-forward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.

88,255 citations


"Characterization Of A Novel Hydroly..." refers methods in this paper

  • ...Phylogenetic analysis was done using neighbor-joining method using the software MEGA 5.0 (Saitou and Nei 1987; Altschul et al. 1990; Thompson et al. 1994; Waterhouse et al. 2009)....

    [...]

Journal ArticleDOI
TL;DR: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved and modifications are incorporated into a new program, CLUSTAL W, which is freely available.
Abstract: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.

63,427 citations


"Characterization Of A Novel Hydroly..." refers methods in this paper

  • ...Phylogenetic analysis was done using neighbor-joining method using the software MEGA 5.0 (Saitou and Nei 1987; Altschul et al. 1990; Thompson et al. 1994; Waterhouse et al. 2009)....

    [...]

Journal ArticleDOI
TL;DR: The neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods for reconstructing phylogenetic trees from evolutionary distance data.
Abstract: A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.

57,055 citations


"Characterization Of A Novel Hydroly..." refers methods in this paper

  • ...Phylogenetic analysis was done using neighbor-joining method using the software MEGA 5.0 (Saitou and Nei 1987; Altschul et al. 1990; Thompson et al. 1994; Waterhouse et al. 2009)....

    [...]

Journal ArticleDOI
TL;DR: The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models, inferring ancestral states and sequences, and estimating evolutionary rates site-by-site.
Abstract: Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.

39,110 citations

Journal ArticleDOI
TL;DR: Jalview 2 is a system for interactive WYSIWYG editing, analysis and annotation of multiple sequence alignments that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server.
Abstract: Summary: Jalview Version 2 is a system for interactive WYSIWYG editing, analysis and annotation of multiple sequence alignments. Core features include keyboard and mouse-based editing, multiple views and alignment overviews, and linked structure display with Jmol. Jalview 2 is available in two forms: a lightweight Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server. Availability: The Jalview 2 Desktop application and JalviewLite applet are made freely available under the GPL, and can be downloaded from www.jalview.org Contact: g.j.barton@dundee.ac.uk

7,926 citations


"Characterization Of A Novel Hydroly..." refers methods in this paper

  • ...Phylogenetic analysis was done using neighbor-joining method using the software MEGA 5.0 (Saitou and Nei 1987; Altschul et al. 1990; Thompson et al. 1994; Waterhouse et al. 2009)....

    [...]