Subscribe Now Subscribe Today
Review Article
 

An Overview of Cold-active Microbial α-amylase: Adaptation Strategies and Biotechnological Potentials



Mohammed Kuddus, Roohi , Jamal M. Arif and Pramod W. Ramteke
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

Absolutely the largest proportion of the Earth's biosphere is comprised of organisms that thrive in cold environments are known as psychrophiles and psychrotrophs. Their ability to proliferate in the cold is predicated on a capacity to synthesize cold-adapted enzymes like amylases, proteases, lipases, pectinases, cellulases, etc. that could be used in low-energy processes. Amylases have most widely been reported to occur in microorganisms, although they are also found in plants and animals. Cold-active α-amylases confer low activation energies and high activities at low temperature which are favorable properties for the production of relatively insubstantial compounds. In addition, these enzymes have an advantage under extreme low temperature conditions due to their inherent greater membrane fluidity, production of cold-acclimation proteins and the mechanism of freeze tolerance. The low temperature stability of cold-active amylases has been regarded as the most important characteristics for use in the industry because of considerable progress towards energy savings but unfortunately these enzymes have largely been overlooked. Now this situation is changing which recently fascinated the scientific community to focus in many fields, such as clinical, medicinal and analytical chemistries, as well as their widespread biotechnological applications such as food processing, additive in detergents, waste-water treatment, biopulping, environmental bioremediation in cold climates and molecular biology applications. This review addresses the present status of knowledge on the source, structure, production and molecular characteristics of cold-active α-amylases and their biotechnological applications.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Mohammed Kuddus, Roohi , Jamal M. Arif and Pramod W. Ramteke, 2011. An Overview of Cold-active Microbial α-amylase: Adaptation Strategies and Biotechnological Potentials. Biotechnology, 10: 246-258.

DOI: 10.3923/biotech.2011.246.258

URL: https://scialert.net/abstract/?doi=biotech.2011.246.258
 
Received: May 09, 2011; Accepted: May 19, 2011; Published: August 11, 2011



INTRODUCTION

Extremophiles are microorganisms that can grow and thrive in extreme environments, like high or low temperature, high or low pH, high salinity, very low water activity, high pressure, low oxygen etc. Extremophiles are structurally adapted at the molecular level to withstand these harsh conditions and among them biocatalysts play a major role which are called as extremozymes produced by these microorganisms. There are various extremozymes such as cellulases, amylases, xylanases, proteases, pectinases, keratinases, lipases, esterases, catalases, peroxidases and phytases which function under extreme conditions. The application of cold-active enzymes enables lowering of temperature without loss of efficiency which results in saving of energy consumption and have great potential for various biotechnological processes (Ramteke and Bhatt, 2007; Kuddus and Ramteke, 2008a). Currently, only ~2% of the microorganisms on the earth have been commercially exploited and amongst these there are only a few examples of extremophiles (Gomes and Steiner, 2004). Around 85% of earth is occupied by cold ecosystems including the ocean depths, polar and alpine regions. Out of which ~70% is covered by oceans that have a constant temperature of 4-5°C, irrespective of the latitude. Remaining 15% included Polar regions to which the glacier and alpine regions must also be added. Extremophiles successfully colonized on these eternally cold environments which we can call as psychrophiles (Feller and Gerday, 2003). The classical definition of psychrophiles is given by Morita (1975) which is frequently used in the literature. This definition proposes that psychrophilic microorganisms have optimum growth temperatures of <15°C. But a psychrotrophic term is used for those cold-adapted organisms that have an optimum growth temperature of ~15-20°C but are able to grow upto 30°C. During the past decade it has been recognized that cold-adapted microorganisms and their enzymes provide a wide biotechnological potential and offering numerous economic and ecological advantages over the thermophilic enzymes/microbes (Kuddus and Ramteke, 2008b).

Alpha amylases (endo-1,4-α-D-glucan glucohydrolase, EC 3.2.1.1) belongs to the enzyme class of hydrolases which randomly cleaves the 1,4-α-D-glucosidic linkages between the adjacent glucose units in linear amylose chain of starch. Most of the α-amylases are metallo-enzymes which require calcium ions for their activity, stability and structural integrity. They belong to family 13 (GH-13) of the glycoside hydrolase group of enzymes (Bordbar et al., 2005). The specificity of the bond attacked by α-amylases depends on the sources of the enzymes. Currently, two major classes of α-amylases are commercially produced through microbial fermentation. Based on the points of attack in the glucose polymer chain, they can be classified into two categories, liquefying and saccharifying. Bacterial α-amylase randomly attacks only the α-1,4 bonds, it belongs to the liquefying category. On the other hand, the fungal α-amylase belongs to the saccharifying category and attacks the second linkage from the non-reducing terminals (i.e. C4 end) of the straight segment, resulting in a disaccharide called maltose. The bond breakage is thus more extensive in saccharifying enzymes than in liquefying enzymes. Thus, amylases have emerged as one of the leading biocatalysts with proven potential to find usage in a wide array of industrial applications, such as additives in processed food industries, additives in detergents, waste-water treatment, biopulping, bioremediations and in molecular biology. These enzymes account for about 30% of the world’s enzyme production (Maarel Van der et al., 2002).

Cold active amylases: Cold-adapted amylolytic microorganisms produce cold-active amylases which function effectively at cold temperatures with high rates of catalysis in comparison to the amylases from mesophiles or thermopiles which shows little or no activity at low temperature. These amylases have evolved a range of structural features that confer a high level of flexibility, particularly around the active site are translated into low activation enthalpy, low-substrate affinity and high specific activity at low temperatures. Moreover, the maximum level of activity of these amylases is shifted towards lower temperatures with a concomitant decrease in thermal stability (Kuddus and Roohi, 2010).

Structural features of α-amylase
General α-amylase structure: The α-amylase family is the largest family of glycoside hydrolases, transferases and isomerases comprising nearly 30 different enzyme specificities (Henrissat, 1991). A large variety of enzymes are able to act on starch. These enzymes can be divided basically into four groups: endoamylases, exoamylases, debranching enzymes and transferases (Maarel van der et al., 2002):

Endoamylases: Cleave internal α-1,4 bonds resulting in α-anomeric products
Exoamylases: Cleave α-1,4 or α-1,6 bonds of the external glucose residues resulting in α-or β anomeric products
Debranching enzymes: Hydrolyze α-1,6 bonds exclusively leaving long linear polysaccharides and
Transferases: Cleave α-1,4 glycosidic bond of the donor molecule and transfer part of the donor to a glycosidic acceptor forming a new glycosidic bond

Alpha amylase is classified as family 13 of the glycosyl hydrolases. The catalytic mechanism of the α-amylase family is that of the α-retaining double displacement. α-retaining mechanism is the characteristic feature of the enzymes from the α-amylase family. They vary widely in their reaction specificities. The attachments of different domains to the catalytic site or to extra sugar binding sub sites around the catalytic site is the prime reason for these differences (Maarel van der et al., 2002). The catalytic domain-A is the most conserved domain in the α-amylase family. It consists of an amino terminal (β/α)8-barrel structure as revealed in three dimensional structure of amylase from Aspergillus sp. (Matsuura et al., 1984).

Structural modifications for cold adaptation: A reduction in temperature, one of the most important environmental factors for life as it influences most biochemical reactions, slows down most physiological processes, changes protein-protein interactions, reduces membrane fluidity and provokes an increased viscosity of water. Moreover, enzymes are also subject to cold denaturation, leading to the loss of enzyme activity at low temperatures (Somero, 1981; Yancey and Somero, 1978). Psychrophilic microorganisms producing cold-active amylases are structurally modified by an increasing flexibility of the polypeptide chain enabling an easier accommodation of substrates at low temperature. They must modify their lipid composition to maintain membrane fluidity at environmental temperatures. This can be done in many ways.

Unlike cold-adapted proteins which improve their structural mobility, the thermal adaptation of membrane lipids does not involve the synthesis of fatty acyls that have increased degrees of freedom but rather the introduction of steric constraints that reduce the packing of acyl chains in the membrane. These steric constraints destabilize the membrane and reduce the lipid viscosity (Margesin et al., 2002; Russell and Hamamoto, 1998; Russell, 1997)
Presence of a cis-unsaturated double bond in the chain that induces a 30° bend. Such bending creates a cavity in the lipid layer and perturbs the packing density. Trans-unsaturated double bonds are also observed but are less efficient as they only produce a modest kink of the acyl chain
The occurrence of branched lipids mainly methyl-branched fatty-acyl chains also perturbs the compactness of neighbouring chains owing to the steric hindrance that is caused by the side-chain group. The position of this branching along the chain also modulates the gel-phase transition temperature
Finally, shorter fatty-acyl chains reduce the contacts between adjacent chains and increase fluidity (Fong et al., 2001; Jagannadham et al., 2000)

Besides the variations in membrane structure and its lipid composition, another important feature in these cold-adapted microorganisms is the presence of Cold-acclimation Proteins (CAPs). These are a set of ~20 proteins which is permanently synthesized during steady-state growth at low temperatures but not at milder temperatures (Hebraud and Potier, 2000; Berger et al., 1996; Hebraud et al., 1994). Interestingly, some of the CAPs that have been identified in cold-adapted bacteria actually acting as cold-shock proteins in mesophiles, such as the RNA chaperone CspA (Berger et al., 1997). It has been proposed that these CAPs are essential for the maintenance of both growth and the cell cycle at low temperatures (Hebraud and Potier, 2000) but their function is still poorly understood. Cold-shock proteins, Csp, are an additional type of adaptation for psychrophilic organisms in cold regions. These proteins act mainly on the regulation of cellular protein synthesis, particularly at the level of transcription and the initiation of translation; and they also act as chaperone by preventing the formation of mRNA secondary structures. Advantage of these Csp’s is that the synthesis of housekeeping gene products is not inhibited by cold-shock which is normally occurring in their mesophilic and thermophilic homologues (Cavicchioli et al., 2000; Berger et al., 1996; Mayr et al., 1996).

Antifreeze proteins, AFPs, are more frequently occurring in fishes, insects, plants, fungi and some microorganisms which decrease the freezing point of cellular water by binding to ice crystals and prevent the destruction of cell membranes and the disruption of osmotic balance. Besides contributing to freeze resistance and freeze tolerance, AFPs also helped to increase species diversity in some of the harshest and most inhospitable environments (Barrett, 2001; Jia and Davies, 2002). Although antifreeze proteins have been reported in several eukaryotes, there is no supporting evidence for the occurrence of such glycopeptides in psychrophilic prokaryotes.

Structure of antarctic psychrophile Alteromonas haloplanctis α-amylase: The cold-active α-amylase from the Antarctic psychrophile, Alteromonas haloplanctis has been studied extensively (Feller et al., 1992). The enzyme has a molecular mass of 49 kDa with few salt bridges, aromatic interactions, small hydrophobic cluster, few arginine residues and weak stabilisation of helix dipoles. It is the first cold-active α-amylase which has been successfully crystallized and the 3-D structure resolved at 1.85 Å (Aghajari et al., 1996; Ramteke and Bhatt, 2007). Also, this α-amylase was successfully expressed in mesophilic host E. coli preserving genuine properties of a psychrophilic enzyme (Feller et al., 1998).

The overall fold of A. haloplanctis α-amylase is very similar to those reported for mesophilic α-amylases (Aghajari et al., 1998a). Three characteristic domains as well as ion-binding sites are found: domain A (residues 1-86 and 147-356); the central N-terminal domain with a (β/α)8-barrel fold; a minor domain B (residues 87-146, an insertion between α3 and β3) that protrudes from domain A and comprises a loop structure, short β strands and a short α helix; and the C-terminal domain C consisting of eight β strands that form a Greek-key motif (the number of β strands in other α-amylases varies from five in barley to ten in human salivary). The largest variations in primary structures between these enzymes from different species have been found in domain C (Jespersen et al., 1993; MacGregor, 1988) and domain B (Janecek et al., 1997) but it should also be mentioned that, throughout the α-amylase family, only eight residues are invariant in the (β/α)8 barrel (Svensson, 1994). These include seven residues at the active site and a structurally important glycine. As in the mammalian α-amylases, binding sites for calcium and chloride ions have been located in the structure of A. haloplanctis α-amylase. The structure of psychrophilic A. haloplanctis α-amylase exposed that amino acid residues (Gln58 and Ala99) replace the cysteines involved in the disulfide bridge between domains A and B (Aghajari et al., 1998b).

Production of cold active α-amylase
Sources of cold active amylases: Cold-active enzymes can be produced by prokaryotic as well as eukaryotic organisms. Till now, most of them originate from bacteria and fish living in polar regions, especially in Antarctic sea water which represent a permanently cold (0±2°C) and constant temperature habitat (Feller et al., 1994). Psychrophilic (cold-loving) or psychrotolerant (cold-adapted) micro-organisms are found inhabiting the low temperature environments of the Earth, including polar regions, high mountains, glaciers, ocean deeps, shallow subterranean systems (i.e., caves), the upper atmosphere, refrigerated appliances and the surfaces of plants and animals living in cold environments, where temperatures never exceed 5°C. In fact, deep oceans which cover over 70% of the Earth’s surface, represent the major ecosystem on the planet. Many psychrophiles live in biotopes having more than one stress factors, such as low temperature and high pressure in deep seas (piezo-psychrophiles) or high salt concentration and low temperature in sea ice (halo-psychrophiles). A diverse range of psychrophilic microorganisms, belonging to bacteria, archaea, yeast and fungi have been isolated from these cold environments. These psychrophiles are able to degrade a wide range of polymeric substances such as starch, cellulose, xylan, pectin, chitin, protein and lipid and produce enzymes like amylase, cellulase, xylanase, pectinases, chitinase, protease and lipase, respectively (Georlette et al., 2004; Feller and Gerday, 2003; Van den Burg, 2003; Margesin et al., 2002; Deming, 2002; Demiorijan et al., 2001; Eichler, 2001; Cavicchioli et al., 2000). In addition, some recent examples of cold-active amylase producing bacteria are shown in the Table 1.

Fermentation conditions for cold active α-amylase production: Cold active amylases are mostly extra cellular and are highly influenced by nutritional and physicochemical factors such as temperature, pH, agitation, nitrogen source, carbon source, inducers, inorganic sources and dissolved oxygen. To meet the demand of industries, low-cost medium is required for the production of α-amylase. Both Solid State Fermentation (SSF) and Submerged Fermentation (SmF) could be used for the production of α-amylases, although traditionally these have been obtained from submerged cultures because of ease of handling and greater control of environmental factors such as temperature and pH. SSF has been used for long to convert moist agricultural polymeric substrates such as wheat, rice, soy, cassava, etc. (Table 2) into fermented food products including industrial enzymes such as α-amylase (Pandey et al., 1995). SSF is generally defined as the growth of microorganisms on moist solid substrates with negligible free water (Selvakumar et al., 1998). The solid substrate may provide only support or both support and nutrition.

Table 1: Cold-active amylase producing microbes

Table 2: Agro substrates used for α-amylase production

SSF is preferred to SmF because of simple technique, low capital investment, lower levels of catabolite repression and end-product inhibition, low waste water output, better product recovery and high quality production (Zadrazil and Puniya, 1995; Regulapati et al., 2007). Although, emerging trends highlight that semi-solid and solid-state fermentation systems have real prospective for the sensible management of bacteria in industry (Joseph et al., 2011).

Temperature and pH: The influence of temperature on amylase production is related to the growth of the organism. Thus, the optimum temperature for enzyme production depends on whether the culture is mesophilic, thermophilic or psycrophilic. Among the fungi, most amylase production studies have been done within temperature range of 25-37°C (Francis et al., 2003; Ramachandran et al., 2004). Bacterial α-amylases are produced at a much wider range of temperature. Bacillus amyloliquefaciens, B. subtilis, B. licheniformis and B. stearothermophilus are among the most commonly used Bacillus sp. reported to produce α-amylase at temperatures 37-60°C (Mishra et al., 2005; Mendu et al., 2005; Syu and Chen, 1997; Mielenz, 1983). A cold active α-amylase from Antarctic psychrophile Alteromonas haloplanktis was reported to exhibit maximum α-amylase production at 4°C (Feller et al., 1998). pH is also one of the important factors that determine the growth and morphology of microorganisms as they are sensitive to hydrogen ions concentration present in the medium. pH is known to affect the synthesis and secretion of α-amylase just like its stability. Earlier studies have revealed that fungi required slightly acidic pH and bacteria required neutral pH for optimum growth. Bacterial cultures such as B. subtilis, B. licheniformis and B. amyloliquefaciens required an initial pH of 7.0 (Saban-Tanyildizi et al., 2005; Ikram-ul-Haq et al., 2005; Syu and Chen, 1997). Hyperthermophilic archae such as Pyrococcus furiosus, P. woesei and T. profundus yielded optimum α-amylase at pH 5.0 (Vieille and Zeikus, 2001). The amylase from L. brunescens is active from pH 5.0 to 7.5 (Tigerstrom and Stelmaschuk, 1987). A list of various cold active α-amylase producing psychrophillic and psychrotrophic bacteria and their production parameters are presented in Table 3.

Carbon and nitrogen sources: These are necessary for the growth and metabolism of organisms. Various carbon sources are tried to optimize the maximum production of cold-active α-amylase for different bacterial species (Table 4). For enhanced growth and metabolism of organisms, nitrogen also play very important role just like carbon. A large variety of nitrogen sources for bacterial species are available which give rise to maximum production of cold-active α-amylase (Table 5).

Other fermentative conditions: In the fermentation medium surfactants are known to increase the production of extracellular amylase enzymes by increasing cell membrane permeability. Some common surfactants are Tween 80, polyethylene glycols, Cholic acid etc. which are used in different concentrations.

Table 3: Production parameters for cold active amylase
NM: Not mentioned

Table 4: Carbon sources used for maximum α-amylase production

Table 5: Nitrogen sources used for maximum α-amylase production

Table 6: Methods of one-step purification of α-amylases

Supplementation of salts of certain metal ions provided good growth of microorganisms and thereby better enzyme production (as most α-amylases are known to be metalloenzymes). Some frequently used metal ions are CaCl2, NaCl, MgSO4, FeCl3, Mn2+, Zn2+ etc. In SSF system some additional fermentative conditions plays very vital role viz. selection of a sui substrate and microorganism; pre-treatment of the substrate; particle size (inter-particle space and surface area) of the substrate; water content; relative humidity; type and size of the inoculum; removal of metabolic heat; period of cultivation; maintenance of uniformity in the environment of SSF system and the gaseous atmosphere, i.e. oxygen consumption rate and carbon dioxide evolution rate.

Purification and characterization of cold active α-amylases: Most of the purification schemes for amylases are based on multistep strategies. However, in these years new techniques have been developed that may yield high recovery. Based on the nature of amylase produced by the organism one has to design the protocol for purification and in the downstream processing the purification process depends on the market need, processing cost, final quality and available technology. There are also various methods are available in which α-amylases can be purified in one-step. The necessity for low cost, large-scale, effective purification of enzymes has resulted in progression of techniques that provide rapid, competent and economical protocols in fewer processing steps. Few purification techniques that produce homogeneous preparation of α-amylases in a single step are given in Table 6.

The purification efficiency is determined by total yield and purification factor (Kademi et al., 2005). Pre-purification steps involve concentration of the protein containing amylases by ammonium sulphate precipitation and ultra filtration by dialysis. The characterization of cold active α-amylases can be studied in terms of optimum pH and pH stability, optimum temperature and thermo-stability and effect of metal ions, chelating agents, inhibitors, nature of substrate, substrate concentration, enzyme concentration, solvents and stabilizing agents. One can think that whether cold-active enzymes do not follow the general principles of biochemistry viz., enzyme activity generally decreases approximately one-half with each decrease of 10°C but in actual fact they also do not disobey this concept rather they simply shift their peak activities to temperature ranges lower than those generally observed for enzymes from mesophilic organisms; just as enzymes from thermophiles often have optimal activity at temperatures higher than found for mesophilic enzymes (Feller and Gerday, 2003). The optimal activity temperature of Nocardiopsis sp. 7326 amylase was 35°C and the enzyme was stable between pH 5 and 10 with maximal activity at pH 8 (Zhang and Zeng, 2008). The activity of α-amylase from Streptococcus bovis JB1 was optimal at pH 5 to 6. The enzyme was relatively stable at temperatures below 50°C (Freer, 1993). Ueda et al. (2008) purified and characterized the novel cold-adapted α-amylase (Amy I and Amy II) which are most active at pH 5.5 and stable at pH 7-9. Both Amy I and II exhibited activities at 10°C. The optimal temperature and pH for the purified amylase from Micrococcus antarcticus were 30°C and 6, respectively (Fan et al., 2009). It still showed high activity at low temperature 10-15°C. It was sensitive to high temperature but was stable at pH 6.0-10.0 with at least 70% activity remained.

Biotechnological approaches in cold active α-amylase: An emerging area of research in the field of enzymology is to develop radically different and novel biocatalysts through various molecular approaches including r-DNA technology, protein engineering, directed evolution and the metagenomic approach. As a whole, amylase biotechnology has just reached the end of lag phase and the beginning of the exponential phase: it demands extension in terms of both quality and quantity. Qualitative improvements in restructuring amylase gene and its protein can be achieved by employing already established r-DNA technology and protein engineering. Quantitative enhancement needs strain improvement, especially through site-directed mutagenesis and standardizing the nutrient medium for the overproduction of cold active α-amylase.

Gene cloning: To date, a very few number of cold active α-amylase genes were isolated and the related studies have been carried out. Early successes in the production of heterologous proteins were achieved using Escherichia coli as host and various kinds of proteins were expressed in E. coli. However, expression of eukaryotic proteins in E. coli became very difficult due to formation of inclusion bodies, protein misfolding and safety issues. Other expression systems were developed among yeasts, fungi, plants and animals. Cloning and expression of the cold-active amylase gene from Alteromonas haloplanctis has been reported (Feller et al., 1992, 1998). The α-amylase secreted by the Antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic α-amylase. The psychrophilic α-amylase is however characterized by a seven fold higher kcat and kcat/Km values at 4°C and a lower conformational stability with respect to the porcine enzyme.

Protein engineering: Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes. Although α-amylase carry significant commercial value, biotechnologically produced or engineered cold active α-amylases may represent the focus of industrial interest in future. Cold active α-amylases could generate avenues for industrial applications, once their specific properties are improved through enzyme engineering. Determination of three-dimensional structures of more cold active amylases would allow the detailed analysis of protein adaptation to temperatures at molecular level. This may include increased thermolabile nature and/or catalytic activity at low temperatures or the modification of pH profiles. Cold active α-amylases from microorganisms retaining high catalytic activity at low temperatures are successfully produced using site directed mutagenesis and directed evolution. α-amylase from the Antarctic psychrophile Alteromonas haloplanctis is synthesized at 0±2°C by the wild strain. This heat-labile α-amylase folds correctly when over expressed in Escherichia coli (Feller et al., 1998). It was found that this heat-labile enzyme is the largest known multi domain protein exhibiting a reversible two-state unfolding (Feller et al., 1999).

Patents in cold active α-amylases: The number of companies involved in funding cold α-amylase research, nevertheless the high risk and cost concerned of this unexplored field. Even though some noteworthy discoveries based on Antarctic amylases and with potential commercial applications were made in collaboration with industrial partners. One important patent is concern to B. licheniformis amylase in which specific activity was increased at temperatures from 10 to 60°C (US Patent number 6673589) by Borchert and coworkers with industrial partner Novozymes. Most patents are process, rather than product based on an isolate from an organism. Though, it appears that none of these discoveries has led to commercialization yet.

Applications of cold active α-amylases
Desizing operation (removal of starch) in textile industry: The desizing of grey fabric with crude amylase is very promising in textile industry. The strength of the textile is improved by warping the starch paste to textile weaving. After weaving the cloth, the starch is usually removed by application of α-amylase and goes to scouring and dyeing. By using cold-active α-amylase the required temperature may be decreases that will reduce energy consumption. It was observed that the desizing of fabric increased with increase in enzyme concentration up to certain limit. But as the concentration of the enzyme increased than the optimum level, it showed adverse effects on the desizing of the fabric. It might be due to the fact that appropriate enzyme to substrate ratio is essential to obtain optimal results (Allan et al., 1997).

Application in detergent industry for cold washing: Cold-active enzymes can be very useful for domestic processes. The activity at low temperature indicates the possibilities of these enzymes as a detergent additive for cold washing. Washing with detergents also use a lot of energy particularly when done at high temperature because peroxide-based bleaches need higher temperature (60°C) to work properly. Thus, lowering the wash temperature by using cold-active enzymes can save lots of energy and may be used to protect environment because it is biodegradable (Kuddus and Ramteke, 2009). Cold-active alkaline amylases can be used in detergents since washing clothes at low temperatures protect the colors of fabrics and reduce energy consumption.

Direct fermentation of starch to ethanol: For large-scale processing, the bioconversion of biomass resources, especially starchy materials, to ethanol, is very useful because it can be used as a biofuel and as the starting material for various chemicals. However In the present scenario its cost of production is very high because of three main reasons; ethanol production from starchy materials via fermentation consists of two or three steps, large quantity of α-amylase is needed and starchy materials need to be cooked at a high temperature (140 to 180°C). However methods are developed in which cost of production can be minimized; by fermenting starch to ethanol in one step using co-cultures of two different strains and by using low-temperature-cooking fermentation systems (that succeeded in reducing energy consumption by approximately 50%) (Matsumoto et al., 1985, 1982).

Manufacture of maltotetraose syrup: Maltotetraose syrup (G4 syrup) is produced by breaking of starch into maltotetraose by the action of amylase enzyme. The sweetness of the syrup is as low as 20% of sucrose. Therefore in foods, G4 syrup can be successfully used in place of sucrose which reduces the sweetness without altering their inherent taste and flavor. It has high moisture retention power which maintains integrity of starch particles and retains suitable moisture in foods. G4 syrup improves the food texture because of its high viscosity than sucrose. It further lowers down the freezing point of water than sucrose or high fructose syrup, so can be used to control the freezing points of frozen foods (Aiyer, 2005).

Manufacture of maltose: Maltose, a disaccharide, is made up of its monomeric units called glucose. It is the main component of maltosugar syrup (Sugimoto, 1997). Maltose is commonly used as sweetener and also as intravenous sugar supplement. It has a great value in food industries since it is non-hygroscopic and does not easily crystallize. For the manufacturing of maltose potato, sweet potato, corn and cassava starches are frequently used. Cold-active α-amylase from psychrophilic organisms may be used.

Manufacture of high molecular weight branched dextrins: High molecular weight branched dextrins are used as extender for production of powdery foods. These are produced by the action of α-amylase on corn starch. Degree of hydrolysis depends on the type of starch and the physical properties desired. Branched dextrins can be collected as powder after chromatography and spray drying.

Treatment of starch containing waste water: Food processing wastewater offers a unique challenge to any treatment system. Biotechnological treatment of food processing starch waste water can produce valuable products such as microbial biomass protein and also purifies the effluent at low temperature (Aiyer, 2005).

Other applications: In food industry cold active α-amylase can be used for the reduction of haze formation in juices and retardation of staling in baking industry. Cold active α-amylase is also very useful for paper industry as it reduces the viscosity of starch for appropriate coating of paper. In pharmaceutical industry they can be used as a digestive aid. Psychrophilic microorganisms have also been proposed for the bioremediation of polluted soils and waste waters during the winter in temperate countries, when the degradative capacity of the endogenous microflora is impaired by low temperatures. Glycosidases are often used in the baking industry but can retain residual activity after cooking that alters the structure of the final product during storage; this can be avoided by the use of psychrophilic glycosidases. Lactose intolerance is a problem for approximately two-thirds of the world’s population. The removal of lactose from milk by a psychrophilic β-galactosidase during cold storage has recently been patented. 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 (Tutino et al., 2001): expression at low temperatures prevents the formation of inclusion bodies and protects heat-sensitive gene products. Using enzymes with high activity below 20°C in food processing to limit the growth of other contaminating microorganisms, shorten the process times and avoid designing expensive heating steps. Cold-active α-amylases could be used in the brewing industry to speed the mashing phase at low temperatures. Psychrophilic microorganisms and their enzymes are already crucial to nutrient cycling and biomass degradation and production. We can take advantage of the natural role of psychrophiles and use ones producing useful enzymes in waste-water treatment and biopulping in cold climates (Joseph et al., 2008). Cold active enzymes, as a result of high catalytic efficiency and unique specificity at low and moderate temperatures and its producing microorganisms may also be ideal for bioremediation purposes (Ramteke et al., 2005).

Conclusions and future prospects: Analysis of the literature reveals that cold-active enzymes offer several advantages over mesophilic/thermophilic enzymes. Cold active α-amylases are promising enzymes to replace the conventional enzyme processes of the biotechnological industries. Although, a more extensive exertion is required to overcome several bottlenecks such as high enzyme cost, low activity and/or stability under environmental conditions and the low biodiversity of psychrophilic/psychrotrophic microbes explored so far. The comparatively latest introduction and progress of novel r-DNA technologies such as, metagenomics and site-directed mutagenesis have an intense positive effect on the expression and production of greater and greater amounts of recombinant proteins which means more competitive prices, by introducing new or tailored catalytic activities of these enzymes at low temperature. Therefore, efforts have to be made in order to achieve economical over production of cold active α-amylase in heterologous hosts and their alteration by chemical means or protein engineering to obtain more robust and active amylases. Genetically improved strains, appropriate for specific cold-active enzyme production, would play an important role in various industrial and biotechnological applications.

REFERENCES
1:  Abou-Elela, G.M., A.N. El-Sersy and S.H. Wefky, 2009. Statistical optimization of cold adapted α-amylase production by free and immobilized cells of Nocardiopsis aegyptia. J. Applied Sci. Res., 5: 286-292.
Direct Link  |  

2:  Aghajari, N., R. Haser, G. Feller and C. Gerday, 1996. Crystallization and preliminary X-ray diffraction studies of α-amylase from the antarctic psychrophile Alteromonas haloplanctis A23. Protein Sci., 5: 2128-2129.
CrossRef  |  

3:  Aghajari, N., R. Haser, G. Feller and C. Gerday, 1998. Crystal structures of the psychrophilic α-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci., 7: 564-572.
CrossRef  |  Direct Link  |  

4:  Aghajari, N., G. Feller, C. Gerday and R. Haser, 1998. Structures of the psychrophilic Alteromonas haloplanctis α-amylase gives insights into cold adaptation at a molecular level. Structure, 6: 1503-1516.
CrossRef  |  PubMed  |  Direct Link  |  

5:  Aiyer, P.V.D., 2004. Effect of C:N ratio on α-amylase production by Bacillus licheniformisSPT 27. Afr. J. Biotechnol., 3: 519-522.
Direct Link  |  

6:  Aiyer, P.V., 2005. Amylases and their applications. Afr. J. Biotechnol., 4: 1525-1529.
Direct Link  |  

7:  Akpan, I. and F.A. Adelaja, 2004. Production and stabilization of amylase preparations from rice bran solid medium. World J. Microbiol. Biotechnol., 20: 47-50.
CrossRef  |  

8:  Allan, S., B. Torbenvedel and B.F. Henrick, 1997. Recombinant α-amylase mutants and their use in textile desizing, starch liquefaction and washing. J. Applied Microbiol., 12: 205-210.

9:  Amritkar, N., M. Kamat and A. Lali, 2004. Expanded bed affinity purification of bacterial α-amylase and cellulase on composite substrate analogue-cellulose matrices. Process Biochem., 39: 565-570.
CrossRef  |  

10:  Babu, K.R. and T. Satyanarayana, 1995. α-amylase production by thermophilic Bacillus coagulans in solid-state fermentation. Process Biochem., 30: 305-309.
CrossRef  |  

11:  Barrett, J., 2001. Thermal hysteresis proteins. Cell Biol., 33: 105-117.
CrossRef  |  PubMed  |  Direct Link  |  

12:  Berger, F., N. Morellet, F. Menu and P. Potier, 1996. Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55. J. Bacteriol., 178: 2999-3007.
Direct Link  |  

13:  Berger, F., P. Normand and P. Potier, 1997. capA, a cspA-like gene that encodes a cold acclimation protein in the psychrotrophic bacterium Arthrobacter globiformis SI55. J. Bacteriol., 179: 5670-5676.
PubMed  |  

14:  Bordbar, A.K., K. Omidiyan and R. Hosseinzadeh, 2005. Study on interaction of α-amylase from Bacillus subtilis with cetyl trimethylammonium bromide. Colloids Surf. B: Biointerfaces, 40: 67-71.
CrossRef  |  

15:  Van den Burg, B., 2003. Extremophiles as a source for novel enzymes. Curr. Opin. Microbiol., 6: 213-218.
CrossRef  |  

16:  Cavicchioli, R., T. Thomas and P.M.G. Curmi, 2000. Cold stress response in Archaea. Extremophiles, 4: 321-331.
CrossRef  |  Direct Link  |  

17:  Chandra, A.K., S. Medda and A.K. Bhadra, 1980. Production of extracellular thermostable α-amylase by Bacillus licheniformis. J. Ferment. Technol., 58: 1-10.
Direct Link  |  

18:  Chessa, J., G. Feller and C. Gerday, 1999. Purification and characterization of the heat-labile α-amylase secreted by the psychrophilic bacterium TAC 240B. Can. J. Microbiol., 45: 452-457.
Direct Link  |  

19:  Deming, J.W., 2002. Psychrophiles and polar regions. Curr. Opin. Microbiol., 5: 301-309.
CrossRef  |  

20:  Demiorijan, D.C., F. Moris-Varas and C.S. Cassidy, 2001. Enzymes from extremophiles. Curr. Opin. Chem. Biol., 5: 144-151.
CrossRef  |  

21:  Dettori-Campus, B.G., F.G. Priest and J.R. Stark, 1992. Hydrolysis of starch granules by the amylase from bacillus stearothermophilus NCA 26. Process Biochem., 27: 17-21.
CrossRef  |  

22:  Eichler, J., 2001. Biotechnological uses of archaeal extremozymes. Biotechnol. Adv., 19: 261-278.
CrossRef  |  

23:  Fan H.X., Y. Liu and Z.P. Liu, 2009. Optimization of fermentation conditions for cold-adapted amylase production by Micrococcus antarcticus and its enzymatic properties. Huan. Jing. Ke. Xue., 30: 2473-2478.
PubMed  |  

24:  Feller, G., O.L. Bussy and C. Gerday, 1998. Expression of psychrophilic genes in mesophilic hosts; Assessment of the folding state of a recombinant α-amylase. Applied Environ. Microbiol., 64: 1163-1165.
Direct Link  |  

25:  Feller, G., D. d'Amico and C. Gerday, 1999. Thermodynamic stability of a cold-active α-amylase from the Antarctic bacterium Alteromonas haloplanctis. Biochemistry, 38: 4613-4619.
CrossRef  |  PubMed  |  Direct Link  |  

26:  Feller, G. and C. Gerday, 2003. Psychrophilic enzymes: Hot topics in cold adaptation. Nat. Rev. Microbiol., 1: 200-208.
CrossRef  |  PubMed  |  

27:  Feller, G., T. Lonhienne, C. Deroanne, C. Libioulle, J. Van Beeumen and C. Gerday, 1992. Purification, characterization and nucleotide sequence of the thermolabile α-amylase from the Antarctic psychrotroph, Alteromonas haloplanctis A23. J. Biol. Chem., 267: 5217-5221.
Direct Link  |  

28:  Feller, G., F. Payan, F. Theys, M. Qian, R. Haser and C. Gerday, 1994. Stability and structural analysis of α-amylase from the antarctic psychrophile Alteromonas haloplanctis A23. Eur. J. Biochem., 222: 441-447.
CrossRef  |  

29:  Fong, N.J., M.L. Burgess, K.D. Barrow and D.R. Glenn, 2001. Carotenoid accumulation in the psychrotrophic bacterium Arthrobacter agilis in response to thermal and salt stress. Applied Microbiol. Biotechnol., 56: 750-756.
CrossRef  |  

30:  Francis, F., A. Sabu, K.M. Nampoothiri, S. Ramachandran, S. Ghosh, G. Szakacs and A. Pandey, 2003. Use of response surface methodology for optimizing process parameters for the production of α-amylase by Aspergillus oryzae. Biochem. Eng. J., 15: 107-115.
CrossRef  |  Direct Link  |  

31:  Freer, S.N., 1993. Purification and characterization of the extracellular alpha amylase from Streptococcus bovis JBI. Applied Environ. Microbiol., 59: 1398-1402.
PubMed  |  Direct Link  |  

32:  Georlette, D., V. Blaise, T. Collins, S. D'Amico and E. Gratia et al., 2004. Some like it cold: Biocatalysis at low temperatures. FEMS Microbiol. Rev., 28: 25-42.
CrossRef  |  

33:  Gomes, J. and W. Steiner, 2004. The biocatalytic potential of extremophiles and extremozymes. Food Technol. Biotechnol., 42: 223-235.
Direct Link  |  

34:  Goyal, N., J.K. Gupta and S.K. Soni, 2005. A novel raw starch digesting thermostable α-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme Microb. Technol., 37: 723-734.
CrossRef  |  

35:  Gupta, A., V.K. Gupta, D.R. Modi and L.P. Yadava, 2008. Production and characterization of α-amylase from Aspergillus niger. Biotechnology, 7: 551-556.
CrossRef  |  Direct Link  |  

36:  Gurudeeban, S., K. Satyavani and T. Ramanathan, 2011. Production of extra cellular α-amylase using Bacillus megaterium isolated from white mangrove (Avicennia marina). Asian J. Biotechnol., 3: 310-316.
CrossRef  |  Direct Link  |  

37:  Hamilton, L.M., C.T. Kelly and W.M. Fogarty, 1999. Purification and properties of the raw starch degrading α-amylase of Bacillus sp. IMD 434. Biotechol. Lett., 21: 111-115.
CrossRef  |  Direct Link  |  

38:  Ikram-ul-Haq, H. Ashraf, M.A. Qadeer and J. Iqbal, 2005. Pearl millet, a source of α amylase production by Bacillus licheniformis. Bioresour. Technol., 96: 1201-1204.
Direct Link  |  

39:  Hebraud, M., E. Dubois, P. Potier and J. Labadie, 1994. Effect of growth temperatures on the protein levels in a psychrotrophic bacterium, Pseudomonas fragi. J. Bacteriol., 176: 4017-4024.
Direct Link  |  

40:  Hebraud, M. and P. Potier, 2000. Cold Acclimation and Cold-Shock Response in Psychrotrophic Bacteria. In: Cold Shock Response and Adaptation, Inouye, M. and K. Yamanaka (Ed.). ASM Press, Norfolk, England, pp: 41-60.

41:  Henrissat, B., 1991. A classification of glycosyl hydrolases based on amino acid Sequence similarities. Biochem. J., 280: 309-316.
CrossRef  |  PubMed  |  Direct Link  |  

42:  Jagannadham, M.V., M.K. Chattopadhyay, C. Subbalakshmi, M. Vairamani, K. Narayanan, C.M. Rao and S. Shivaji, 2000. Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum. Arch. Microbiol., 173: 418-424.
CrossRef  |  

43:  Janecek, S., B. Svensson and B. Henrissat, 1997. Domain evolution in the α-amylase family. J. Mol. Evol., 45: 322-331.
CrossRef  |  Direct Link  |  

44:  Jespersen, H.M., E.A. MacGregor, B. Henrissat, M.R. Sierks and B. Svensson, 1993. Starch and glycogen debranching enzymes: Prediction of structural features of the catalytic (βα)8-barrel domain and evolutionary relationship to other amylolytic enzymes. J. Protein Chem., 12: 791-805.
PubMed  |  

45:  Jia, Z. and P.L. Davies, 2002. Antifreeze proteins: An unusual receptor-ligand interaction. Trends Biochem. Sci., 27: 101-106.
CrossRef  |  

46:  Joseph, B., P.W. Ramteke and G. Thomas, 2008. Cold active microbial lipases: Some hot issues and recent developments. Biotechnol. Adv., 26: 457-470.
CrossRef  |  PubMed  |  

47:  Joseph, B., S. Upadhyaya and P.W. Ramteke, 2011. Production of cold-active bacterial lipases through semisolid state fermentation using oil cakes. Enzyme Res., 2011: 1-6.
CrossRef  |  Direct Link  |  

48:  Kademi, A., L. Danielle and H. Ajain, 2005. Lipases. In: Enzyme Technology, Pandey, A., C. Webb, C.R. Soccol and C. Larroche (Eds.). Asiatech Publishers Inc., India, pp: 297-318.

49:  Kuddus, M. and Roohi, 2010. Microbial Cold-Active α-Amylases: From Fundamentals to Recent Developments. In: Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, Mendez-Vilas, A. (Ed.) Vol. 2, Formatex Research Center, Spain, pp: 1265-1276.

50:  Kuddus, M. and W.R. Ramteke, 2008. Purification and properties of cold-active metalloprotease from Curtobacterium luteum and effect of culture conditions on production. Chin. J. Biotechnol., 24: 2074-2080.
PubMed  |  Direct Link  |  

51:  Kuddus, M. and P.W. Ramteke, 2008. A cold-active extracellular metalloprotease from Curtobacterium luteum (MTCC 7529): Enzyme production and characterization. J. Gen. Applied Microbiol., 54: 385-392.
PubMed  |  

52:  Kuddus, M. and P.W. Ramteke, 2009. Cold-active extracellular alkaline protease from an alkaliphilic Stenotrophomonas maltophilia: Production of enzyme and its industrial applications. Can. J. Microbiol., 55: 1294-1301.
CrossRef  |  PubMed  |  

53:  Lealem, F. and B.A. Gashe, 1994. Amylase production by a Gram-positive bacterium isolated from fermenting tef (Eragrostis tef). J. Applied Bacteriol., 77: 341-352.
CrossRef  |  

54:  Liao, Y.C. and M.J. Syu, 2005. Novel immobilized metal ion affinity adsorbent based on cross-linked b-cyclodextrin matrix for repeated adsorption of α-amylase. Biochem. Eng. J., 23: 17-24.
CrossRef  |  

55:  Van der Maarel, M.J.E.C., B. van der Veen, J.C.M. Uitdehaag, H. Leemhuis and L. Dijkhuizen, 2002. Properties and applications of starch-converting enzymes of the α-amylase family. J. Biotechnol., 94: 137-155.
Direct Link  |  

56:  MacGregor, E.A., 1988. α-Amylase structure and activity. J. Protein. Chem., 7: 399-415.
CrossRef  |  

57:  Margesin, R., G. Feller, C. Gerday and N. Russell, 2002. Cold-Adapted Microorganisms: Adaptation Strategies and Biotechnological Potential. In: The Encyclopedia of Environmental Microbiology, Bitton, G. (Ed.). John Wiley and Sons, New York, pp: 871-885.

58:  Matsumoto, N., O. Fukushi, M. Miyanaga, K. Kakihara, E. Nakajima and H. Yoshizumi, 1982. Industrialization of a noncooking system for alcoholic fermentation from grains. Agric. Biol. Chem., 46: 1549-1558.
CrossRef  |  Direct Link  |  

59:  Matsumoto, N., H. Yoshizumi, S. Miyata and S. Inoue, 1985. Development of the non-cooking and low temperature cooking systems for alcoholic fermentation of grains. Nippon Nogeikagaku Kaishi, 59: 291-299.

60:  Matsuura, Y., M. Kusunoki, W. Harada and M. Kakudo, 1984. Structure and possible catalytic residues of taka-amylase A. J. Biochem., 95: 697-702.
Direct Link  |  

61:  Mayr, B., T. Kaplan, S. Lechner and S. Scherer, 1996. Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201. J. Bacteriol., 178: 2916-2925.
PubMed  |  Direct Link  |  

62:  Mendu, D.R., B.V.V. Ratnam, A. Purnima and C. Ayyanna, 2005. Affinity chromatography of α-amylase from Bacillus licheniform. Enzyme Microb. Technol., 37: 712-717.
CrossRef  |  

63:  Smith, M.R. and J.C. Zahnley, 2005. Production of amylase by Arthrobacter psychrolactophilus. J. Ind. Microbiol. Biotechnol., 32: 277-283.
CrossRef  |  PubMed  |  

64:  Mielenz, J.R., 1983. Bacillus stearothermophilus contains a plasmid-borne gene for α-amylase. Proc. Natl. Acad. Sci., 80: 5975-5979.
CrossRef  |  

65:  Mihaela, C., N. Teodor, B. Gabriela and S. Peter, 2009. Cold adapted amylase and protease from new Streptomyces Antarctic strain. Innovative Rom. Food Biotech., 5: 23-30.
Direct Link  |  

66:  Mishra, S., S.B. Noronha and G.K. Suraishkumar, 2005. Increase in enzyme productivity by induced oxidative stress in Bacillus subtilis cultures and analysis of its mechanism using microarray data. Process Biochem., 40: 1863-1870.
CrossRef  |  

67:  Morita, R.Y., 1975. Psychrophilic bacteria. Bacteriol. Rev., 39: 144-167.

68:  Morita, Y., T. Nakamura, Q. Hasan, Y. Murakami, K. Yokoyama and E. Tamiya, 1997. Cold-active enzymes from cold-adapted bacteria. J. Am. Oil Chem. Soc., 74: 441-444.
CrossRef  |  

69:  Nahas, E. and M.M. Waldemarin, 2002. Control of amylase production and growth characteristics of Aspergillus ochraceus. Rev. Latinoam. Microbiol., 44: 5-10.
PubMed  |  Direct Link  |  

70:  Najafi, M.F. and A. Kembhavi, 2004. One step purification and characterization of an extracellular α-amylase from marine Vibrio sp. Enzyme. Microb. Technol., 36: 535-539.
CrossRef  |  

71:  Narayana, K.J.P. and M. Vijayalakshmi, 2008. Production of extracellular α-amylase by Streptomyces albidoflavus. Asian J. Biochem., 3: 194-197.
CrossRef  |  Direct Link  |  

72:  Nguyen, Q.D., J.M. Rezessy-Szabo and A. Hoschke, 2000. Optimisation of composition of media for the production of amylolytic enzymes by Thermomyces lanuginosus ATCC 34626. Food Technol. Biotechnol., 38: 229-234.
Direct Link  |  

73:  Pandey, A., L. Ashakumary and P. Selvakumar, 1995. Copra waste: A novel substrate for solid-state fermentation. Biores. Technol., 51: 217-220.
CrossRef  |  

74:  Pedersen, H. and J. Nielsen, 2000. The influence of nitrogen sources on alpha amylase productivity of Aspergillus oryzae in continuous culture. Applied Microbiol. Biotechnol., 53: 278-281.
CrossRef  |  Direct Link  |  

75:  Poornima, R., M.K. Sahu, K. Sivakumar and V. Pushpavalli, 2008. Optimization of α-amylase production by actinomycete strain AE-19 isolated from shrimp pond. Trends Applied Sci. Res., 3: 45-52.
CrossRef  |  Direct Link  |  

76:  Ramachandran, S., A.K. Patel, K.M. Nampoothiri, F. Francis, V. Nagy, G. Szakacs and A. Pandey, 2004. Coconut oil cake-A potential raw material for the production of α-amylase. Bioresour. Technol., 93: 169-174.
CrossRef  |  Direct Link  |  

77:  Ramteke, P. and M.K. Bhatt, 2007. Cold active polysaccridases and their potential industrial applications. Res. Signpost, 37: 661-673.

78:  Ramteke, P.W., B. Joseph and M. Kuddus, 2005. Extracellular lipases from Antarctic anaerobic microorganisms. Ind. J. Biotechnol., 4: 293-294.

79:  Reddy, P.V.V., S.S.S.N. Rao, M.S. Pratibha, B. Sailaja and B. Kavya et al., 2009. Bacterial diversity and bioprospecting for cold-active enzymes from culturable bacteria associated with sediment from a melt water stream of Midtre Lovenbreen glacier: An Arctic glacier. Res. Microbiol., 160: 538-546.
CrossRef  |  PubMed  |  

80:  Russell, N.J., 1997. Psychrophilic bacteria-molecular adaptations of membrane lipids. Comp. Biochem. Physiol. Physiol., 118: 489-493.
PubMed  |  

81:  Russell, N.J. and T. Hamamoto, 1998. Psychrophiles. In: Extremophiles: Microbial life in Extreme Environments, Horikoshi, K. and W.D. Grant (Eds.)., Wiley-Liss, Chichester, pp: 25-45.

82:  Safarikova, M., I. Roy, M.N. Gupta and I. Safarik, 2003. Magnetic alginate microparticles for purification of α-amylases. J. Biotechnol., 105: 255-260.
CrossRef  |  

83:  Selvakumar, P., L. Ashakumary and A. Pandey, 1998. Biosynthesis of glucoamylase from Aspergillus niger by solid-state fermentation using tea waste as the basis of a solid state. Bioresour. Technol., 65: 83-85.
CrossRef  |  

84:  Smita, H.P., R.S. Mnas, K. Shaktimay, C.R. Ramesh and M. Dider, 2008. Statistical optimization of α-amylase production by probiotic Lactobacillus plantarum MTCC1407 in submerged fermentation. Polish J. Microbiol., 57: 149-155.
PubMed  |  Direct Link  |  

85:  Sodhi, H.K., K. Sharma, J.K. Gupta and S.K. Soni, 2005. Production of a thermostable α-amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production. Process Biochem., 40: 525-534.
CrossRef  |  Direct Link  |  

86:  Somero, G.N., 1981. pH-temperature interactions on proteins: principles of optimal pH and buffer system design. Mar. Biol. Lett., 2: 163-178.

87:  Srinivas, T., S.S.N. Rao, P.V.V. Reddy, M.S. Pratibha and B. Sailaja et al., 2009. Bacterial diversity and bioprospecting for cold-active lipases, amylases and proteases, from culturable bacteria of kongsfjorden and ny-alesund, svalbard, arctic. Curr. Microbiol., 59: 537-547.
CrossRef  |  PubMed  |  Direct Link  |  

88:  Srivastava, R.A.K. and J.N. Baruah, 1986. Culture conditions for production of thermostable amylase by Bacillus stearothermophilus. Applied Environ. Microbiol., 52: 179-184.
Direct Link  |  

89:  Sugimoto, K., 1997. Denpunkagaku Handbook. Asakura, Tokyo, pp: 450-461.

90:  Suman, P., T.N.R. Srinivas, P.K. Pindi, K.H. Kishore and Z. Begum et al., 2010. Bacterial biodiversity from Roopkund Glacier, Himalayan mountain ranges, India. Extremophiles, 20: 59-64.
CrossRef  |  

91:  Svensson, B., 1994. Protein engineering in the α-amylase family: Catalytic mechanism, substrate specificity and stability. Plant Mol. Biol., 25: 141-157.
PubMed  |  Direct Link  |  

92:  Syu, M.J. and Y.H. Chen, 1997. A study on the α-amylase fermentation performed by Bacillus amyloliquefaciens. Chem. Eng. J., 65: 237-247.
Direct Link  |  

93:  Saban-Tanyildizi, M., O. Dursun and M. Elibol, 2005. Optimization of α-amylase production by Bacillus sp. using response surface methodology. Process Biochem., 40: 2291-2296.
Direct Link  |  

94:  Groudieva, T., M. Kambourova, H. Yusef, M. Royter, R. Grote, H. Trinks and G. Antranikian, 2004. Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, spitzbergen. Extremophiles, 8: 475-488.
CrossRef  |  PubMed  |  

95:  Tigerstrom, R.G.V. and S. Stelmaschuk, 1987. 1987. Purification and partial characterization of an amylase from Lysobacter brunescens. J. Gen. Microbiol., 133: 3437-3443.
CrossRef  |  Direct Link  |  

96:  Tutino, M., A. Duilio, E. Parrilli, E. Remaut, G. Sannia and G. Marino, 2001. A novel replication element from an Antarctic plasmid as a tool for the expression of proteins at low temperature. Extremophiles, 5: 257-264.
CrossRef  |  Direct Link  |  

97:  Vieille, C. and G.J. Zeikus, 2001. Hyperthermophilic enzymes: Sources, uses and molecular mechanisms for thermostability. Microbiol. Moll. Biol. Rev., 65: 1-43.
CrossRef  |  Direct Link  |  

98:  Viswanathan, P. and N.R. Surlikar, 2001. Production of alpha amylase with flavus on Amaranthus grain by solid state fermentation. J. Basic Microbiol., 41: 57-64.
Direct Link  |  

99:  Yancey, PH. and G.N. Somero, 1978. Temperature dependence of intracellular pH: Its role in the conservation of pyruvate apparent Km values of vertebrate lactate dehydrogenases. J. Comp. Physiol. B., 125: 129-134.
CrossRef  |  Direct Link  |  

100:  Zadrazil, F. and A.K. Puniya, 1995. Studies on the effect of particle size on solid-state fermentation of sugarcane bagasse into animal feed using white-rot fungi. Bioresour. Technol., 54: 85-87.
CrossRef  |  Direct Link  |  

101:  Zhang, J. and R. Zeng, 2008. Purification and characterization of a cold-adapted α-amylase produced by Nocardiopsis sp. 7326 isolated from prydz bay, antarctic. Mar. Biotechnol., 10: 75-82.
CrossRef  |  

102:  Ueda, M., T. Asano, M. Nakazawa, K. Miyatake and K. Inouye, 2008. Purification and characterization of novel raw-starch-digesting and cold-adapted α-amylases from Eisenia foetida. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 150: 125-130.

103:  Regulapati, R., N.P. Malav and N.S. Gummadi, 2007. Production of Thermostable α-amylases by solid state fermentation: A review. Am. J. Food Technol., 2: 1-11.
CrossRef  |  Direct Link  |  

104:  Baysal, Z., F. Uyar and C. Aytekin, 2003. Solid state fermentation for production of α-amylase by a thermotolerant Bacillus subtilis from hot-spring water. Process Biochem., 38: 1665-1668.
CrossRef  |  Direct Link  |  

©  2020 Science Alert. All Rights Reserved