Halophiles as a source of polyextremophilic α-amylase for industrial applications

Halophiles are perceived as an excellent source of novel enzymes possessing inherent ability to function under saline and hypersaline environment conditions. The article covers and puts in perspective the structural and biocatalytic features of α-amylases from halophilic sources. The choice of α-amylase as the target enzyme is based on the fact that this is among the largest selling enzymes. Oligosaccharide synthesis is favored in presence of organic solvents and at high temperature. For this reason, the demand for α-amylases that are functional at high temperature and salt as well as stable towards organic solvents, is on the rise in recent years. Halophilic α-amylases are deemed to possess all the above characteristics. They are generally salt stable. In terms of water activity, saline environments are similar to non-aqueous systems. Therefore halophilic α-amylases also exhibit stability in organic solvents. In this context, the review encompasses α-amylase producing predominant halophilic microorganisms from saline habitats; strategies adopted for purification of halophilic α-amylase; their salient structural features and unique functional characteristics. Halophilic α-amylase applications and future aspects in research are also analyzed.


Introduction
The search for new enzymes endowed with novel activities and enhanced stability continues to be a desirable pursuit in enzyme research.This is fuelled by industrial requirements and necessity of enzymatic interventions in new and challenging bioprocesses.Industrial applications of enzymes necessitate them to be stable under harsh operational conditions.In this context, enzymes from extremophiles are often useful because they withstand and carry out catalysis under extreme physiological conditions.Extremophiles are therefore perceived as an excellent source of novel enzymes possessing inherent ability to function under extreme conditions [1][2][3][4][5].
Halophiles, a class among extremophiles, have the unique ability to thrive in environments rich in salt.The word halophile is derived from the Greek, meaning "salt loving".They are salt-loving organisms found in all three domains of life: Archaea, Bacteria and Eukarya.Halophiles have been mostly isolated and characterized from the habitat like saline water, saline soil, salt lakes, soda lakes, salted foods and salterns [6,7].Their metabolic and physiological activities are therefore adapted to function under high salt conditions.Research on halophiles has gained considerable attention because of their potential usefulness in food industries, biodegradation of toxic pollutants, as a source of organic osmotic stabilizers, bioplastics, enzymes and bacteriorhodopsin [8][9][10][11][12].
Their enzymes possess unique features to exhibit high activity and stability in saline environment [13][14][15].In general, halophilic enzymes are active at salt concentrations ranging from 0.2 M to 5.2 M and have been documented to be better catalysts for peptide synthesis, enhanced oil recovery and hypersaline waste treatment, wherein normal enzymes may not function optimally or may even get denatured [10,16].
Hydrolases from halophiles have been of particular interest in recent years [14,17].Many of them have been investigated in details and characterized as novel biocatalyst.The present review summarizes the halophilic hydrolases in general and encompasses the α-amylases in particular.Halophilic α-amylases have been less studied so far but hold tremendous potential for the production of oligosaccharides, which are high in demand as prebiotics.They have the ability to catalyze reaction in high salt/ organic solvents, which favors oligosaccharide synthesis much better as compared to mesophilic amylases [18,19].

Enzymes from Halophiles
Enzymes from halophiles are stable in presence of high concentrations of salt.They perform the same function as their non-halophilic counterparts but in presence of high salt concentrations.The potential of halophilic enzymes has been extensively reviewed [10,[20][21][22][23].Some of their interesting attributes include: (i) optimum activity and stability at high salt concentrations, (ii) protective role of salt in maintaining the structure, (iii) higher resistance towards denaturation, and (iv) ability to catalyze in low water or non-aqueous medium [20,24,25].One of the distinct points of halophilic enzymes is reports of highly reversible refolding from denaturations in some cases, such as betalactamase, nucleoside diphosphate kinase and Kocuria varians α-amylase [25][26][27].High reversibility was caused by high solubility of halophilic proteins due to the "highly acidic properties/ nonaggregative characteristics" of them, even under denatured conditions.
It is quite apparent that they are stable under a variety of extreme conditions.These are therefore recommended as catalysts of choice for applications in (i) hypersaline waste treatment (ii) peptide synthesis (iii) detergents (iv) textile industry (v) pharmaceuticals and (vi) food [10,52,53].
α-Amylase is part of family 13 (GH-13) of the glycoside hydrolase.They share certain common characteristics such as (i) catalytic domain is formed by (β/α) 8 or TIM barrel, (ii) catalytic triad consists of one glutamatic acid and two aspartatic acid residues and (iii) presence of four conserved sequences involved in catalysis and substrate binding [56,59].
α-Amylase from wide range of sources with distinct characteristics are available.Yet search continues for novel α-amylase to increase the realm of processes where it can be used.In this context, isolation and screening of extremophilic organisms for α-amylase of desired trait is a contemporary research area.The use of halophilic α-amylase in bioprocesses presents the advantage to obtain optimal activities at high salt concentrations.Halophilic α-amylases also might be particularly resistant to organic solvents because they function under conditions where water activity is low.

Isolation of amylase producer halophiles
Good and Hartman, screened about ten halophiles as early as 1970, for amylase activity.They reported Halobacterium salinarum (formerly Halobacterium halobium), to be the best producer [65].Later Onishi [66] isolated amylase producing moderate halophilic Micrococcus from unrefined solar salt.An amylase producing Acinetobacter sp.strain was isolated by the same group from sea-sands [67].
Amylase producing halophiles have been isolated during screening and isolation of hydrolase producers from different ecosystems.In one such study, amylase producers represented by the genus Salinivibrio, Bacillus and Halomonas were isolated from hypersaline environments in south Spain [68].Hundred seventy seven amylase producing halophilic strains were isolated from Howz Soltan Lake, Iran.Amylase producers were mainly Gram-positive rods followed by Gram-negative rods and Gram-positive cocci.They belonged to Halobacillus, Gracilibacillus, Thalassobacillus, Oceanobacillus and Halomonas genus [69].Starch hydrolyzing bacteria, majority of which belonged to genus Bacillus and could tolerate upto 10% (w/v) sodium chloride (NaCl) concentration have been isolated from Ethiopian soda lakes [70].Similar culture dependent diversity studies on halophiles for hydrolase producers have been done from Tuzkoy salt mine, Turkey [71], deep-sea sediments of the Southern Okinawa Trough [72], Tunisian Solar Saltern [73], Atacama Desert, Chile [74] and Argentinean salterns [75].
There are relatively fewer studies on halophilic amylase producing actinomycetes and fungi.In a biodiversity study on actinomycetes from salt lakes in Hami, China, out of total sixty three isolates, forty seven were halophilic actinomycetes.Amylase was the predominant hydrolase produced by forty six strains [76].During another screening, sixty eight amylase producing marine actinomycetes were isolated from the Bay of Bengal, India [77].Table 2 summarizes the major amylase producing halophiles reported so far, along with the place of their isolation.

Amylase production by halophiles
α-Amylase production levels in halophiles are generally low and very few studies have been done where many factors have been optimized to increase it.Carbon sources, nitrogen sources, salts and metal ions effects have been commonly investigated for amylase production.In halophiles, amylase production is usually growth dependent starting in exponential phase and reaching maximum in stationary phase [64].An exception to this was observed in case of Halorubrum xinjiangense amylase production, which was growth independent starting in early exponential phase and reaching maximum in mid of this phase [80].Amylase production was also growth independent for marine actinomycetes Streptomyces sp.D1 [88].

Nitrogen sources
Effect of nitrogen sources on amylase production among halophiles has not been much investigated.Peptone has been suggested to support better production, in case of Halobacterium salinarum (formerly Halobacterium halobium) [91] and Halorubrum xinjiangense [80].Yeast extract at 8.3 g/L was optimum for production by Rhodothermus marinus ITI 990 [93].Combination of yeast extract and tryptone worked best for production in case of Bacillus sp.strain TSCVKK.In this study tryptone, peptone and urea failed to induce production and there was no growth in absence of organic nitrogen source [83].Amylase production by Chromohalobacter sp.TVSP101 was best in presence of tryptone while ammonium choride and urea did not back up production [84].Casein enzyme hydrolysate at 1% (w/v) concentration was found to be best for amylase production by Marinobacter sp.EMB8 [64].Thus no single nitrogen source may be termed as universally good.

Salts and metal ions
Salt is vital for the growth of halophiles, so in most of the cases growth as well as amylase production diminishes in absence of salt.Sodium chloride is the preferred salt for growth and amylase production.Optimum concentration of salt varies from 5-25% (w/v) for maximum production.Optimized salt concentrations in some of production studies were 5% (w/v) NaCl for Halomonas meridiana [61]; 10% (w/v) NaCl for Bacillus sp.strain TSCVKK [83]; 20% (w/v) NaCl or 15% (w/v) KCl for Chromohalobacter sp.TVSP101 [84]; 25% (w/v) NaCl for Halobacterium halobium [91].Sodium sulphate and potassium chloride are suggested to be an alternate replacement and better for amylase production by Halobacillus sp.strain MA-2 [82] and Bacillus dipsosauri [81] respectively.In Micrococcus sp. 4 though, amylase production was induced by other salts such as sodium nitrate, sodium sulphate and potassium nitrate but maximum production was attained with 1 M sodium chloride containing medium [92].
Divalent metal ions have shown stimulatory effect on amylase production in halophiles.Production by Bacillus sp.strain TSCVKK was enhanced by 0.2% (w/v) CaCl 2 [83].Presence of 50 mM CaCl 2 increased the production by 29% in case of Chromohalobacter sp.TVSP101 [84].Addition of zinc sulphate (0.1 mM) not only stimulated amylase production but also decreased the time for maximum production from 10 to 5 days for Halobacterium halobium [91].In case of Halobacillus sp.strain MA-2 sodium arsenate served as best metal ion source, while copper sulfate decreased and lead nitrate failed to induce any change [82].

Effect of pH, temperature and aeration
The effect of above factors has not been investigated much for amylase production in halophiles.Halobacillus sp.produced maximum amylase at pH 7.8, temperature 30 °C and aeration rate of 200 rpm [82].In Bacillus sp.strain TSCVKK amylase production was maximum at 30 °C and pH 8.0 [83].For Chromohalobacter sp.TVSP101 optimum conditions were pH 9.0 and 37 °C [84].Maximum α-amylase production was observed at pH 7.0-7.5,35 °C and shaking speed of 200 rpm in case of Marinobacter sp.EMB8 [64].In general, a slightly alkaline pH and a temperature range of about 30-37 °C supports amylase production.

Purification, characterization and specific properties of halophilic α-amylase
There have been very limited studies on the purification and characterization of halophilic amylases.The reason for this could be that halophilic strains produce rather very low amounts of α-amylase, as evident from the production data in the previous section.The purification strategies and characteristics of amylase from various halophiles are compiled in Table 4.A combination of chromatographic matrices has been used in multistep for halophilic amylase purification.The purification methods used suggest that there is no generic protocol for obtaining purified amylase.They are monomeric proteins with molecular weight mostly in the range of 50-100 kDa.Compared to other halophilic amylases Thalassobacillus sp.LY18 has a lower molecular mass of 31 kDa [85].In some cases two amylases were obtained after purification.For instance, two halotolerant extracellular amylases have been purified from Chromohalobacter sp.TVSP 101 by ultrafiltration, ethanol precipitation, hydrophobic interaction chromatography on Butyl Sepharose 4B and Sephacryl S-200 size exclusion chromatography.They were designated as amylase I and amylase II having molecular mass of 72 and 62 kDa respectively.Both had maximum activity at pH 9.0 and temperature 65 °C.They showed activity in 0-20% (w/v) NaCl and even at 30% NaCl concentration 50% activity was retained.In absence of NaCl, amylase I was more stable compared to amylase II [84].Acinetobacter sp. was the first halophilic amylase to be purified and characterized.Purification process employing Glycogen-complex formation, DEAE-Sephadex A-50 chromatography and Sephadex G-200 gel filtration led to two pure amylases namely, amylase I and amylase II of molecular masses 55 kDa and 65 kDa respectively.Both enzymes had pH optima at 7.0 in 0.2-0.6 M NaCl or KCl.The maximum activity was observed at 50-55 °C [67].
Salt is an important additive, modulating activity and stability of halophilic α-amylase.Salt requirement is more in cases of archaea as compared to bacteria.Archaeal amylase from Natronococcus sp.strain Ah-36 loses its activity irreversibly at low salt concentration, therefore 2.5 M NaCl concentration was maintained throughout the purification process.Optimum 2.5 M NaCl is required for its activity and stability.No activity is detectable below 1.0 M NaCl and less than 20% activity is recordable at 5.0 M and above.KCl, RbCl and CsCl at higher concentration could replace NaCl for optimum activity.Complete loss of activity was observed below 1.3 M NaCl whereas; at higher salt concentration it was quite stable with retention of 70% activity at 4.4 M NaCl.In presence of 0.5% soluble starch, amylase was completely stable from 2.1 to 3.6 M NaCl concentration [90].The amylase from Haloferax mediterranei was stable in the salt range of 2-4 M NaCl with maximum activity at 3 M NaCl [63].Similar to other haloarchaeal amylases, Haloarcula hispanica amylase also worked well at high salt concentrations with optimum activity at 4-5 M NaCl.Interestingly, it was active even at lower salt concentrations and showed 30% activity even in absence of NaCl.Activity loss in absence of salt is reversible as it was recovered as soon as optimum salt concentration was restored [79].
Among bacteria, Rhodothermus marinus was active in 0-4.0 M NaCl, optimum being 0.5 M NaCl [95].Extracellular amylase from Nesterenkonia sp.strain F was active in 0-4.0 M NaCl with optimum at 0.5 M. It was stable in 1 to 4 M NaCl [33].
Their pH optima were mostly in the neutral range and some of them showed better activity at alkaline pH.A few of them showed better activity and stability at higher temperature.Micrococcus halobius amylase showed optimum activity at 50-55 °C and at pH 6.5.The enzyme was stable in the pH range of 6.5 to 7.5 [94].Amylase from Natronococcus sp.strain Ah-36 has pH optimum of 8.7 with good stability in the pH range of 6.0-8.6.The activity declined sharply in acidic range.Maximum amylase activity is attained at 55 °C, although 50% of activity is lost at this temperature [90].Extracellular amylolytic enzyme from Rhodothermus marinus showed maximum activity at pH 6.0 and was highly thermostable with temperature optima at 80 °C.At 80 °C and 85 °C, it had a half-life of 73.7 and 16.7 minutes respectively [95].Amylase from Saccharopolyspora sp.A9 haloalkaliphilic marine actinomycetes was alkaline in nature with optimum pH at 11.0 and stability in buffers of pH 8.0 to 12.0.It was quite heat stable and maximum activity was shown at 55 °C [32].A haloalkaline and thermostable amylase with stability in the temperature range (30-90 °C), pH range (6.0-12.0),and NaCl concentrations (0-20%) has been reported from Thalassobacillus sp.LY18, while optimum activity was at pH 9.0, 70 °C and 10% (w/v) NaCl [85].
α-Amylases are metalloenzymes, well-known to contain calcium ion.Similar behavior has also been observed in case of halophiles.Calcium ions acted as activator for α-amylase in some studies and dialysis against ethylenediaminetetraacetic acid (EDTA) has resulted in loss of activity.Mercury ions are potent inhibitor for this class of amylase.Micrococcus halobius amylase activity was completely lost against dialysis with distilled water and 0.01 M EDTA in 24 and 8 h respectively.It could not be restored by dialysis against CaCl 2 .The enzyme was dependent on calcium ions for stability [94].Addition of EDTA resulted in irreversible loss of amylase activity of Haloferax mediterranei.Among bivalents, magnesium chloride acted as an inhibitor, while calcium chloride activated the enzyme [63].Calcium seems to support the catalysis of Haloarcula hispanica amylase.Presence of calcium ions in assay mixture enhanced activity, optimum being 2 mM CaCl 2 .In absence of calcium ions, the activity is considerably reduced.EDTA treatment caused complete loss of activity, which was restored by the addition of calcium ions thus confirming its role in the enzyme activity [79].Calcium ions activated the amylase from Saccharopolyspora sp.A9 to greater extent.Other divalent metal ions such as Mg 2+ , Mn 2+ , Co 2+ and Cu 2+ also led to increase in activity but to a lesser extent as compared to calcium.Activity was inhibited by Hg 2+ , Zn 2+ and Fe 3+ ions.Incubation with EDTA caused decrease in amylase activity confirming the role of calcium ions in catalysis [32].Calcium ions enhanced activity while mercury inhibited it in case of Thalassobacillus sp.LY18 amylase.Other metal ions did not have any significant effect on activity.EDTA inhibited activity significantly confirming amylase to be a metalloenzyme [85].
α-Amylases are endolytic enzymes and show preferential activity on starch.Among halophiles, favored substrate is starch though they are active on amylose, amylopectin and glycogen.They do not use cyclodextrin and pullulan as substrate.Haloarcula hispanica [79], Marinobacter sp.EMB8 [19] and Halorubrum xinjiangense [80] amylase was active on starch, amylose, amylopectin and glycogen but no activity was detected towards pullulan and β-cyclodextrin.
In some cases the properties of crude amylases have been studied without any attempt to purify it e.g.Halomonas meridiana, Halobacillus sp.strain MA-2, Bacillus sp.strain TSCVKK.Crude H. meridiana amylase showed maximum activity at pH 7.0, temperature 37 °C and 10% (w/v) NaCl concentration.It was more active in alkaline pH range.Maltose and maltotriose were formed as major end products after starch hydrolysis by this enzyme [61].Optimum assay conditions for Halobacillus sp.amylase were pH 7.5 to 8.5, 50 °C and 5% NaCl [82].Bacillus sp.strain TSCVKK amylase was partially purified by acetone precipitation and was optimally active at pH 7.5, 55 ºC and 10% NaCl concentration.This amylase did not show activity in the absence of salt and was stable in various surfactants and detergents [83].

Organic solvent stability
Stability in presence of organic solvents has been observed as a generic feature for enzymes from halophiles [87].This is because of the fact that presence of salts decreases the water activity of medium; the same effect is caused by organic solvents.So, it is hypothesized that if the enzyme is stable under presence of salts, it will also be stable in organic solvents.Organic solvent stability of αamylase from halophiles is reported in some studies.Haloarcula sp.strain S-1, a halophilic archaea isolated from a commercial French solar salt has been described to produce an organic solvent tolerant extracellular amylase.The activity and stability of enzyme was maintained in various organic solvents such as n-decane, n-nonane, n-octane, xylene, styrene, toluene, benzene and chloroform.However, the activity is lost in presence of hydrophilic solvents [78].As a useful feature, Thalassobacillus sp.LY18 amylase showed activity and stability in hydrophobic solvents of log P ow ≥2.13 [85].Marinobacter sp.EMB8 α-amylase exhibited stability in 25% (v/v) concentration of DCM (dichloromethane), benzene, toluene, hexane, cyclohexane and decane up to 24 h.The enzyme was effectively utilized for maltooligosaccharide synthesis in presence of solvents [19].Halorubrum xinjiangense amylase was stable in a range of organic solvents and as a unique feature was able to hydrolyze raw starches in aqueous/ hexadecane two phase system [80].Organic solvent tolerant amylases from halophiles have also been reported from Exiguobacterium sp.DAU5 [31] and Amphibacillus sp.NM-Ra2 [30].

Polyextremophilicity
Halophiles inhabit environments of various extreme conditions in addition to salts.Such circumstances can be extremes of pH, temperature and pressure.It can be said that they live in polyextreme situations.Their metabolic machinery and enzymes are stable and functional under these conditions.Similar property of activity and stability under polyextreme conditions viz.increased salt concentration, high pH, elevated temperature, presence of organic solvents, detergents and surfactants has been exhibited in some reports of α-amylase from halophiles.α-Amylase from Marinobacter sp.EMB8 was stable up to 20% NaCl (w/v) as well as in broad pH range of 6.0-11.0.It showed considerable thermal stability with half-life of 80 minutes at 80 °C.In addition to the above, it was stable in various organic solvents, detergents and surfactants [19].A haloalkaline thermally stable extracellular amylase from haloarchaea H. xinjiangense was stable in SDS, detergents and a range of organic solvents [80].Analogous polyextremophilic performance was given away by Amphibacillus sp.NM-Ra2 amylase having additional pullulanase activity.Enzyme showed stability in salts, high pH, elevated temperature, organic solvents, surfactants and oxidizing agents [30].

Cloning and overexpression of halophilic α-amylase
Evidently, the halophilic enzymes have typical enzymatic properties with additional polyextremities viz.salt requirement for activity and stability, alkaline inclination and resistance to unfolding even with high concentration of chaotropic reagents.This raises interest in their structure and adaptive features at molecular level.The nucleotide sequence, cloning and overexpression of halophilic enzymes have been studied only scantly.E. coli does not serve well as suitable host for halophilic protein expression, although attempted in few cases.The lack of NaCl/ KCl/ osmolytes in the cytoplasm of E. coli (being mesophilic), may affect the correct folding of translated nascent protein.Albeit, the Haloferax volcanii has been a successful host in quite many cases of halophiles.The progress on the molecular characterization of halophilic amylase, available so far is compiled in Table 5. Haloferax volcanii α-Amylase gene was of 1512 bp with signal peptide of 43 amino acids; The activity of recombinant amylase was over 100 times higher than that of native Natronococcus sp.strain Ah-36 [97] Halomonas meridiana pML122/123 pVK102 pMJC21-28 Halomonas elongata; E. coli Amylase protein (AmyH) contained a high content of acidic amino acids as well as the four highly conserved regions in amylases; First 20 codons of the amylase precursor protein constituted signal peptide [98] Halothermothrix orenii pSK5A6 pTrcHisB pTH5A6

E. coli strain TOP10
α-Amylase gene was of 1545 bp encoding signal peptide of 25 amino acid and a 490 amino acid mature protein; Over 90% activity was observed at high salt concentration [62] Kocuria varians pTAF E. coli BL21 (DE3) The kva gene of 2211 bp codes 736 amino acids residue protein; presence of starch binding domain (SBD) enables the enzyme to hydrolyse raw starches [27] Exiguobacterium sp.DAU5 pET-AmyH-sp.pET-32a The 1545 bp ORF encodes 514 amino acid protein; Amylase was highly stable in presence of organic solvents [31] Haloarcula japonica pET-21b pWL102

Haloarcula japonica
The ORF of 1989 nucleotides encodes an intracellular α-amylase of 663 amino acids; It shows high activity on soluble starch, amylose and amylopectin [99] Escherichia coli JM109 pSE380 E. coli XL10-Gold A halophilic α-amylase was obtained from a non-halophilic microorganism and retains activity in high salt concentrations [100] Zunongwangia profunda Gene of 1785 bp encodes an αamylase of 594 amino acids; A cold active and salt stable amylase [101] The first report of α-amylase gene cloning and overexpression from archaea Natronococcus sp.strain Ah-36 came in the year 1994 by Kobayashi et al. [97].Though amylase was purified and characterized previously [90].Amylase was expressed in Haloferax volcanii with correct cleavage of signal peptide.The heterologous protein expression was growth associated and enhanced by presence of starch in medium.Purified expressed protein showed properties similar to native.The α-amylase gene of Natronococcus sp.strain Ah-36 was sequenced and showed an open reading frame of 1,512 base pairs [97].Extracellular amylase from moderate halophile H. meridiana was characterized by Coronado et al. [61].The H. meridiana was the first moderate halophile α-amylase gene to be cloned.The α-amylase gene amyH was sequenced and showed high degree of homology with amylase from Alteromonas haloplanktis.Further in this study thermostable α-amylase from Bacillus licheniformis were expressed in H. meridiana and H. elongata.This established that moderately halophilic bacteria can be used as cell factories for heterologous protein expression [98].
Haloarcula japonica is an extremely halophilic archaea whose genome sequence has been determined recently.A cytoplasmic α-amylase gene malA was identified from genome sequence and was subsequently cloned and expressed.The His-tagged expressed amylase was purified by Niaffinity column.The amylase was of 663 amino acid residues and the catalytic domain showed homology to GH13 family.Amylase showed optimum activity at pH 6.5, 45 °C with 2.6 M NaCl.The enzyme activity spans over broad salt range, viz.about 83% and 95% activity in 0.6 and 4.2 M NaCl respectively.It was unaffected by calcium ions and EDTA.Examination of primary protein sequence revealed that calcium binding residues were replaced by other amino acids, thus the amylase activity was calcium-independent [99].The study on Escherichia coli JM109 was unique in sense that for the first time a halophilic α-amylase was reported from a non-halophilic bacteria.This α-amylase (EAMY) gene was expressed in E. coli XL10-Gold cells and later purified and characterized.Amylase showed maximum activity at pH 7.0, temperature 55 °C in 2 M NaCl.It is activated in presence of NaCl.With specific activity of about 1,087 U/mg, it is quite superior over typical halophilic amylases.The K m and K cat are represented as 4.3 mg/mL and 825/s respectively [100].Halophilic α-amylase with psychrophilic character has been reported from Zunongwangia profunda.The recombinant amylase exhibited maximum activity at 35 °C and retained 39% activity even at 0 °C [101].Cold active α-amylase with salt tolerance property has investigated in detail from Pseudoalteromonas haloplanktis (formerly Alteromonas haloplanctis) [102,103].
α-Amylase from bacterial halophiles have shown properties and phylogenetic similarity with amylase of eukaryotic origin.Amylase from Halomonas meridiana exhibited substantial homology with amylase from insects and mammals and was a member of family 13 of glycosyl hydrolase.It contained four conserved regions found in this class of enzyme.Amino acids involved in catalysis, substrate, calcium ion and chloride ion binding were also conserved [98].Kocuria varians amylase catalytic domain (468 amino acids) showed similarity with human salivary and the porcine pancreatic α-amylases with presence of four conserved regions of amylase family.It is inhibited by proteinaceous inhibitor from S. nitrosporeus.This amylase inhibitor acts on animal amylases, whilst ineffective on B. subtilis α-amylase [27].Bioinformatic analysis of Marinobacter α-amylase revealed its phylogenetic closeness with mammals.Other members of halophilic Gammaproteobacteria such as Halomonas meridiana, Kocuria varians and Nesterenkonia sp.strain F also clustered with animal α-amylase.Marinobacter and other halophilic α-amylase having sequence similarity with mammals showed existence of N-terminal signature sequence [104].Previous study has revealed that presence of eukaryotic α-amylase domain among bacteria results from horizontal gene transfer [105].
An additional property demonstrating similarity between halophilic and mammalian α-amylase is chloride activation.Chloride ions play a vital role in activating numerous α-amylases and similar property is encountered in mammalian amylase, such as human salivary amylase.Chloride ions are reported to act as allosteric activator in case of human as well as halophilic α-amylases such as Pseudoalteromonas haloplanktis.Binding of chloride ions lead to interaction with catalytic residues and ultimately activation of α-amylase activity [106,107].Chloride activation was encountered in halophilic amylase of Marinobacter sp.EMB8.It was also activated with other anions of similar size to chloride such as acetate, nitrate and azide [64].Bromide and iodide ions which are of comparable size to chloride ions also acted as activators but to a lesser extent.Analogous performance was also demonstrated by Natronococcus sp.strain Ah-36 amylase.Among the various anions, activity was best in citrate followed by chloride and acetate, while no activity was detected in NaF, NaBr, and NaClO 4 [90].

Structure and function relationship
Normally high concentrations of salt result in precipitation of proteins due to salting out effect.On account of differential amino acid composition and their precise structural orientation halophilic proteins maintain their solubility, structural integrity and activity in high salt environment.They comprise of high proportion of acidic amino acids compared to their non-halophilic counterpart [20,108,109].Presence of acidic aspartic and glutamic amino acids on surface helps in binding to water dipoles.This enables proteins of halophiles in maintaining essential water molecules and neutralizes their surface charge to make them soluble even under high salt conditions [110].Apart from excess acidic amino acids, halophilic proteins have low lysine content, increased small hydrophobic amino acids instead of large hydrophobic residues and increased number of salt bridges [111].Parallel pattern is also observed in α-amylase from halophiles.Amino acid composition of Natronococcus sp.strain Ah-36 amylase revealed high frequency of glutamic acid or glutamine and glycine.Lysine, serine and threonine content were low.The study thus indicated role of acidic residues in activity and stability [90].Amylase from Halomonas meridiana too exhibited typical halophilic inclination viz.high content of acidic amino acids, lower number of basic amino acids and lower pI [98].Likewise Kocuria varians amylase showed surplus of acidic amino acids over basic and had lower pI of 3.97 [27].Marinobacter α-amylase characteristics were also similar with greater proportion of acidic amino acids, low lysine content and excess of small hydrophobic amino acids as compared to larger ones [104].On the contrary Halothermothrix orenii amylase did not demonstrate surplus of acidic amino acids [62].
There is a lack of structural studies on halophilic amylase to ascertain basis of stability under multitude of harsh conditions.Report on Marinobacter sp.EMB8 α-amylase established the role of salt in proper folding and structure maintenance.Solvent stability of α-amylase was established by structural studies demonstrating preserved tertiary structure in presence of hydrophobic solvents [104].Loss of structure with decrease in fluorescence intensity in absence of salt was also encountered for α-amylase of Haloarcula hispanica [79].The non-availability of three dimensional structures restricts our understanding about structure-function relationship of halophilic α-amylases.Three dimensional structure of halophilic α-amylase has been deduced only in case of Halothermothrix orenii [112].M. algicola α-amylase 3D structure was modelled using Pseudoalteromonas haloplanktis α-amylase as a template.Presence of a higher proportion of acidic residues on α-amylase surface imparts stability in saline environment [104].The three dimensional structure of α-amylase from halophilic archaea is not available.To understand the haloadaptation mechanism in archaea structural modeling of Haloarcula marismortui, Haloarcula hispanica and Halalkalicoccus jeotgali α-amylase were done.The haloarchaeal α-amylases have increased proportion of coil forming region as compared to helix formation.Further, excess acidic amino acids, low hydrophobicity, augmented salt bridges and diminished hydrophobic interaction on the surface provide them stability at increased salt concentrations [113].

Applications of halophilic α-amylase
α-Amylase is one among the top selling industrial enzymes.Whereas, mesophilic α-amylases have been widely used in diverse sectors like food, detergent, textile and pharmaceuticals [54,114].Halophilic amylases have been least explored, even though they offer advantage of working better towards saline samples, due to their activity and stability in presence of varying concentrations of salt.Other features of industrial importance encountered in this class of amylases are stability in alkaline pH, high temperature and low water activity (presence of organic solvents) conditions.Table 6.Starch hydrolysis products of various halophilic α-amylase.

Source of α-amylase
Hydrolysis products Reference(s)
In one report on immobilization, Marinobacter sp.EMB8 amylase immobilized on silica nanoparticle demonstrated better starch hydrolysis compared to free enzyme [64].Other then hydrolysis of soluble starches, raw starch hydrolyzing capability has been shown in halophilic amylase from Nesterenkonia sp.strain F [33] and Amphibacillus sp.NM-Ra2 [30].Halorubrum xinjiangense also showed this property as it was able to hydrolyse raw starches in aqueous/ hexadecane two phase system [80].Raw starch hydrolysis saves energy as heating process employed for starch solubilisation is energy intensive.
Stability and activity at high temperatures desired for starch liquefaction have been observed in many halophilic amylases.α-Amylase from hyperthermophilic bacterium Thermotoga maritima showed maximum activity at 90 °C on α -1, 4-linked carbohydrates [115].Rhodothermus marinus amylase can be used for production of branched oligosaccharides at high temperature and salinity [95].Cold active amylase from Psychromonas antarcticus and Pseudoalteromonas haloplanktis could prove beneficial for bioprocessing of starch at low temperature [103,116].
Since saline environment has low water activity, stability in non-aqueous medium has been observed among many halophilic enzymes.Haloarcula sp.strain S-1 α-amylase was the first report of halophilic α-amylase showing stability in presence of n-decane, n-octane, xylene, toluene, benzene and chloroform [78].Later, organic solvent stability was seen in amylases from Nesterenkonia sp.strain F [117], Thalassobacillus sp.LY18 [85], Exiguobacterium sp.DAU5 [31] and Halorubrum xinjiangense [80].Enzymes active in organic sovents/ low water activity are required in many biotransformation processes.In recent years, halophilic amylases have been envisaged to be uniquely suitable for bioremediation of hypersaline wastes containing starch and organic solvents.Polyextremophilic -Amylase from Aspergillus gracilis showed potential for application in the bioremediation of saline and low water activity effluents.The performance of enzyme in synthetic waste water remediation was better as compared to the commercial one at higher salt concentrations [96].

Conclusions
α-Amylases among halophiles have mostly been reported from bacteria and archaea.They are highlighted as efficient catalysts under high salt, alkaline pH and in presence of organic solvents.The polyextremophilic characteristics make halophilic α-amylases as prospective entrant for starch hydrolysis, food and bioremediation applications.The new directions in their research are envisaged as (i) metagenomic approach to investigate the α-amylases from non-culturable halophiles will add to existing repository.(ii) Their adaptive structural features yet to be completely comprehended will enable better functional understanding of such enzymes.