THERMOPHILIC BACTERIA AND THEIR ENZYMES-A REVIEW IS

DEVELOPED IN BIOTECHNOLOGY AND MOLECULAR BIOLOGY A

MODERN AND CLINICAL APPROACH.

ABSTRACT

In general, thermostable thermophile bacteria or extremophiles from a hot environment are

able to generate the enzyme. Protease, lipase, and amylase are a few examples. We all know
that bacteria can create the enzyme amylase, which can hydrolyze starch into sugar.
Because of its physiologic features, the thermostable of amylase can withstand high
temperatures and acidic environments. It is utilised in several industrial processes,
including those that produce food, fermented beverages, textiles, alcohol, paper,
pharmaceuticals, and detergents. Because hot springs have the potential to be a source of
bacteria that produce thermostable amylase, it is necessary to screen for thermo-amylase
bacteria there. The activity of the thermostable amylase that naturally occurring bacteria
generate is often not very high. The objective of this work is to get thermophile bacteria
that generated thermostable amylase and other enzymes that have an clinical and
commercial value in biotechnology and molecular biology.

Key words : Thermophiles, Thermostable amylase, Thermotoga, P. furiosus protease etc.

Introduction:
Researchers and natural chemists have been interested in organic entities created for high
temperature survival since they can endure temperatures where proteins and nucleic acids
are anticipated to be denatured. Thermophilic microorganisms, also known as
extremophiles, are among the extremophiles that have been studied the most. It has long
been held that the arrangement of living things according to how they respond to
temperature is the most important feature of biological chemistry. For the most part,
earthbound microorganisms can thrive best in a temperature range of 25 to 35 °C.
According to their preferred temperature, microorganisms were divided into three groups:
A thermophile prefers temperatures between 50 and 80 °C, a mesophile prefers
temperatures between 20 and 40 °C, and a psychrophile prefers temperatures below 20 °C.
The term "thermophile" has traditionally been used to describe living forms with a
maximum development temperature Tmax higher than 50 °C. In 1969, Brock advocated
basing the definition of the upper limit of thermophiles on two claims. In nature,
temperatures below this threshold are usual; higher temperatures, on the other hand, are
often linked to geothermal and modern activity. Furthermore, no multicellular creatures or
plants have been shown to be able to endure temperatures beyond 50 °C. Archaea and
bacteria live alongside thermophilic microorganisms, which have extreme temperature
fluctuations that can reach 121 °C, jeopardising normal life. Thermophiles are divided into
different types based on how they need to be kept at a certain temperature. Thermophiles
were classified as organisms that can survive at temperatures as low as about room
temperature (25 °C), Orth thermophiles as bacteria that can survive at temperatures as high
as the temperature at which protein coagulates (60–70 °C), and Thermotolerant as
organisms that can survive at temperatures as high as 50–55 °C while still thriving the
temperature of room. Thermophiles are further classified into two classes : The first
category includes real thermophiles, which showed no growth or follow development
below 40–45 °C and optimum development above 60–70 °C. The second category is
facultative thermophiles, which started to grow at a temperature of 25 °C and whose
optimal growth and endurance temperature ranges from 50 to 55 °C, with 60 °C being the
highest. Thermophiles are sometimes known as severe thermophiles since they developed
above 55°C, While most thermophiles formed at 55 °C but not at 37 °C, thermotolerant
species thrived between 40 and 50 °C. A recent classification of thermophiles includes
three categories: moderate thermophiles (ideal development 50–60°C), outrageous
thermophiles (ideal development 60–80°C), and hyperthermophiles (ideal development
80–110°C). Most thermophiles have been kept away from manures, solar-heated soils,
subsurface aquifers, underwater aqueous vents, and geothermally heated oil wells.
Examples of bacterial thermophiles include Thermotoga maritima (phylum Thermotogae),
Thermus aquaticus, and Thermus thermophilus. Both of the latter two are classified as
eubacteria under the Deinococcus-Thermus phylum. It is believed that thermophilic
bacteria were among the first types of eubacteria. The majority of known thermophiles,
however, are archaebacteria. For instance, Methanopyrus kandleri, the "hottest"
thermophile archaeon, can survive at temperatures as high as 250°F. Thermus aquaticus
was the first extremophile capable of growing at temperatures more than 70°C. This
bacteria would eventually enable the unavoidable use of a cutting-edge invention called
the polymerase chain reaction. The first hyperthermophile was in this time period.

Nature and habitats of thermophiles:

Geothermal zones are often distributed all over the world. Due to flaws caused by structural
development or volcanic ejection, hot water develops from the outer world and frames
natural aquifers. Natural aquifers with pH values that are close to neutral, acidic and
sulphurous, or iron-rich are present on Earth's surface. Different types of hot underground
places exist, ranging from those that are warmed by volcanic activity to those that, like
Australia's Great Artesian Basin, are warmed by the ethics of their depth. It's possible for
natural aquifers to be fundamental, impartial, or acidic. The most diverse, however, are
those with soluble pH, which are frequently associated with volcanic or structural activity.
Active volcanoes or shallow lava pools are linked to acidic aquifers beneath the earth's
surface, as the one in the Yellowstone caldera. Acidophiles like Sulfulobus flourish in
acidic subsurface aquifers, whereas other thermophiles like Thermoproteus, Pyrobaculum,
Methanothermus, Desulforococcus, and Thermofilum survive in neutral to mild natural
aquifers, demonstrating how the variety of geothermal springs is mirrored in their pH..
Volcanic and aqueous vents are present in underwater environments.

Thermophilic bacteria:
The term "thermophilic" refers to a broad category of prokaryotes (bacteria and
archaea) that tolerate and typically require high temperatures for their development
and survival. microorganisms. Within the last few decades, a wide variety of these
microorganisms have been isolated and characterised in heated conditions. In both
natural (such as geothermal sites, terrestrial hot springs, deep-sea hydrothermal
vents, etc.) and man-made habitats (such as waste treatment facilities, biological
wastes, self-heated compost piles, etc.), thermophilic microbial species have been
identified. The thermogenic phase (50–80 °C) of the composting process uses
thermophilic microbes directly to handle organic solid waste. The composting
processes begin right away, and as time passes, the temperature of the compost rises
until it reaches its peak in the centre of the compost pile within a few days.The
population of thermophiles grows and that of mesophiles shrinks as the temperature
changes. Extremely high temperatures impede the breakdown of organic waste and
prevent the majority of the bacteria present from growing. Autotrophic,
heterotrophic, and mixotrophic thermophilic bacterial strains have been discovered
from practically every type and form of compost, and each scientific study has
highlighted a different trait. has drawn attention to certain traits .At temperatures
higher than 60 °C, Bacillus, Thermus, and Clostridium exhibit metabolic activity .
According to the scientists, only a few species of thermophilic sporogenous bacteria
(Bacillus stearothermophilus and Bacillus subtilis) and the genus Thermus Gram-
negative, aerobic, non spore producing bacteria displayed metabolic activity above
70 °C during the thermogenic phase of compost. Eighty-seven percent of the
microbiota is made up of Bacillus species, which include B. licheniformis, B.
subtilis, B. coagulans type B and B. stearothermophilus. Purple microbes or
proteobacteria, thermophilic taxa, are common and coexist between mesophilic and
thermophilic preparations. Genus Proteobacteria are more restricted under anaerobic
conditions. The sulfur and hydrogen oxidizing bacteria Hydrogenophilus and
Thermothrix and the sulfur and hydrogen reducing bacteria Desulfurella and
Thermodesulfobacterium are thermophilic. The maximum temperature of
developmental representatives is moderate thermophilic, usually in the range of 55-
65 °C. Geobacillus species (isolated from deep-sea waste, fertilizer, geothermal soil,
crude oil and subterranean aquifers) have been disclosed.

Fig-1

Source of thermophiles:
High heat regions:
Inside the active volcanic zones are high temperature fields, and the magma chamber serves
as the source of heat Most of the time, they are in important positions. The main hotspots
include Torfajokull east of Hekla, Grimsvotn in the Vatnajokull ice sheet, Hengill near
Reykjavik, Kerlingarfjoll, Namafjall near Myvatn, Kverkfjoll on the north side of
Vatnajokull, and Krisuvik south of Reykjavik. The water there is between 150 and 350 °C,
and At the surface, steam and volcanic gases are produced. 386 °C was the temperature that
garnered the greatest media attention. In Icelandic subterranean aquifers, high sulphide
fixations (30 mg/L-1) and dense bacterial mats are framed with accelerated sulphur, which
produces stunning bright yellow or white tones.
 Fig-2
Fig-1

Enzymatic activities of bacteria during the thermophilic stage :

In an effort to degrade natural waste via composting, microbial groups produce and secrete
a extensive variety of thermostable enzymes, broadly speaking hydrolytic and
oxidoreductase activities . Enzymes like proteases, cellulases, hemicellulases, and lignin-
modifying enzymes are one of the fundamental factors using the composting procedures
and could have many capability industrial packages, therefore representing a area of studies
in industrial enzymology. studied the variant of thermophilic bacteria's enzymatic pastime
in high-temperature dairy cattle manure compost, and discovered that the best bacterial
activity became at 54 °C, observed by way of a lower at 60 °C, a brand new boom at 70 °C,
and a reduction in organic count decomposition due to the high temperature itself.
Considering those enzymatic sports impact the modifications that take place and
manipulate how easily organic materials degrade, extra consciousness has been placed on
how they arise during the composting method. At every step of the system, they range
relying at the composition of the compost, the physico-chemical parameters, and the
dynamics of the microbial population .
Thermoenzymes:
Thermoenzymes, which are produced by thermophiles and hyperthermophiles, are enzymes
that are most active between 60 and 120 °C and resistant to irreversible inactivation at high
temperatures. Thermozymes have a number of modern and biotechnological benefits over
mesophilic proteins. They can withstand higher substrate concentrations, have superior
resilience to synthetic denaturants (solvents and guanidinium hydrochloride), and are
simpler to heat-treat to disinfect. Thermozyme responses often exhibit greater response
rates than mesozyme catalysed responses because to their robustness at elevated
temperatures, which makes them less vulnerable to microbial contamination. Given these
major advantages, thermozymes are currently generating a lot of interest. Thermozymes
may also be used as models to understand thermo soundness. As a result, it's crucial to
recognise the underlying factors that contribute to thermozyme strength in order to imagine
how physico-compound standards can contribute to protein strength or collapse
Additionally, This comprehension is essential for creating proteins that are more stable for
modern cycles. The main advantage of thermozymes is their high strength at elevated
temperatures, which is useful for a broad range of contemporary cycles. Numerous
polymeric substrates or natural substrates have altered bioavailability and enhanced
dissolvability as a result of temperature increases . The expansion of natural mixes'
dispersion, a decrease in thickness, more developed exchange rates, and afterwards
enlarged reaction rates are all connected to temperature. Another advantage is that it also
lowers the rate of developed exchange, which leads to higher response rates.risk of
microbial contamination since all saprophytes and harmful microbes die at temperatures
over 70 °C, reducing the quantity of microbes that contaminate food production. A strong
overall conformational structure, such as more rigid, high pressing thickness,
conformational strain discharge stability of the a-helix, reduced unfurling entropy, ideal
charge example, or particle pair and oligomer development, as well as additional
intermolecular cooperations (such as hydrogen bonds, electrostatic interactions,
hydrophobic associations, disulphide bonds, and metal restricting) are factors that
contribute to stability.. In comparison to mesophilic proteins, proteins from thermophiles
have more salt extensions due to electrostatic interactions. The amount of Glu, Arg, and
Lys in thermophilic proteins is larger in the helices, which causes control buildups to
expand and improves the proteins' temperature resistance in hyperthermophilic bacteria.
Atomic collapse and thermostability are strongly supported by hydrophobic forces . By
repairing protein centre holes and increasing the hydrophobicity of the centre, a catalyst
can achieve its maximum pressing efficiency. The protein core of the Escherichia coli
ribonuclease HI has a pit. The presence of a methyl group in the cavity improved protein
security by increasing hydrophobic cooperation within the protein core. Today's bio
cleaners use varieties of proteins including amylase, protease, cellulase, and lipase that are
resistant to harsh environments. Aside from these processes, lipases are also used in natural
biosynthesis, fat hydrolysis, esterification, interesterification, and trans-esterification.
Other applications for lipase include the removal of pitch from delivered mash in the paper
industry, the hydrolysis of milk fat in the dairy industry, the removal of non-cellulosic
contaminants from raw cotton before further processing into coloured and finished items,
the removal of subcutaneous fat in the cowhide industry, and the assembly of medications
in the pharmaceutical industry.

Application of thermoenzymes and their commercial value:

The rapid popularisation of PCR-related technologies and the characterisation of T.

aquaticus Taq DNA polymerase both contributed to the scientific and industrial
communities' increased interest in thermophilic and hyperthermophilic enzymes. The
increasing usage of thermophilic and hyperthermophilic enzymes in a wide range of
applications is predicted by the increasing number of enzymes identified from
hyperthermophilic animals and the recent development of potent protein engineering
techniques.

Application in molecular biology:

DNA polymerase:
The T. aquaticus Taq DNA polymerase was cloned and expressed in E. coli, which was
crucial in the advancement of PCR technology. Since then, thermophilic DNA
polymerases from a variety of thermophiles and hyperthermophiles have been cloned and
characterised. The processivity and fidelity of these DNA polymerases, two of their key
characteristics, are utilised in a variety of PCR applications . Taq DNA polymerase is the
preferred enzyme for sequencing or detection methods due to its high processivity. When
high fidelity is required, proofreading enzymes like Vent and Deep Vent polymerases are
recommended. Although mesophilic enzymes have been largely supplanted by
thermophilic DNA polymerases in a few applications, most applications—such as PCR
in situ hybridization and reverse transcription-PCR—were created after the invention of
PCR.

DNA ligase:
Commercially available thermophilic DNA ligases are available. They are a great
complement to PCR technology and are most active between 45 and 800C (Mesbah and
Sarmiento, 2016). These enzymes' ability to bind can be exploited for ligase chain
 reactions, oligonucleotide ligation assays, mutational analysis, and gene synthesis (from
overlapping oligonucleotides).

Thermostable protease:
There are currently very few commercially available thermostable proteases. Alcalase,
which was isolated from Bacillus licheniformis, is one of them. This preparation's main
component is subtilisin, a serine-type endoprotease with peak activity at 60 °C and a pH
of 8.3. Due to their limited selectivity towards various proteins from both plant and
animal sources, alcalase finds several uses in the food sector. For instance, this enzyme
plays a crucial role in the digestion of soy meal to produce soluble, non-bitter hydrolyzate,
which is utilised as a component of protein-fortified soft drinks and diet foods . Alcalase
is also helpful for recovering proteins from waste from the manufacturing of chitin and
from the by-products of the meat and fish industries. Anionic or non-ionic surfactants that
are active at temperatures exceeding 60°C are used in dishwashing detergents (Banerjee
et al,1999; Niehaus et al,1999). The efficiency of the process can be increased by using
these enzymes to completely clean ultrafiltration membranes at high temperatures.

Various alternative enzymes:

Nowadays, molecular biology and biochemistry techniques utilise a range of thermophilic
and hyperthermophilic proteases. At temperatures between 20 and 600 °C, some proteins,
particularly thermophilic proteins, can withstand proteolytic digestion.Only beyond 700C
do they begin to unfurl and become vulnerable to proteolytic assault . Thermus-like
proteases .In DNA and RNA purification methods, EGTA-rapidly inactivates Rt41A
serine protease PRETAQ Once inactivated, PRETAQ won't obstruct other enzymes from
treating the DNA or RNA in a subsequent step . The P. furiosus protease is used to
fragment proteins prior to peptide sequencing since it has a broad specificity. There are
currently several thermophilic restriction endonucleases available for purchase. The
majority of them, which were identified from Bacillus and Thermus strains, are most
active between 50 and 650C.

A few illustrations of thermophilic and hyperthermophilic enzymes used as

molecular biology tools.

Table-1
 Enzymes properties source application

Taq polymerase PCR technologies Thermus Optimal activity at 75°C, pH 9.0

aquaticus
DNA polymerase Roche molecular Thermus Reverse transcriptase activity
biochemical thermophilus
Pfu DNA ligase Ligase chain reaction and Pyrococcus Active at 45-80°C; t1/2-60 min
DNA ligations furiosus (95°C)
Serine protease DNA and RNA Thermus strain Optimal activity at 90°C, pH 8.0
(PRETAQ) purifications; cellular Rt41A
structure degradation prior
to PCR
Protease S Protein fragmentation for Pyrococcus Optimal activity at 85-95°C
sequencing furiosus
Carboxypeptidase C-terminal sequencing Sulfolobus Broad specificity (can release
solfataricus basic, acidic, and aromatic
residues); stable in solvents at
40°C
Alkaline Enzyme-labeling Thermococcus Optimal activity at 85°C, pH 9.9;
phosphatase applications where high neapolitana t1/2- 4h (90°C)
stability is required

Fig-3 Numerous uses for thermophilic bacteria

cleaning up textile dyes
The laccase enzyme that turned into received from the thermophile Geobacillus
thermocatenulatus MS5 has a very high catalytic pastime, is cheap, is extraordinarily
strong at a variety of temperatures and pH levels, and may be used significantly and
efficiently to dispose of the dyes that pollute the surroundings. Verma and Shirkot regarded
into Remazol exquisite Blue R (RBBR), Indigo carmine, Congo red, amazing
inexperienced, and Bromophenol blue elimination the usage of the natural laccase enzyme.
After 48 hours of incubation, 99% of the indigo carmine and Congo pink dyes decoloured,
observed by using RBBR dye, Bromophenol Blue, and splendid green at ninety eight, 70,
and 60% respectively. From soil, digested slurry, and plant waste, thermophilic ligninolytic
fungal cultures were isolated after which dealt with to Remazol wonderful Blue (RBB)
acclimatisation at 0.05% concentration in malt extract broth (MEB). The findings
confirmed that the isolates can be used to assist degrade reactive dyes .
conversion of agricultural wastes to sugar
The best fungal isolate Sporotricum thermophile LAR5 may use unprocessed agricultural
waste as a supply of carbon and nitrogen to create a extensive cellulase titre. From the
perspective of business applications, cellulase has preferred traits such hobby and stability
for the duration of a huge pH variety and high temperatures, as well as precise
saccharification capability on acid-pretreated rice straw. Enzymatic hydrolysis of acid-
pretreated solids has been shown to create big amounts of sugars (3.five, 5.7, 7.nine
micromoles/ml from 1, 3, and seven% acid-pretreated solids, respectively). A terrific
property of recombinant S. thermophile cellulase is that it has the ability to hydrolyze a
variety of cellulosic substrates, with the presence of lignin in some substrates growing the
diploma of saccharification.
Recovery of heavy metals
Heavy metal pollution is an issue since industrial activity is growing. A range of
interactions between microorganisms and heavy metals can reduce the mobility and
solubility of the metal. The physiology of the metal and sulfate-reducing bacteria is
conducive to metal precipitation and immobilisation. These microorganisms'
activities produce metabolic byproducts including iron and hydrogen sulphide,
which result in the creation of minerals. According to Chalaal and Islam , these
minerals have the ability to precipitate when in contact with heavy metals, hence
detoxifying the body. Chalaal and Islam employed two strains of thermophilic
Bacillus family bacteria that were obtained from hot water streams to remove
strontium from aqueous stream systems in order to study the removal of such sorts
of toxins. These microorganisms had the capacity to focus.
Thermophilic bacteria in the preparation of dairy
In the context of dairy processing, thermophilic bacilli are utilised as markers of processed
product cleanliness. this is a result of these lines' capacity to create biofilms and
endospores. Thermophilic micro organism, inclusive of Geobacillus species and
Anoxybacillus flavithermus, constitute a enormous elegance of pollutants in the dairy area.
even though those bacilli are regularly not dangerous, their presence in dairy merchandise
is an indication of uncleanliness, and excessive concentrations are too excessive for
customers. additionally, their enlargement may produce milk product flaws due to the
formation of acids or enzymes, which might bring about off flavours. it's been validated
that several strains of the genera Lactobacillus and Bifidobacterium, as well as sure
enterococci and yeasts, showcase probiotic qualities with the potential for prevention and
treatment of a spread of conditions.
In cancer therapy
The thermophile Aspergillus terreus turned into used to perceive Asperjinone, a nor-
neolignan, and Terrein, a suppressor of ABCG2-expressing breast cancer cells. these
compounds can repair drug sensitivity and can preserve the key to developing greater
powerful treatments for breast most cancers. Terrein proven giant cytotoxicity in opposition
to MCF-7 breast most cancers cells. Breast cancer cells that expressed ABCG2 had been
substantially slowed of their proliferation after terrein treatment. The activation of the
caspase-7 pathway and inhibition of the Akt signalling device, which resulted inside the
induction of loss of life, had been used to generate this suppressive effect, which reduced
the number of ABCG2-expressing cells and the aspect-populace phenotype. traditional
chemotherapeutic drug treatments usually do not goal malignant cells specifically and
forestall any dividing cells from proliferating. due to the incidence of severe facet outcomes
and drug resistance, the therapeutic capacity of anticancer medicines is seriously restricted.
consequently, retailers which might be more efficient, greater centered, and in all likelihood
free of drug resistance are needed. A compound comprising bacteriocin generated from
lactic acid bacteria and a carrier that may be used to forestall the increase of malignant cells
is disclosed.

CONCLUSION
The fact that there are more patents shows that humans are getting greater interested by
using thermophiles for enterprise functions. In current years, there has been a sharp upward
thrust within the demand for thermostable enzymes. There appear to be many
hyperthermophilic catalysts with unusual features nonetheless undiscovered as simplest a
small variety of species from this category of microorganisms have been diagnosed thus
far.

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