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ISSN: 2155-9910
Journal of Marine Science: Research & Development

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Psychrozymes- The Next Generation Industrial Enzymes

K.K. Pulicherla1, Mrinmoy Ghosh2, P. Suresh Kumar2 and K. R. S. Sambasiva Rao2*

1Department of Biotechnology, R.V.R. and J.C. Collage of Engineering, Chowdavaram, Guntur-522019, Andhra Pradesh, India

2Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur-522510, Andhra Pradesh, India

*Corresponding Author:
Dr. K.R.S. Sambasiva Rao
Department of Biotechnology
Acharya Nagarjuna University
Nagarjuna Nagar, Guntur- 522510
Andhra Pradesh, India
Tel: +91 9440869477
E-mail: [email protected]

Accepted date April 26, 2011; Published date April 29, 2011

Citation: Pulicherla KK, Ghosh M, Kumar PS, Sambasiva Rao KRS (2011) Psychrozymes- The Next Generation Industrial Enzymes. J Marine Sci Res Development 1:102. doi:10.4172/2155-9910.1000102

Copyright: © 2011 Pulicherla KK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The earth is dominated by low-temperature environments. Over 80% of the earth’s biosphere is cold, and about 90% of the oceans are colder than 5°C. This cold marine environment is characterized by challenging conditions for the survival of native microorganisms and are classified as psychrophilic or psychrotolerant organisms. The biocatalysts, produced by these microorganisms are called as cold adapted enzymes, function under extreme cold condition and display a high specificity. It is associated with a relatively extreme stability provides new opportunities for the potential application of these cold adapted enzymes in various industrials processes. The main objective of current review is to discuss the sources, possible industrial applications and current scenario of the cold adapted enzymes.

 

Keywords

Extremophiles; Psychrophiles; Cold adapted enzyme; Anti freezing protein

Abbreviations

FP: Freezing Point; CSDS: Cold Shock Domains; CAPS: Cold-Acclimated Proteins; HSPS: Heat Shock Proteins; AFPS: Anti Freezing Proteins; GOS: Galacto-Oligosaccharides; ToMO: Toluene-O-Xylene Monooxygenase; LPUFA: Long-Chain Polyunsaturated Fatty Acids.

Introduction

Nature is the platform of varsity. Among all organisms, microbial world gives a true example, reflecting through their taxonomical characters, psychological properties, genomic and proteomic expression. Many of them are adapted to grow harmoniously under hazardous environments such as acidic or alkaline condition, hot spring environments, dry rock surfaces or deserts, cold water or deep in the sea. Based on their growth conditions, microorganisms have been divided into three broad categories: thermophiles, mesophiles and psychrophiles.

Approximately 71% of the earth's surface i.e. 361 million square kilometers is covered by the oceans which is believed to contain a total of approximately 3.67 × 1030 microorganisms [1]. According to Bruun's (1959) ecological zonation of the deep sea, the psychrosphere occupied at least 90% marine environment and their average annual temperature is in between 10°C to 5°C. However, based on the growth temperature, psychrospheric organisms are again sub-divided into two categories such as psychrophiles (optimal growth temperature below 15°C) and psychrotrophs or psychrotolerants (grow at optimal temperature of around 20-25°C) [2]. Moreover, this environment represents an enormous pool of potential microbial biodiversity, range from Gram-negative bacteria (e.g. Pseudoalteromonas, Moraxella, Psychrobacter, Polaromonas, Psychroflexus, Polaribacter, Moritella, Vibrio and Pseudomonas), Gram-positive bacteria (e.g. Arthrobacter, Bacillus and Micrococcus), Archaea (e.g. Methanogenium, Methanococcoides and Halorubrum), Yeast (Candida and Cryptococcus) and Fungi (Penicillium and Cladosporium) [3-7], collectively all these organisms revolutionized the cold marine biotechnology.

There is an increasing demand for the use of microbial biocatalysts in industrial application because of their withstanding nature at various robust processing conditions. Most of the enzymes which are in use in today's industrial processes are sourced from mesophiles. Despite their many advantages, the application of the mesophilic enzymes is restricted due to their limited stability at the extremes of temperature, pH and ionic strength [8]. Moreover the drive for cost-cutting lies in the heating or cooling steps of industrial processes and increase in the recovery of the products of enzymatic reaction [9] can give a much attention to the use of proteins isolated from cold loving microorganisms.

The documented data of psychrophiles revealed the ability to degrade a wide range of polymeric substances and the producer of various enzymes like amylases, cellulases, pectinases, β- Galactosidase, oxidases, protease and lipase etc. Due to the potential properties, these cold-active enzymes has led to accelerate the interest in investment of a huge amount of money and effort world wide in the research and development for exploring more number of industrially important cold active enzymes in the fields of food industry (such as pectinase, β-galactosidase), biopolishing and stone washing of textile products and detergent formulation industries (such as lipases, amylases, and cellulases). Moreover these psychrozymes are successfully used in bioremediation (such as oxidases) and for biotransformations (methylases and aminotransferases) [10] as well as in biomedical applications. This review broadly covers the Microbial resources of the cold adapted enzymes and effect of the various potential factors on its activity and their industrial applications.

Cryo-defense strategy of marine cold loving microorganisms

Temperature is one of the most important environmental factors for life as it influences most biochemical reactions. Research has been in progress to reveal the biochemical and molecular mechanisms of various microorganisms that can help to establish the thermal sensitivities, optimal and tolerance limits of differently adapted species and differently acclimated or acclimatized populations of a single species. These studies have given clues about the changes in membrane and temperature dependent lipid compositions [11], antifreeze and icenucleating molecules involved in freeze avoidance or freeze tolerance [12,13], temperature dependent pH regulatory process [14,15], evolutionary and acclamatory changes in structural proteins.

The active life always require liquid water; but the cold loving organisms are facing the physical limitations due to the super-cool or frozen [16] property of water at subzero environment. If cooling is sufficiently slow and ice is formed at extra-cellularly, the cell loses water rapidly enough by exosmosis resulting in increasing the concentration of intracellular solutes that maintains the chemical potential of intracellular water in equilibrium with the extracellular water, by that the cells are not dehydrated and freezed intra-cellularly. But if the cell is cooled too rapidly, it is not able to lose water fast enough to maintain equilibrium and can eventually freeze intra-cellularly [17,18]. This lower temperature limitation is commonly defined as the freezing point (FP) for cellular water which can drop significantly below 0°C for many organisms. However, the low temperatures and freezing conditions influence the lives of all organisms in multiple ways, e.g., reduced biochemical reaction rates, increased viscosity of the medium, changes in membrane fluidity and protein conformation, nutrient availability, ability to reproduce successfully and need for protection against freezing.

For maintaining the membrane stability, the marine psychrophilic organisms show important adaptations in the cell membrane, such as the production of polyunsaturated fatty acids to maintain fluidity and synthesis of enzymes, which are capable of catalysis at low temperatures [19]. Moreover as temperature is decreased, fatty acid side chains in membrane bi-layers undergo a change from a liquid crystalline state to a gel-like state [20]. Suutari and Laakso reported that at low temperature the ratio of unsaturated fatty acids to saturated fatty acids is increased in the membrane. It can modulate the activities of the enzymes involved in fatty acid and lipid biosynthesis[21] and help to synthesize the short-chain fatty acids, branched chain fatty acids and anteiso-fatty acids to long chain fatty acids, straight-chain fatty acids and iso fatty acids respectively [22]. Some investigators believed that cryoprotectants prevent the cold induced aggregation of proteins and maintain the optimum membrane fluidity even at low temperature. The evidence on cryoprotective role of glycine which act as a betaine in bacteria was mentioned by Chattopadhyay [23].

The genomes of psychrophiles contain the cold shock domains (Csds) or folds that have putative roles in RNA stabilization [24]. Degradosome, a protein complex consists of polynucleotide phosphorylase and RNA helicase that makes sure of the stability of cellular RNA [25]. CsdA had also been identified as an essential multifunctional protein at low temperatures, which is involved in biogenesis of the 50S ribosomal subunits. The data showed that CsdA associates with 50S precursors at low temperatures and can complement the ribosome defect. Moreover, it was examined that deletion of CsdA gene from mutant E. coli can reduce the growth rate and exhibit filamentous growth at low temperatures [26].

Cold-acclimated proteins (Caps) is a set of 20 proteins, which seem to be an important and general feature of the cold-tolerant microorganisms. It can over express under low temperature to prolong the growth phase [27]. The Caps have been well found both in mesophiles and psychrophiles. The recent study has demonstrated that expression of CspA-like proteins give an immediate response against the temperature downshift in all the psychrotrophic bacteria [28].

The ongoing research on psychrophiles reveals the secrets of cold loving microorganisms at genomic as well as proteomic level. Heat shock proteins (Hsps) are believed to protect the organisms from thermal stress. However, it was found that Hsps also play a significant role in cold adapted bacteria [29] for increasing cell survivability. Under various types of stress conditions, particularly under oxidative stress, Hsps help to stabilize the native state of cellular proteins.

So far at low temperature, five different types of anti freeze proteins (AFPs) have been reported so far in fish’s blood. Duman and Olsen were the first to demonstrate the presence of thermal hysteresis proteins in bacteria [30]. In 2002, Yamashita demonstrated the presence of AFPs in Antarctic bacteria named Moraxella sp [31]. Unlike many other extracellular bacterial AFPs, the AFP activity in Moraxella sp was found only in the supernatant of cell lysate [32]. Hence, it was suggested that these proteins might be localized at the periplasmic space. The discovery of the AFPs appears to be a significant addition at the state of knowledge on anti freeze strategy of psychrophilics. The summarization of ice nucleating proteins exhibit four main effects on cold loving species [33], such as a) the formation of ice is controlled by the organism and progressively formed by that enabling appropriate metabolic adjustments, b) in this way cryoprotectants can also be produced concomitantly preventing excessive dehydration, c) ice is confined into the extracellular space, d) the formation of extracellular ice induces limited cell dehydration and that also contributes to depress the freezing point of the intracellular fluid.

Psychrozymes and their stability

The stability details of any enzyme are related to the three dimensional structural features of that particular protein molecule. In the case of psychrozymes, it seems that all weak interactions (ion pairs, aromatic interactions, hydrogen bonds and helix dipoles) are less abundant and non-polar core clusters have a weaker hydrophobicity, making the interior of the protein less compact. The diminishing numbers of proline residues can restrict the rotation of enzymes and thearginine residues for developing salt bridge and hydrogen bonds are less where as the clusters of glycine residues which essentially have no side chains provide the localization of chain mobility. Moreover, stabilizing cofactors bind weakly and loose or relaxed protein extremities seem to be unzipping. It was reported that in the case of multimeric enzymes, the cohesion between monomers is also reduced by decreasing the number and strength of interactions that are involved in the association [34].

Enzymes from marine cold loving microorganisms

Studies on marine microorganisms not only provide the information on the key role that they play in marine food webs and biogeochemical cycling in marine ecosystems but also help in exploiting their ability to produce novel enzymes and metabolites or compounds with potential biotechnological applications [35]. Hence, the research on psychrophiles mostly concentrates on four main areas;

(i) Enzymes in industrial processes including food technology;

(ii) Bioremediation and other pollution control technologies;

(iii) Use of Anti-freeze proteins;

(iv) Medical and other Pharmaceuticals uses;

Enzymes in industrial processes including food technology: Of course from many decades the uses of enzymes in industries are well known and nowadays the enzymatic methods occupy an important and essential role in modern industrial processes. Almost all bacterial enzymes for the industrial products are isolated from mesophiles, simultaneously due to the heat-stability, the thermophilic enzymes are also frequently used in industries as ideal biocatalysts. But in some industrial applications, the enzymatic reactions have to be carried out at low temperature. Therefore, in such cases cold adapted enzymes could be more applicable over mesophilic or themophilic enzymes. Hence, with all other benefits such as high specific activities at low temperature, less ion bonds, the cold adapted enzymes can offer many other advantages like energy saving, saving of labile or volatile compounds, prevention of contamination and easy inactivation of enzymes.

Due to these advantages, most of the food industries like to treat the products with cold adapted enzymes for maintaining the quality during transportation and storage. Moreover detergent and textile industries are other two areas where the cold adapted enzymes are utilized frequently (Table 1).

Enzymes Application Advantages
Pectinase, Cellulase Clarification of fruit juice Keeping fragrance
Protease Removal of fish skin Maintaining the product quality
Lysozyme, Oxidase Preservation For volatile and heat sensitive material
β- Galactosidase Modification of constitution Keeping freshness
Protease,Lipase,Cellulase Detergent formulation Used in tap water
Lipase Treatment of waste water For volatile and heat sensitive materials

Table 1: Applications of cold active enzymes.

Pectinase: Pectinases naturally degrade pectic substances therefore, it has vast use in industries where the elimination of pectin is essential such as in fruit juice processing, coffee and tea processing, macerating of plants and vegetable tissue, degumming of plant fibers, extraction of vegetable oil, haze removal from wine and are also used for the production of low methoxy pectin for diabetic foods [36].

The raw pressed juice is rich in insoluble particles mainly made up of pectic substances. When the tissue is grounded, the pectin is found in the liquid phase (soluble pectin) causing an increase in viscosity and the pulp particles. Therefore, it is difficult to extract the juice by pressing or using other mechanical methods. The addition of pectinase can reduce the viscosity of fruit juice and improve the pressing ability of the pulp causing the disintegration of jelly structure thus the fruit juice is easily obtained with higher yields.

Pectinase hydrolyses the residual pectin and hemicelluloses. The pectic enzymes are classified based on their attack on the galacturonan of the pectic substance molecule. There are three types of pectic enzymes are observed; de-esterifying enzymes (pectin esterase) catalyzing the hydrolysis of methyl to produce pectic acid and methanol, depolymerizing enzymes consisting of hydrolases and lyases. Lyases are also called as transaminases, which split the glycosidic bonds of either pectate (polygalacturonate) or pectin (polymethylgalacturonate) and protopectinases [37].

Pectinasess from psychrophiles increase the storage capacity. Godfrey reported that addition of pectinases to the fruit mash i.e. about 40-200 g enzyme/ton for 30-60 min at 15-30°C decreases the viscosity by pectin hydrolysis and thus increases the yield. Among the sources of enzymes for the food industry particularly in the processing of fruit juices, the psychrophilic microorganisms have attracted much attention [38]. Investigations suggested that the hydrolysis of juice by cold adapted pectinase allows to concentrate the sugar in juice which can reduce the risk of contamination simultaneously it can also help to reduce viscosity, clarify fruit juices [39], minimize the cost of product processing and allow to preserve the product for longer duration with maintained quality.

ß-Galactosidase: Glycosidases hydrolyse glycosidic bonds in oligoor polysaccharides and heteroglycosides. Out of these glycosidases, ß galactosidase has tremendous potential in research and also having applications in food, bioremediation, biosensor, diagnosis and treatment of disorders especially in dairy technologies for preparation of lactose free milk and biosynthesis of galacto-oligosaccharides (GOS). In recent years number of cold-active ß -galactosidase sources have been reported such as Arthrobacter psychrolactophilus, Bacillus subtilis KL88, Carnobacterium piscicola BA, Pseudoalteromonas haloplanktis, and Planococcus.

Low activity of β-galactosidase causes digestive insufficiency, called lactose intolerance. Lactose intolerance is a problem for approximately two-thirds of the world's population. So, lactose free milk is prescribed which is available in today's market processed by various chemical and mechanical means. Most preferred way of degrading the lactose in the milk is by treating with β-galactosidase sourced from psychrophiles. Moreover removal of lactose from milk provides higher solubility, suppression of lactose crystallization in sweet condensed milk and ice creams, increase of sweetness, decrease in the hydroscopicity of dried dairy products etc. For treatment of milk, pH and temperature are the most important conditions for sustained enzyme activity. An ideal β -galactosidase should be active at pH 6.7 to 6.8 and at 4 to 8°C during shipping and long storage conditions for eliminating any contamination with mesophilic microorganisms and to avoid nonenzymatic browning products formed at higher temperature.

Currently for lactose hydrolysis, β-Galactosidase isolated from mesophilic Kluyveromyces lactis has performed poorly at 20°C, hence under these conditions the enzyme activity decreases 5-10 times and is not suitable for long time storage [40], therefore it can be replaced with a psychrophilic β-Galactosidase that can shorten the process of lactose hydrolysis. The main highlighted products derived from cold adapted β-galactosidase by lowering the lactose processing are; low lactose milk, low lactose concentrates for ice cream, food syrups and sweetener manufacture, low lactose yoghurt and sweetened yoghurt of acid and sweet whey.

Kur reported that recombinant cold-adapted β-galactosidase from Antarctic bacterium Pseudoalteromonaus haloplankic displayed high yielding in lactose hydrolysis in the temperature range of 0-30°C; approximately 90% of the milk lactose was hydrolyzed by this enzyme after 6 hrs at 30°C and after 28 hrs at 15°C, respectively [41].

Proteases: Due to the inefficiency of non enzymatic detergents to remove proteins from textile fibers, nowadays, proteases are the most widely used enzymes in the detergent industry and is a useful catalyst for "peeling" of leather. Moreover, the recent developments on cold adapted enzymes suggested that cold proteases possessing great catalytic efficiency at low temperatures even allow washing of textiles at room temperature [42]. In industries, for dehairing of hides and skins using psychrophilic proteases not only save energy but also reduce the impacts of toxic chemicals used in de-hairing.

In comparison to mesophilic enzymes, the specific activity of the psychrophilic protease is four time higher at 4°C and 25°C, and its half time of denaturation at 45°C (2 min) is 10 times shorter [43]. Thus the major drawback in the use of cold proteases for such purposes is their weak stability at temperatures higher than 25°C. However, in recent years with the growing interest on cold enzymes, researchers are focusing more on the thermal stability and molecular adaptation of these protein molecules[44,45]. Taguchi demonstrated that sequential random mutagenesis exhibited 70% higher catalytic efficiency in comparision with wild type at 10°C when N-succinyl-L-Ala-L-Ala-LEnzymes Pro-L-Phe-p-nitroanilide was used as a synthetic substrate. Moreover it was found that increase in substrate affinity was mostly responsible for the increase in the activity [46]. Hence in coming day's crystallographic study and genetic manipulation on those cold adapted enzymes can exploit the prospects for industrial use of such proteins.

Cellulases: Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis (i.e. the hydrolysis) of cellulose. The definition suggested that the enzymes which hydrolyze hemicellulose are usually referred to as hemicellulase and are usually classified under cellulase. However, enzymes that cleave lignin are occasionally classified as cellulase, but this is usually considered erroneous. Cellulose composed of linear polymers of thousands of glucose residues linked by β-1, 4-glycosidic bonds. The rigid structure makes the cellulose to insoluble form in water and help to resist against the natural biological degradation. In most cases of cellulase activity, the enzyme complex breaks down cellulose to beta-glucose. This type of cellulase is produced mainly by symbiotic bacteria in the ruminating chambers of herbivores. Besides ruminants, most animals (including humans) do not produce cellulase in their bodies, and are therefore unable to use most of the energy contained in plant material.

The carboxymethylcellulase was not affected by laundry components such as surfactants, chelating agents, therefore it proves to be a pioneer application in the detergent industries. The application of cellulase in laundry detergents exhibited color brightness and 'biopolishing' of fabric give the softness and finishing look of the product. The small amount of cellulase application can replace the use of large quantity of pumice stones for production of "faded" jeans. Moreover use of cellulase can reduce the processing cost. Nilsson reported that cellulase enzymes are used to weaken the cellulose polymer and helps in dislodging the weakened fiber by mechanical process well known as "Biopolishing" [47].

In food industries, Cellulase is used commercially at coffee processing. It performs hydrolysis of cellulose during drying of beans. Furthermore, cellulases are widely used in textile industry and in laundry detergents. They are also used in the pulp and paper industry for various purposes, and they are even used for pharmaceutical applications. Cellulase is used for treatment of Phytobezoars, a form of cellulose bezoar found in the human stomach.

The cold adapted cellulase displays the usual properties as like other psychro enzymes produced by cold loving microorganisms, i.e., a high specific activity at low and moderate temperatures significantly at higher rate than the mesophilic counterparts. Microbial cellulases are made up of a three-domain structure consisting of a catalytic module (CM) and a carbohydrate-binding module (CBM), this two region separated by a distinct linker region (LR) comprising a simple or repetitive sequence often rich in proline, threonine, serine or glycine residues. However, in psychrophilic cellulases less number of proline residues is present. The LR (109 residues) is rich in cysteine, aspartate, glycine, valine, serine, threonine, and asparagine residues, whereas the LR of the mesophilic Cel5A, is a peptide of 34 residues rich in serine and threonine. The structural difference in CMs of Cel5G with small-angle X-ray scatter revealed that LR of Pseudoalteromonas haloplanktic is long, extended and highly flexible structure, thereby the cold adaptation of Cel5G could arise not only from the specific properties of the CM, but also from the unusual properties of the LR [48,49].

α-Amylases: Psychrophilic α-amylases are first reported enzyme, on which detailed crystallization studies have been done. Amylases widely distributed in bacteria, fungi, animals, and plants. It catalyze the hydrolysis of starch, glycogen, and related polysaccharides by cleaving internal α-1, 4-glycosidic bond. The psychrophilic α-amylases has been synthesized in three forms such as a signal peptide composing preproenzyme (24 residues), the mature enzyme (453 amino acids, 49 kDa), and a long C-terminal propeptide (192 residues, 21 kDa) which constitutes a structurally independent domain that does not exhibit any folds function or affect the amylase catalytic activity [50].

It is quite interesting that α -amylases from Antarctic bacterium displays approximately 66% amino acid similarity with porcine pancreatic α -amylase [51]. However, the characterization of psychropilic α-amylases at 4°C showed seven folds more kcat and kcat/Km values where as the stability was 10 kJ.mol-1 compared with porcine enzyme [52]. The detailed proteomic study on psychrophilic α-amylases found that it consist of four disulfide bonds such as Cys20- Cys74, Cys120-Cys137, Cys328-Cys355, and Cys402-Cys416 which make an important difference between bacterial and mammalian structure [53]. The flexibility of psychrophiles over mesophiles defined that an extra disulfide bridge connecting in between domain A with domain B limits the movement of the mesophilic domain.

Amylases have wide range of application in industrial processes such as food processing, fermentation and pharmaceutical industries. Although α-amylase is produced by SmF and SSF process but due to optimization of different fermentative parameters, mostly SmF process is preferable at industrial production. The production of α-amylase is essential for conversion of starches into oligosaccharides. The wide spread application is to convert starch into fructose and glucose. The second vast use of this enzyme is in the detergent industries for detergent formulation to enhance the detergent workability and ensure environmental safety. However, the data shows that 90% of all liquid detergents contain this enzyme, where as in textile industries it can be used for desizing process without hampering the fiber quality. For bread making, addition of this enzyme can degrade the starch in the flour into smaller dextrins, which are subsequently fermented by the yeast and generate additional sugar in the dough. Beside that, α-amylase also has anti-staling effect in bread making thus increasing the shelf life and softness of these products. Moreover, α-amylases are used in the pulp and paper industry for the modification of starch of coated paper. α-amylases from cold sources have certain limitations. At lower temperature under oxidizing environment, enzyme's stability is very less; hence their use as a detergent in the industry is very limited. Therefore, it is expected that in coming days the change in structural domain may possibly solve the problem.

Lipase: Lipases have potential utilization in a broad range of biotechnological applications due to the ability to catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. But at industrial production, thermo stable lipases are regarded as the most important. However, the psychrophilic lipase having high activity at low temperature attracted attention for synthesis of organic substances due to their inherent greater flexibility whereas the activity of mesophilic and thermophilic enzymes are severely impaired by excess rigidity.

The study on cold adapted lipase revealed that the marine psychrophilic microorganisms are the potential sources for lipase. Compared with mesophilic and thermophilic lipases, relatively smaller number of cold active bacterial lipases are well studied. Cold active lipases cover a broad application in biotechnological processes like detergents formulation, environmental bioremediations, and additives in food industries, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies [54]. In fact, today cold active lipases are a good choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, and microbiologists.

Bioremediation and other pollution control technologies

Oxidases: With an urgent need of improving biological remediation techniques, the enzyme technology has been receiving increased attention. Human activities in the polar area often involved the use of petroleum hydrocarbons such as the petroleum contamination. Number of studies revealed that cold-adapted microorganisms as well as their enzymes (e.g. oxidase, peroxidase, and catalase) are potential for the low-temperature biodegradation and alternatives to physicochemical methods for the bioremediation of solids and waste waters polluted by hydrocarbons, oils and lipids [55]. However, the biodegradation with cold active enzyme seems to have several advantages over other existing methods. It has been noted that treatment of contaminated soil with psychrophilic enzymes are far more cost efficient than traditional methods such as incineration, storage or concentration. Several studies have been carried out to isolate biodegradable bacterium from unpolluted Antarctic marine water. A study revealed that recombinant Antarctic Pseudoalteromonas haloplanktis express toluene-o-xylene monooxygenase (ToMO) which can efficiently convert several aromatic compounds into their corresponding catechols in a broad range of temperature. It has been suggested that the use of this engineered Antarctic bacterium in the bioremediation of chemically contaminated marine environments and/or cold effluents [56]. Studies have been carried out to analyze the physiology, genetics and ecology of degradative cold-adapted bacteria and the utilization of this knowledge for decontaminating the polluted area.

Application of anti-freeze proteins: The cold loving organisms developed various defense systems to exist in cold environment, AFPs is one of that. As mentioned earlier, marine psychrophiles express high levels of AFPs to adapt thermal hystersis and the prevention from ice formation. Hence numbers of cold adapted bacteria were reported (Pseudomonas putida, Pseudomonas fluorescens, Marinomonas protea, a Moraxella species) for AFPs activity. The studies on AFPs reported that it will be useful in cryosurgery and cryopreservation of cells and tissues. Moreover, in food industries AFPs can be used to improve the quality of frozen food, preservation of food texture and flavour in frozen food. Reduction or prevention of microbial contamination of frozen food can be used as ice nucleators to inhibit recrystallization of ice during freezing and thawing and maintaining the fluidity of ice slurry. Moreover, the direct injection of AFPs can improve the cold tolerance in fishes.

Medical and other pharmaceuticals uses: Studies of psychrophilic bacteria from the Antarctic genus Shewanella and Colwellia have shown that high proportion of cold-adapted bacteria from sea ice possess the ability to produce PUFA, such as eicosapentaenoic acid or docosahexaenoic acid. The traditional source for long-chain polyunsaturated fatty acids (LPUFA) has numerous properties to fight against several diseases such as atherosclerosis, diabetes, high blood pressure. But obtaining LPUFA from fish oil comes with several problems at the risk of large scale production and purification. Moreover, the commercial fish stocks are likely to decline in the future. Algal-derived oils require a relatively high investment of technology and expense compared to the prospect of bacterial fermentation, although bacteria contain a lower proportion of lipid. In bacteria, PUFA are component fatty acids of certain phospholipids which occur in the cell membrane. Generically, long-chain PUFA consists of a long chain of carbon atoms (usually C20 or C22) containing a number of (usually 4-6) methylene interrupted double bonds. A key advantage of bacterial PUFA production is that only a single PUFA is produced, rather than the complex mixture yielded from fish or algal oils.

R & D on potential marine psychrophilic enzymes: The high activity of these cold adapted enzymes at low and moderate temperatures offers potential economic benefits that build up the interest on psychrophilic enzymes [57]. As an example, the "peeling" of leather by cold adapt protease can be done with normal water instead of 37°C. The heat-liability of these enzymes also ensures their fast, efficient and selective inactivation in complex mixtures. It had revealed that only minor structural modifications are needed to adapt a mesophilic homologue to cold temperatures. Moreover, an important achievement in the field has been the construction of a host-vector system that allows the over expression of genes in psychrophilic bacteria even at low temperatures which prevents the formation of inclusion bodies and protects heat-sensitive gene products.

The estimation shows that approximately forty three companies are involved in R&D and/or sale of products sourced from arctic area. The activities of these companies fall into 9 broad categories of specialization. Moreover, thirty one patents or patent applications from US and European database have been identified in relation to inventions based on or derived from the genetic resources of the Arctic. The data shows that 34% of the patents/patent applications identified from enzymes with life science research applications in DNA research, 23% of patents/patent applications identified in Medicine and pharmaceutical categories, 10% of patents/patent applications identified under Cosmetics and skin care, Nutraceuticals, Dietary supplements and other health products, Animal health products including aquaculture, Food technology categories and enzymes with industrial applications constitute 3% of patents identified.

Conclusion

The recent increasing interests on psychrophilic microorganisms not only focus on the genomic and proteomic study to establish the relationships but also on production of industrial important substitutions from psychrophiles. Although the psychrophilic enzymes have high specific activity but short half life makes a major drawback in the use of these enzymes at commercial aspect. One of the most desirable targets in investigations on bacterial cold adaptation is genetically engineered strains that are capable of fulfilling the industrial expectations. Thus, it is necessary to identify the key feature of these cold adapted protein molecules to evaluate their biotechnological potential.

Acknowledgements

The authors are grateful To theDepartment of Biotechnlogy (DBT), Govt. of India for the financial support provided for the ongoing project on cold active enzymes. The authors acknowledge the Acharya Nagarjuna University and R.V.R & J.C. C.E. Guntur, India for providing the facilities to carry out the work.

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