Miraculous Adaptations in Extremophiles

International Journal of Applied Research and Studies (iJARS) ISSN: 2278-9480 Volume 2, Issue 4 (April- 2013) www.ijars.in

Research Article

Miraculous Adaptations in Extremophiles Authors: 1

Urvashi Kohli *, 2Zeeshan Fatima, 3 Saif Hameed, 4SMP Khurana Address For correspondence:

Amity Institute of Biotechnology, Amity University Haryana, Gurgaon (Manesar), India.

Abstract— Extremophiles are the organisms that thrive under extreme environments such as high or low temperature, high or low pH, high salinity, very low water activity, and high pressure. Extremophiles include representatives of all three domains (Bacteria, Archaea, and Eucarya) however, the majority of these are from Archaea group of microorganisms. The enzymes known as “extremozymes” are produced by these microorganisms and hence function under extreme conditions. These microorganisms have developed many molecular adaptations to survive under challenging environments. There exist various classes of extremophiles that range all around the globe where each class corresponds to the way its surrounding niche differs. Depending upon the environmental conditions they may be classified as thermophiles, acidophiles, alkalophilies, halophiles, piezophiles to name few of them, however, these classifications are not exclusive and one may fall under multiple categories. This article focuses on the natural occurrences of extremophiles followed by their diverse ranges of adaptation strategies they adopt to survive the extreme environmental conditions. Keywords- component; Extremophiles; extremozymes; acidophiles; thermophiles; barophiles; alkaliphiles; psychrophiles. I.

INTRODUCTION

Extremophiles are eccentric microorganisms capable of growing and thrive under extreme environmental conditions, which were formerly considered too hostile to support life. These bacteria were discovered in the 1970’s in places such as Yellowstone National Park, a hot spring with water temperatures up to 90oC (188oF) and around hydrothermal vents on the ocean floor. The term ‘extremophile’ was introduced by MacElroy [1]. They belong to group archaea.

Cavities and cracks in rocks are the natural habitat of these microbes. Although, scientists have found these bacteria in sever extremities like liquid water pockets lodged about twelve feet deep in “solid” lake ice and even in groundwater 5 kilometers below the surface in deep gold mines of the Witwatersrand Basin in South Africa. Furthermore, researchers are engaged in investigating life within and below permafrost in northern Canada. Extremophiles have been categorized according to their niche, such as thermophiles that thrives under temperature exceeding 600C, psychrophiles capable of growing below 00c temperatures, barophiles or piezophiles withstands hydrostatic pressures as high as about 1,000 atmospheres, halophiles tolerant to salt concentrations exceeding the salinity of the sea water by more than a factor of ten, acidophiles flourish at pH values close to 0 and alkaliphiles grow at pH values exceeding 10. Organisms showing tolerance to high concentrations of heavy metals or ionising radiations have also been placed under extremophiles by some researchers. Although this is not a universal fact because, by and large, these organisms are capable enough to grow even in the absence of extreme conditions. Certain other terms encountered in the area involve xerophile, or, more appropriately, xerotolerant to describe organisms tolerant to very low water activity whether they are inside rocks i.e. endoliths, halophiles or simply inhabitant of extremely dry environments. Extremophiles also with stand a combined set of extreme environmental conditions for example a high temperature and high acidity, high temperature and high alkalinity, low temperature and high pressure. For instance, members of the Sulfolobus genus are known with optimal growth parameters around 85◦C and pH 2.0.

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Table 1: The current described limits for the extremophiles. Thermophile

Archaeal ‘Strain 121’

121 ◦C

Psychrophile

Himalayan Midge

−18 ◦C

Acidophile

Ferroplasma acidarmanus Alkaliphilus transvaalensis Moritella spp.

pH 0

Alkaliphile Barophile

pH 12.5 80 Mpa

There are representatives of all kingdoms in the extremophiles, although they are most strongly associated with the Archaea which were earlier thought to be morphologically similar to bacteria. Although in 1970s by DNA sequence analysis, found to be distinct from both the Bacteria and Eukarya [2]. The Himalayan Midge [3] is capable to grow at −18◦ C and various bacteria and algae can withstand less than 1.0 pH range and certain bacteria are there that show growth at pH 12.5 [4]. II.

ACIDOPHILES

Acidophiles are the microorganisms that resides in acidic environments that is less than pH 3, which may be natural or man-made, including sulfidic mine areas and marine volcanic vents. Acidophiles are widely spread in the bacterial and archaeal domains and found to contribute numerous biogeochemical cycles [5]. The iron and sulfur cycles are the most vital cycles for biotechnological applications [6], that involve metal extraction from ores [7]. This property of acidophile is becoming increasingly significant [8] due to its reduced and containable pollutant outputs [9]. Acidothermophiles are the extremophiles which live under conditions of low pH and high temperature. For example, Sulfolobus solfataricus ,an acidothermophile, grow at pH=3 and 80 °C. True acidophiles belonging to the archaea Picrophilus torridus and Picrophilus oshimae show optimum growth at pH values as low as 0.7 and at 60 °C and reported to produce starch-hydrolyzing enzymes (amylases, pullulanases, glucoamylases and glucosidases) [10].It is well documented that the microorganisms often withstand a broad range of pH for the growth. Despite of the broad range pH tolerance, there are some limits for their sustained growth. Marked variations in cytoplasmic pH can injure microorganisms by disrupting their plasma membrane or inhibiting the activity of enzymes and membrane transport proteins. Microorganisms die if the internal pH drops much below 5.0 to 5.5. Neutrophiles use an antiport transport system to exchange potassium for protons. Shift in the external pH also might alter the ionization of nutrient molecules and thus

impair their availability to the organism. Various mechanisms for the maintenance of a neutral cytoplasmic pH in acidophiles have been put forward. The plasma membrane is relatively impermeable to protons. Acidophiles have highly impermeable cell membrane that maintains change in pH by restricting influx of protons into the cytoplasm. It is the membrane proton permeability that determines the rate at which protons drop inward, the balance between proton permeability, influx of proton through energetic and transport systems, and the rate at which proton pump outward determines whether a cell can sustain an appropriate proton motive force (PMF). The archaeal cells have highly impermeable cell membrane composed of tetraether lipids as compared to the ester linkages present in bacterial and eukaryal cell membranes. The archaeal-specific structures has been identified in Thermoplasma acidophilum [11], Ferroplasma acidiphilum YT and Y-2 [12-14], Ferroplasma acidarmanus [15], Sulfolobus solfataricus [16] and Picrophilus oshimae [17]. Indeed, different optimal growth pH in extreme acidophiles ‘F. acidarmanus’ and F. acidiphilum is due to the differences in lipid head-group structures and ion permeability. The studies of liposomes derived from P. oshimae membrane lipids [16], establish several factors are responsible for the low permeability of acidophile membranes. These are for instance firstly the fixed nature of the monolayer so that fracturing of these membranes does not cleave the two opposite lipid layers and opposing polar head groups [16]; then secondly a bulky isoprenoid core [17]; and lastly the ether linkages characteristic of these membranes are less sensitive to acid hydrolysis than ester linkages [9]. It has been reported that the membrane channels also contribute to homeostasis as they have a reduced pore size [18]. Internal buffering/cytoplasmic buffering may also maintain pH homeostasis [18]. All microbes contain an array of cytoplasmic buffer molecules that have basic amino acids viz. lysine, histidine and arginine, capable of sequestering protons. For example, the decarboxylation of glutamate and arginine in E. coli is involved in cell buffering by consuming protons, which are then transported out of the cell. Other buffering molecules present within the cell include phosphoric acid (H3PO4) and at near-neutral pH the addition or removal of protons has a negligible effect on the pH of this molecule. The microorganisms acclimatize to the environmental pH changes for their survival. Small variations in pH may possibly be précised by potassium/proton and sodium/proton antiport systems in bacteria. Although, other mechanisms come into play,when pH becomes too acidic. A protontranslocating ATPase regulates this defense mechanism, either by making more ATP or by pumping protons out of the cell. If the external pH reduces to 4.5 or less, chaperones such as acid

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shock proteins and heat shock proteins are synthesized. Most likely these prevent the acid denaturation of proteins and assist in the refolding of denatured proteins. Microorganisms usually change the pH of their own environment by producing acidic or basic metabolic waste products. It has been reported that the fermentative microbes form organic acids from carbohydrates, whereas chemolithotrophic microorganisms like Thiobacillus carry out oxidation of reduced sulfur components to sulfuric acid. The studies demonstrate that in acidophiles intracellular enzymes might be stabilized by ‘iron rivets’. For example, Ferroplasma acidiphilum proteome contains a uniquely high proportion of iron proteins that might confer to the pH stability of enzymes at low pH [19]. III.

ALKALOPHILES

Alkaliphiles are the mesophilic microorganisms that involve two main physiological groups: alkaliphiles and haloalkaliphiles [20-22]. The alkaliphiles include different groups of bacteria, archaea, cyanobacteria,yeast and filamentous fungi. The major assemblage of alkaliphiles is found in the bacterial genera Bacillus, Micrococcus, and Pseudomonas. Alkalithermophiles as well as alkaliphiles have been isolated mainly from alkaliphilic and thermobiotic environments such as alkaline hot water springs, the new alkaline hydrothermal vents of the »Lost City« and alkaline lakes in Africa, Egypt and Israel. However, various alkalithermophiles have been isolated from mesobiotic, slightly acidic to neutral habitat and sometimes even from acidic soil samples and sewage. Haloalkaliphiles have been mainly found in extremely alkaline saline habitat, such as the Rift Valley lakes of East Africa and the western soda lakes of the United States [21] . Alkaliphiles need an alkaline pH of 8 or more for their growth with an optimal growth pH of about 10, whereas haloalkaliphiles require both an alkaline pH (pH>8) and high salinity (up to m/V (NaCl) = 33 %). The term alkalithermophilic has been used for microorganisms that grow optimally under two extreme conditions: at pH values of 8 or above and at high temperatures (50–85 °C). Some of the alkalithermophiles require additional salt concentrations for their growth. One of the most attracting properties of alkalophiles is that they maintain a neutral pH internally by the use of proton pumps and therefore their intracellular enzymes rescue from the adaptation to extreme growth conditions. Extreme alkalophiles for example, Bacillus alcalophilus maintain their internal pH closer to neutrality by exchanging internal sodium ions for external protons. Alkalophiles maintain low alkaline level of pH 8 inside the cells by constantly pumping in hydrogen ions (H+) across the cell membranes into their cytoplasm. These hydrogen ions in the form of hydronium

ions (H3O+) were pumped inside the cytoplasm. Alkaliphiles thrive in extreme alkaline environment depending upon the mechanism viz. active and passive regulation. Sodium ion channel drive the active regulation, whereas polyamines in cytoplasm and low membrane permeability are two modes of passive regulation. . These polyamines are rich in amino acids that contain positively charged side groups (such as arginine, lysine and histidine), that exist in some cells and because of its presence, the cells maintains the pH of their cytoplasm in alkaline environment. In order for the cells to survive in the aggressive conditions of pH, alkaliphiles utilize several alternative strategies. In addition to peptidoglycan, alkaliphiles have negatively charged cell-wall polymers which may reduce the charge density at the cell surface and help to stabilize the cell membrane [21-22]. Cellular fatty acids in alkaliphilic bacterial strains contain primarily saturated and mono-unsaturated straight-chain fatty acids [21-22, 24]. In spite of all this, the extracellular enzyme proteins of alkaliphiles require to function at alkaline pH. The mechanism of action of these extracellular proteins at high pH values is yet not clear. The mechanism of adaptation of alkaline enzymes is been elucidated on the basis of comparison between the structures of alkaline and non-alkaline enzymes. Several experimental and theoretical physico-chemical studies have been performed and the current theories of protein high-pH adaptation appear to fall into three major categories, namely, pKa modulation, Asp+Glu gain(+DE), and Asp+Lys loss-Glu+Arg gain(DK+ER) strategies. The adaptation of enzyme function to alkaline condition is modulation of the pKa of catalytic residues toward a higher pH. This can be achieved by modifying the hydrogen bonds of catalytic residues, shielding the catalytic residues from the solvent or changing the net charge of the protein molecule. The comparative studies from alkaline and non-alkaline phosphoserine aminotransferases have shown that the ratio of surface- exposed Asp+Glu to Lys+Arg was higher for the alkaline enzymes ( { Asp+Glu}/{ Lys+Arg}=44/32 and 43/34 for BALC PSAT from B.alcalophilus and BCIR PSAT from B.circulans, respectively) compared to the E.coli homologue ({ Asp+Glu}/{Lys+Arg}. The crystal structure analysis of the high – alkaline M-protease, the alkaline cellulase CelK, and the alkaline α- amylase AmyK, together with ancestral sequence evolutionary trace (ASET) was done to find the mode of adaptation to alkaline environment. Lys and Asp tended to decrease and Arg,His, and Glu tended to increase in the alkaline – adaptation process. Some hydrophobic amino acid contents also showed significant changes. Leu was lost and the relatively smaller hydrophobic residues Ala and Val appeared to be acquired in the adaptation process, which

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suggests that changes in hydrophobic interactions were involved in the alkaline adaptation [25]. Thermoalkaliphiles and alkaliphiles are good sources of alkaliphilic enzymes like amylases, cellulases, xylanases, proteases, lipases, peroxidase ,pectinases, chitinase, catalase and oxidoreductase [21-22] .Thermoalkaliphilic enzymes have great biocatalytic potential in processes that are carried out at alkaline pH and higher temperatures. For example, lipases, proteases, and cellulases are used as additives in laundry and dishwashing detergents. Proteases has been used for dehairing of hides, skins and to improve smoothness and dye affinity of wool [20-22, 26]. Cellulase-free xylanases are employed for biobleaching of pulp and paper , catalase and peroxidase or oxidoreductase may be used to remove residual hydrogen peroxide from effluent streams of the textile processing industry [21-22, 27-29]. Due to their versatile applications, alkaliphilic enzyme-producing bacteria and archaea have received great attention in recent years [21-22]. IV. PSYCHROPHILES The growth of microorganisms is significantly affected by the environmental temperature .It is well documented that microorganisms are particularly responsive because they are usually unicellular and their temperature varies with that of the external environment. Both function and cell structure is affected when microorganisms are above the optimum temperature. If temperatures are very low, function is affected but not essentially chemical composition and structure of the microbial cell. Psychrophiles are the microorganisms which grow well at 0°C and have an optimum growth temperature of 15°C or lower; the maximum is around 20°C. These microorganisms have adapted to their environment in various ways. Their protein synthetic machinery, enzymes and transport systems function well at low temperatures. The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids that allow them to remain semifluid under cold climatic conditions. Even, many psychrophiles begin to leak cellular constituents at temperatures higher than 20°C because of disruption of cell membrane. In permanently cold environment like Antarctica, temperatures never exceed 5oC. The communities in this region include photosynthetic eukarya, particularly algae and diatoms, as well as a diverse number of bacteria. Polaromonas vacuolata, is a prime representative of a psychrophile: its optimal temperature for growth is 4 o C, and it finds temperatures above 12 oC too warm for reproduction. Extremozymes from psychrophilic or ‘cold-loving’ microorganisms could find use in numerous applications as

they are able to function with reduced energy requirements. Adequate washing in cold water could be allowed by the addition of psychrophilic polymer-hydrolyzing extremozymes such as β-glycanases to detergents. Psychrophilic polymerdegrading enzymes could also find its use in the paper industry, aiding in manipulation of pulp or in bioremediation processes [30]. Cold environments, nonetheless, expose proteins with a number of physical challenges. Although molecular studies of the alterations adopted by psychrophilic extremozymes are in their blastoff, several properties are apparent. Compare to their mesophilic counterparts cold adapted proteins contain fewer disulfide bonds and salt bridges, specific sequence modifications, increased solvent interactions, helix dipole structures of lower net charge, a decreased number of hydrogen bonds at domain interfaces and a lower degree of hydrophobic interactions in the core of the protein [31]. Together, these adaptations yield proteins that are structurally flexible and consequently catalytically effective in the cold. Indeed, these modifications are essentially the opposite of those alterations that impart heat stability to thermophilic proteins. V.

THERMOPHILES

Thermophilic microorganisms can grow at temperatures of 55°C or higher. The minimum temperature falls around 45°C and generally with an optimum range between 55 and 65°C. Majority of them are procaryotes although a few algae (Cyanidium caldarium) and fungi (Mucor pusillus )are also thermophilic. These organisms flourish in many habitats involving composts, self-heating hay stacks, hot water lines, and hot springs. In comparison to mesophiles thermophiles contain much more heat-stable enzymes and protein synthesis systems that are capable to function at high temperatures. Also their membrane lipids are more saturated than those of mesophiles and have higher melting points; consequently thermophile membranes remain intact at higher temperatures. A few thermophiles can grow at 90°C or above and some have maxima above 100°C. Procaryotes having growth optima between 80°C and about 113°C are called hyperthermophiles. They commonly do not grow well below 55°C. In hot areas of the seafloor certain marine hyperthermophiles Pyrococcus abyssi and Pyrodictium occultum has been reported. Though extreme thermophiles include both archaeal and bacterial species, whilst the vast majority of hyperthermophiles are members of the Archaea [32]. Microorganisms found at the highest temperatures (103–113 o C) are solely archaeal [33] .Hyperthermophiles essentially rely on the reduction of elemental sulfur for energy generation, that result, in most cases, in the generation of H2S and due to this cultivation of hyperthermophiles is problematic. Consequently the large number of studies on the extremozymes of hyperthermophilic archaea has bring into

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line the strains belonging to the genera Pyrococcus and Thermococcus, which are also capable of showing growth in the absence of sulfur [32]. The focus has been turned towards the application of thermophilic extremozymes because of their relevance in biotechnologically related processes which require to be carried out at higher temperatures. For example, in chemical reactions comprising organic solvents, higher reaction rates occur at elevated temperatures as there is decrease in viscosity and increase in diffusion coefficient [34-35]. Such applications are relevant to a variety of processes including those involving hydrophobic compounds that normally display low solubilities. Increased temperatures can also augment the availability of such compounds for biodegradation efforts. Furthermore, achieving reactions at higher temperatures reduces the possibility of complications resulting from contamination. Hence, the various molecular approaches adopted by thermophilic extremozymes in adapting to extremely high temperatures are apparently genetically ciphered. As explained by biophysical and structural studies [36], such adaptations (relative to their mesophilic counterparts) include modification of sequences, like replacement of conformationally strained residues by glycines, salt bridges addition, additional ion pairing and hydrogen bond formation ,increased hydrophobic interactions, improved core packing and shortening of loops. These approaches, used to differing dimensions by different thermophilic proteins, provide not only higher thermal stability to proteins but also reinforce rigidity and resistance to chemical denaturation. VI.

PIEZOPHILES

Piezophiles are broadly defined as organisms that show optimal reproduction rates at pressure >0.1Mpa. The majority of piezophiles can also be classifed by the temperatures where they flourish: thermopeizophiles grow in high temperatures and pressure while psychropeizophiles grow in low temperatures and high pressure [37].Piezophiles are found underwater, at effectually all depths. Depending on the location of their underwater habitat, piezophiles deal with different temperatures. For example, the thermopiezophiles would be found around deep-sea vents [36].The fluidity of biological membrane get reduced at low temperature and high hydrostatic pressure because of increased of fatty acyl chains [38].Some reports has shown that a pressure of 100 Mpa (1000 atm) with a temperature of 2◦ C exert a similar impact on membranes as a temperature of −18◦ C at atmospheric pressure. This harmonious effect is significant in many deep sea environment with the exception of at hydrothermal vents. Deep-sea microbes preserve membrane functionality at high pressure and low temperature by increasing the percentage of unsaturated fatty acids in their lipids. The study on piezosensitive, piezotolerant as well as obligately piezophilic

Shewanella strains [39], implied a trend in amino acid composition of a single-stranded DNA binding protein, indicative of pressure adaptation. The increasing pressure optimum for the source strain was associated with the reduction in glycine and proline composition. It was proposed that a reduction in the helix-breaking amino acid proline and helix destabilizing amino acid glycine residues reduces the flexibility of single-stranded-DNA-binding protein (SSB) from Shewanella PT99. A proline to glycine substitution in staphylococcal nuclease which both increased the stability of the protein to elevated pressure as well as decreased chain mobility has also been reported [38]. This modification could reflect a greater compression in the folded state. The first genomic comparison was performed determining amino acid substitutions in 141 aligned orthologous proteins obtained from Pyrococcus furiosus, a pressure-sensitive archea and Pyrococcus abyssi [40]. The statistically significative deviations of the single amino acid substitution suggest that arginine, glycine, valine and aspartic acid have the most piezophilic behavior, while tyrosine and glutamine the least piezophilic. High pressures of a few hundred MPa can be used during processing and sterilization of food materials to induce the formation of gels or starch granules, the denaturation/coagulation of proteins or the transition of lipid phases.The use of high pressure results in better flavor and color preservation than the use of high temperature to accomplish the same ends [23, 41-43]. In addition, enzymes that can act at increased pressure and temperature have great advantages in biotechnological applications. Despite of numerous possible biotechnological applications of piezophiles and piezophilic enzymes, there are few known practical applications of piezophiles or piezophilic enzymes [23],[41].The reason may be the extreme high pressure conditions in which they are cultivated. This property made piezophilic enzymes and other cellular components less explored by the researchers. VII.

HALOPHILES

Halophiles are salt-loving organisms that populate hypersaline habitats. Also, a term “halotolerant,” has been given to some organisms indicating that the organism has the ability to grow in both the hypersaline environments and non-saline environments, but for optimum growth saline is not required. They include mainly prokaryotic and eukaryotic microorganisms having the ability to balance the osmotic pressure of the environment and combat the denaturing properties of salts. Halophilic microorganisms include diverse number of heterotrophic and methanogenic archaea; heterotrophic, lithotrophic and photosynthetic bacteria; and photosynthetic and heterotrophic eukaryotes. Halobacterium species (archaea group), Aphanothece halophytica

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(cyanobacteria) and the Dunaliella salina (green alga) are the examples of well-adapted and widely distributed extremely halophilic microorganisms. The salt concentrations at which halophilic organisms grow can be anywhere from 3% to 35% [44]. These organisms can be found in environments such as sea water and hypersaline lakes (the Dead Sea, the Great Salt Lake) .Much like thermophiles, halophiles can also be split into three different groups; instead of optimum growth temperature, the groups are placed on optimum salinity. These are slight halophiles that grow at an optimum salinity 2% to 5%, moderate halophiles that grow at an optimum salinity of 5% to 20%, and finally extreme halophiles that grow at an optimum salinity of 20% to 30% [45]. An interesting property of hypersaline environments is the formation of gradients in concentration with respect to time. As small form of hypersaline waters vaporize, the salinity gradually increases. The salinity of water can initiate at 1M NaCl, but with time the salinity can increase to over 5M NaCl [46]. This results in natural fluctuations in the halophilic species that inhabit that particular body of water. As, for example, when water is around 1M to 3M NaCl, the environment conduce to be filled with algae, protists, and yeasts [45]. However, as evaporation occurs, and the salinity increases 5M, those microrganisms diminish because they cannot bear such high salt concentrations. Microrganisms that can withstand at these higher salt concentrations, such as red-orange halobacteria, exceptionally increase in numbers until the body is completely dried up or reduced back to a lower concentration [45]. The accumulation of red-orange halobacteria populations are very striking and blooms can be as dense as 108 cells per mL [45]. However, all halophiles face difficulties in surviving, no matter what level of salinity the organism can bloom at.One of the considerable problem faced by halophiles in maintaining homeostasis is the balance of osmotic pressure. Halpophiles maintain proper osmotic pressure in their cytoplasm by using two strategies. The first comprises the accumulation of molar concentrations of potassium and chloride. This strategy depends upon the comprehensive adaptation of the intracellular enzymatic machinery to the presence of salts, as the proteins from these halophiles should maintain their appropriate conformation and activity at nearsaturating salt concentrations. The proteome of such organisms is profoundly acidic, and most proteins denature when suspended in low salt. By and large, the microorganisms depending on such "salt-in" strategy are obligate halophilic Archaea. Another strategy of haloadaptation is based on the biosynthesis and/or accumulation of organic osmotic solutes (osmolytes) like ectoin, glycine, betaine or others [46]. Cells entrusting on this strategy exclude salts from their cytoplasm as much as possible. These organic "compatible" solutes even at high concentrations do not largely interfere with normal enzymatic activity. Therefore

fewer adaptations of the cells proteome are required. Such organisms can often adapt to a broad range of salt concentration [47]. The halophilic proteins from "salt-in" organisms are differentiated from their non-halophilic homologous proteins by showing remarkable instability in low salt concentration and by maintaining soluble and active conformations in an environment generally detrimental to other proteins. Undoubtedly, hypersaline conditions encourage protein aggregation and collapse, hinder the electrostatic interactions between protein residues, and are accountable for a general decrease in the availability of water molecules [48]. Instead of being folded by these conditions, halophilic proteins, appears to depend on the presence of salts. In recent years, detailed investigations have been performed to uncover the correlation between structure and stability in halophilic proteins [49]. In particular, the findings proposed that the halophilic proteins bind significant amounts of salt and water in solvent conditions similar to their physiological environment. The distinct ability of halophilic proteins to bind large amount of salts is largely dependent on the number of acidic amino acids on protein surface [50-56]. Electrostatic interactions are the key determinant of haloadaptation as they play a role in the stability and folding of halophilic proteins [57-58]. Moreover, it was discovered that halophilic proteins are characterized by a general decrease in hydrophobic amino acid frequency and a greater ability to form random-coil structures, instead of α-helices [54]. Negative charges on the halophilic proteins bind significant amounts of hydrated ions, thus reducing their surface hydrophobicity and decreasing the tendency to aggregate at high salt concentration. The strategy of haloadaptaion not by osmolytes but by the presence of molar salt concentration weakens the hydrophobic interactions, more importantly at the level of conserved hydrophobic contacts. These weakening of hydrophobic interactions maintain their strengthening by the presence of salts in solution. This helps to prevent structural aggregation and/or loss of function in hypersaline conditions. The major sources of extremely halophilic enzhymes are the halophiles belonging to archaeal domain. Some halophiles belonging to the genera Acinetobacter, Haloferax, Halobacterium, Halorhabdus, Marinococcus, Micrococcus, Natronococcus, Bacillus, Halobacillus and Halothermothrix has been reported to produce halophilic enzymes, such as xylanases, amylases, proteases and lipases [20, 49-50, 59, 6063]. Although these enzymes can perform enzymatic functions identical to those of their non-halophilic counterparts, but they exhibit peculiar properties in the sense that these enzymes require high salt concentrations (1–4 M NaCl) for activity and stability and a high amount of acidic over basic amino residues

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[50] . The presence of high negative surface charge in halophilic proteins allows them to be more soluble and renders them more flexible at high salt concentrations, environments under which non-halophilic proteins tend to aggregate and become rigid. This high negative surface charge is neutralized mainly by tightly bound water dipoles [49-50, 62-63].These halophilic enzymes require high salt concentration for the stabilization bacause of low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thereby stabilizing the active conformation of the protein [50]. The dependence of halobactrerial proteins on salt concentration for stability and the catalytic properties has been the subject of studies for many years. The unique sparingly soluble halophilic enzymes are advantageous as they are employed in aqueous/organic and non- aqueous media [64-65]. The use of halophilic enzymes in organic solvents has been limited to very few enzymes [60]. To explore the nature of these extremozymes in organic media, different strategies have been adopted, involving the dispersal of the lyophilized enzyme or the use of reverse micelles [65-66] . Indeed, the use of reverse micelles in maintaining high activities of halophilic extremozymes under unfavourable conditions could unveil new fields of application such as the use of these enzymes as biocatalysts in organic media. The reverse micelles are considered as micro-reactors, they are very dynamic and vary with the environmental conditions [65-66]. Halophilic enzymes encapsulated in reverse micelles maintain catalytic properties in organic solvents, even at low salt concentrations. For example, the effluents generated during manufacture of chemicals such as pesticides, herbicides, explosives and dyes contain complex mixtures of xenobiotic compounds, salt and organic solvents. Halophilic extremozymes encapsulated in reverse micelles can be used in bioremediation as to reduce or remove environmental hazards resulting from accumulation of toxic chemicals and other hazardous wastes [65-66]. CONCLUSION Thermophiles, acidophiles, alkaliphiles, halophiles and piezophiles all these organisms grow under extreme conditions. These organisms have filled niches on Earth through their different mode of adaptations to face almost any adverse conditions. Through the ongoing research on extremophiles and extremozymes, understanding of protein folding, stability, structure and function has added great information regarding their adaptability. In many industrial processes (food and feed, agriculture, detergents, leather, textile, pulp and paper), several research groups are investing money and time for exploiting the potential of these microbes and extracting extremozymes from these microorganisms.

Although there are potential difficulties that are associated with the cultivation of extremophiles, genetic engineering of the extremozymes of interest into mesophilic hosts will allow production of these extremozymes at large- scale. These new findings may revolutionize biotechnology through novel applications of extremozymes. ACKNOWLEDGMENT We acknowledge the Dr. Poonam N. Singh for assisting in preparation of the manuscript. REFERENCES 1. 2.

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