Chapter 2
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2. Review of Literature
2.1 Thermophiles
Organisms capable of living at high temperatures have held a particular fascination
for biologists and biochemists, as they exist at temperatures where their proteins and
nucleic acids would be expected to be denatured.
Heat-loving microbes, or thermophiles, are among the best studied of the
extremophiles. The classification of living organisms based on their relation to
temperature has always been considered as the most basic element of biological
systematic (Kristjansson and Stetter, 1992). Mostly, the optimum temperature for
growth of terrestrial microorganisms is between 25-35ºC. Microorganisms were
grouped into three categories based on their optimum temperature (Topt),
psychrophiles that have a Topt below 20°C, mesophiles that grow optimally between
20°C to 40°C and thermophiles between 50 to 80°C (Madigan et al, 1997).
Traditionally, organisms with a maximal growth temperature Tmax (i.e., above which
no growth occurs) higher than 50°C have been described as thermophiles. Brock
(1969) suggested setting the boundary of thermophiles above 60°C based on two
arguments. First, temperatures below this boundary are common in nature, whereas
higher temperatures are mainly associated with geothermal and industrial activities.
Second, certain invertebrates can survive exposures to temperatures close to 100°C,
but cannot grow above 50°C (Table 2.1). The thermophilic world would therefore
only be prokaryotic. Further, no multicellular animals or plants have been found to
tolerate temperatures above 50°C (122°F). Thermophilic microorganisms belong to
Archaea and Bacteria are unique in adaptation to as high as 121ºC temperature, which
is hostile to ordinary life. Thermophiles are classified in different categories according
to their temperature requirement as shown in table 2.1. Thermophiles were classified
as organisms with a temperature minimum of about room temperature (25°C),
Orthothermophiles as microorganisms with a temperature maximum above the
temperature of protein coagulation (60 – 70°C) and the thermotolerants as organisms
with a temperature maximum of 50 – 55°C, but also grow well at room temperature.
Thermophiles are also divided into two groups as first is true thermophiles, which
showed optimum growth at 60 – 70°C and no growth, or trace growth, below 40 to
45°C. The second is facultative thermophiles, which exhibited growth at 25°C and
have their optimum temperature for growth and survival is 50 – 55°C, and maximum
Faculty of Applied Sciences and Biotechnology
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60°C (Bergey, 1919). Thermophiles are also classified as strict thermophiles, which
showed growth above 55°C; thermotolerant showed optimum growth at temperature
between 40-50°C (Morrison and Tanner, 1922) and obligate thermophiles exhibited
growth at 55°C, but not at 37°C (Cameron and Esty, 1926). Currently, thermophiles
are classified into moderate thermophiles (optimum growth 50 – 60°C), extreme
thermophiles (optimum growth 60 – 80°C) and hyperthermophiles (optimum growth
80 – 110°C). Some scientists made a single group of hyperthermophiles and extreme
thermophiles (Imanaka and Atomi, 2002).
Table 2.1: Classification of thermophiles (Stetter, 1998)
Category
Facultative thermophiles
Temperature (oC)
50-60˚C
Obligative thermophiles
60-70˚C
Extreme thermophiles
60-80˚C
Hyperthermophiles
> 80˚C
Based on their optimum temperature for growth, several thermophilic bacteria
and archaebacteria have been classified as moderate thermophiles (Bacillus
caldolyticus,
Geobacillus
Clostridium
stearothermophilus,
thermohydrosulfuricum,
Thermoplasma
acidophilum),
Thermoactinomyces
Thermoanaerobacter
extreme
themophiles
vulgaris,
ethanolicus,
(Thermus
aquaticus,
Thermodesulfobacterium commune, Sulfolobus acidocaldarius, Thermomicrobium
roseum Dictyoglomus thermophilum, Methanococcus vulcanicus, Sulfurococcus
mirabilis, Thermotoga mritima) and hyperthermophiles (Methanoccus jannaschii,
Acidianus infernos, Archaeoglobus profundus, Methanopyrus kandleri, Pyrobaculum
islandicum,
Pyrococcus
Thermococcus
littoralis,
furiosus,
Pyrodictiumoccultum,
Ignicoccus
islandicum,
Pyrolobus
fumarii,
Nannoarchaeum
equitans
Geobacillus sp.) (Kristjansson et al., 1994; Stetter, 1998 ; Gonzalez, 1999; Nazina et
al., 2001; Reysenbach et al., 2002; Ghosh et al., 2003). Most of the thermophiles have
been isolated from composts, sun-heated soils, terrestrial hot springs, submarine
hydrothermal vents and geothermally heated oil reserves and oil wells (Waring, 1965;
Sonne-Hansen and Ahring, 1999). The first extremophile capable of growth at
temperatures greater than 70°C was a bacterium, now called Thermus aquaticus, that
would later make possible the widespread use of a revolutionary technology—the
polymerase chain reaction (PCR). About the same time, the first hyperthermophile
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was found in an extremely hot and acidic spring. This Archaea, Sulfolobus
acidocaldarius grows at temperatures as high as 85°C. Most hyperthermophiles
belong to the archaeal domain. Among bacteria, there are only few species that can be
called hyperthermophiles, such as Thermotoga and Aquifex, which have a Topt in the
range of 90 to 95°C (Huber, 1995). Until now, no hyperthermophilic microorganisms
in the domain Eukarya have been reported. For a long time, the record for highest
Tmax was held by Pyrolobus fumarii, isolated from a deep-sea thermal black smoker
vent chimney. P. fumarii has a Tmax of 113°C and a Topt of 106°C and is unable to
grow at temperature < 90°C (Blochl et al., 1997). Strain 121, a Fe(III)-reducing
Archaea isolated from a hydrothermal vent along the Juan de Fuca Ridge, is reported
to have a doubling time of 24 h at 121°C and remains viable after exposure to
temperature as high as 130°C (Kashefi and Lovley, 2003).
Various thermophilic fungi have been isolated from soils, nesting materials of
birds, composts and wood chips (Satyanarayana et al., 1979; Mouchacca, 1999).
These includes Zygomycetes (Rhizomucor miehei, R. pusillus), Ascomycetes
(Chaetomium
thermophile,
Dactylomyces
thermophilus),
Basidiomycetes
(Phanerochaete chrysosporium) and Hyphomycetes (Acremonium alabamensis and
Myceliophthora thermophila,). Some algae (Achanthes exigua, and Cyanidium
caldarium) and protozoa (Cothuria sp. Oxytricha falla, Cercosulcifer hamathensis,)
showed growth and reproduce at elevated temperatures.
Table 2.2: Growth characteristics of thermophiles (Cowan and Morgan, 1981;
Brocklesbury and Morgan, 1986; Huber et al., 1986; Weigel and Ljungdahl, 1986)
Bacterial Strains
T opt (°C)
T max
(°C)
78
pH opt
55-60
T min
(°C)
40
Bacillus
stearothermophilius
Bacillus acidocaldarius
Bacillus caldolyticus
Thermus aquaticus
Thermus sp. T351
Thermus sp. T4-1A
Caldocellum
saccharolyticum
Thermotoga muritima
Geobacillus sp
Methanothermus fervidus
Sulfolobus acidocaldarius
60-65
72
70
75-80
75-80
68
45
40
44
47
-
78
76
79
80
2.0-5.0
6.3-8.5
7.5-7.8
8.7
7.2
7.0
80
75
85
70-75
55
50
70
-
90
85
100
85-90
6.5
7
-
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2.2 Habitats of thermophiles
Natural geothermal areas are widely distributed across the globe. Hot springs are
formed as a result of the movement of hot water from the earth’s crust via faults
created by tectonic movement or volcanic eruption. Terrestrial surface environments
include hot springs that are close to neutral pH, or acidic and sulphurous, or rich in
iron. Hot subterranean areas are diverse and ranges from volcanically heated
environments to those, such as the Great Artesian Basin in Australia that are heated
by virtue of their depth. Hot springs could be basic, neutral or acidic. However, the
most numerous are those with alkaline pH, usually associated with volcanic or
tectonic activity. Acidic hot springs such as that of the Yellowstone caldera are
associated with active volcanoes or shallow magma pools. The pH of geothermal
springs reflect their biodiversity, for instance, in acidic hot springs, acidophiles such
as Sulfulobus thrive and in neutral or moderate hot springs a diverse number of
thermophiles
such
as
Thermoproteus,
Pyrobaculum,
Methanothermus,
Desulforococcus and Thermofilum exist (Huber and Stetter, 1998). Submarine
environments include volcanic and hydrothermal vents. The latter are often described
as black smokers owing to the precipitation of minerals when hot, mineral-rich
volcanic fluids come into contact with cold ocean waters (5°C). The present record
for high-temperature growth is held by the archaeon Pyrolobus fumarii, which can
grow at 113°C (Blochl et al., 1997). Well known hot springs of India are given in
table 2.3.
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Fig 2.1 Collage showing hot springs situated in USA, New Zealand, China,
Thailand, Japan and India.
Table 2.3: Hot springs of India (Panda et al., 2012)
Name of the hot springs
Ganeshpuri, Akloli, Vajreshwari
Manikaran, Khirganga, Tapri, Tattapani,
Garam Kund, Vashisth
Bendrutheertha, Irde, Bandaru
Chavalpani, Dhunipani, Tatapani
Suryakund
Phurchachu (Reshi), Yumthang, Borang,
Ralang, Taram-chu and Yumey Samdong
Bakreshwar of Birbhum, Tantloi,
Kendughata, Bholeghata, Tantni
Gaurikund, Tapt Kund, Surya Kund
Hotspring of Dirang area
Taptapani, Atri, Deulajhari, Tarabalo
Tatta, Jarom, Brahma Kund, Ram Kund
Ushnagudam
Mannargudi
Faculty of Applied Sciences and Biotechnology
States
Maharastra
Himachal Pradesh
Karnataka
Madhya Pradesh
Gaya, Bihar
Sikkim
West Bengal
Uttarakhand
West Kameng,
Arunachal Pradesh
Orissa
Jharkhand
Andhra Pradesh
Tamil Nadu
15
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2.2.1 Terrestrial hot springs
Terrestrial geothermal areas can be generally divided into two classes according to the
nature of the heat source and pH.
High-temperature fields
High temperature fields are located within the active volcanic zones and magma
chamber serves as the heat source. They are mostly on high ground. Torfajokull east
of Hekla, Grimsvotn in the Vatnajokull glacier, and Hengill near Reykjavik,
Kerlingarfjoll, Namafjall near Myvatn, Kverkfjoll on the north side of Vatnajokull
and Krisuvik south of Reykjavik are the main high-temperature areas. In these areas,
steam and volcanic gases are emitted at the surface and the water temperature reaches
150 to 350°C. The highest recorded temperature was 386°C. Icelandic hot springs
-1
have high sulphide concentrations (30 mg L ) and thick bacterial mats are formed
with precipitated sulphur and make spectacular bright yellow or white colours
(Skirnisdottir et al., 2000).
Low-temperature fields: The low temperature hot spring fields are located outside
the active volcanic zones. The surface of low-temperature fields is mostly covered
with hot springs and geysers. The hot water of these areas is alkaline. Mostly, the
chemical contents of low-temperature water are similar to the chemical contents of
freshwater therefore it is possible to use the low-temperature water. Deep lava flows
and dead magma chambers serve as heat sources and the water temperature is usually
below 150°C at depths of 500 to 3000 m.
The warmest water holes in low-
temperature fields having temperature 75 – 100°C are called hot springs.
Subterranean hot springs: Deep sediments, rocks and minerals offer environments
for life that are very different from terrestrial and aquatic habitats. Water usually
contains H2, CH4 and CO2 that promote chemolithoautotrophic life but microbial
growth rates is low (Bachofen et al., 1998). Steam and water are collected in
boreholes 1500 to 2000 m deep and temperatures are ranging between 50 to 130°C
(Takai and Horikoshi, 1999).
2.2.2 Marine and terrestrial oil reservoirs: Oil fields are considered as new habitats
for thermophiles. Depending on the geographic location, reservoirs have temperatures
ranging from 60 to 130°C and pressures between 15 and 40MPa (Beeder, 1994).
Deep-sea hydrothermal vents
In these tectonically active areas, seawater infiltrates through cracks in the
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ocean floor, down to several kilometers. The fluid is very hot (250 to 400°C), acidic,
rich in metals (iron), CH4 and H2S (Prieur et al., 2001). If there is mixing of
hydrothermal fluid and cold seawater just before emission, its temperature is 5 to
100°C and if there is no mixing , the temperatures of fluid vents is 350°C or above.
The deep sea vents are also known as black smokers, due to the constant discharge of
precipitated minerals in seawater that take the aspect of thick, black clouds. Most of
the known hydrothermal vent can be found at depths greater than 3500 m and as
shallow as 400 m.
2.2.3 Other geothermal habitats
Constant hot habitats other than geothermal are very few in nature. Solar-heated
ponds and biologically-heated composts, hay and manure may cause high
temperature. Man-made, hot environments such as hot water pipelines burning coal
refuse piles, wastes from treatment plants or industrial processes in the food or
chemical industry have also been created... Several well-known thermophiles, such as
several Thermus species (Williams, 1992), Thermoplasma acidophilum (Darland,
1970), have been primarily isolated from those man-made hot systems (Kristjansson
and Stetter, 1992). For instance, Thermus scotoductus, a pigment-producing rod was
isolated first from hot tap water in Iceland (Kristjansson et al., 1994).
2.3 Phylogeny, taxonomy, and physiology of thermophiles
The phylogeny of extremophiles has been very closely linked to questions on the
origin and early evolution of life on Earth. Many thermophiles and hyperthermophiles
seem to have very “old” (or deep) lineages in the so-called Tree of Life (Fig. 2.2).
This is particularly evident among the archaeal thermophiles. It is also very striking
that 11 of the 23 major cultivated bacterial phyla contain thermophilic representatives.
Four of these, i.e., the Aquificae, the Thermotogae, the green-non-sulphur bacteria
including the Thermales and the Thermodesulfobacter group comprise all of the
deepest phylogenetic branches in the bacterial Phylogenetic tree. This supports the
hypothesis that thermophiles and hyperthermophiles represent the most ancient forms
of life now present on Earth (Madigan, 1997)
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Figure 2.2: Rooted universal phylogenetic tree as determined by comparative
analysis of ribosomal genes sequences. The data supports the discrimination of three
domains, two of which contain prokaryotic representatives (Bacteria and Archaea). In
dashed lines are indicated phylogenetic groups which are exclusively thermophilic or
contain few thermophilic representatives (Madigan et al., 1997).
2.3.1 Archaea
Archaea (from the Greek archaios, ancient or primitive) are quite diverse in
morphology and physiology. Archaea like bacteria are prokaryotes, are microscopic,
lack nucleus and can be identified by Gram-staining. They can be either Gram
positive or negative and may be rod-shaped, spherical, plate-shaped, spiral, lobed,
irregularly shaped, or pleiomorphic. Some are single cells, whereas other form
filaments or aggregates. Their diameter is in range of
0.1 to 15 μm, and some
filaments can grow up to 200 μm in length. Gram positive archaea have a cell wall
made up of Pseudo-murein instead of peptidoglygan (N-acetyl muramic acid and Nacetyl glucosamine). Gram negative archaea do not possess a cell wall and outer
membrane like Gram negative bacteria, but have a thick protein outer coat.
Multiplication may be by binary fission or budding (Prescott et al., 1999). Another
distinguishing feature is that archaea share some similarities with Eukaryotes such as
DNA transcription and translation mechanism and also binding of histones to DNA
(Gerday and Glansdorff, 2007). All known Archaea belong to four taxonomic phyla,
Crenarchaeota, Euryarchaeota, Korarchaeota and the very recently described
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Nanoarchaeota (Huber et al., 2002). The most commonly detected archaeal sequence
from hot springs are those of Crenarchaeota, followed Euryarchaota (Barns et al.,
1994; Marteinsson et al., 2001), even though archaea do not form the dominant
population in hot springs and in some cases they are not detected. Hugenholtz (1998)
and Reysenbach (1994) could not find any archaea in the Obsidian pools and Octopus
spring respectively, of the Yellow Spring National Park. Nanoarchaeota have only
been found in Yellow Stone National park in the USA and Russia. Korarchaeote have
been detected by 16S rRNA studies (Barns et al., 1994; Reysenbach et al., 2000) as
well as by isolation in the Yellow Stone hot spring (Huber et al., 2002).
The kingdom Crenarchaeota, consists entirely of extreme thermophiles and
hyperthermophiles. The Crenarchaeota are thought to resemble the ancestor of the
Archaea, and all well-characterized species are thermophiles and hyperthermophiles.
They are adapted to the marine environment and represented by the crenarchaeal
genera Archaeoglobus, Pyrodictium, Thermodiscus, Staphylothermus, Hyperthermus,
Methanopyrus, Pyrococcus, Thermococcus, and some members of Methanococcus.
Hyperthermophilic genera contained within the Crenarchaeota are Sulfolobus,
Desulfurococcus, Pyrodictium, Thermofilum, Thermoproteus and Pyrolobus. These
organisms have optimum growth temperatures range from 75° - 105°C, and the
maximum temperature of growth can be as high as 110°C (Pyrodictium occultum) or
even up to 113°C (Pyrolobus). They are unable to grow below 80°C (Stetter, 1996).
There are three major branches among the Crenarchaeota. The Thermoproteales are
Gram-negative
anaerobic,
facultative
and
hyperthermophiles
can
grow
chemolithoautotrophically by reducing sulphur to hydrogen sulphide. The
thermoacidophiles Sulfolobales are coccoid-shaped with optimum temperature 70 –
80°C and optimum pH 2 – 3. The order Desulfurococcales contains coccoid or discshaped anaerobic, facultative anaerobic and aerobic hyperthermophiles.
Euryarchaeota:
The
thermoacidophiles
are
represented
by
the
order
Thermoplasmatales, which contains only two genera, Thermoplasma and Picrophilus.
Thermoplasma are found in extremely acidic and moderately hot conditions (pH 1 – 2
and temperature 55 – 65°C). They grow on refuse piles of coal mines. Thermoplasma
takes the form of an irregular filament at temperature 59°C, whereas at lower
temperatures it is spherical (Prescott et al., 1999).
In terms of affinity to acid conditions, it is the most extreme archaeon. Thermococcae
is hyperthermophilic representatives of the Euryarchaeota. It is divided into three
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orders,
Review of Literature
Archaeoglobales,
Thermococcales,
and
Methanopyrales.
The
Archaeoglobales can take electrons from a variety of electron donors (H2, lactate,
glucose) and reduce sulphate, sulphite or thiosulphate to sulphide. The
Thermococcales have their Topt ranging from 88 to 100°C and are anaerobic
fermentatives (Prescott et al., 1999). They release H2 gas as a by-product of
fermentation, which is detoxified by reducing elementary sulphur to sulphide. The
third order, Methanopyrales is exclusively restricted to deep hydrothermal
ecosystems.
Korarchaeota: This is the most ancient phylum among the Archaea. It was
discovered by the use of molecular techniques in hot springs biomass samples
originating from the Yellowstone Park (Barns et al., 1994). The Korarchaeota has
been postulated on the basis of PCR amplification of 16S rRNA genes from
environmental DNA, but has not been confirmed by the pure cultivation of any
organisms (Skirnisdottir et al., 2000).
Nanoarchaeota: phylum is represented only by one species, the symbiont
Nanoarchaeum equitans. It grows in co-culture with a new chemolithoautotrophic
Ignicoccus species. N. equitans harbours the smallest archaeal genome, which is only
0.5 megabases in size. It has anaerobic mode of life and showed growth at elevated
temperature. The Nanoarchaeota are possible primitive form of microbial life (Hohn
et al., 2002; Huber et al., 2002).
2.3.2 Bacteria. The thermophilic bacterial representatives showed optimal growth
below temperature 75°C, except Thermotogae and Aquificae have optimum
temperature above 85°C. The pH range for bacterial thermophiles is 5 - 9, with few
exceptions like Hydrogenobaculum spp. or Bacillus species. The Aquificae,
Thermotogae,
Thermodesulfobacteria,
Thermo-microbia
and
Thermales
are
thermophilic families. Aquificae species are thermophilic aerobic, obligate,
chemolithoautotrophic bacteria and used H2 or reduced sulphur compounds as energy
sources. The Thermotogae, are anaerobic and fermentative species. They are Gram
negative cells with a distinct outer sheath-like envelope of “toga” (Boone et al.,
2001). The Thermodesulfobacteria cells are rod-shaped, strictly anaerobic,
chemoheterotrophic exhibiting a dissimilar sulphate-reducing metabolism. The
phylum Thermomicrobia cells are obligatory aerobic and grow only on complex
nutrients. The order Thermales represents Thermus aquaticus, isolated from the
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Chapter 2
Yellowstone Park in the USA (Brock and Freeze, 1969).
Review of Literature
The Thermales are
predominantly aerobic and heterotrophic. (Skirnisdottir et al., 2001; Miroshnichenko
et al., 2003). The Thermus genus can be found in various environments such as hot
tap water, thermally polluted streams or compost piles and natural hydrothermal
areas, marine hydrothermal vents and shallow marine hot springs (Marteinsson et al.,
1995; Marteinsson et al., 1999). Other genera compose the order Thermales, such as
Meiothermus, and Marinithermus, Oceanithermus and Vulcanithermus all are isolated
from deep marine vents (Miroshnichenko et al., 2003; Sako et al., 2003). The
thermophilic Gram-positives are scattered among 22 genera, and 20 are exclusively
thermophilic. The Cyanobacteria are mesophilic oxygenic photosynthetic prokaryotes
with the few exceptions of moderate thermophiles such as Fischerella or Oscillatoria
and Synechococcus which grows optimally above 55°C. The genera Rhodothermus
and Thermonema are the unique thermophilic representatives among the Cytophaga /
Flexibacter / Bacteroides group. Rhodothermus grows above 70°C and requires at
least 1 % salt concentration for growth. It has been used extensively as a source of
new enzymes (Alfredsson et al., 1988; Thorbjarnardottir et al., 1995; NordbergKarlsson et al., 1997). Thermonema is a moderate thermo- halophile with a Topt 65°C.
The genus has been reported from New Zealand, Italy and Iceland (Tenreiro et al.,
1997).
The purple bacteria or Proteobacteria, thermophilic genera scattered among
the, β, γ, δ and ε-subdivisions co-exist with both mesophilic and psychrophilic
representatives. The β-proteobacterial genera are growing strictly under anaerobic
conditions. The thermophilic ones are the hydrogen-oxidizing Hydrogenophilus and
the sulphur-oxidizing Thermothrix, as well as the sulphur-reducers Desulfurella and
Thermodesulfobacterium. Representatives of the ε-subdivision are mostly moderate
thermophiles with Topt varying around 55 to 65°C (Alain et al., 2002). The
Geobacillus spp (isolated from hot springs, compost, geothermal soil, crude oil, gold
mine and deep ocean sediments) reported worldwide and reviewed by Daniel (2014).
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Chapter 2
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Table 2.4: Worldwide distribution of Geobacillus sp.
Geographic
location
Germany
Isolation source
Isolate name
Japan
Japan
Agricultural produce
(fiber)
Compost
Compost
U.S.A.
Italy
Compost
Compost
India
Mariana
Trench
USA
(Yellowstone
NP)
Italy
Greece
Crude oil (parrafinic)
deep ocean sediment
(10,897 m)
Geothermal soil
Geobacillus thermoglucosidasius strain
PB94A
Geobacillus sp. kpuB3
Geobacillus thermodenitrificans strain
TSAA1
Geobacillus sp. WSUCF1
Geobacillus galactosidasius strain
CF1BT
Geobacillus sp. TERI NSM
Geobacillus kaustophilus HTA426
South Africa
China
Iran
Iran
Malaysia
USA
(Montana)
Pakistan
Turkey
Turkey
Turkey
Russia
China
Thailand
Pacific Ocean
Canada
Turkey
China
China
Japan
Lithuania
China
Singapore
U.K.
Geothermal soil
Geothermal soil,
sediment, seawater
Gold mine (50°C water)
Hot spring
Hot spring
Hot spring
Hot spring
Hot spring
Hot spring
Hot spring
Hot spring
Hot spring
Hot spring (microbial
mat)
Hot spring
Hot spring
Hydrothermal field
Compost (manure)
Oil well (pipeline
sediment)
Oil well (production
water)
Oil well (production
water)
Deep subterranean
petroleum reservoir
Oilfield
Oilfield reservoir (deep
subterranean)
Sewage sludge
Silage (Italian rye grass)
Gene bank
acc. No
FJ491390.1
AB292801.1
JX262152.1
GU289510.1
AM408559.1
EF199739.2
NR_074989.1
Geobacillus sp. M-7
FJ896054.1
Geobacillus sp. A1
Geobacillus sp. SP24
HM776457.1
JN692241.2
Geobacillus thermoleovorans strain GE-7
Geobacillus sp. TC-W7
Geobacillus sp. MKK-2005
Geobacillus sp. LH8
Geobacillus thermoleovorans
CCB_US3_UF5
Geobacillus thermoglucosidasius strain
AUT-01
Geobacillus sp. SBS-4S
Geobacillus caldoxylosilyticus strain TK4
Geobacillus kaue strain NB
Geobacillus sp. Ge1
Geobacillus gargensis strain Ga
AY450926.1
GQ866911.1
DQ309334.1
DQ192572.1
NR_074931.1
Geobacillus sp. 6k51
Geobacillus sp. H6a
Geobacillus sp. MT-1
Geobacillus thermodenitrificans strain
CMB-A2
Geobacillus thermodenitrificans subsp.
calidus strain F84b
Geobacillus sp. SH-1
DQ141699.1
EU248957.1
DQ288898.1
GQ293454.1
Geobacillus sp. XT15
HQ891030.1
Geobacillus thermoleovorans
AB034902.1
Geobacillus lituanicus
Geobacillus sp. MH-1
AY044055.1
FJ874632.1
Geobacillus sp. SF03
Geobacillus sp. DDS012
AY327448.1
EF426762.1
Faculty of Applied Sciences and Biotechnology
|GU356031.1
AB306519.1
AY248718.1
AF411066.1
HE613733.2
NR_115167.1
EU477773.2
DQ839487.1
22
Ge
Chapter 2
Germany
Northern
Ireland
Northern
Ireland
Iran
Taiwan
South Korea
South Korea
Japan
China
Review of Literature
Soil
Soil
Geobacillus thermodenitrificans HRO10
Geobacillus debilis strain F10
AJ785764.1
AJ564608.1
Soil
Geobacillus debilis strain TfT
AJ564616.1
Soil
Sugar refinery
wastewater
Wood chips composted
with swine manure
Wood chips composted
with swine manure
Hot spring
Hot spring
Geobacillus zalihae strain T1
Geobacillus thermoleovorans
AY166603.1
AY074879.1
Geobacillus thermodenitrificans strain
SG-01
Geobacillus thermodenitrificans strain
SG-02
Geobacillus thermodenitrificans
Geobacillus thermodenitrificans strain T2
FJ481105.1
FJ481104.1
AB546234.1
EF570295.1
* Adapted from Daniel (2014) and modified
2.4 Adaptation of Bacteria and Archaea to High Temperature
Thermophiles thrive at temperatures from 60°C and above. Their systems are
designed in such a way that they can tolerate the extreme environments they inhabit.
High temperature leads to a corresponding rise in membrane permeability; therefore
hyperthermophiles must possess stable membranes, nucleic acids and proteins in
order to survive at these temperatures and pressure (Lopez, 1999; Robb and Clark,
1999; Daniel and Cowan, 2000; Kumar and Nussinov, 2001; Cavicchioli, 2007;
Gerday and Glansdorff, 2007).
Hyperthermophilic/ thermophilic membranes are less permeable to solutes
compared to mesophiles. Passive transport across the membrane increases at high
temperature, which makes the membranes more permeable to solutes (Daniel and
Cowan, 2000). As a result, solutes can easily move across the plasma membrane or
inter-membrane space. To survive at high temperature, these bacteria developed
means of controlling the movement of solutes across their membranes. The typical
fatty acid bilayer structure of the bacterial cytoplasmic membrane would become
disrupted at extreme temperatures, where as in thermophiles membrane are composed
of saturated fatty acids that provides a hydrophobic environment for the cell and
maintains the cell rigidity even at elevated temperatures (Herbert and Sharp, 1992).
This rigidity is due to the methyl side groups, attached to the hydrocarbon (fatty acid)
part of the cell membrane. The methyl side group restricts mobility of the
hydrocarbon chains. Archaea or hyperthermophiles monolayer membrane composed
of phytanyl chains connected to glycerol with ether linkage on their cell membrane
and are more heat resistant than bacterial thermophiles in which membrane are
Faculty of Applied Sciences and Biotechnology
23
Chapter 2
Review of Literature
formed of fatty acids (De-Rosa et al., 1994). Hyperthermophiles also contain a
periplasmic–like region which is believed to buffer pH and other harsh external
conditions (Cavicchioli, 2007).
Nucleic acid thermostabilization involves methylation of the bases to protect
against base loss. For replication at high temperature DNA binds with salts and
histone-like basic proteins (Daniel and Cowan, 2000). Binding with K+, Na+ and Mg2+
also prevents chemical thermodegradation of the phosphodiester bonds and binding of
these cations help in protecting the phosphate backbone (Gerday and Glansdorff,
2007). DNA packaging by histones is also prominent in hyperthermophiles. This help
to preserve the duplex structure of DNA. Such proteins increase the fusion
temperature of DNA up to 40ºC above normal values and protect DNA from thermal
damage than their mesophilic counterparts (Todorova et al., 2004).
Another mechanism involved in DNA stabilization is by producing a
particular type of DNA topoisomerase, called DNA reverse gyrase, which introduces
positive super coils in the DNA molecule at the expense of ATP, this conferring a
greater stability by elevating the melting point of DNA strands and rendering it more
resistant to thermal denaturation (Lopez,1999). In addition methanogenes of large
amounts of cyclic 2, 3-potasium-bi-phospho-glycerate are present in cytoplasm,
which avoids chemical damage such as DNA depurinization (Grogan, 2004). Proteins
of thermophiles have interactions at the primary, secondary and tertiary levels. Protein
interactions include high packing efficiency, network of ion pairs and reduction of
conformational strain and disulphide bonds (Daniel and Cowan, 2000). The loss of
protein stability is due to the unfolding of the monomer into inactive or denatured
form. The formation of intermolecular interactions will therefore prevent
denaturation. One such example is the chorismate mutase from Methanocaldococcus
Jannaschii is believed to have evolved as an adaptation to heat. In addition, proteins
of thermophiles have increased surface charge and less exposed thermolabile amino
acids such as Cysteine, Glutamine and Arginine (Robb and Clark, 1999). Thus,
increased ionic interaction and hydrogen bonds, increased hydrophobicity, decreased
flexibility and smaller surface loops confer stability on the thermophilic protein.
Thermophiles produce special heat shock proteins known as “chaperonins”, which are
thermostable and resistant to denaturation and proteolysis. Proteins of thermophiles,
denatured at high temperature, are refolded by the chaperonins, thus restoring their
native form and function (Kumar and Nussinov, 2001).
Faculty of Applied Sciences and Biotechnology
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Chapter 2
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Thermophiles and hyperthermophiles are able to proliferate at high
temperatures because their nucleic acids, membranes and proteins are adapted to such
environments and are not harmed by these extremities. In cases where there is harm
due to high temperature, the cell devised fast repair mechanism that counters the
damage.
2.4.1 Cellular and molecular basis of protein thermostability
To cope with high temperatures, microorganisms have specific adaptations on the
levels of physiology and metabolism, structure and functions of macromolecules and
cellular functions and regulation of gene expression. Under extreme conditions of
temperature, pH and pressure, amino acids can be damaged irreversibly by
deamination, β-elimination, hydrolysis, Maillard reaction, oxydations, and disulphide
interchange. As a result, at temperatures above the boiling point of water, the half-life
of some amino acids is significantly shorter than the generation time of
hyperthermophiles. A large number of enzymes produced by thermophilic
microrganisms are stable and active at elevated temperatures (temperature exceeding
the upper growth limits of the producing organism) (Sterner and Liebl, 2001). It is at
high temperature range where thermal decomposition of amino acids and protein
backbones become significant. The cellular strategies have been developed to enhance
the stability of proteins by additional factors such as chaperone proteins, protein
repair enzymes and high concentrations of small stabilizing solutes (Sterner and Liebl,
2001). Chaperones are involved in the proper folding of newly synthesized protein.
Chaperones prevent the aggregation of proteins that unfold after exposure of the cell
and cell compounds to high temperature and also prevent misfolding and aid in the
refolding of denatured proteins. The molecular basis of protein thermostability has
been extensively reviewed (Vielle and Zeikus, 2001; Fujiwara 2002). The forces that
keep thermophilic and hyperthermophilic enzymes functional and stable at higher
temperatures are apparently similar to those in mesophilic proteins; apart from the
proteins in thermophilic microorganisms have variations in their sequences and
secondary structures. The comparison of the amino acid content in partial and
complete genomes of mesophilic and thermophilic microorganisms showed that
thermophilic protein contain fewer residues than mesophilic proteins, with a larger
proportion of charged residues (Arg, Lys, His, Asp, Glu) and smaller proportion of
uncharged polar residues (Ser, Thr, Gln, Asn, Cys). The main characteristics of
Faculty of Applied Sciences and Biotechnology
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Chapter 2
Review of Literature
thermophilic proteins are the hydrophobic core of the protein is strengthened by
increasing the number of hydrophobic amino acids with branched side chains. Slight
changes of amino acids distribution and sequences increase the number of stabilizing
interactions in the folded protein, such as: additional ion-pairs, disulphide bridges,
hydrogen bonds and hydrophobic interactions (Hartley and Payton 1983; Mozhaev,
1993; Kumar and Nussinov, 2001; Farias and Bonato, 2003). Other strategies for
stabilizing proteins include filling cavities in the molecular structure of the proteins,
shortening of the loops and reduction of accessible hydrophilic surface area (Voght et
al., 1997; Stetter, 1998; Thompson and Eisenberg, 1999; Fukuchi and Nishikawa,
2001; Shiraki et al., 2001). Other modifications include: metal ion binding and
diminished amount of residues susceptible to deamidation or oxidation. Moreover,
some thermozymes contain thermolabile residues in location in which they are not
susceptible to degradation (Zeikus et al., 1998).
Table 2.5 Isolation sources of thermophiles and their growth characteristics
Organisms
Isolation sites
Pyrococcus furiosus
shallow marine
sediments,
Vulcano Island,
Italy
solfatara, Naples,
Italy
solfatara,
Kodakara Island,
Japan
shallow marine
sediments,
Vulcano Island,
Italy
Submarine
hydrothermal vent
Submarine
hydrothermal vent
Deep sea
hydrothermal vent
Deep sea
hydrothermal vent
Hot spring
Sulfolobus
solfataricus P2
Thermococcus
kodakaerensis KOD1
Thermotoga maritima
MSB8
Nanoarchaeum
equitans
Methanocaldococcus
villosus
Aciduliprofundum
boonei
Geoglobus
acetivorans
Acidilobus
saccharovorans
Pyrococcus yayanosii
Deep sea
hydrothermal vent
Topt
(°C)
98
Growth physiology
References
Fermentative
anaerobe
Frock and Kelly,
2012
80
Aerobic, extreme
Thermoacidophile
Fermentative
anaerobe,
Frock and Kelly,
2012
Frock and Kelly,
2012
80
Fermentative
anaerobe,
Frock and Kelly,
2012
70–98
Parasitic
80
Chemolithoautotroph
70
Fermentative
anaerobe
Chemolithoautotroph
Huber et al.,
2002
Bellack et al.,
2011
Reysenbach et
al., 2006
Slobodkina et
al., 2009
Prokofeva et al.,
2009
Birrien et al.,
2011
85
81
80–85
98
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Fermentative
anaerobe
Fermentative
anaerobe
26
Chapter 2
Review of Literature
2.5 Applications of thermophiles
The ability of thermophilic microorganism to grow and reproduce at high temperature
and to produce extracellular enzymes with unique and valuable properties was due to
their ability to manipulate their genetic composition. Therefore, these thermophilic
microorganisms are considered as the most valuable bio prospecting microbes for
industrial and biotechnological applications (Norashirene et al., 2013). A major
attraction for thermophilic micro-organisms is the production of solvents such as
ethanol, butanol and acetone. The evaporation of volatile products at high
fermentation temperatures supplies a solution to the problem of removal of potentially
inhibitory products in the culture medium. In contrast to yeast and Zymomonas,
thermophilic ethanol producers may be inhibited by as little as 1 % ethanol
(Sonnieitner and Fiechter, 1983; Hartley and Payton, 1983).
2.5.1 Thermozymes
Enzymes produced by thermophiles and hyperthermophiles are optimally active at
high temperatures, between 60 and 125°C and resistant to irreversible inactivation at
elevated temperatures are known as thermoenzymes. Thermozymes offer various
biotechnological and industrial advantages over mesophilic enzymes. They are easier
to purify by heat treatment, higher resistance to chemical denaturants (solvents and
guanidinium hydrochloride) and withstand higher substrate concentrations. Because
of their stability at elevated temperature, thermozyme reactions are less susceptible to
microbial contamination and often display higher reaction rates than mesozyme
catalyzed reactions. In view of these important advantages, thermozymes are
attracting much industrial interest (Becker et al., 1997). Furthermore, thermozymes
can be used as models for understanding thermo stability. Thus, identifying structural
features involved in stability of thermozymes is essential for a theoretical description
of the physico-chemical principles contributing to protein stability and folding.
Furthermore, this information is essential also for designing more stable enzymes for
industrial processes.
The main advantage of thermozymes is their high stability at elevated
temperatures which is beneficial for a large variety of industrial processes. The
increase of temperature has a significant influence on the bioavailability and increased
solubility of many polymeric substrates or organic substrate. The elevation of
temperature also related with increase in diffusion of organic compounds, decrease in
viscosity, improved transfer rates and consequently increased reaction rates (Krahe et
Faculty of Applied Sciences and Biotechnology
27
Chapter 2
Review of Literature
al., 1996; Becker, 1997). Besides, the another advantage is that it also reduces the
risk of microbial contamination as all the pathogenic bacteria and saprophytes are
killed at temperature above 70°C and reduce number of bacteria which cause
contamination of food processes.
Factors contributing to stability include additional intermolecular interactions
(e.g. hydrogen bonds, electrostatic interactions, hydrophobic interactions, disulfide
bonds, metal binding) and good general conformational structure (i.e. more rigid, high
packing density; conformational strain release; stability of a-helix; reduced entropy of
unfolding, optimum charge pattern or ion pair and oligomer formation). Electrostatic
interactions increase the number of salt bridges in proteins of thermophiles as
compare to mesophilic proteins (Karshikoff and Ladenstein, 2001). In thermophilic
proteins, the amount of Glu, Arg and Lys are higher in the helices, which lead to
increase in charge residues, enhancing thermo tolerance of proteins in
hyperthermophilic microorganisms (Das and Gerstein, 2000).
Hydrophobic forces are major contributors to molecular folding and
thermostability (Goodenough and Jenkins, 1991). Maximum packing efficiency of an
enzyme can be achieved by filling cavities in the protein core and lead to increase the
core hydrophobicity. The ribonuclease HI from Escherichia coli has a cavity near Val
74 within the protein core. Introduction of a methyl group in the cavity increased
hydrophobic interaction within the protein core and therefore enhanced protein
stability (Ishikawa et al., 1993). Higher amount of alanine, isoleucine and proline
provides extra stability to loops and tighter the packing in hydrophobic cores.
Proteins are stabilized by disulfide bridges through an entropic effect.
Disulfide bridges decrease the entropy of protein’s unfolded state (Matsumura et al.,
1989). The disulfide bond connects the C- terminus of helix1 at 20 and 27 position,
following β-turns which lead to the thermo stability of glucoamylase (Li et al., 1998).
Metals are known to stabilize and activate enzymes. A study of B. licheniformis
xylose isomerase (BLXI) showed stabilizing forces are associated with the presence
of metals in the holoenzyme. The energy of activation for irreversible inactivation
was influenced by the presence of metal ion. The activation energy ranged from 342
(apoenzyme) to 1166 kJ/mol (Co2+enzyme) (Vieille et al., 2001). Generally,
mesophilic proteins are inactivated at elevated temperature due to covalent
modifications. This type of inactivation may be prevented by substitution of specific
Faculty of Applied Sciences and Biotechnology
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surface amino acids such as glutamine, aspargine, cysteine, tryptophane and
methionine with residues that are less susceptible to degradation (Nosoh and
Sekiguchi, 1990; Vieille and Zeikus, 1996).
Various kinds of polysaccharide degrading enzymes such as amylases,
cellulases, pullulanases, xylanases, mannanase, pectinases and chitinases, lipases,
esterases, proteases and phytases have been characterized from extremely
thermophilies and hyperthermophiles. A large number of thermozymes are required
by industries. Starch, textile, pharmaceutical, leather, pulp and paper, detergent, food
and feed industries are the main user of thermophilic enzymes (Adams et al., 1995;
Van der Maarel et al., 2002). The starch industry is one of the largest users of
thermostable amylolytic enzymes. Amylases, glucoamylases and isoamylases or
pullulanases are used in starch industries for the hydrolysis and modification of starch
to produce glucose and various other products (Leveque et al., 2000). Amylolytic
enzymes are also used in baking, textile and paper industries. Cellulolytic enzymes
are employed in detergents for colour brightening and softening of textiles, biostoning of jeans, removal of polyphenolic substances from juices, pulp and paper
industries and pre-treatment of plant biomass. Nowadays, bio detergents contain
enzymes like amylase, protease, cellulase and lipase, using variants that are resistant
to harsh conditions. Lipases are also used in various processes, for example, fat
hydrolysis,
esterification,
interesterification,
trans-esterification
and
organic
biosynthesis. Other applications of lipase include the removal of pitch from pulp
produced in the paper industry, hydrolysis of milk fat in the dairy industry, removal of
non-cellulosic impurities from raw cotton before further processing into dyed and
finished products, removal of subcutaneous fat in the leather industry and
manufacturing of drugs in the pharmaceutical industry (Gomes and Steiner, 2004).
The synthesis of polymer intermediates, pharmaceuticals, specialty chemicals
and agrochemicals is often hampered by expensive processes that suffer from low
selectivity and undesired by-products. Mesophilic enzymes are often not well suited
for the harsh reaction conditions required in industrial processes (biocatalysts in
organic reactions) because of the lack of enzyme stability (Demirjian et al., 2001).
However, the discovery of thermostable enzymes has resulted in a revolution and rebirth of a large number of industrial processes because of the overall inherent stability
of the enzymes, and implying higher applications potentials. The applications of
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Chapter 2
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thermozymes have been extensively reviewed (De-Vos et al., 1998; Zeikus et al.,
1998; Hough and Danson, 1999; Niehaus et al., 1999; Eichler, 2001). Table 4.1
summarizes some of the major applications of thermostable enzymes for commercial
applications. While the most widely used thermostable enzymes are the amylases in
the starch industry, a number of other applications are in various stages of
development: in the food-related industry for the production of amino acids, in the
petroleum, chemical, and pulp and paper industries for the elimination of sulphur
containing pollutants, in the fine chemical industry by the production of chiral
products, and amplification of DNA (Taq DNA polymerase from Thermus aquaticus)
(Haki and Rakshit, 2003).
Thermostable enzymes are gaining wide industrial and biotechnological
interest due to the fact that these enzymes are better suited for harsh industrial
processes (Diane et al., 1997; Zeikus et al., 1998; Haki and Rakshit, 2003).
Table 2.6 Industrial applications of thermozymes (Diane et al., 1997; Zeikus et al.,
1998; Haki and Rakshit 2003).
Enzyme
Cellulases
Microorganism
Salfolobale sp.
Properties
100°C
Xylanase
Pyoidictium abyssi
110°C
Alcohol
dehydrogenase
Pectinase
Thermococcus
litoralis
Sporotrichum
thermophile
98-85oC
Proteases
Pyrococcus furiosus
105°C
α-Amylas, ßAmylase
Amylopullulanase ,
Glucoamylase
Desulfurococ-cus
100°C
mucosus;
Pyrococcus furiosus;
Thermotoga
maritima
Taq polymerase
Pfu DNA polymerase
Thermus aquaticus
Pyrococcus furiosus
55°C
> 75° C
> 80° C
Faculty of Applied Sciences and Biotechnology
Applications
Conversion of cellulose to
soluble sugars, e.g. for
production of biofuel.
Release lignin and reducing
sugars from kraft pulp,
enhancing pulp bleach ability.
Synthesis of optically active
alcohols.
Fruit juice clarification, juice
extraction, manufacture of
pectin-free starch.
Laundry detergent, silk
degumming, biopolishing of
wool and silk, baking, protein
processing.
Starch liquefaction and
saccharification, laundry
detergent, textile processing,
clarification of beer and fruit
juices, anti-staling in bread,
ethanol production.
PCR technology
High fedility in PCR
30
Chapter 2
Review of Literature
2.6 Amylases
Amylases are enzymes which utilize and hydrolyse starch and glycogen as substrate.
2.6.1 Classification of amylase (Fogarty and Kelly, 1979).
α Amylase
The α amylases (EC 3.2.1.1, 1,4-α-D-glucan glucanohydrolase) breaks down longchain carbohydrates by acting at random locations along the starch chain ultimately
yielding maltotriose and maltose from amylose, or maltose, glucose and "limit
dextrin" from amylopectin. In animals, it is a major digestive enzyme. In human
physiology, both the salivary and pancreatic amylases are α amylases. α-Amylases are
produced by microorganism isolated from various sources such as, hyper saline
environment, hot spring, deep sea hydrothermal vent and psychrophilic regions.
β Amylase
β amylase (EC 3.2.1.2, 1, 4-α-D-glucanmaltohydrolase) is synthesized by bacteria,
fungi and plants. β amylase catalyzes the hydrolysis of the second α-1, 4 glycosidic
bond from non-reducing end, cleaving off two glucose units (maltose) at a time.
During the ripening of fruit, β amylase breaks starch into sugar, resulting in the sweet
flavour of ripe fruit. Many microbes also produce amylase to degrade extracellular
starches.
γ-Amylase
In addition to cleaving the last α (1-4) glycosidic linkages at the nonreducing end of
amylose and amylopectin, yielding glucose, γ-amylase (EC 3.2.1.3, Glucan1, 4-αglucosidase) will cleave α (1-6) glycosidic linkages (Maton et al., 1993).
Figure 2.3: Hydrolysis of starch by amylase
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2.6.2 Thermophilic α-amylase and their applications in the conversion of starch.
The pullulanases, isoamylases, β-amylases and glucoamylases used today in industrial
starch processing originate from mesophilic organisms. There is also a need for
thermostable enzymatic equivalents because increasing the temperature of the
saccharification process would be beneficial. This includes:
(i)
higher substrate concentration,
(ii)
decreased viscosity
(iii)
limited risks of bacterial or fungal contamination,
(iv)
increased reaction rates and decrease in operation time,
(v)
lower costs of enzyme purification, and
(vi)
longer catalyst half-life due to the inherent enzyme thermostability
Thus finding of extremely thermostable starch-hydrolyzing enzymes (α-amylases and
pullulanases) that are active at high temperature will improve significantly the
industrial
bioconversion
of
starch,
i.e.,
liquefaction,
saccharification
and
isomerization. The bioconversion of starch to glucose is a multi step process
(liquefaction: pH 6-6.5, 95 to 105°C; saccharification: pH 4.5, 60 to 62°C;
isomerization: pH 7-8.5, 55 to 60°C) performed at different temperatures and under
different pH conditions. This multistep process is accompanied by the formation of
undesirable high concentrations of salts, which have to be removed by expensive ion
exchangers (Niehaus et al., 1999). Ideally, for conversion of starch, α-amylase is
required which is able to work at low pH that would reduce substantially the cost of
pH adjustments, simplify the process and reduce the formation of high pH byproducts (Crabb and Mitchinson 1997). Thus novel thermostable enzymes are
required, which are active and stable above 100°C and at acidic pH values. The
thermal stability of purified enzymes from extreme thermophiles is quite dramatic in
comparison to that of their mesophilic counterparts: for example, the half life of
Bacillus subtilis α-amylase
is a few seconds at 90°C, but the α-amylase from
Bacillus caldolyticus has a half life of about 20 minutes at 95°C. Enzymes from
thermophiles and hyperthermophiles are regarded as interesting candidates for use in
the starch industry and intensive research has been performed aimed at the isolation
of thermostable and thermoactive amylases from those microorganisms (Niehaus et
al., 1999). As shown in Table 4.4, a large number of amylases are present with wide
diversity of microbes belonging to both Bacteria and Archaea. The molecular cloning
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Chapter 2
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of the corresponding genes and their expression in heterologous hosts, e.g.,
Escherichia coli or Bacillus subtillis, allowed circumvention of the problem of
insufficient expression in the original host (Archaea). The thermophilic and
hyperthermophilic exoamylases (β-amylases, glucoamylases, and α-glucosidases)
show good potential for the saccharification step.
Thermophilic pullulanases have been isolated from aerobic thermophiles.
These pullulanases are optimally active at low pH but their application for the starch
processing remains to be tested. Thermococcale α -amylases are generally active in a
broad temperature range, between 40-140°C (Leveque et al., 2000) whereas amylase
enzyme produced by Pyrococcus strain DSM3638 showed maximum activity at
100°C (Brown et al., 1990). Extracellular α-amylase activity from Pyrococcus
furiosus (Koch et al., 1990) and P. woesei (Koch et al., 1991) was also reported.
Thermococcale α -amylases are generally active in a broad pH range 3.5–9 with the
optimal activity between pH 5 - 6.5. Pyrococcus sp. KOD1 showed maximum
amylase activity at 6.5–7.5 (Laderman, 1993). Mamo et al., (Mamo et al.,1999) also
reported optimal temperature of 75–80°C for amylase from Bacillus sp. WN11. A
hyperthermostable α-amylase activity was exhibited by Geobacillus sp. (Dheeran et
al., 2010) and Bacillus thermoleovorans (Malhotra et al., 2000; Narang and
Satyanarayana 2001). The thermostability amylases from B. licheniformis,
Thermococcus litoralis and Sulfolobus solfataricus have been reported. (Brawn and
Kelly, 1993; Haseltine et al., 1996; Dong et al., 1997). The thermostable
Actinomycetes including Thermomonospora and Thermoactinomyces are versatile
producers of the enzymes (Ben et al., 1999).
Table 2.7 Vari ou s ther mop h i l i c b acteri a p rod u ci n g α -amyl ase
Micro-organism
Bacillus flavothermus
Bacillus spTF2
Bacillus sp WNII
B. amyloliquefaciesm
B. licheniformis
Pyrococcous woesei
P. furious
Thermo coccous celer
Fervidobacterium pnennavorans
Desulfurococcous mucosus
D. mucosus strain TY
D. mucosus strain TYS
Thernotoga maritime
Faculty of Applied Sciences and Biotechnology
Optimum
Temperature(oC)
55
90
65
70
90
100
100
95
90
90
105
100
100
33
Chapter 2
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2.7 Proteases
Protease (peptidase) is an enzyme that catalyses the digestion of protein in to small
peptides and amino acids by cleavage of peptide bonds leading to total hydrolysis of
proteins. This enzyme found in a wide diversity of sources such as plants, animals,
and microorganisms. They are required for a diverse range of physiological processes
including cell division, regulation of protein turnover, activation of zymogenic
preforms, blood clotting, and lyses of blood clot, processing and transport of secretory
proteins across membrane, nutrition, regulation of gene expression and pathogenesis
(King et al., 1996; Pahl and Baeuerle, 1996; Martoglio, 1999). The inabilities of the
plant and animal proteases to meet current world demands have led to an increased
interest in microbial proteases. Microorganisms elaborate a large array of proteases
and they account for two-third share of commercial protease production in the world
(Kumar and Takagi, 1999). Depending on their site of action, proteases are grossly
subdivided into two major groups, exopeptidases and endopeptidases.
Fig 2.4: Cleavage of peptide bond. Reaction catalysed by protease.
2.7.1 Classification of proteases
Proteases are mainly classified according to the chemical nature of active site (Rao et
al., 1998). Based on the catalytic site on the substrate, proteases are classified into two
types.
a. Exopeptidases
Exoproteases preferentially act at the end of the polypeptide chain. Exoproteases are
further classified into two subclasses: amino proteases and carboxypeptidase.
Aminopeptidases 4.6 EC 3.4.13 acts at a free N terminus of the polypeptide chain and
liberate a single amino acid residue, a dipeptide 4.7 EC 3.4.14, or a tripeptide EC
3.4.11.4. Aminopeptidases occur in a wide variety of microbial species including
bacteria and fungi (Watson, 1976). In general, aminopeptidases are intracellular
enzymes, except for a single report on an extracellular aminopeptidase produced by A.
oryzae (Labbe, 1974). The carboxypeptidases (EC number 3.4.16 - 3.4.18) act at C
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Chapter 2
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terminus of the polypeptide chain and liberate a single amino acid or a dipeptide.
Carboxypeptidases can be divided into three major groups, serine carboxypeptidases,
metallocarboxypeptidases, and cysteine carboxypeptidases, based on the nature of the
amino acid residues at the active site of the enzymes. (Felix and Brouillet, 1966; Rao
et al., 1998)
b. Endoproteases
Endoproteases preferentially act in the inner regions of the polypeptide chain away
from the N and C termini. Endoproteases are classified into the following types based
on the functional group present in their active site (Hartley, 1960).
Serine proteases
Serine proteases are proteases having a serine residue (-OH) in their active site.
Examples include trypsin and subtilisin. They are present in all the organisms like
viruses, bacteria, and eukaryotes. On the basis of structural similarities, serine
proteases have been divided into 20 families, which have been further, subdivided
into six clans with common ancestors (Barett, 1994). These proteases are generally
active at neutral and alkaline pH (pH 7-11) and isoelectric points of these proteases
ranges between pH 4 and 6. On the basis of their substrate preference, they are
divided into three types; i) trypsin like which cleave after positively charged residues;
ii) elastase-like, which cleave after small hydrophobic residues, and iii) chymotrypsinlike, which cleave after large hydrophobic residues. They have broad substrate
specificities, including significant esterolytic activity toward many ester substrates,
and are generally of low molecular weight (18.5 – 35 kDa). Largest serine proteinase
reported is the Blackeslea trispora enzyme, with a Mr = 126 kDa (Govind et al.,
1981).
Cysteine proteases
Cysteine proteases have a thiol (-SH) group in their active site. Examples include
papain and bromolain. These are present in all the organisms. These proteases are
mostly active at neutral pH but some show activity in acidic conditions e.g.,
lysosomal proteases.
Metalloproteases
Metalloproteases require a divalent metal ion like Zn2+ or Co2+ for their catalytic
activity. Examples includse elastase in humans and thermolysin in bacteria. There are
reports of 30 families of metalloprotease, of which 17 are endopeptidases, 12 are
exopeptidases, and 1 contaning both endo and exopeptidases (Barett, 1994). All
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Chapter 2
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metalloproteases have pH optimal 5 – 9. These proteases are characterized by
inhibition of enzyme activity by ethylenediamine tetra acetic acid (EDTA) but are
unaffected by serine proteinase inhibitors. Many of the EDTA inhibited enzymes can
be reactivated by ions such as zinc, calcium, or cobalt. Based on the specificity of
their action, they are divided into four types: (i) neutral (these show specificity for
hydrophobic amino acids), (ii) alkaline (possess broad specificity), (iii) Myxobacter I
(show specificity for small amino acid residues on either Side of the cleavage bond),
and (iv) Myxobacter II (show specificity for lysine residue) (Mala et al., 1998).
Aspartic protease
Aspartic proteases possess an aspartate residue in their catalytic active site. They are
widely distributed in fungi, but are rarely found in bacteria or Protozoa. The most
common examples include pepsin and chymosin. Enzymes show maximum activity at
low pH (pH 3-4), and therefore, are also known as acidic proteases. Most aspartic
proteinase has molecular weights in the range 30-45 kDa and their isoelectric points
in the range of pH 3-4. These proteases are inhibited by pepstatin, diazoacetyl-DLnorleucine methyl ester (DAN), and 1, 2-epoxy-3-(p-nitrophenoxy) propane (EPNP)
in the presence of copper ions (Fitzgerald, 1990).
Table 2.8 Families of proteolytic enzymes (Neurath, 1984).
Family
Serine protease I
Serine protease II
Cysteine proteinases
Aspartic proteases
Metallo- protease I
Metallo-proteses II
Representative example(s)
Chymotrypsin
Trypsin
Elastase
Pancreatic kallikrein
Subtilisin
Papain
Actinidin
Penicillopepsin
Rhizopus chineses and
Endothia parasitica,
acid proteases
Renin
Bovine carboxypeptidase A
Thermolysin
Based on the optimal pH for activity, proteases are classified as follows:
Acid proteases
Acid proteases are proteases that are active in the pH range of 2-6 (Rao et al., 1998)
and are mainly of fungal origin, e.g., pepsin, chymosin.
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Neutral proteases
Neutral proteases are proteases which are active at neutral pH (7.0), weakly alkaline
pH (7.5) or weakly acidic pH (6.5). They are mainly of plant origin, except a few
fungal and bacterial neutral proteases, e.g., thermolysin.
Alkaline proteases
Alkaline proteases are optimally active in the alkaline pH range (8-11) (Horikoshi,
1999). They are obtained from neutralophilic and alkaliphilic microorganisms such as
Bacillus and Streptomyces species, e.g., subtilisin
2.7.2 Applications of proteases
Proteases are essential for several physiological processes in all living organisms.
They are ubiquitous, being found in a wide diversity of sources such as plants,
animals, and microorganisms. Among all sources, microorganisms represent an
excellent source of protease enzymes due to their broad biochemical diversity and
their amenability to genetic manipulation. Proteases are important industrial enzymes
accounting for nearly 40 % of the total worldwide enzyme market (Chen et al., 2004;
Chu, 2007). They are widely used in industries involving detergent, brewing, meat,
photographic, leather, dairy, membrane cleaning and waste treatment (Chu, 2007).
In addition to these major applications, alkaline proteases have other
applications, such as contact lens cleaning (Nakagawa, 1994), isolation of nucleic
acids (Kyon et al., 1994), pest control (Kim et al., 1999) and degumming of silk
(Vaithanomsat and Kitpreechavanichb, 2008). Most commercial proteases, mainly
neutral and alkaline, are produced by organisms belonging to the genus Bacillus. The
applications of proteases in various industries are summarized in table 2.3.
Table 2.9 Industrial application of proteases.
INDUSTRY
Photographic industry
Detergent
Food industry
Bread / confectionery
Cheese production
Meat
Beverage
Leather Industry
Management of industrial
and
household waste
Silk degumming
Chemical industry
APPLICATIONS OF PROTEASES
Bioprocessing of used X-ray or photographic films for silver
recovery.
To remove protein - based stains from clothes.
To modify gluten elasticity.
Casein coagulation and cheese ripening.
Meat tenderization.
Solubilisation of grain proteins and stabilization of beer.
To dehair hides and soften leather.
Lowering the biological oxygen demand of aquatic systems.
Management of waste feathers from poultry slaughterhouses.
Proteases are used for degumming of silk.
Sucrose-polyester synthesis (biodegradable plastic)
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The best characterized thermostable protease is thermolysin produced by
Bacillus thermoprtiolyticus (Endo, 1962). Thermostable proteases are also used in
peptide synthesis because of their compatibility with organic solvents (Wilson et al,
1992). Thermolysin is used in synthesis of dipeptide (N-CBZ-Asp-L-Phe) methyl
ester which is precursor in preparation of sweetener aspartame (Isowa et al, 1979). In
addition Thermus protease, Pretaq, is used to clean DNA before amplification in
Polymerase chain reaction. Protease can also be used in cleaning ultra filtration
membranes and may allow these products to be reused, leads to reduction in chemical
use and waste reduction. Thermophilic bacteria are used in the decomposition of these
hard-to-degrade animal proteins. At the elevated temperature range thermophilic
bacteria can grow and such proteins tend to gain plasticity, resulting in more
susceptibility to protease attack (Sugiyama et al., 2005). However, the temperature
range (over approximately 80°C) suitable for growing extremely thermophilic
bacteria too rapidly induces thermal denaturation of the proteins (Van der Plancken et
al., 2003). Geobacillus collagenovorans MO-1 strain can optimally grow at
temperatures between 50–70°C and efficiently degrade collagens in a neutral pH
range (Okamoto, 2001). It produces two types of collagen-degrading enzymes
(Miyake et al., 2005). One is a collagenolytic protease and other enzymes are two
isozymes of oligo peptidases that recognize the collagen-specific tripeptide unit, GlyPro-X, contained in collagen and cleave the peptide bond proceeding to Gly (Miyake
et al, 2005). Fervidobacterium pennivorans, isolated from a hot spring of the Azore
islands in the Atlantic Ocean. The strain grows optimally at 70°C and pH 6.5 and
belongs to the anaerobic Thermotogales (Friedlich and Antranikian, 1996). This is the
first known extreme thermophile which is able to degrade native feathers at high
temperatures. The strain produces an extracellular subtilisin-like serine protease,
Fervidolysin, which is composed of a signal peptide and a proteolytic part containing
a catalytic region (Kluskens et al., 2002). Thermoanaerobacter keratinophilus was
isolated from the same region (Riessen and Antranikian, 2001). The properties of its
keratinolytic protease are similar to those of Fervidolysin.
Fervidobacterium islandicum AW-1 is another native-feather-degrading
Fervidobacter species, isolated from a geothermal hot stream in Indonesia (Nam et
al., 2002). The strain grows anaerobically at 40–80°C and at pH 5–9, whereas the
keratinolytic protease that is dimeric membrane-bound (>200 kDa) shows optimal
protease activity at 100°C and pH 9 and has a half-life of 90 min at 100°C. In
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contrast, keratinases (KRT) from mesophiles it takes several days to degrade native
KRT completely. Moreover, mesophilic bacteria and dermatophytes producing strong
keratinases are mostly pathogenic. (Gradisar et al., 2005).
2.8 Cellulases
Cellulose is the most abundant organic compound on earth and has extensively used
as a substrate for the production of single cell proteins, biofuels, and various other
chemicals through microbial enzymatic degradation. The conversion of cellulosic
biomass to fermentable sugars requires different types of cellulase namely, β-1, 4
endoglucanase (EC 3.4.1.4), β-1, 4 exoglucanase (EC 3.2.1.91) and β-1, 4 glucosidase
(EC 3.2.1.21) (Yi et al., 1999). Various bacteria, fungi and yeast synthesize these
enzymes, but the most extensively studied cellulases are those produced by efficient
lignocellulose degrading fungi, particularly Trichoderma (Narsimha et al., 2006) and
Aspergillus (Baig, 2005). Dozens of novel mesophilic strains with improved
characteristics have been engineered and successfully applied in industrial production
since the 1980’s.
Fig 2.5: Hydrolysis of Cellulose: Reaction catalysed by cellulases
In nature, fungi tend to produce more cellulases than bacteria, however,
cellulases produced by bacteria are better catalyst as they encounter less feedback
inhibition. The bacterial cellulases are thermostable and also active at an alkaline pH
in comparison to fungal cellulases (Macedo et al., 1995). The cellulolytic activity was
also reported in Bacillus spp. (Mawadza et al., 2000) and in thermophilic anaerobic
bacterium Clostridium thermocellum (Mori, 1992). The optimal activity of
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thermophilic cellulolytic Bacillus strains from Bakreshwar hot spring, West Bengal,
India, was observed at 60°C (Acharya and Chaudhary, 2011). Endocellulase, with the
ability to hydrolyze microcrystalline cellulose, was isolated from the extremely
thermophilic bacterium Anaerocellum thermophilum and maximal activity was
observed at pH 5.0-6.0 and 85-95°C. Thermophilic bacteria strain, Geobacillus
pallidus was isolated from Empty Fruit Bunch (EFB) and Palm Oil Mil Effluent
(POME) compost and have cellulase activity (Azhari et al, 2010). Thermostable
cellulases of archaeal origin include those isolated from Pyrococcus furiousus
(Kengen et al., 1993) and Pyrococcus horikoshi (Ando et al., 2002) has an optimum
temperature of enzyme activity of 97 - 105 °C.
Sulfolobus solfataricus MT4,
Sulfolobus acidocaldarius and Sulfolobus shibatae were also described as producers
of β-gucosidases (Grogan, 1991). Thermotoga maritema MSB8 showed optimally
active cellulase activity at 95 °C and pH 6-7.0 (Bronnenmeier et al., 1995). Other
endocellulases (CelA and CelB) from Thermotoga neapolitana, were purified and
characterized (Bok et al., 1998). Optimal pH for CelA was 6.0 at 95 -106°C. CelA,
with the ability to hydrolyze microcrystalline cellulose, was isolated from the
extremely thermophilic bacterium Anaerocellum thermophilum (Zverlov et al., 1998)
and maximal activity of this enzyme was observed at pH 5.0–6.0 and 85–95°C. A
highly thermostable cellobiose (115 °C at pH 6.8–7.8) was also produced from
Thermotoga sp. FjSS3-B1 (Ruthersmith and Daniel, 1991). The thermophilic
cellulases
of
Clostridium
thermocellum,
Dictyoglomus
thermophilum,
and
Acidothermus cellulolyticus hydrolyze a wide range of substrates including β-glucans,
amorphous cellulose, and crystalline celluloses
2.8.1 Current industrial application of cellulases
In paper industries cellulases are used to decrease the viscosity of the processed
material during the pulping process (bio-mechanical pulping) and to improve sheetstrength properties of the end-product. In textile industry, they are extensively used in
the bio-stoning of denim fabrics. Cellulases are used in the production of
environmentally friendly washing powders. They improve the detergent performance
as they restore the softness and brightness of cotton fabric by selectively removing
small and fuzzy fibrils from the surface. In wine production cellulases are applied to
obtain better fruit skin degradation, improved colour extraction, easier clarification,
better extraction, and improved quality and stability of the end product.
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Cellulases are widely used to supplement monogastric and ruminant feed. Their role
is to improve nutritional factors present in grains and vegetables and to improve feed
conversion rate (Voragen et al., 1980; Thomke et al., 1980; Chesson, 1987; Voragen,
1992). Cellulase production is also found to be the most expensive step during ethanol
production from cellulosic biomass, and accounted for approximately 40 % of the
total cost (Spano et al., 1975). Thermophilic microorganism Clostridium
thermocellum have been used to ferment cellulose and cellobiose to ethanol. The
cellulose-fermenting
co-culture
(Clostridium
thermocellum
and
Clostridium
thermohydrosulfuricum) is an extremely stable at 60°C. The direct fermentation of
cellulose to ethanol by the co-cultures has several advantages over proposed
saccharification and fermentation processes using T. reesei cellulase and
Saccharomyces cerevisiae. By using thermophilic cellulose-fermenting co-culture,
cellulase is produced directly in one step (both saccharification and fermentation
proceed at same temperature) instead of requiring different temperature.
2.9 L-Glutaminase (L-glutaminase amidohydrolase)
L-glutaminase (EC.3.5.1.2) is an amidohydrolase which catalyses the hydrolytical
deamination of L-glutamine resulting in the production of L-glutamic acid and
ammonia.
Fig 2.6: Hydrolysis of Glutamine: Reaction catalysed by glutaminase
L- Glutaminases are ubiquitous in the biological world and organisms ranging
from bacteria to human beings have the enzyme (Ohshima et al., 1976; Iyer and
Singhal, 2010). L-Glutaminase has a central role in mammalian tissues (Errerra and
Greenstein, 1949). The enzyme catalyzes the hydrolysis of γ amido bond of lglutamine. These are generally categorized as the kidney type and liver type
glutaminases and both the types have been purified and characterized (Svenneby et al,
1973; Cuthoys et al., 1976; Heini et al, 1987). Interest on amidohydrolases started
with the discovery of their anti-tumour properties (Broome, 1961; El-Asmar and
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Greenberg, 1966; Santana et al, 1968; Roberts et al, 1970) and since then, a lot of
efforts have gone into extensive studies on microbial L-glutaminases with the
intention of developing them as antitumor agents. Microbial L-glutaminases have
found several potent applications in various industrial sectors in the recent years. The
enzyme, though originally identified as a potent anti-cancer drug with possible
applications in enzyme therapy, has been used in food industry for flavour
enhancement. Recent applications of the enzyme include its use in biosensors.
2.9.1 Microbial sources of L-glutaminase
Initially the research work on L-glutaminase enzyme was started in the year 1956.
The importance of L-glutaminase enzyme was accidently found when scientist were
working on the measurement of the total uric acid-N15 enrichment and the N15
abundance of each of the four uric acid nitrogen’s, found that an abnormality of
glutamine metabolism in primary gout is implicated. It is suggested that the
abnormality in glutamine metabolism may have significance for the pathogenesis of
primary gout. Thus, from then the study on this enzyme was focused. Glutaminase
activity is widely distributed in microorganisms including bacteria, yeast and fungi
(Imada et al., 1973; Yokotsuka et al, 1987). L-Glutaminase synthesis has been
reported from many bacterial genera, particularly from terrestrial sources, like
Pseudomonas sp. (Kabanova et al., 1986), Acinetobacter (Holcenberg et al., 1978),
and Bacillus sp. (Cook et al., 1981). Other microorganisms, such as Proteus
morganni, Xanthmonas juglandis, Erwnia carotovora, Serratia marcescens,
Enterobacter cloacae, Klebsiella aerogenes and Aerobacter aerogenes (Wade et al.,
1971; lmada et al., 1973; Novak and Philips, 1974) were also reported to have
glutaminase activity.
Among other groups of bacteria, species of Pseudomonas, especially, P.
aeruginosa (Greenberg et al., 1964; Ohshima, 1976), P. aureofaciens (lmada et al.,
1973), P. aurantiaca (Kabanova et al., 1986) are well recognised for the production
of glutaminase. Among yeasts, species of Hansenula, Cryptococcus, Rhodotorula,
Candida scottii were observed to produce significant levels of glutaminase under
submerged fermentation. Species of Tilachlidium humicola, Verticillum malthoasei
and fungi imperfecti were recorded to possess glutaminase activity (lmada et al.,
1973). Glutaminase activity of soy sauce fermenting Aspergillus sojae and A. oryzae
were also reported (Yano et al., 1988).
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A. oryzae is observed to secrete L-glutaminase both extracellularly and
intracellularly (Gilbert et al., 1949). Japanese, while making traditional food, soya
sauce, add some yeast and fungus mold to increase the taste of sauce. L-glutaminase
produced by the microorganism play a vital role for improving the taste (Yamamoto
and Hirooka 1974). Among bacteria, P. fluorescens, Vibrio costicola, and V. cholerae
are observed to produce extracellular L-glutaminases, and the extracellular secretion
is about 2.6 to 6.8 times higher than intracellular production (Renu and
Chandrasekaran, 1992). L-Glutaminase is secreted extracellularly also by strains of
Bacillus subtilis and B. licheniformis (Cook et al., 1981), Debaryomyces sp. (Dura et
al., 2002), Beauveria sp and Streptomyces rimosus (Ivakumar et al., 2006). Among
yeast species, Zygosaccharomyces rouxii is halophilic yeast which is well studied for
the extracellular L-glutaminase production (Kashyap et al, 2002; Iyer and Singhal,
2008; 2010). Microbial L-glutaminases were produced in both submerged and solid
state fermentation.
2.9.2 Applications of L-glutaminase
Therapeutic applications of L-glutaminase
Several attempts were made towards using L-glutaminase in cancer therapy (Santana
et al., 1968; Spires et al., 1979). L-glutaminase was also used against adult leukemia
(Warrell, 1981). The enzyme causes selective death of glutamine dependent tumour
cells by depriving these cells of glutamine. One of the major problems encountered in
the treatment with microbial L-glutaminase is the development of immune responses
against the enzyme. Clinical application of A. glutaminase as a drug in the treatment
of leukaemia was described (Spires et al., 1979). A number of glutaminases with
antitumour activity have been isolated from Acinetobacter glutaminasificans,
Pseudomonas aureofaciems, P. aeruginosa, Pseudomonas 7 A and Achromobacter
(Roberts, 1976; Spires et al., 1979). One of the most promising therapeutic
applications ever proposed for L-glutaminase is in the treatment of HIV, where Lglutaminase from Pseudomonas sp. 7A is administered so as to inhibit HIV
replication in infected cells (Kumar and Chandrasekaran, 2003; Roberts et al., 2001).
Application of L-glutaminase in food industry
L-Glutaminase enhances the flavor of fermented foods by increasing their glutamic
acid content thereby imparting a palatable taste (Yokotsuka, 1987). The pleasant and
palatable tastes of fermented foods like soy sauce, miso and sufu is due to high
content of L-glutamic acid in them (Chou and Hwan, 1994) which is accumulated by
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hydrolysis of a protein component catalysed by proteolytic enzymes, including Lglutaminase, protease and peptidases (Lu et al., 1996). The quality of soy sauce and
miso has been improved by the action of microbial L-glutaminases (Yokotsuka et al,
1987; Nakadai and Nasuno, 1989). L-glutaminase from Koji mould and Cryptococcus
albidus was used for increasing the L-glutamate content of soy sauce (Yamamoto and
Hirooka 1974; Yokotusa et al, 1987; Iwasa et al, 1987; Nakadai and Nasuno, 1989).
In addition, peptidoglutaminase of Bacillus circulans and L-glutaminase from
Actinomucor taiwanensis is used to improve the flavour of soy sauce (Kikuchi et al.,
1973) and to increase the L-glutamate content of Sufu (Chou and Hwan 1994)
respectively. The use of L-Glutaminase as a flavour enhancing agent in Chinese foods
has replaced the use of monosodium glutamate, which is considered allergic to some
individuals (Sabu, 2000). Since at high temperature, no pathogenic microbes can grow
and reproduce, thus the glutaminase activity and stability at high temperature enhance
the food quality and prevent it from microbial contamination. Glutaminase enzyme
from thermophilic microorganism has not yet been reported. Therefore it is essential
to explore thermophilic microbes for glutaminase activity.
Analytical applications
L-glutaminase for biosensor application to determine the L-glutamine levels was
investigated by Kikkoman Corporation, Japan, (Sabu et al, 2000), mammalian cell
culture media (Huang et al, 1995; Mulchandani and Bassi, 1996) and hybridoma
culture media (Meyerhoff, 1995) by flow injection analysis. L-Glutaminases are used
currently both in free enzyme or immobilized forms as biosensors for monitoring
glutamine and glutamate levels of fluids. Free enzyme was used in the determination
of glutamine in insect cell culture media.
2.10 Lipase as Biocatalysts
Lipases (EC.3.1.1.3, triacylglycerol acylhydrolases) are group of enzymes which have
the ability to hydrolyze triacylglycerols at an oil-water interface to release free fatty
acids and glycerol (Reis et al., 2009). Lipases are truely defined as carboxylesterases
that catalyze both the hydrolysis and synthesis of long-chain acylglycerols (Jaeger et
al., 1999). Lipases are present in microorganisms, plants and animals (Jisheng et al.,
2006). Lipases catalyze a wide range of reactions, including hydrolysis, alcoholysis,
esterification, inter-esterification, acidolysis, and aminolysis (Joseph et al., 2008). In
the presence of organic solvents, various inter-esterification and trans-esterification
reactions
are
effectively
catalysed.
Furthermore,
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lipases
show
44
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regiospecificity and chiral selectivity and have different substrate specificities (Gupta
et al., 2003). Many species of bacteria, yeast and molds are lipases producing.
Lipases are used for the conversion of less desirable lipids to higher value
lipids by modifying their properties, by altering the location of fatty acid chains in the
glyceride and replacing one or more of the fatty acids with new ones (Pabai et al.,
1995). Microbial lipases are used to obtain PUFAs (Poly Unsaturated Fatty Acids),
which are essential for normal synthesis of lipid membranes and prostaglandins.
PUFA are derived from animals and plant lipids by the action of microbial lipases.
PUFAs are used as pharmaceuticals, neutraceuticals, and food additives (Gill and
Valivety, 1997; Belarbi et al., 2000). In addition, lipases have been used for
development of flavors in cheese ripening, bakery products, and beverages and also
used to remove fat from meat and fish products (Kazlauskas, 1998). Lipases are
widely used as a detergent additive for the removal of fat and lipid based strain from
clothes. In paper and pulp manufacturing hydrophobic components (Pitch) like wax
(Jaeger and Reetz, 1998) can be removed up to 90% by the application of lipase.
Lipases are widely used in oleochemical industries for minimizing thermal
degradation during glycerolysis, alcoholysis and hydrolysis (Hoq et al., 1985). The
enzyme can also be used for the synthesis of biodegradable aromatic polyesters
(Linko et al., 1998). Vitamin A and its derivatives have great potential in cosmetics as
skin care products. Water soluble Vitamin A derivatives are produced by the action of
immobilized lipase (Maugard et al., 2002). Lipases have been used as digestive aids.
They activate tumour necrosis factors and hence used for the treatment of cancer
(Kato et al., 1989). In addition lipase derived from bacterial monoculture can be used
for the bio augmentation of lubricant contaminated water or soil (Vasileva and
Galabova, 2003).
Due to the physiological importance of the n-3 polyunsaturated fatty acids (n3 PUFAs), various techniques have been used for concentrating these compounds,
especially eicosapentaenoic acid and docosahexaenoic. One of the most promising
techniques is the use of lipase-catalyzed enzymatic hydrolysis reactions, which are
generally more productive, compared to other concentration methods such as
esterification and inter-esterification (Xuebing et al., 2000). The utilization of lipases
in this process is based on the fatty acids-specific selectivity that lipases exhibit
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toward saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs),
leaving n-3 PUFAs intact on the glyceride moiety (Wanasundara et al., 1998).
Commercially available free lipases derived from microorganisms such as species of
the genera Candida, Aspergillus, Mucor and others have been studied to be used in
this reaction (Linder et al., 2005; Zheng et al., 2007). However, the use of pure
soluble enzymes in chemical and biochemical reactions is expensive. Thus, in order to
recover the enzyme, it is necessary to immobilize the enzyme in supports. Therefore,
improvements in enzyme immobilization are a current focus of research in the fat and
oil industries (Bastida et al., 1998; Palomo et al., 2004).
Figure 2.7: Hydrolysis of lipids: Reaction catalysed by lipase
Biotechnological uses of lipase
The extracellular microbial lipases are an important group of biotechnologically
relevant enzymes as their bulk production is much easier and they find applications in
food, diary, detergent, and pharmaceutical industries (Gupta et al., 2004). Lipase
catalysed reactions are widely used in the manufacturing of fats and oils, degreasing
formulations, synthesis of fine chemical, paper manufacture and production of
cosmetics. Lipases are also used to accelerate the degradation of fatty wastes and
polyurethane (Jisheng et al., 2006). Some important lipase-producing bacterial species
are Bacillus, Pseudomonas and Burkholderia (Svendsen, 2000).
Usually enzymes are not stable in organic solvents and they tend to denature
and lose their activities. But lipases remain stable and active in organic solvents
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without any stabilizer. Substrates of lipase are often insoluble or partially soluble in
water and thus the use of organic solvents or organic–aqueous solutions is in favour of
some reactions (Zhao et al., 2008). Because of its ability to catalyze in organic
solvents, many new biotechnological applications of lipases have been identified. One
of the applications is the synthesis of chirally important drugs and drug intermediates
(Singh and Benarjee, 2007). Reactions catalyzed by lipases are carried out in organicaqueous interface. Lipases are generally produced in the presence of lipid source such
as oil, triacylglycerols, fatty acids, hydrolyzable esters, tween and glycerol.
2.10.1 Properties of Lipase
Lipases are acyl hydrolases and that play a key role in fat digestion by cleaving longchain triglycerides. Because of an opposite polarity between the enzyme (hydrophilic)
and their substrates (lipophilic), lipase reaction occurs at the interface between the
aqueous and the oil phases (Reis et al., 2009). Generally, mesophilic bacterial lipases
are neutral or alkaline and show activity in a broad pH range (pH 4 – 11) and thermal
stability of lipases ranging from 20 – 60°C (Gupta et al., 2004). According to
substrate specificity, microbial lipases are divided into three categories; nonspecific,
regiospecific and fatty acid-specific. Nonspecific lipases behave randomly on the
triacyglyceride molecule and produce complete breakdown of triacyglyceride to fatty
acid and glycerol. Conversely, regiospecific lipases are 1, 3-specific lipases which
hydrolyze only primary ester bonds and hydrolyse triacylglyceride to give fatty acids.
Fatty acid-specific lipases display activity in presence of fatty acid. Thermophilic
bacteria are able to produce thermostable lipolytic enzymes (capable of degradation of
lipid) at temperatures higher than mesophilic bacteria.
Several thermophilic lipases has been isolated and characterized. Biological
treatment at high temperature is advantageous, since fats above their melting point are
in liquid state are more accessible to enzyme. A thermophilic microorganism, Bacillus
thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular
lipase activity and high growth rates on lipid substrates at elevated temperatures. The
enzyme showed optimal activity at 70 -75°C and pH 7.5 and exhibited 50 % of its
original activity after 1 h incubation at 60°C (Cho et al., 2000). Recently, several
thermophilic lipases have been purified and characterized from thermophilic Bacillus
sp. (Sidhu et al., 1998; Sharma et al., 2002) like Bacillus thermoleovorans
(Markossian et al, 2000; Lee et al, 1999), Bacillus stearothermophilus (Sinchaikul et
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al., 2001) and Bacillus circulans (Kademi et al., 2000). Thermostable lipolytic
enzymes from the archaeon Sulfolobus acidocaldarius, Clostridium saccharolyticum,
Pyrococcus furiosus and many Pseudomonas sp. have also been reported.
2.11 Chitinase
In addition chitinase enzyme or chitin deacetylases has been isolated from fungal or
bacterial that could promote a better deacetylation of chitin (Kafetzopoulos et al.,
1993) Chitin degradation is suggested to occur via the deacetylation of chitin into
chitosan by chitin deacetylases. Thermostable chitinases have been isolated from
moderately thermophilic microorganisms such as Bacillus spp. (Takayanagi et al.,
1991; Sakai et al., 1998) Microbispora spp. (Nawani et al., 2002); Streptomyces
thermoviolaceus (Tsujibo et al., 1993) Chitinases are also reported from
hyperthermophilic Archaea such as Thermococcus kodakaraensis and Thermococcus
chitonophagus. Pyrococcus furiosus represent extremely thermostable chitinases of
GH18. The enzyme showed optimal temperatures > 90°C and half-lives of 30 min up
to 1 h at 120°C (Tanaka et al., 2001; Gao et al., 2003; Andronopoulou and Vorgias,
2004). These enzymes showed resistance to long exposure to heat, pH variations and
denaturants.
2.12 Xylanase
There is a great interest in the enzymatic hydrolysis of xylan, the major constituent of
hemicelluloses, due to possible applications in clarification of juices, preparation of
dextran for use as food thickeners, production of juices from plant materials,
manufacture of liquid coffee, feedstock, fuel, chemical production and paper
manufacturing (Coughlan and Hazelwood, 1993). α- 1, 4 xylanases (1, 4 â xylanxylanohydrolase, E.C. 3.2.1.8) catalyzes the hydrolysis of xylan to xylooligosaccharides and xylose. The occurrence of xylanases in extreme thermophilic
bacteria was first reported by Bragger et al., (1989). Thermotoga strains were able to
degrade xylan. The endoxylanase of Thermotoga sp. strain FjSS3B.1 exhibited
maximum activity at 105°C. An archaeal xylanase has been detected in extracts of the
hyperthermophilic archaeon Pyrodictium abyssi (Andrade, 1996). The enzyme
exhibited optimal activity at 110°C and pH 6.0, and thermostable showing activity
even after 100 min of incubation at 105°C.
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2.13 Phytase
Phytases are considered to be potential candidate in enhancing the nutritional quality
of phytate-rich feed and foods for animal and human consumption, respectively
(Greiner and Koneitzny, 2006; Ugwuanyi et al., 2009). In addition, phytase would be
an eco-friendly product, reducing the amount of phosphorus entering the environment
or problems resulted by eutrophication. Phytases has been isolated from a host of
sources including plants (Maugenest et al., 1997), animals (Craxton et al., 1997) and
microorganisms (Dassa et al., 1990). A wide variety of microbial phytases have been
characterized from Aspergillus spp. and Peniophora lycii, being used as feed additives
(Simon and Igbasan, 2002; Haefner et al., 2005). A novel phytase producing
thermophilic strain of Bacillus licheniformis was isolated. The optimum temperature
and pH of the phytase from B. licheniformis (Phy C) were 55℃ and 7.0, respectively.
These thermophilic enzymes can be cloned and expressed in mesophilic hosts
without losing their thermostability and active conformation (Bouzas et al., 2006).The
number of genes from thermophiles encoding amylolytic, proteolytic and lipolytic
enzymes that have been cloned and expressed in mesophiles. (Bertoldo and
Antranikian, 2001). These thermostable proteins expressed in mesophilic hosts
maintain their thermostability, are correctly folded at low temperature, are resistant to
host proteolysis. They can be easily purified by using thermal denaturation of the
mesophilic host proteins. The degree of enzyme purity obtained is generally suitable
for most industrial applications. Thermostable lipases from hyperthermophilic archaea
Pyrobaculum calidifontis, Pyrococcus furiosus and Pyrococcus horikoshii have been
isolated and cloned in E. coli (Ando et al., 2002). Hyperthermophilic xylanases from
Dyctioglomus sp., Thermotoga sp., and Pyrococcus furiosus and serine proteases from
P. furiosus and Coprothermobacter proteolyticus have been cloned and expressed in
E. coli (Halio et al., 1996; Xue and Shao 2004; Toplak et al., 2013).
2.14 Molecular identification of bacterial species
The development of molecular markers has been prominent in the expansion of
population genetic approaches. However, different types of molecular markers may
exhibit different responses to different evolutionary histories. For example, the short
tandemly repeated arrays of microsatellites (or simple sequence repeats, SSRs) show
extremely rapid evolution (Charlesworth et al., 1994), whereas many amino acid
coding sequences (e.g., rbcL; Olmstead and Palmer, 1994) usually exhibit relatively
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Different forms of an enzyme that carry out the same
catalytic function are called isozymes. When the genes encoding them occur at a
common locus, they are called allozymes. Though allozymes represent coding DNA
sequences, polymorphism is usually considered to be nearly neutral (Mitton, 1994),
and accordingly they are often used to study evolutionary processes such as genetic
drift and mating systems (Hamrick and Godt, 1989).
Advances in molecular biology and genetic engineering have provided a new
class of genetic markers that allowed looking at the genetic variability at the DNA
level. These were commonly called the DNA markers and the first category of them
to be utilized was the restriction fragment length polymorphisms (RFLPs) (Botestien
et al., 1980). The RFLPs markers were recognized by autoradiography after
hybridization with suitably labelled probes. However, Restriction fragment length
polymorphisms (RFLPs) are relatively time consuming and require the use of labelled
DNA probes. These were soon followed by other DNA markers whose recognition
depends on using short oligonucleotide primers to selectively amplify the target DNA
and visualize it on electrophoretic gels by staining with a DNA-specific dye. The
application of this type of DNA markers was made possible by the discovery in the
early 1980’s of the polymerase chain reaction (PCR) technique (Mullis et al., 1986).
Molecular characterization techniques are now widely used both for
ecological, epidemiological analyses and molecular typing of a wide range of
bacterial species. The randomly amplified polymorphic DNA (RAPD) markers,
developed by Williams et al., (1990) and by Welsh and Mc-Clelland (1990), widely
used to study genetic diversity at the DNA level and for linkage mapping and
detection of relatedness between species (Rafalski, 1997; Bardakci, 2001; Tahir,
2008). RAPDs access genomic regions poorly sampled by other types of markers
(e.g., repetitive DNA; Williams et al, 1990), and resolve a high level of
polymorphism. Randomly amplified polymorphic DNA (RAPD) is a PCR-based
technique, using single short oligo nucleotide primers are arbitrarily selected to
amplify a set of DNA segments distributed randomly throughout the genome and
detect changes in the DNA sequence at sites in the genome. Only minute quantities
(picograms) of template DNA are required, markers are easily scored with nonradioactive staining techniques, and unlimited numbers of markers are available due
to the random nature of primers. The polymorphism within the set of DNA fragments
generated has been used in discriminating microorganisms both at the inter species
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and intra species level (Welsh and Mc-Clelland, 1990). In several Vibrio spp. this
technique has been used in diversity studies (Shangkuan et al., 1997; Somarny et al.,
2002). In V. harveyi it has been used in differentiating pathogenic and non pathogenic
strains (Somarny et al., 2002; Hernandez and Olmos, 2004; Alavandi et al., 2006).
The PCR technique has been used in a large number of molecular markers in
addition to RAPDs. Some of these have built on the RAPD technique (e.g., SCARS),
while others target specialized genomic regions including microsatellites (Rafalski
and Tingey, 1993). Conserved sequences have provided information for the
construction of primers to assay nearby polymorphic regions (e.g., the internal
transcribed spacer of nuclear ribosomal DNA; Baldwin et al., 1995), and recently has
lead to novel cytoplasmic markers (Taberlet et al., 1991, Demesmure et al., 1995),
which may exhibit useful polymorphism within populations (Mc-Cauley et al., 1996).
The 16S ribosomal RNA (16S rDNA) is a component of the 30S small subunit
of prokaryotic ribosomes. The genes encoding for its rRNA are referred to as 16S
rDNA (Fig. 2.4), and are used in reconstructing phylogenies. In the 1970’s, Carl
Woese and his colleagues determined the sequence of 16S rRNA of more than 400
organisms (Woese, 1970). They discovered characteristic sequences, called signature
sequences (Nester et al., 2001). Carl Woese during the 1970, established a molecular
sequence-based phylogenetic tree by comparison of ribosomal RNA sequences that
would be used to relate all organisms and reconstruct the history of Life (Pace, 1987).
Ribosomal RNA turned out to be an excellent evolutionary chronometer
because it functionally constant, universally distributed and moderately well
conserved across broad phylogenetic distances (Madigan et al., 1997). In addition,
there is no evidence of lateral gene transfer of rRNA genes between different species
and hence rRNA genes can bring true information regarding evolutionary
relationships (Pace, 1987).
There are 3 types of RNAs found in the microbial ribosomes, 5S rRNA, 16S rRNA
and 23S rRNA (Madigan et al., 1997). The first attempts to characterize microbes
began by extracting the 5S rRNA molecules directly from the cells. However, the
information content in the approximately 120-nucleotide long molecule is relatively
small as compared to 1,500 nt long 16S rRNA gene, and to a lesser extent to the 3,000
nt long 23S rRNA. The 16S rRNA molecule has several advantages. 16S rRNA gene
sequences are used to study bacterial phylogeny and taxonomy for a number of
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reasons. These reasons include (i) its presence in almost all bacteria, often existing as
a multigene family, or operons; (ii) the function of the 16S rRNA gene over time has
not changed, suggesting that random sequence changes are a more accurate measure
of time (iii) the 16S rRNA gene (1,500 bp) is large enough for informatics purposes
(Patel, 2001). Some regions of the gene are universally conserved and suitable for
phylogenetic studies of distantly related organisms. Other regions are semi-conserved
and are more useful for the analysis of phylogenetic relationship between phyla and
families. Variable and hyper-variable regions in the 16S rRNA enable us to
discriminate between organisms belonging to the same genus or even between species
(Amann et al., 1995). The length of the gene is convenient so PCR and sequencing are
easy. Furthermore, the ends of the gene are highly conserved across all bacterial and
archaeal domains; therefore almost the entire gene can be amplified by PCR. 16S
rRNA genes or gene fragments can be selectively amplified by PCR from complex
DNA mixtures obtained directly from the environment. The method circumvents then
the need to cultivate microorganisms in order to identify them.
The 16S rRNA of major phylogenetic groups has one or more signature sequences.
These oligonucleotide signature sequences are specific that occur in most or all
members of a particular phylogenetic group. They are rarely or never present in other
groups, even closely related ones. Thus, signature sequences can be used to place
microorganisms in the proper group. Signature sequences have been identified for
bacteria, archaea, eukaryotes, and many prokaryotic groups (Prescott et al., 2005).
The most common primer pair used in sequencing was devised by Weisburg
(Weisburg et al., 1991) and is currently referred to as 27F and 1492R.
2.14.1 Role of bioinformatics tools to study phylogenetic relations
Stackebrandt and Goebel (1994) correlated similarity values of complete 16S rDNA
sequences of isolates distributed throughout the bacterial kingdom with their DNADNA hybridization results. They concluded that, when similarity values fall below 97
%, one can generally assume that the isolate represents a new species. This is the
minimal value to be considered as an evidence for two organisms to be recognized
and established of being from the same species. Sequences with less than 93 %
similarity will be considered as being in a new genus (Madigan et al., 2000). As a
final output, a phylogenetic tree is created, gathering all information regarding the
relationship between the newly obtained sequences and the reference sequences.
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Large number of thermophiles has been isolated from different hot springs.
The correlation of their evolution is not well studied. Analysis of 16S rDNA gene
could be explored to construct a phylogenetic tree that would shed light into evolution
of thermophiles.
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