Lec-4 [Bioprospecting of novel extremozyme from prokaryotes]

Extremophiles are remarkable organisms that thrive in the harshest environments on Earth. Thus,
they are among the best model organisms to study adaptive mechanisms that lead to stress tolerance.
Furthermore, extremophiles can be a valuable resource for novel biotechnological and
biomedical products due to their biosynthetic properties.
✓ Enzymes, as natural catalysts, have shown remarkable abilities that have revolutionized the
chemical, biotechnological, bioremediation, agricultural, and pharmaceutical industries.
✓ The narrow range of stability of most described biocatalysts from mesophilic organisms
limits their use for many applications. Enzymes derived from microorganisms thriving
under harsh conditions, called extremophiles, can overcome these restrictions, and today,
such biocatalysts are in higher demand.
✓ Extremophiles are present in all three domains of life (bacteria, archaea, and eukarya).
✓ Polyextremophiles are perfect candidates as a source of novel enzymes for industrial needs.

ENZYMES: A SUSTAINABLE SOURCE FOR GREEN CHEMISTRY

• Enzymes are broadly used in biotechnology and a variety of industries (e.g., agriculture,
food, textiles, chemicals, pharmaceuticals, and biofuels) as catalysts, therapeutic agents,
analytic reagents, and diagnostic tools.
• In general, enzymatic reactions are safer, faster, less hazardous, and generate less waste,
thus following the twelve rules of green chemistry.
• Although enzymes have vast potential in biotechnological applications, they have been used
only in very few specific reactions. Most described enzymes can be used only for a
limited number of industrial processes.
• This limitation is caused by the narrow ranges of enzymatic stability, including a majority
of essential parameters for chemical reactions, such as temperature, pressure, pH, and the use
of organic solvents.

Green chemistry's 12 principles

Green chemistry is the design of chemical products and processes that reduce or eliminate the
use or generation of hazardous substances. These principles demonstrate the breadth of the concept
of green chemistry:
1. Prevent waste: Design chemical syntheses to prevent waste. Leave no waste to treat or clean up.
2. Maximize atom economy: Design syntheses so that the final product contains the maximum
proportion of the starting materials. Waste few or no atoms.
3. Design less hazardous chemical syntheses: Design syntheses to use and generate substances
with little or no toxicity to either humans or the environment.
4. Design safer chemicals and products: Design chemical products that are fully effective yet have
little or no toxicity.
5. Use safer solvents and reaction conditions: Avoid using solvents, separation agents, or other
auxiliary chemicals. If you must use these chemicals, use safer ones.
6. Increase energy efficiency: Run chemical reactions at room temperature and pressure whenever
possible.
7. Use renewable feedstocks: Use starting materials (also known as feedstocks) that are renewable
rather than depletable. The source of renewable feedstocks is often agricultural products or the
wastes of other processes; depletable feedstocks are often fossil fuels (petroleum, natural gas, or
coal) or mining operations.
8. Avoid chemical derivatives: Avoid using blocking or protecting groups or any temporary
modifications if possible. Derivatives use additional reagents and generate waste.
9. Use catalysts, not stoichiometric reagents: Minimize waste by using catalytic reactions.
Catalysts are effective in small amounts and can carry out a single reaction many times. They are
preferable to stoichiometric reagents, which are used in excess and carry out a reaction only once.
10. Design chemicals and products to degrade after use: Design chemical products to break down
to innocuous substances after use so that they do not accumulate in the environment.
11. Analyze in real time to prevent pollution: Include in-process, real-time monitoring and control
during syntheses to minimize or eliminate the formation of byproducts.
12. Minimize the potential for accidents: Design chemicals and their physical forms (solid, liquid,
or gas) to minimize the potential for chemical accidents including explosions, fires, and releases
to the environment.

Halophiles and Xerophiles

Halophilic and xerophilic microorganisms exhibit extraordinary adaptability to extreme
environments. Their enzymes and bioactive compounds hold significant promise for diverse
industrial, medical, and environmental applications, making them valuable assets in
biotechnology and modern scientific research.
Halophilic Organisms:
• Industrial and Commercial Applications: Halophilic organisms and their enzymes are utilized
in various fields:
✓ Production of fermented foods
✓ Solar salt manufacturing from seawater
✓ Leather industry
✓ Environmental bioremediation
✓ Textile and pharmaceutical industries
• Biochemical Properties and Uses: Molecules, enzymes, and compatible solutes synthesized by
halophiles have potential implications in fine chemicals, medicines, bioimplants.
• Source of Extremozymes: Halophiles are potential sources of novel extremozymes, including
amylases, proteases, nucleases, cellulases, chitinases, xylanases, esterases, alcohol
dehydrogenases, & lipases.
• Example of Commercial Use: Nuclease from Micrococcus varians has been commercially used
for producing the flavoring agent 5′-guanylic acid (5′ GMP).
• Polyextremophilicity: Some halophilic enzymes show polyextremophilicity, meaning they are
stable under multiple extreme conditions:
✓ High salt concentrations
✓ Elevated or low temperatures
✓ Alkaline or acidic pH
✓ Non-aqueous media
This makes them highly suitable for industrial and biotechnological processes.
• Bioactive Molecules: In addition to enzymes, halophiles produce several bioactive molecules
with diverse applications.
• Ectoine:
✓ A biocompatible solute produced by halophiles
✓ Has potential use in respiratory medicine
✓ Can reduce cell stress in nanoparticle-induced lung inflammation by inhibiting stress signals
• Polyhydroxyalkanoates (PHAs):
✓ Certain halophiles (e.g., Halobacterium, Haloferax) accumulate PHAs
✓ PHAs are biopolyesters with applications in medical & environmental fields, industrial sectors

• Antimicrobial Compounds:
✓ Halophiles such as Natronococcus occultus and Naloterrigena hispanica produce
anti-microbial peptides & di-keto-piper-azines
Xerophilic Organisms:
• Industrial Applications: They exhibit unique adaptations that make them suitable for use in:
✓ Microbial electrochemical systems
✓ Next-generation industrial biotechnology
• Can be used to treat long-chain fatty acids, cellulose, chitin, rubbers, & other complex compounds.

Thermophiles, psychrophiles, acidophiles, and alkaliphiles represent unique groups of extremophiles

with significant potential in industrial, medical, and biotechnological applications. Their enzymes,
capable of operating under extreme environmental conditions, offer innovative solutions for a variety
of industries, including pharmaceuticals, food, textiles, and more.

Thermophilic Organisms and Enzymes:

✓ Along with polymerases, various thermophilic enzymes are commercially available, including
lipases, laccases, & xylanases
✓ These thermophilic enzymes make industrial processes more environmentally friendly, as they
often reduce the need for harsh chemicals and extreme conditions.
Psychrophiles:
▪ Psychrophiles are organisms that thrive at low temperatures.
▪ Their cell membranes contain surfactants that help sustain stability in cold environments, making
them promising for pharmaceuticals and medicine.
▪ Some psychrophilic organisms include:
✓ Pandalus borealis ✓ Moraxella spp
✓ Euphausia superba ✓ Flavobacterium spp
▪ These organisms have been found to produce anticancer and antitumor agents.
▪ Psychrophilic enzymes have several important features:
✓ High catalytic activity at low temperatures
✓ Stability at low temperatures
✓ Heat lability, meaning they are sensitive to heat
▪ These enzymes may be useful in various industries, including:
✓ Pharmaceutical science ✓ Food processing
✓ Molecular biology ✓ Feed technologies
✓ Textiles ✓ Detergents
✓ Paper production ✓ Cosmetics
▪ Psychrophilic bacteria adaptation mechanism:
i. Unsaturated fatty acids, cyclopropane-containing fatty acids, and short chain fatty acids in
their membranes, which prevent the loss of membrane fluidity.
ii. Cold-shock proteins (CSPs) and chaperones to protect the synthesis of RNA and proteins;
iii. Antifreeze proteins (AFPs) that bind to ice crystals and create a state of thermal hysteresis;
and
iv. Mannitol and other compatible solutes that act as cryoprotectants to prevent cell damage by
ultraviolet (UV) radiation and ice formation.
Acidophiles
▪ Acidophilic enzymes have specific therapeutic and industrial applications:
✓ They can block the activity of matrix metallopeptidases (MMPs), which are essential
for tumor metastasis.
✓ MMP inhibitors derived from an acidophilic Penicillium species isolated from
Berkeley Pit Lake show promise as a therapeutic approach for cancer.
▪ Other Uses of Acidophilic Enzymes:
✓ Proteolytic enzymes from acidophiles are reported as non-allergenic preservatives in
medicines.
✓ Amylolytic enzymes, such as tre-halase isolated from the acidophilic Sulfolobus
solfataricus, are used as preservatives and stabilizers in medicine.
Alkaliphiles
Alkaliphilic enzymes have diverse industrial uses, including:
▪ Tannery water treatment ▪ Cosmetic manufacturing
▪ Food production ▪ Pharmaceutical production

Radiophiles
Radiophiles produce both primary and secondary metabolic products that help protect their DNA from
radiation damage. These metabolic products have important applications, including:
✓ Manufacture of anticancer drugs
✓ Antioxidants
✓ Sunscreens
• Extremolytes are compounds produced by radiophiles that enhance the organism's ability to
survive extreme conditions. Mycosporin-like amino acids (MAAs) from the red alga
Porphyra rosengurttii are commercially utilized for:
✓ Enhancing the UV protective properties of sunscreens
✓ Therapeutic use as preventive agents against UV radiation-induced cancers, e.g. melanoma
• Bacterio-ruberin, isolated from Halobacterium and Rubro-bacter, and deino-xanthin, isolated from
Deino-coccus radio-durans, are therapeutic candidates for treating cancer diseases.
Polyextremophiles:
▪ Polyextremophiles are organisms that can thrive in multiple extreme conditions, such as:
✓ High and low temperatures
✓ High salt concentrations
✓ Alkaline or acidic pH
▪ These organisms have a diverse range of uses and applications due to their extreme adaptability.
▪ Halothermophiles and halopsychrophiles are specific types of polyextremophiles that show
great promise as sources of useful enzymes. Examples:
a) Alkali-thermophilic serine proteases from Alkali-bacillus sp. NM-Da2 have potential
applications in biotechnological & pharmaceutical industries.
b) Alkali-psychrophilic esterase from the marine bacterium Rhodococcus sp. has potential use
in the food industry.

Lec-5 [Microbial diversity in extremophile]

Types of extremophiles
▪ Their superpower will be related to a special resistance towards a given physical, chemical, and
biological factor that normally acts to limit the growth and function of regular living forms.
▪ These factors include temperature, availability of liquid water, pH of their surroundings, energy,
nutrients, and trace elements available.

Oligotrophs
▪ Oligotrophs refer to organisms adapted to use low-nutrient concentrations efficiently. Oligotrophic
bacteria can survive in extremely nutrient-depleted environment.
 ▪ There are speculations that production of exo-polymeric substances by bacteria growing under
extremely nutrient-poor conditions (where these nutrients are available at levels below
threshold concentrations) might aid in concentrating the nutrient for sustenance.
▪ Klebsiella variicola are facultative oligotrophic strains from River Mahananda, Siliguri, India,
which produce exo-polysaccharide in oligotrophic media.

Extremolytes
▪ Extremolytes, such as glycerine, betaine, ectoine, & hydroxy-ectoine, produced from
radio-resistant extremophiles, halophiles, and halo-alkaliphiles are widely adopted in the
food industry, cosmetology, pharmaceutics, and molecular biology.
▪ The high compatibility, lack of toxicity, and macromolecule-stabilizing properties
of extremolytes make them promising candidates for the use as pharmaceutical excipients.
▪ A major application area established today is in cosmetics, in which ectoine is now used
in a growing range of skin care products.
▪ Additionally, ectoine can also open a new area of application for extremolytes as
functional food ingredients, once it also occurs in food and has known protein and
cell protection properties.
▪ One application could be the use of ectoine as food freshness stabilizer, with
additional potential health benefits due to its cytoprotective properties.
▪ To date, several UVR-protective compounds have been isolated from
UVR-resistant extremophiles, including mycosporine-like amino acids, scytonemin,
bacterioruberin, melanin, and carotenoids.
▪ The carotenoid biosynthetic enzymes could be used to enhance the resistance of genetically
engineered strains and genetically modified economic crops to resist DNA damage.
▪ Furthermore, carotenoids could also benefit human health if administrated as
natural antioxidants.
▪ cancer detection can be done using a protein from H. halobium as an antigen.
▪ to detect antibodies against the human c-Myc oncogene product in the sera of cancer
patients suffering from leukemia.
Microbial diversity in extreme environments: Temperature
( ) Few thermophilic fungi belonging
• Zygomycetes (Rhizomucor miehei, R. pusillus),
• Ascomycetes (Chaetomium thermophile, Thermoascus aurantiacus, Dactylomyces
thermophilus,Melanocarpus albomyces, Talaromyces thermophilus, T. emersonii
 • Basidiomycetes (Phanerochaete chrysosporium) and
• Hyphomycetes (Acremonium alabamensis, A. thermophilum, Myceliophthora thermophila,
Thermomyces lanuginosus, Scytalidium thermophilum, Malbranchea cinnamomea).
( ) Algae
• Achanthes exigua, Mougeotia sp. and Cyanidium caldarium
( ) Protozoa
• Cothuria sp. Oxytricha falla, Cercosulcifer hamathensis, Tetrahymena pyriformis, Cyclidium
citrullus, Naegleria fowleri
( ) Bacteria and archaebacteria
They have been classified based on their optimum temperature requirements:
a) MODERATE: Bacillus caldolyticus, Thermoactinomyces vulgaris, Clostridium
thermohydrosulfuricum, Thermoanaerobacter ethanolicus, Thermoplasma acidophilum
b) EXTREME: Thermus aquaticus, T. thermophilus, Thermodesulfobacterium commune,
Sulfolobus acidocaldarius, Thermomicrobium roseum,Dictyoglomus thermophilum,
Methanococcus vulcanicus, Sulfurococcus mirabilis, Thermotoga mritima
c) HYPERTHERMOPHILES: Methanoccus jannaschii, Acidianus infernos, Archaeoglobus
profundus, Methanopyrus kandleri, Pyrobaculum islandicum, Pyrococcus furiosus, Pyrodictium
occultum, Pyrolobus fumarii, Thermococcus littoralis, Ignicoccus islandicum.
The hyperthermophilic extreme acidophiles, with pH optima for growth at or below 3.0,
sulfolobus, sufurococcus, desulfurolobus and acidianus produce sulphuric acid from the
oxidation of elemental sulphur or sulphidic ores, in solfataras of yellowstone national park.
• Other microbes that occur in hot environments including metallosphaera that oxidizes sulphidic
ores and stygiolobus sp. which reduces elemental sulphur.
• Thermoplasma volcanicum that grows at pH 2 and 55°C, has also been isolated from
solfataric fields.
• Thermoplasma acidophilum was isolated from self heating coal refuse piles.
• Thiobacillus caldus was isolated from hot acidic soils.

Lec-6 [Psychrophile]
Psychrophiles are cold- loving organisms that grow at temperature of 15° C or lower. They are able
to grow at temperature close to the freezing point of water but fastest growth rate at above 20° C.
Biotype:
• It refers to a group of organisms having the same genotype or sharing similar physiological and
ecological characteristics. In the context of cold environments, it includes microorganisms that are
specially adapted to thrive in low-temperature habitats.
• They are found inhabiting a wide range of low-temperature environments on Earth. These include:
polar regions, glaciers, ocean deeps, snow covered regions, shallow subterranean regions, upper
atmosphere, refrigerated appliances and on and in plants and animals inhabiting cold regions.
• Approximately 90% of the water in the oceans has temperatures around 5 °C, indicating a vast cold
habitat suitable for psychrophilic and psychrotolerant microorganisms.
• Around 20% of the terrestrial region of the Earth is covered by glaciers & ice sheets, polar sea ice,
& snow-covered regions.
Psychrophiles: Examples ▪ Kocuria Polaris
▪ Psychrobacter aquaticus ▪ Sporosarcina mcmurdoensis
▪ Pseudomonas antarctica ▪ Cyanobacteria Oscillatoria, Phormidium and Nostoc
▪ Pseudomonas proteolytica ▪ Arthrobacter flavus
▪ Halomonas variabilis ▪ Leifsonia aurea
▪ Methanogens, members of Archaea, are the only group known to be Psychrophiles
(Eg. Methanococcoides burtonii).
▪ A Nematode Panagrolaimus davidi- can withstand freezing of all body water.
Adaptation Mechanism of Psychrophile
To survive and remain metabolically active in such extreme conditions, they have evolved several
structural and biochemical adaptations:
1. Maintaining Membrane Fluidity
• Unsaturated-cis-Fatty Acids: Psychrophiles incorporate unsaturated-cis-fatty acids into their
cell membranes, which helps maintain membrane fluidity at low temperatures.
• Carotenoids: These compounds help prevent membrane freezing, ensuring membrane
integrity is preserved in extreme cold.
2. Cold Adaptive Proteins
• Cold Shock Proteins (CSPs) and Cold Acclimatization Proteins (CAPs):
These small proteins are specifically adapted to cold environments. They:
✓ Bind to RNA
✓ Preserve its single-stranded conformation
✓ Facilitate proper transcription and translation at low temperatures
3. Protection Against Freezing
• Antifreeze Proteins: Prevent the formation of ice crystals inside the cells.
• Ice Nucleating Proteins: Control ice formation externally to protect the internal cell structure.
• Compatible Osmotic Solutes: These molecules stabilize cellular structures and prevent
damage due to osmotic stress under freezing conditions.
4. Protein Structure Adaptation
• Psychrophilic proteins have a higher content of α-helices relative to β-sheets. This structural
feature enhances protein flexibility and functionality at low temperatures.
5. Cryoprotectants
• Trehalose and Exopolysaccharides (EPSs):
o Act as cryoprotectants
o Prevent protein denaturation and aggregation
o Play a critical role in maintaining cell viability in freezing and thawing conditions
Prospective Applications of Cold-Adapted Microbial Proteases
Cold-adapted microbial proteases, primarily produced by psychrophilic microorganisms, exhibit high
catalytic efficiency at low temperatures. Their unique characteristics, such as thermal instability and
broad substrate specificity, make them highly valuable across various industries. Industrial sectors
using cold-adapted proteases are food, detergent, textile, pharmaceutical, leather, brewing and wine,
paper and pulp industries. Below is a detailed overview of their prospective applications:
A. Food Industry
Cold-adapted proteases are thermally unstable and can be selectively and rapidly inactivated, making
them ideal for controlled food processing.
• Protease from Pseudoalteromonas sp. improves the taste of frozen meat by releasing more
taste-enhancing and essential amino acids than mesophilic proteases.
 • Another Pseudoalteromonas sp. protease releases more free amino acids from milk protein
at 4 °C than mesophilic papain, indicating better substrate specificity for low-temperature
food processing.
• Metalloprotease from Enterococcus faecalis is safe for oral administration with no side
effects. It improves solubility and stability of health foods.
• Alkaline peptidase from Penicillium chrysogenum is suitable for cheese manufacturing,
preferred over mesophilic/thermostable enzymes.
• Aspartic protease from Geomyces pannorum is also suitable for cheese-making.
• Proteases from Arsukibacterium ikkense are generate bioactive peptides by extensively
degrading casein and also useful in dairy products and functional foods.
• Serine protease from Chryseobacterium sp. are active at low temperature, salt tolerant, and
suitable for meat and other food processing industries
B. Detergent Industry
Cold-adapted proteases, when combined with detergents, are more effective in cold washing than
enzyme-free detergents. They exhibit-
✓ High activity and stability at low temperatures
✓ Broad alkaline pH tolerance
✓ Compatibility with commercial detergents, surfactants, and bleaches
• They can efficiently remove proteinaceous stains such as chocolate, tea, blood, egg yolk, grass.
• They are ideal for laundry and dishwashing applications
C. Textile Industry
Cold-adapted proteases might find applications in the textile industry because their actions on fabrics
can reduce the harmful effects of chemicals. Other applications-
• Extend the life of woolen and silk fabrics
• Retain fabric quality after cold washing
• Improve surface appearance and reduce bristling in wool
• Help preserve finishing in silk cloth
D. Feed Additives
Proteases due to their extensive substrate specificity as well as reasonably advantageous activity levels
at a physiologically applicable temperature and pH can be used as an eco-friendly feed additive for
improving the manufacturing performance of animal farms.
• Cold adapted proteases which possess keratinolytic activity can facilitate and endorse
biotechnological processing of biomaterials consisting of keratinous waste from leather and
poultry industries.
E. Polymer Degradation
• Flavobacterium species regulate fatty acid composition to maintain membrane fluidity in cold
• Capable of organic polymer degradation
• Produce extracellular proteases in cold
• Important in mineralization of organic materials in freshwater sediments during cold seasons
F. Bioremediation
• Anaerobic psychrophiles (e.g., from Antarctica):

o Thrive in cold and produce proteases on diverse substrates

o Useful for degrading protein-rich waste like night soil
 • Psychrophilic Pedobacter cryoconitis:
o Utilizes various organic compounds:
✓ Oil hydrocarbons
✓ Carbohydrates
✓ Proteins
o Effective in treating wastewater in cold conditions

Lec-7 [Hyperthermophiles]
Hyperthermophiles
• They’re microorganisms that thrive at extremely high temperatures, generally above 80 °C.
• They were first discovered in 1965 by Thomas D. Brock in the hot springs of Yellowstone
National Park, Wyoming. As of now, approximately 90 species of hyperthermophilic
archaea and bacteria have been identified.
Notable Hyperthermophilic Organisms
Organism Temperature tolerance Notable feature
Pyrolobus fumarii Up to 113 °C Thrives in extreme heat
Methanopyrus kandleri Up to 122 °C Highest known tolerance
Sulfolobus acidocaldarius ~80 °C, pH ~3 Hyperthermophile & acidophile
Geogemma barosii (Strain 121) — Survives autoclaving
Thermus aquaticus — Source of Taq polymerase
Nanoarchaeum equitans — Smallest known archaeon
Pyrococcus furiosus — Biotechnological relevance

Sulfolobus acidocaldarius was the first Identified hyperthermophile which is both a hyperthermophile
and an acidophile.
Biotopes (Habitats) of Hyperthermophiles
Hyperthermophiles inhabit both natural and artificial high-temperature, water-containing
environments.
( ) Natural Habitats
• Hot springs: Including neutral to slightly alkaline types
• Solfataric fields: Sulfur-harboring and acidic
• Marine biotopes: They are shallow or deep-sea hot sediments, & hydrothermal systems,
including deep-sea hydrothermal vents (also called “black smokers”)
• Volcanic exhalations: They rise from deep magma chambers, heating soils & surface waters
( ) Artificial Habitats
• Hot outflows from geothermal power plants
( ) Recently Discovered Non-Volcanic Habitats
• About 3500 meters below the North Sea seabed
• Below the permafrost soil of the Alaskan North Slope

Hydrothermal Vents
• First discovered in 1977 along the mid-ocean ridge in the eastern Pacific Ocean.
• Formation Process:
✓ Formed when seawater meets hot magma beneath the ocean floor.
✓ Commonly occur at both diverging and converging plate boundaries.
✓ Magma rises, cracking the ocean floor and overlying sediments, releasing heat.
✓ Seawater enters these fractures, becomes superheated, and dissolves minerals,
concentrating sulfur and other compounds.
✓ The hot, mineral-rich water exits the crust and mixes with the cool seawater, leading to
rapid precipitation of minerals.
✓ This process creates tall towers or chimneys from the precipitated minerals.
• Plumes of Water: Two types of hydrothermal plumes stream upward from vents, often rising
1,000 feet above:
i. Black Smokers:
✓ Emit the hottest, darkest plumes
✓ High in sulfur content
✓ Form chimneys up to 55 meters (180 feet)
ii. White Smokers:
✓ Emit light-colored plumes
✓ Rich in barium, calcium, and silicon
✓ Plume temperature is lower than black smokers
Hot Springs
• Formation in Non-Volcanic Areas:
o Water is heated upon contact with hot rocks in the Earth's crust.
o Heated water rises back to the surface to form hot springs.
• Types Based on Water Movement:
o If heated water rises slowly, it forms a hot spring
o If it rises quickly, it results in a geyser
Case Study: Octopus Spring (Yellowstone National Park, USA)
• Type: Alkaline hot spring
• pH range: 8.3–8.8
• Water temperature:
o Source: ~95 °C
o Outflow channel: Cools down to 83 °C
• Microbial Zonation (as temperature decreases):
o At 83 °C: Dominated by pink filamentous Thermocrinis ruber
o At 65 °C: A microbial mat forms:
▪ Upper layer: Thermophilic cyanobacterium Synechococcus
▪ Lower layer: Photosynthetic bacterium Chloroflexus

Adaptation Mechanisms of Thermophiles

Thermophiles are heat-loving microorganisms that thrive in environments with temperatures above
45°C. To survive in such extreme heat, they have evolved a variety of unique physiological,
structural, and biochemical adaptations that help protect their cellular components, especially
proteins, membranes, and genetic material.
1. Nutritional Adaptations
▪ Most thermophiles are chemolithoautotrophs, which means they derive energy from inorganic
redox reactions and use CO₂ as their sole carbon source to build cellular material. Inorganic redox
reactions serve as energy sources and CO₂ is the only carbon source required to build up organic
cell material.
▪ They can perform both aerobic and anaerobic respiration. Oxygen-respiring hyperthermophiles
are microaerophilic and grow only at reduced oxygen concentrations (as low as 10 ppm). In
anaerobic conditions, they use various electron acceptors like nitrate, ferric iron, sulfate, sulfur,
CO₂.
▪ The main electron donor is H₂, but sulfide, sulfur, and ferrous iron are also used.
▪ Some thermophiles are facultative heterotrophs, which means they can switch to using organic
compounds when available.

Energy-Yielding Reactions in Chemolithotrophic Hyperthermophiles

Reaction Process/Products Representative Genera
Sulfolobus, Acidianus,
2S⁰ + 3O₂ + 2H₂O → 2H₂SO₄ Sulfur oxidation
Metallosphaera, Aquifex
2FeS₂ + 7O₂ + 2H₂O → Pyrite oxidation (Metal Sulfolobus, Acidianus,
2FeSO₄ + 2H₂SO₄ leaching) Metallosphaera
Hydrogen oxidation Aquifex, Acidianus,
H₂ + ½O₂ → H₂O
(aerobic) Metallosphaera
Hydrogen + nitrate
H₂ + HNO₃ → HNO₂ + H₂O Aquifex, Pyrobaculum
(nitrate reduction)
4H₂ + HNO₃ → NH₄OH + Nitrate reduction to
Pyrolobus
2H₂O ammonium
 H₂ + 6Fe(OH)₃ → 2Fe₃O₄ +
Iron reduction Pyrobaculum
10H₂O

2FeCO₃ + HNO₃ + 5H₂O → Iron carbonate oxidation

Ferroglobus
2Fe(OH)₃ + HNO₂ + 2H₂CO₃ + nitrate reduction
4H₂ + H₂SO₄ → H₂S + 4H₂O Sulfate reduction Archaeoglobus
Acidianus, Stygiolobus,
Elemental sulfur
H₂ + S⁰ → H₂S Pyrobaculum, Thermoproteus,
reduction
Pyrodictium, Igneococcus

Methanopyrus,
4H₂ + CO₂ → CH₄ + 2H₂O Methanogenesis Methanothermus,
Methanococcus

2. Membrane Adaptations
▪ Thermophiles have highly stable cell membranes made of ether-linked lipids, which are more
heat-resistant than ester-linked lipids found in other organisms.
▪ Membrane lipids have ether linkage—more branched, more saturated and are of high
molecular weight. These lipids increase the melting point of the membrane, helping it remain
intact at high temperatures.
▪ They also form monolayer membranes with isoprenoid tetraether structures, which are more
rigid and thermally stable than typical bilayers.

 Top layer depicts the phospholipid layer of

thermophiles – ether linkages – isoprenoid diether
or tetraether structures.
 Lipid bilayer labelled 9 – eukaryotes and bacteria.
 Lipid monolayer labelled 10 – Thermophiles.

3. Protein Stability Mechanisms

Thermophilic proteins are incredibly stable due to multiple features:
• Amino Acid Composition: Though they use the same 20 amino acids as other organisms,
these undergo modifications at high temperatures like deamidation, β-oxidation, hydrolysis,
and disulfide bond formation.
• Hydrophobic Packing: Their proteins have tightly packed hydrophobic cores that help
maintain structure. Efficiently packed hydrophobic core is a common feature of stable globular
proteins.
• Salt Bridges and Disulfide Bonds: These proteins have more salt bridges and disulfide
linkages, both of which enhance thermal stability.
• Ionic Networks: Thermophilic enzymes often have strong ionic interactions between acidic
• and basic residues, which are stable even when water structure is altered by heat. Ionic
interactions act over longer ranges and are relatively immune to alterations in water structure.
 Salt bridge

• Oligomerization: Many proteins that are monomeric in mesophiles become oligomeric in

thermophiles (e.g., dimers), adding to their stability. Chorismate mutase in hyperthermophiles
develops a dimeric quaternary structure.
• Terminal Protection: Thermophilic proteins often have immobilized N- and C-termini to
prevent unraveling. The C-termini of the dimeric citrate synthases of hyperthermophiles has
an intertwined structure.
4. DNA Stability Mechanisms
DNA is prone to denaturation at high temperatures, but thermophiles protect it through:
• High GC Content: GC pairs have three hydrogen bonds, which makes DNA more heat-stable.
High GC content than AT content in nucleic acid structure.
• Reverse DNA Gyrase: This enzyme introduces positive supercoils into DNA, making it more
resistant to heat damage. Positively supercoiled DNA appears to resist degradation more than
negatively supercoiled DNA.
• Histone-like Proteins: DNA is wrapped and stabilized by histone-like proteins.
• Protective Salts: High levels of potassium and magnesium ions help prevent DNA backbone
degradation. Salts like potassium and magnesium... protect DNA from phosphodiester bond
degradation.
5. Cell Surface and Structural Protection
Thermophiles are often surrounded by a thick, pseudo-crystalline proteinaceous S-layer, which acts
like a protective armor against extreme environmental conditions. Thick pseudo-crystalline
proteinaceous surface layer (S-layer) surrounding cell.
6. Energy Source Independence
Unlike most organisms, thermophiles do not require sunlight or organic food. They can live without
sunlight or organic carbon as food—survive on sulfur, hydrogen, iron sulfide and other materials that
other organisms cannot metabolize. This makes them highly versatile and capable of inhabiting
environments where no other life forms can thrive.
The remarkable survival of thermophiles in high-temperature environments is the result of an intricate
set of molecular, structural, and metabolic adaptations. Their unique proteins, lipids, and nucleic acid
protection strategies not only help them endure extreme heat but also make them valuable in
biotechnology, industry, and evolutionary studies.
Industrial Applications of Thermophilic Enzymes
Thermophilic enzymes, due to their high thermal stability, have become vital tools in various
industrial and biotechnological applications. Their ability to function at elevated temperatures
provides numerous advantages over mesophilic enzymes.
1. Use in Molecular Biology: The PCR Revolution
• The first major application of thermophilic enzymes was Taq DNA polymerase, isolated
from the bacterium Thermus aquaticus.
 • This enzyme allowed for the automation of Polymerase Chain Reaction (PCR) by withstanding
high denaturation temperatures.
• It reduced cost and accelerated research in molecular biology and biochemistry labs.
2. Alternative Thermostable DNA Polymerases
• In place of T. aquaticus, DNA polymerases from archaeal species are now used for greater
fidelity and efficiency. These include pfuTurbo, DeepVent®, Therminator™.
These polymerases offer higher accuracy and thermostability, making them ideal for advanced PCR
applications.
3. Thermostable Amylase Applications
• A thermostable amylase from Pyrococcus furiosus is another key enzyme used industrially.
• A mutation in this amylase (Pf-amylase) increased the production of maltoheptaose from
β-cyclodextrin, a compound used in food and pharmaceutical industries.
4. Non-Catalytic Use at Ambient Temperatures
Interestingly, thermophilic enzymes are also valuable because they do not catalyze reactions at room
temperature. This unique property is exploited in optical nanosensor technology, where:
o The enzyme binds a substrate without converting it into a product.
o The substrate-enzyme complex changes fluorescence, which can be measured.
o This allows for quantification of the substrate in a sample.
This has major potential in biotechnology, medical diagnostics, & drug discovery.
5. High-Temperature Saccharification Process
Raising the temperature during saccharification (sugar breakdown) offers multiple process advantages
when using thermophilic enzymes:
• Higher substrate concentration tolerance
• Reduced bacterial contamination risks
• Longer catalyst (enzyme) half-life
• Faster reaction rates and shorter process time
• Lower viscosity → reduced pumping cost
• Lower enzyme purification costs