Review of the enzymatic machinery of Halothermothrix orenii with special reference to industrial applications

Enzyme and Microbial Technology 55 (2014) 159–169

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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt

Review

Review of the enzymatic machinery of Halothermothrix orenii with special reference to industrial applications Abhishek Bhattacharya, Brett I. Pletschke ∗ Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, PO Box 94, Grahamstown 6140, South Africa

a r t i c l e

i n f o

Article history: Received 7 August 2013 Received in revised form 23 October 2013 Accepted 25 October 2013 Keywords: Extremophile Extremozymes Glycosyl hydrolases Halothermothrix orenii

a b s t r a c t Over the past few decades the extremes at which life thrives has continued to challenge our understanding of physiology, biochemistry, microbial ecology and evolution. Innovative culturing approaches, environmental genome sequencing, and whole genome sequencing have provided new opportunities for the biotechnological exploration of extremophiles. The whole genome sequencing of H. orenii has provided valuable insights not only into the survival and adaptation strategies of thermohalophiles but has also led to the identification of genes encoding biotechnologically relevant enzymes. The present review focuses on the purified and characterized enzymes from H. orenii including amylases, ␤-glucosidase, fructokinase, and ribokinase – along with uncharacterized but industrially important enzymes encoded by the genes identified in the genome such as ␤-galactosidases, mannosidases, pullulanases, chitinases, ␣-L-arabinofuranosidases and other glycosyl hydrolases of commercial interest. This review highlights the importance of the enzymes and their applications in different sectors and why future research for exploring the enzymatic machinery of H. orenii should focus on the expression, purification, and characterization of the novel proteins in H. orenii and their feasible application to pertinent industrial sectors. H. orenii is an anaerobe; genome sequencing studies have also revealed the presence of enzymes for gluconeogenesis and fermentation to ethanol and acetate, making H. orenii an attractive strain for the conversion of starch into bioethanol. © 2013 Elsevier Inc. All rights reserved.

Contents 1.

2. 3.

The extremophilic realm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Halophilic ecology and physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Halothermophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Importance of extremozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Halothermothrix orenii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Halophilic proteins and unique features of H. orenii proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. orenii glycosyl hydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Alpha–Amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Pullulanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Alpha-glucosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Beta-mannosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Alpha-mannosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Beta-glucosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Chitinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Beta-galactosidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Beta-1,3-glucanases and glucan endo 1,3-beta-glucosidases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10. Fructokinases and ribokinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11. Xylose isomerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12. Alpha-L-arabinofuranosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. E-mail address: [email protected] (B.I. Pletschke). 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.10.011

160 160 160 160 160 160 162 162 162 164 164 164 164 165 165 165 165 165 165

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4. 5. 6. 7. 8.

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Other enzymes of industrial and biotechnological significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complete enzyme machinery for starch hydrolysis and fermentation in one organism, H. orenii, for consolidated bioprocessing . . . . . . . . . . . . . . . Organic solvent tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improving enzyme application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. The extremophilic realm Extremophiles are organisms that are well adapted to extreme environmental conditions. Organisms that live at the extremes of pH (pH > 8.5, pH < 5.0), temperature (>45 ◦ C, <15 ◦ C), pressure (>500 atmospheres), salinity (>1.0 M NaCl) and in high concentrations of recalcitrant substances or heavy metals (extremophiles) represent one of the last frontiers for biotechnological and industrial discovery. Extremophiles are physiologically heterogenous. They include aerobic and anaerobic chemoorganotrophs, photoautotrophs, chemolithotrophs, and photoheterotrophs [1]. 1.1. Halophilic ecology and physiology Halophilic microorganisms inhabit saline and hypersaline environments with sodium chloride concentrations up to saturation. These organisms are adapted to high salt concentrations and the high osmotic pressure of their environment. Halophiles thrive in hypersaline lakes and other environments with high salt concentrations. Diverse prokaryotic communities have been found over a wide range of salt concentrations from seawater (0.5 M or 3.3% w/v) to lakes saturated with sodium chloride (5 M or 33% w/v). Halophilic microorganisms require very high salt (NaCl) concentrations for growth. They are found in salterns and hypersaline lakes, such as the Great Salt Lake, the Dead Sea and solar lakes in Africa, Europe and the USA, and have also been reported in Antarctic lakes [2]. Hypersaline environments are divided into two broad categories: thalassohaline and athalassohaline. Thalassohaline water bodies have similar salt composition to seawater with sodium and chloride being the dominant ions; common examples include the Great Salt Lake in Utah, and brine springs from underground salt deposits and solar salterns [3]. In contrast, athalassohaline water bodies such as the Dead Sea, Lake Magadi in Kenya, Wadi Natrun in Egypt, the sodalakes of Antarctica and Big Soda Lake and Mono Lake in California are dominated by potassium, magnesium, or sodium [3]. Hypersaline water bodies are commonly 9–10 times more concentrated than sea water, which is generally defined as having 3.5% (w/v) dissolved salts [4]. Halophiles are spread all over the phyla and orders of the domain Bacteria and appear to be an individual adaptation to high salt concentrations. The halophilic bacteria are physiologically heterogeneous; they include the aerobic moderate halophiles of the family Halomonadaceae [5], the fermentative obligately anaerobic halophilic Haloanaerobiales [6], the halophilic anoxygenic photosynthetic sulfur bacteria of the genus Halorhodospira and Ectothiorhodospira [7] and the photosynthetic Cyanobacteria [8]. 1.2. Halothermophiles A halothermophile exhibits characteristics unique for growth optima of both halophiles and thermophiles. Various categories within the halophiles and thermophiles also exist. Obligate and extreme halophiles and halotolerant organisms are found in addition to moderate and hyperthermophiles. A halothermophile is defined as an organism requiring at least 1.5 M NaCl and a

165 166 166 167 167 167 167

temperature close to or above 50 ◦ C for optimal growth [9]. Only a small number of halothermophiles have been described thus far (Table 1). 1.3. Importance of extremozymes The majority of the enzymes used to date originate from mesophilic organisms and, despite their many advantages, the applications of these enzymes are restricted due to their limited stability at extremes of temperature, pH and ionic strength. In contrast, extremophiles are a potent source of extremozymes that exhibit extraordinary stabilities under extreme conditions. Thus, biocatalysis using extremophiles as well as extremozymes is rapidly being transformed from a fundamental science to an industrially viable technology. Each group of the extremophiles has unique features, which can be harnessed to provide enzymes with a wide range of applications [2,10]. Currently, a large amount of money is being invested worldwide in the development of industrial as well as biomedical applications of extremophiles and their extremozymes [10,11]. 2. Halothermothrix orenii Halothermothrix orenii is a true halophilic and thermophilic anaerobic bacterium that was isolated from a Tunisian salt lake [12]. It is an anaerobic bacterium with a typical Gram negative cell wall growing optimally at 60 ◦ C (minimum 42 ◦ C–maximum 70 ◦ C) with 10% NaCl (growth range between 4 and 20%) and optimal pH range of 6.5–7.0 (growth within pH range of 5.5–8.2). It is currently classified under the order Haloanaerobiales in the class Clostridia of the Firmicutes (low G + C Gram positive) phylum on the basis of 16S rRNA sequence analysis [12]. The genome consists of one circular chromosome of 2,578,146 bp with a %GC content of 38%. A total of 2366 of the identified 2451 genes were predicted as protein coding genes. The central metabolism of H. orenii contains all the genes necessary for the glycolytic degradation of monosaccharides. Enzymes of the TCA cycle are absent, but the cellular machinery is equipped with enzymes for gluconeogenesis and fermentation of ethanol to acetate as well as production of butyrate from branched amino acids. Genes involved in the metabolism of cellobiose (but not cellulose), starch, glucose, galactose, fructose, fucose, xylose, ribose, and citrate were identified [13]. In H. orenii the amino acid composition of the proteins bear a resemblance to the amino acid profile of thermophilic organisms and is quite divergent from that of the salt-in halophilic profiles, suggesting that its proteins have been adapted to high temperatures and a salt-out strategy. This type of adaptation to salinity is an indication of the versatility of the organism which allows it to survive in a diverse environment with fluctuations in salt concentration [14]. 2.1. Halophilic proteins and unique features of H. orenii proteins Glycosyl hydrolases include a diverse range of enzymes that are industrially very important. According to a report by the BCC Research (a publisher of market research reports, reviews and technical newsletters, formerly known as Business Communications

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Table 1 The diversity of halothermophiles isolated from different saline niches. Microorganism

Isolation source

Na+ opt. (M)

Na+ range (M)

Aerobic Dichotomicrobium thermohalophilum Salinibacter ruber

solar lake near Elat (Sinai) saltern crystallizer ponds, Spain

2.43 4.00

1.37–3.80 2.57–

8.5 8.0

Solar saltern, California Salin-de-Giraud, France Lake Magadi, Kenya Wadi An Natrun, Egypt

3.0 2.57 2.05 4.62 3.76 1.71 4.3 3.9 3.9 3.7 3.9

0.5–5.0 0.86–5.13 0.51–2.91 1.71–5.81 1.54–5.14 0.68–3.42 2.9-sat. 3.1–5.0 3.1–4.9 3.1–5.4 3.1–5.3

7.0 7.8 8.5 8.5 7.8 7.0 9.5 10.5 9.5 9.5 9.9

Anaerobic Halanaerobacter chitinivorans Halanaerobacter salinarius Halonatronum saccharophilum Halorhodospira halochloris Halorhodospira halophila Halothermothrix orenii Natranaerobius grantii Natranaerobius jonesii Natranaerobius thermophilus Natranaerobius trueperi Natronovirga wadinatrunensis

Chott El Guettar (lake), Tunisia Lake Magdi, Kenya Lake Magdi, Kenya Wadi An Natrun, Egypt Wadi An Natrun, Egypt Wadi An Natrun, Egypt

pH opt.

pH range

Temp. opt

5.8–9.5 6.0–8.5

50 47

20–65 20–52

[88] [89]

45 45 55 48 50 60 46 66 53 52 51

23–50 10–50 18–60 33–50

[90] [91] [92] [93] [94] [12] [95] [96] [97] [1] [1]

5.5–8.5 7.7–10.3 8.1–9.1 5.5–8.2 7.5–10 8.5–11.5 8.3–10.6 7.8–11.0 8.5–11.5

Temp. range

45–68 31–52 47–71 35–56 26–55 24–58

Reference

Blank cells indicate data not found. Adapted from Bowers et al. [97].

Company Inc.), the total expenditure on the enzyme market, particularly the industrial enzymes, has increased up to US $3.3 billion in 2011, and is expected to rise further up to US $4.4 billion by the end of 2015 [15]. The ever increasing demand of improved and novel enzymes that can withstand harsh conditions such as extremes of pH, temperature, and salinity has led studies to focus on extremophiles and polyextremophiles, as their metabolic machinery allows them to withstand harsh environmental conditions. Consequently, their enzymes (extremozymes) provide a better alternative under such demanding conditions compared to their mesophilic counterparts. The last decade has seen an upsurge in the identification, purification, and characterization of extremophiles and particularly halophiles, due to their ability to perform optimally under different salt concentrations. Some of these extremophiles are also thermostable and exhibit a wide range of pH tolerance [16]. The limited availability of water affects the structural and functional dynamics of most enzymes; adaptation to high salt concentrations also requires proteins to compete with salts for retaining water molecules. In fact, halophilic proteins and enzymes have unique properties that allow them to have multilayered hydration shells and maintain their functional conformation in the presence of high ionic concentration [15,16]. The halophilic proteins exhibit unique characteristics: an abundance of acidic amino acids, notably, glutamatic acid, aspartic acid, serine and threonine; and these halophilic proteins are also characterized by an increase in the concentration of smaller hydrophobic amino acids (glycine, alanine and valine) and a reduction in the number of lysine residues. These characteristics allow a higher degree of cooperation between the electrostatic interactions and formation of higher number of salt bridges that allow halophilic enzymes to perform optimally. A number of halophilic glycosyl hydrolases from both the bacterial and archeal domains have been studied that exhibit similar properties [15,16]. However, the crystal structure study of Amy A from H. orenii indicated the lack of higher number of acidic amino acid residues on the surface of the protein. Genome tag analysis [17] and whole genome sequencing studies [13] have also indicated the presence of an increased number of both positively and negatively charged residues. Genome sequencing studies also indicated the presence of a reduced number of thermolabile amino acids, notably glutamine, histidine and threonine, and an increase in the number of hydrophobic lysine residues (Table 2). These changes are unique to H. orenii proteins and are completely opposite to the well established concept of stabilized halophilic proteins. Such alterations can be explained if we analyse the changes, keeping in mind the thermostability of the H. orenii proteins. The increase in number of positive and

negative residues and decrease in frequency of thermolabile amino acids, glutamine and threonine (that are important for stabilization under saline conditions) is subject to higher thermostability due to an increase in ionic bonds between oppositely charged amino acids, and is an established characteristic for thermostable proteins [18]. Table 2 also indicates that the amino acid composition of H. orenii has a higher degree of resemblance to thermophilic proteins compared to halophiles; however, there are certain characteristics like a higher number of lysine and isoleucine residues that are unique to this species and further studies are required to elucidate their role. H. orenii is a polyextremophile and thus represents an interesting category that involves proteins that are thermostable and halostable. Thus, this organism is unique and as it shares and also differs in both the physical and chemical properties of its proteins compared to that in both thermophiles and halophiles [14]. Amy A does not have an acidic surface and other structural features known to stabilize halophilic proteins; it also lacks the presence of surface exposed salt bridges and metal ion binding sites relevant to thermophilic proteins. The structural insights provided by thermal stability studies at different salt concentrations of Amy A indicate that the protein is extremely thermostable over the entire salt concentration range up to 4.7 M and retains 40% of the activity in the absence of salt with no significant change in its secondary structure [14]. Novel oligomerization of the Amy A protein in the absence of any salt maintains the structural and functional stability of the protein, and with an increase in salinity, the structure opens up into monomers due to binding of salt at the sites responsible for intersubunit interactions. Such phenomena are not observed with other halophilic proteins, as under low salt concentration they have low stability due to repulsive forces exerted by excessive acidic amino acid residues. The oligomerization of Amy A is completely different from previously established oligomerization of halophilic proteins with respect to changes in salt concentration [19]. The unique features of Amy A and other H. orenii proteins indicate a clever evolutionary strategy used to handle two extreme conditions simultaneously, i.e. the fact that the organism was isolated from a hypersaline lake, where water evaporates during summer and fills during rain, resulting in extreme salt and temperature fluctuations. Thus, the evolution of this novel adaptive measure seems logical when the poly-extremophilic existence of H. orenii is considered. The distinctive attributes of H. orenii proteins, notably thermostability, adaptation to a wide range of salt concentration and broad pH stability, promote their favourable application in novel enzymatic processes (biofuel production, foods, textiles, chemicals and pharmaceuticals, etc.) under harsh conditions with attractive biotechnological potential.

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Table 2 A comparison of the differences in significant amino acid residues and their abundance between thermophilic and halophilic bacteria and H. orenii (Modified from Mavromatis et al. [13]. Amino acid

Halophiles (approximate percentage, %)

Thermophiles (approximate percentage, %)

H. orenii (approximate percentage, %)

Properties

Alanine (A) Aspartic acid (D) Histidine (H)

10.0 6.0 2.0

8.0 4.5 1.8

6.0 5.5 1.8

Isoleucine (I) Lysine (K) Glutamine (Q)

5.0 3.0 5.0

6.2 6.5 3.0

9.0 9.0 3.0

Threonine (T)

6.2

5.0

4.6

Tyrosine (Y) Proline (P) Asparagine (N)

3.0 5.0 3.8

4.0 5.0 3.8

4.0 3.8 5.0

Hydrophobic, high frequency in halophiles, important for halostability Acidic, positively charged, important for halostability Basic, negatively charged, thermo-labile, low frequency in thermostable proteins Hydrophobic, role not yet ascertained Hydrophobic, characterized by its low frequency in halophiles. Acid amide, polar charged converts to glutamic acid (acidic), highly desirable for halostability but thermolabile and low frequency in thermostable proteins Polar charged, higher frequency in halostable proteins, but thermolabile and low frequency in thermostable proteins Polar aromatic uncharged Cyclic, non polar hydrophobic Acid amide, polar charged, important for halostability

This review focuses on those glycosyl hydrolases identified and predicted in the genome of H. orenii, it targets the glycosyl hydrolase enzymes that have been characterized to date and also provides a bird’s eye view of the currently uncharacterized glycosyl hydrolases whose genes have been identified in the H. orenii genome and which are well studied in other organisms (Table 3). The focal theme of this review also includes the extremozymes from H. orenii and how they can be applied in different industries.

3. H. orenii glycosyl hydrolases 3.1. Alpha–Amylases Two ␣-amylases, Amy A [20] and Amy B [21] have been cloned from the genomic library of H. orenii generated in E. coli [17]. Analysis of the crystal structures provided important insights into biochemical characteristics along with functional and structural properties of amylases from this anaerobic thermohalophile. The amy A gene is 1545 bp long and encodes a 515 amino acid residue protein containing 25 residues as signal peptide. The mature protein contains 490 amino acid residues with a molecular weight of 56.96 kDa. The enzyme was found to be stabilized at 70 ◦ C in presence of NaCl (10%) and CaCl2 (10 mM). Amy A exhibits a halophilic nature as it requires 5% (w/v) NaCl for optimum activity. The enzyme shows remarkable activity (90%), even at very high salt concentration (25% w/v); interestingly, the enzyme also retains 45% activity even in the absence of NaCl. The enzyme exhibits a broad pH (6–9.5) and temperature profile (37 ◦ C–75 ◦ C), being optimally active at pH 7.5 and a temperature of 65 ◦ C. The gene amy B was identified in one of the recombinant clones from the genomic library of H. orenii and encodes for a 624 amino acid residue. While the first 25 residues comprises the signal peptide, the mature protein consists of 599 amino acid residues with a molecular weight of ∼71.0 kDa and a theoretical pI of 4.4. Similar to Amy A, Amy B is active over a broad range of salt concentrations, retaining 80%, 60%, 12% and >45% activity at 1.7 M, 2.6 M, 4.3 M and 0 M concentrations of NaCl, respectively. The enzyme exhibits optimum activity at pH 7.0 with soluble starch as substrate and retains >20% activity within the pH range 5.2–9.3. The enzyme retains >40% activity over a wide range of temperature (37 ◦ C–80 ◦ C) with an optimum of 65 ◦ C. Similar to Amy A, Amy B also exhibits a strong dependence on calcium ions [20,21]. Certain distinct differences are portrayed between Amy A and Amy B. The ORF of amy B showed high homology in particular with bacterial ␣-amylases from Bacillus stereothermophilus and Bacillus licheniformis but not with amy A. Amy B is characterized by the presence of an N-domain which is absent in Amy A.

Moreover, Amy B is characterized by the presence of three methionine side chains (Met 316, Met 235, and Met 176) that are engaged as sugar platforms resembling sugar aromatic ring stacking. Amy B displays a typical charge distribution pattern with increased negative charged residues over positive residues [21], indicating the presence of excess amino acid residues on the surface of the protein, but Amy A exhibits an even charge distribution of both positive and negative charges and lacks excess acidic amino acid residues [14], thus illustrating two different modes of adaptability required for survival in a habitat that is subjected to extreme fluctuations in salt concentration, temperature variation and random changes in ionic concentration. The fact that H. orenii genome contains four copies of ␣-amylase gene (Hore 02410, Hore 23200, Hore 09700, Hore 14700) illustrates the importance of this enzyme and also indicates starch as a major source of energy in the specific sediment depth at which the bacterium can feed. Halophilic amylases have been reported from both archeal and bacterial domains. These have markedly different enzymatic properties and range from being halotolerant, thermohalotolerant, halophilic and thermohalophilic [11]. Amylases are being employed in different biotechnological applications and comprise a substantial part of industrial enzymes with ∼25% of market share [22]. Alpha-amylases have been extensively used in food industry for bread making, processing of starch for synthesis of highly branched dextrins that are used as food ingredients, in detergents to promote stain removal and also in the paper and pulp industry for the modification of starches for coated paper [3,23].

3.2. Pullulanases The genome analysis of H. orenii reveals two genes, Hore 19550 and Hore 13880, both coding a pullulanase type I (3.2.1.41) enzyme. Hore 19550 encodes for a 640 (1923 bp) amino acid polypeptide with a molecular weight of 72.95 kDa (from nucleotide sequence), while Hore 13880 encodes for a 874 (2625 bp) amino acid polypeptide with molecular weight of 98.79 kDa (from nucleotide sequence). Pullulanases have been characterized from proteins purified from different bacterial and archeal sources. The cloning, purification and characterization of pullulanase type I enzymes from H. orenii may be the first report of a thermohalotolerant pullulanase type I from halothermophilic bacterium. Due to the specific debranching ability, the use of pullulanase in starch processing industry is specifically promoted. Pullulanase is employed in the saccharification process to enhance the efficiency of the process [24]. The products from the starch-processing have wide applications in various industries, such as in the production of beverages, confectionary, canning, ice cream, etc. Pullulanase can also be

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Table 3 Industrially important enzymes identified in H. orenii genome. Gene

Enzyme

Reaction catalyzed

Position on chromosome

Nucleotide sequence length/amino acid sequence/Molecular weight of polypeptide

Hore 19090

␣-glucosidase (EC 3.2.1.20)

2,040,845 ← 2,043,250 79.16 centisomes

2406 bp/801 aa/93.15 kDa

Hore 23190

␣-glucosidase (EC 3.2.1.20) ␣-mannosidase (EC 3.2.1.24) ␣-mannosidase (EC 3.2.1.24) ␣-mannosidase (EC 3.2.1.24) ␣-amylase (EC 3.2.1.1)

(1,4-␣-D-glucosyl)(n) + H2 O <=> (1,4-␣-Dglucosyl)(n−1) + ␣-D-glucose maltotriose + H2 O <=> maltose + ␣-D-glucose ␣-maltose + H2 O <=> 2 ␣-D-glucose (1,4-␣-D-glucosyl)(n) + H2 O <=> (1,4-␣-Dglucosyl)(n−1) + ␣-D-glucose [reactants unspecified] <=> [products unspecified]

2,534,773 ← 2,536,878 98.32 centisomes 2,508,176 ← 2,511,340 97.29 centisomes 1,530,512 ← 1,533,658 59.36 centisomes 1,600,795 ← 1,603,407 62.09 centisomes 248,392 → 249,486 9.63 centisomes

2106 bp/701 aa 79.973 kDa 3165 bp/1054 aa/120.48 kDa

2,536,924 ← 2,538,888 98.4 centisomes 1,054,625 → 1,055,905 40.91 centisomes 1,567,871 ← 1,569,418 60.81 centisomes 433,564 → 436,767 16.82 centisomes 2,211,551 ← 2,213,803 85.78 centisomes

1965 bp/654 aa/75.208 kDa

2,132,901 ← 2,135,120 (82.73 centisomes)

2220 bp/739 aa/81.865 kDa

518,050 → 519,348 20.09 centisomes

1299 bp/432 aa/50.445 kDa

1,407,371 ← 1,409,884 54.59 centisomes 2,371,686 → 2,372,810 91.99 centisomes 450,354 → 454,226 17.47 centisomes

2514 bp/837 aa/98.505 kDa

2,097,006 ← 2,099,630 81.34 centisomes

2625 bp/874 aa/98.798 kDa

1,479,115 ← 1,481,037 57.37 centisomes

1923 bp/640 aa/72.953 kDa

2,092,895 ← 2,093,959 81.18 centisomes

1065 bp/354 aa/40.437 kDa

Hore 22970 Hore 14420 Hore 15000 Hore 02410

Hore 23200 Hore 09700 Hore 14740 Hore 04160 Hore 20490

␣-amylase (EC 3.2.1.1) ␣-amylase (EC 3.2.1.1) ␣-amylase (EC 3.2.1.1) ␤-1,3 glucanase (EC 3.2.1.39) ␤-galactosidase (EC 3.2.1.23)

Hore 19810

␤-glucosidase (EC 3.2.1.23) (EC 3.2.1.74)

Hore 04820

␤-glucosidase (EC 3.2.1.23) (EC 3.2.1.74) ␤-mannosidase (EC 3.2.1.25) chitinases (EC 3.2.1.14) glucan endo-1,3-␤D-glucosidase (EC 3.2.1.39) pullulanases, type 1 (EC 3.2.1.41) pullulanases, type 1 (EC 3.2.1.41) xylose isomerise (EC 5.3.1.5)

Hore 13110 Hore 21810 Hore 04240

Hore 19550

Hore 13880

Hore 19520

[reactants unspecified] <=> [products unspecified] [reactants unspecified] <=> [products unspecified] a maltooligosyl-trehalose <=> a maltodextrin + trehalose starch + H2 O <=> a maltodextrin(n) + maltose + D-glucose a 1,4-␣-D-glucan + H2 O <=> a 1,4-␣-D-glucan + maltopentaose a 1,4-␣-D-glucan + n H2 O <=> a 1,4-␣-D-glucan + maltohexaose starch <=> maltose + glucose starch + n H2 O <=> a large-branched glucan a long-linear glucan + n H2 O <=> n short glucans Same as by Hore 02410 Same as by Hore 02410 Same as by Hore 02410 1,3-␤-D-glucan(n) + H2 O <=> 1,3-␤-D-glucan(n−1) + ␤-Dglucose lactose < => allolactose lactose + H2 O <=> ␤-D-galactose + ␤-D-glucose XXLG xyloglucan oligosaccharide + H2 O <=> XXXG xyloglucan oligosaccharide + ␤-D-galactose XLLG xyloglucan oligosaccharide + 2 H2 O <=> XXXG xyloglucan oligosaccharide + 2 ␤-D-galactose 3’-ketolactose + H2 O <=> 3-keto-␤-D-galactose + ␤-Dglucose a galactosylated galactose acceptor + H2 O <=> a non galactosylated galactose acceptor + ␤-D-galactose XLXG xyloglucan oligosaccharide + H2 O <=> XXXG xyloglucan oligosaccharide + ␤-D-galactose DIMBOA-Glc + H2 O <=> DIMBOA + ␤-D-glucose (1,4-␤-D-glucosyl)(n) <=> (1,4-␤-D-glucosyl)(n−1) + ␤-Dglucose lotaustralin + H2 O <=> (2R)-2-hydroxy-2methylbutanenitrile + ␤-D-glucose cellobiose + H2 O <=> 2 ␤-D-glucose linamarin + H2 O <=> acetone cyanohydrin + ␤-D-glucose cis-coumarinic acid-␤-Dglucoside + H2 O <=> coumarinate + ␤-D-glucose a ␤-D glucoside + H2 O <=> a non glucosylated glucose acceptor + ␤-D-glucose Same as by Hore 19810

a ␤-D-mannoside + H2 O <=> ␤-D-mannose + an organic molecule chitin + n H2 O <=> n a chitodextrin 1,3-␤-D-glucan(n) + H2 O <=> 1,3-␤-D-glucan(n−1) + ␤-Dglucose a large-branched glucan + n H2 O <=> n a long-linear glucan pullulan + n H2 O <=> n maltotriose Same as by Hore 19550

␤-D-glucose < => D-fructose ␣-D-xylopyranose < => D-xylulose

3147 bp/1048 aa/122.02 kDa 2613 bp/870 aa/100.97 kDa 1095 bp/364 aa/43.707 kDa

1281 bp/426 aa/49.994 kDa 1548 bp/515 aa/59.933 kDa 3204 bp/1067 aa/118.73 kDa 2253 bp/750 aa/86.489 kDa

1125 bp/374 aa/42.827 kDa 3873 bp/1290 aa/144.53 kDa

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Table 3 (Continued) Gene

Enzyme

Reaction catalyzed

Position on chromosome

Nucleotide sequence length/amino acid sequence/Molecular weight of polypeptide

Hore 18220

fructokinase (EC 2.7.1.4) galactokinase (EC 2.7.1.6) glucokinase (EC 2.7.1.2) ribokinase (EC 2.7.1.15) carbonate dehydratase (EC 4.2.1.1) asparaginase (EC 3.5.1.1)

␤-D-fructofuranose + ATP <=> D-fructose-6phosphate + ADP + H+ ␣-D-galactose + ATP <=> ␣-D-galactose 1-phosphate + ADP + H+ a D-hexose + ATP <=> D-hexose 6-phosphate + ADP

1,945,042 ← 1,946,004 75.44 centisomes 2,234,999 ← 2,236,213 86.69 centisomes 1,720,418 ← 1,721,386 66.73 centisomes 491,274 → 492,194 19.06 centisomes 1,483,933 ← 1,484,535 57.56 centisomes

963 bp/320 aa/35.391 kDa

Hore 20660 Hore 16050 Hore 04580 Hore 13920

Hore 01750 a

+

D-ribose + ATP <=> D-ribose-5-phosphate + ADP + H bicarbonate + H+ <=> CO2 + H2 O carbonic acid <=> CO2 + H2 O L-asparagine + H2 O <=> L-aspartate + ammonia + H+

179,440 → 180,444 6.96 centisomes

1215 bp/404 aa/45.575 kDa 969 bp/322 aa/33.999 kDa 921 bp/306 aa/33.595 kDa 603 bp/200 aa/23.233 kDa

1005 bp/334 aa/36.536 kDa

adapted from BioCyc, SRI International.

applied in the baking and detergent industries [25]. There are some reports on the application of pullulanase in the synthesis of branched-cyclodextrins (CD) that can contribute to the pharmaceutical field as drug-carriers due to their higher aqueous solubility and cell-targeting abilities [26].

3.3. Alpha-glucosidase The H. orenii genome contains two genes, Hore 19090 (GCCC-2152) and Hore 23190 (GCCC-2571), that encode for ␣glucosidases. The gene Hore 19090 (2406 bp) encodes a 801 amino acid polypeptide with a molecular weight of 93.15 kDa (from nucleotide sequence), while Hore 23190 (2106 bp) codes for a 701 amino acid polypeptide with molecular weight of 79.97 kDa (from nucleotide sequence). Alpha-glucosidase from Aspergillus niger [27] and Bacillus stearothermophillus [28] have been reported to mediate transglycosylation reactions that are being exploited in biotechnology to produce oligosaccharides with applications in the food industry and also for conjugation of sugars with biologically active materials [29]. It will be interesting to understand the significance of these enzymes in H. orenii with special reference to their substrate specificity and also why two genes present in the genome encode for the same enzyme.

3.4. Beta-mannosidase The genome sequencing studies of H. orenii identified a gene, Hore 13110 (GCCC-1541), that encodes for an enzyme ␤-mannosidase (1,4-␤-D-mannopyranosidase hydrolase, E.C. 3.2.1.25); this enzyme constitutes the primary group of mannan degrading enzymes with ␤-mannanase and ␤-glucosidase. The gene constitutes of 2514 bp encoding for 837 amino acid polypeptide with an approximate molecular weight of 98.50 kDa (from nucleotide sequence). The enzyme is yet to be cloned, purified, and characterized. The biotechnological applications of the hydrolysis products, particularly simple sugars that can be used as energy, feed and food sources have been evaluated [30]. Other applications of this enzyme have been reported in the extraction of vegetable oils from leguminous seeds, viscosity reduction in extracts during the manufacture of instant coffee, improvement in beer consistency and in bio-pulping of softwood [31]. The application of ␤-mannosidases and related enzymes for synthesis of oligosaccharides, which are now becoming significant products in medical use, have increased the industrial and medical significance of these enzymes.

3.5. Alpha-mannosidase Alpha-Mannosidase is an enzyme involved in the cleavage of the ␣ form of mannose. Analysis of H. orenii genome has revealed the presence of three genes: Hore 14420, Hore 15000, and Hore 22970. Hore 14420 codes for a 1048 amino acid polypeptide with molecular weight of 122.02 kDa (from nucleotide sequence), Hore 15000 codes for a 870 amino acid polypeptide with a molecular weight of 100.97 kDa (from nucleotide sequence), while Hore 22970 codes for a 1054 amino acid polypeptide with a molecular weight of 120.48 kDa. The enzyme has been used in the medical field where it is involved in experiments that determine the presence or absence of mannose specific molecules such as recombinant proteins that are used in vaccine development [32].

3.6. Beta-glucosidase The H. orenii genome contains two ␤-glucosidase enzyme coding genes. Hore 19810 (GCCC-2224) with nucleotide sequence of 2220 bp encoding a 739 amino acid polypeptide with a molecular weight of 81.86 kDa (from nucleotide sequence). The other gene, Hore 04820, with a nucleotide sequence of 1299 bp, encodes for a 432 amino acid containing polypeptide with a molecular weight of 50.44 kDa (from nucleotide sequence). The ␤-glucosidase A gene (Hore 04820) that encodes for 451 amino acid polypeptide has been cloned, over-expressed in E. coli and has been purified to homogeneity using metal-ion affinity chromatography. The recombinant protein appeared as 53 kDa polypeptide unit as monitored by SDSPAGE under reducing and denaturing conditions [33]. The enzyme has been found to be active at high temperatures (45 ◦ C–60 ◦ C); however, biochemical characterization and substrate specificity analysis are still pending. The preliminary crystallographic analysis of recombinant ␤-glucosidase (BglA) from H. orenii indicates the presence of orthorhombic crystals with a resolution limit of ˚ ␤-glucosidases are widespread in bacterial domains. They 3.5 A. have extensive applications in the field of agriculture, biotechnology, industry and medicine. This enzyme forms the part of the cellulase machinery required for the complete degradation of cellulose [34]. The enzyme has also found valuable application in other industrial processes such as flavour enhancing and production of bio-degradable non-ionic surfactants [35], developmental regulation and chemical defence against pathogen attack [36], in the synthesis of diverse oligosaccharides, glyco-conjugates and alkylaminoglycosides. Application of such biomolecules is gaining interest in the manufacture of pharmaceuticals, fine chemicals and food ingredients, due to the high selectivity of the enzyme and mild reaction conditions involved [37].

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3.7. Chitinases The genome of H. orenii harbours a gene, Hore 21810 (GCCC2433), that encodes for a 374 (1125 bp) amino acid polypeptide with a molecular weight of 42.82 kDa, deduced from the nucleotide sequence. The enzyme has received increased attention due to its application for protoplast preparation from fungi [38], as a protective agent against plant pathogenic fungi [39] and in the production of chito-oligosaccharides as biologically active substances. The application of these biologically active oligosaccharides have extended to the fields of medicine, agriculture and other industries due to their antibacterial, antifungal, hypercholesterolemic and anti-hypersensitive activities, and as food quality enhancers [40,41]. The purification and characterization of H. orenii chitinases could offer potentially new applications for these enzymes due to their extremophilic source. 3.8. Beta-galactosidases The H. orenii genome has one gene, Hore 20490 (GCCC-2292), that encodes for a 750 amino acid (2253 bp) polypeptide with a molecular weight of 86.48 kDa (from nucleotide sequence). The enzyme is yet to be purified and characterized from a true thermohalophile. This enzyme has widespread industrial applications. It has been used in conjugation with glucose oxidase as a biosensor for the quantitative detection of lactose in commercial milk [42]. The associated transglycosylation reaction has enabled the synthesis and production of galacto-oligosaccharides (GOS) that have been used in the field of pre and probiotics as functional foods and as food ingredients in human and animal nutrition [43]. Fucosylated molecules have distinct biotechnological applications including their use as surface active molecules and non-ionic surfactants [44]. Another field of investigation related to ␤-D-fucosylated compounds is the synthesis of molecules with reputed pharmacological potential, such as asterosaponins with antitumor and antiviral activities [45]. 3.9. Beta-1,3-glucanases and glucan endo 1,3-beta-glucosidases The genome sequencing of H. orenii has revealed the presence of two genes: Hore 04160 (GCCC-621) coding for 1067 amino acid (3204 bp) polypeptide with a molecular weight of 118.77 kDa (from nucleotide sequence) and Hore 04240 (GCCC-629) coding for 1290 amino acid (3873 bp) polypeptide with a molecular weight of 144.53 kDa (from nucleotide sequence) with glucan endo 1,3-␤glucosidase activity. Bacterial ␤-1,3 glucanases have been reported to be involved in the breakdown of fungal cell walls and hydrolysis of microalgal cells that can be used as food source [46]. Recent studies have shown that ␤-1,3-glucanases from Delftia tsuruhatensis strain MV01 have potential applications in the field of vinification [47]. 3.10. Fructokinases and ribokinases The genome sequencing of H. orenii has revealed the presence of a gene Hore 18220 (GCCC-2064) encoding a 320 amino acid (963 bp) polypeptide with a molecular weight of 35.39 kDa (from nucleotide sequence) exhibiting fructokinase activity. The enzyme from H. orenii has been cloned, purified and its crystal structure refined to 2.8 A˚ [44]. Hore 18220 exhibits the highest structural similarity with uncharacterized putative sugarkinase PH1489 from Pyrococcus horikoshii (PDB ID: 3 EWM). Structure based homology studies with ribokinase indicate conserved residues predominantly in the substrate and nucleotide binding pockets [48]. The structure of H. orenii FRK provided a homology based model for the study of plant FRKs, the study also indicated that FRKs have smaller side

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chains than corresponding residues in ribokinase, making the FRK pocket larger, thereby potentially allowing the accommodation of larger substrates. The genome sequence of H. orenii indicated the presence of Hore 04580 (GCCC-663) gene encoding for ribokinase, a 306 amino acid polypeptide with a molecular weight of 33.59 kDa (based on nucleotide sequence and SDS-PAGE). The enzyme was cloned, over-expressed and purified using immobilized metal-ion affinity chromatography and crystallized using the sitting drop method [49]. 3.11. Xylose isomerases D-Glucose/xylose isomerase (D-xylose ketolisomerase; EC 5.3.1.5), commonly referred to as glucose isomerase, catalyses the reversible isomerisation of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The H. orenii genome codes for two xylose isomerase genes, designated as Hore 19520 (GCCC-2195) encoding 354 amino acid (1065 bp) polypeptide with a molecular weight of 40.43 kDa (from nucleotide sequence) and the other as Hore 19530 (GCCC-2196), encoding for 439 amino acid polypeptide with a molecular weight of 49.61 kDa (from nucleotide sequence). Isomerisation of glucose to fructose is of commercial importance in the production of high fructose corn syrup (HFCS) [50]. One of the major factors governing the application of this enzyme at commercial scale is its stability in the pH range 6.0–7.0 and increased thermostability [51]. Thus evaluation of H. orenii xylose isomerase under specified parameters could potentially offer a commercial candidate enzyme. This enzyme has very high commercial value; being one of the highest tonnage value enzymes (in addition to amylase and protease) over the past few decades [52]. 3.12. Alpha-L-arabinofuranosidase The genome analysis of H. orenii has revealed the presence of two sequences related to the GH3 family and one sequence related to family GH43. Hore 20580 (GCCC-2301) belonging to family GH43 encodes a 315 amino acid (948 bp) polypeptide with a molecular weight of 35.53 kDa, while the genes related to the GH 3 family have been identified to code for ␤-glucosidase and ␤-Nacetylhexosaminidase, thus indicating that the Hore 20580 gene should be coding for ␣-L-AFases in H. orenii. The presence of a ␣-LAFases in the H. orenii genome has been confirmed [[49], Kori and Patel personal communication]; however, cloning, purification, and characterization reports are still pending. L-arabinosyl residues restrict the hydrolysis of hemicelluloses and pectins [53,54], therefore acting as formidable technological barriers for different industrial processes [55]. ␣-L-AFases are accessory enzymes that act synergistically with other hemicellulases and pectic enzymes for the complete hydrolysis of hemicellulose and pectin [56]. A plethora of applications for this enzyme include the clarification of fruit juices [57], digestion enhancement of animal feed stuff [58], application in the wine industry [59], as a natural improver of bread, for the production of arabinose used as antiglycemic agent [60], production of antimetastatic and anticarcinogenic components [61], treatment of paper and pulp [62], production of fermentable sugars for bioethanol industry [63] and the synthesis of pentose containing compounds such as oligosaccharides and glycoconjugates via enzymatic or chemoenzymatic routes [64]. 4. Other enzymes of industrial and biotechnological significance The genome of H. orenii contains a Zn-dependent alcohol dehydrogenase Hore 03240 (GCCC-526) coding for a 358 amino acid polypeptide with a molecular weight of 39.67 kDa (from nucleotide

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sequence). This enzyme has found considerable interest in the production of chiral alcohols in pharmaceuticals and in fine chemical industries. Stereospecific alcohol dehydrogenases are very interesting from both a scientific and industrial perspective and have the inherent advantage over chemical catalysis in terms of their high chemo-enantio and regioselectivities [65]. The genome of H. orenii contains a nickel-iron dependent form of hydrogenase with two subunits Hyd N (Hore 03850) and Hyd B (Hore 03860), with hydrogenase producing maturation proteins HypA and HypF (Hore 3870, Hore 03920) indicating a fully functional hydrogenase [13]. Currently, there is a huge demand for chemical hydrogen, and today 96% of hydrogen is derived from fossil fuels with 48% from natural gas, 30% from hydrocarbons, 48% from coal and about 4% from electrolysis. Environmental concerns have resulted in an increase in the hydrogen requirement at refineries for gas line and diesel desulfurization [66]. Hydrogen is considered as an alternate for fossil fuels. Industrial fermentation of hydrogen or whole cell catalysis requires a limited amount of energy, allowing hydrogen to be produced from any organic matter that can be derived from whole cell catalysis. The use of H. orenii as hydrogen producers, along with the members of genus Clostridium that are natural hydrogen producers, as mixed culture under thermophilic conditions with a pH range of 5.0–6.5 provides a better alternative as it allows co-operation of different species to efficiently degrade and convert organic waste into hydrogen, accompanied by the formation of organic acids [67]. The H. orenii genome harbours a carbonate dehydratase gene designated as Hore 13720, which encodes a 200 amino acid (603 bp) polypeptide with a molecular weight of 23.33 kDa (from nucleotide sequence). This enzyme is also known as carbonic anhydrase and it catalyses the reversible hydration of carbon dioxide to bicarbonate. Recent studies have demonstrated the application of the ancient enzyme in novel biomimetic approach for CO2 sequestration [68–70]. This strategy involves the conversion of CO2 to carbonic acid mediated by carbonic anhydrase at neutral or slightly alkaline pH, where carbonic acid is converted into carbonate ions, which in the presence of a saturated concentration of calcium ions (seawater or produced water or concentrated brine) are precipitated as calcium carbonate, which can be stored indefinitely [70]. A thermostable enzyme active at high calcium ion concentration is essential for this process. Purification and further characterization of H. orenii carbonic anhydrase could offer a potential candidate for this approach. The H. orenii genome also contains many genes related to several other glycosyl hydrolases, glycosyl transferases, glycosyl esterases and carbohydrate binding modules (Table 4). Their identification and characterization may provide certain other important enzymes with novel traits relevant to different industries.

renewable and sustainable fuel in comparison to other feedstocks [72]. “First Generation” fuel crops, based on sugars/starch from food grains and oil-based crop plants, have limited application, because of the lack of availability of cultivable land and the food-versus-fuel debate [73]. The use of lignocellulosic biomass (termed “second generation biofuels”) as polysaccharide source has some advantages, but it suffers from the high costs associated with different pre-treatment strategies and the enzymatic hydrolysis process. Algal biofuel(s), particularly bioethanol from algae, are gaining worldwide importance and is grouped under “third generation” biofuels. Assimilation of carbohydrate and starch in algae occurs via the photosynthetic conversion of atmospheric carbon dioxide, thus algae act as natural sinks for the removal of greenhouse gases and as bio-refineries for the production of bioethanol [74]. Both microalgae and macroalgae can be used as source of starch containing biomass. Microalgae can thrive under higher levels of carbon dioxide and can thus utilise carbon dioxide emitted from petroleum based power stations or other industrial processes. Algae are not as structurally complex as higher plants and thus they do not require stringent pre-treatment processes for the effective breakdown of biopolymers [74]. Saccharification of marine micro-algae using amylases produced by halotolerant marine bacteria for ethanol production has been successfully reported [75]. Intracellular microalgal starch can be easily extracted and fermented to ethanol, which involves two processes, saccharification and fermentation [76]. Saccharification is achieved using alpha-amylases while fermentation occurs in presence of yeast cells. H. orenii encodes the complete enzymatic machinery required for starch hydrolysis and the enzymes are thermo-halostable, thus marine microalgae can be utilized as biomass source without prior desalination. The process can be carried out at higher temperatures, thus aiding in the mechanical breakdown of the algal biomass and reducing the risk of microbial contamination. The process of fermentation represents the bottleneck as it requires the presence of yeast cells. Simultaneous saccharification and fermentation (i.e. consolidated bioprocessing) has been at the forefront of research as it improves the bioethanol yield greatly. Utilisation of starch degrading ethanol producing microbial strains can reduce the costs involved during the saccharification process. H. orenii is an anaerobe; genome sequencing studies have revealed the presence of enzymes for gluconeogenesis and fermentation to ethanol and acetate, making H. orenii an attractive strain for the conversion of starch into bioethanol. Thus, further research on utilising H. orenii as a novel organism for the industrial production of bioethanol from marine algal sources should be encouraged.

6. Organic solvent tolerance 5. Complete enzyme machinery for starch hydrolysis and fermentation in one organism, H. orenii, for consolidated bioprocessing The production of biofuel (especially bioethanol) from starch/cellulosic biomass has led to studies worldwide devising different mitigatory strategies for reducing greenhouse gas emissions. Biomass assimilation involves the utilisation of atmospheric carbon dioxide thus reducing the greenhouse gas levels. Ethanol is less toxic and biodegradable and its blending with gasoline can significantly reduce the emission of greenhouse gases. Bioethanol can also be used as a fuel for power generation in thermo-chemical fuel cells, in power co-generation systems and in the form of raw materials in chemical industries and also as replacement for octane enhancers [71]. Algae can provide a high yield of bioethanol and can serve as a suitable alternative for

The past few decades have seen an emerging interest in the industrial application of enzymes under non-aqueous conditions, especially the use of organic solvents [77]. The use of organic solvents improves the solubility of non-polar substrates and eliminates the chances of microbial contamination. Loss of enzyme activity in the presence of organic solvents is attributed to the loss of crucial water molecules that restrain the protein conformation, affecting the Km and Vmax values. Activity retention in the presence of organic solvents is possible only when the surface and the active site remain well hydrated [78,79].The ability of halostable enzymes to retain hydration shells under low water concentration can be extended to a range of functions in the nonaqueous environment. Halophilic enzymes such as proteases with organic solvent tolerance have been purified from Salinivibrio sp. strain AF-2004 [80]. Organic solvent tolerance of halophilic ␣amylase from a haloarcheon, Haloarcula sp. strain S-1, in presence of

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Table 4 The different carbohydrate metabolizing families identified in H. orenii genome. Glycoside Hydrolase Family

Glycosyl Transferase Family

Carbohydrate Esterase Family

Carbohydrate-Binding Module Family

Family type

No. of Sequences

Family type

No. of Sequences

Family type

No. of Sequences

Family type

No. of Sequences

GH1 GH2 GH3 GH4 GH13 GH16 GH17 GH18 GH30 GH31 GH32 GH38 GH43 GH57 GH65 GH81 GH94 GH97

03 04 02 01 12 03 01 02 01 03 01 03 01 01 03 01 03 01

GT2 GT4 GT5 GT9 GT19 GT26 GT28 GT30 GT35 GT51 GT83 GTNC

05 09 02 03 01 01 01 01 03 02 01 02

GE04 GE09 GE11

03 01 01

CBM4 CBM6 CBM25 CBM41 CBM48 CBM50

02 01 01 01 03 17

chloroform, has been reported [81]. A halophilic ␣-amylase, purified from the moderately halophilic Nesterenkonia sp. strain F, was found to be stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane [82]. H. orenii ␣-amylases are stable over a wide range of salt concentrations which makes them ideal candidates as organic solvent tolerant enzymes. Characterisation of these ␣-amylases for organic solvent tolerance could therefore provide enzymes with novel properties and potential industrial applications. Industrial application of organic tolerant halophilic ␣-amylases in the field of bioremediation of carbohydrate-polluted salt marshes and industrial waste water contaminated with solvents appears to be an emerging and encouraging prospect [82].

characterized and their structures have been solved. The enzymatic pool of this organism includes certain extremozymes such as amylases, pullulanases, chitinases, ␤-galactosidases, mannosidases, ␣-L-arabinofuranosidases, hydrogenases and ␤-glucosidases that have tremendous potential in different industries such as bioenergy (biofuel) production, lignocellulosic biomass conversion, bioremediation, food and feed ingredients and paper and pulp industries and novel biomedical applications. There is thus an urgent need for the cloning, overexpression, purification and characterization of the industrially relevant enzymes from H. orenii which will not only provide excellent candidate extremozymes, but will also provide an insight to the structural adaptability of these enzymes functioning under such extreme conditions.

7. Improving enzyme application

Acknowledgements

Enzymes that have optimal activities at extreme temperatures and pH are widely used in household detergents and in the food, textile, pulp and paper, leather processing and chemical industries. For each application, the enzymes have to fulfil numerous requirements related to features such as activity and stability, substrate specificity and enantioselectivity. As a result, natural enzymes are often not optimal for a desired biotechnological application. Consequently, a variety of approaches have been used to modify enzyme properties. These include error-prone PCR, saturation mutagenesis, structure-based protein engineering, and in vitro evolution approaches [83,84]. Such approaches are best combined with genetic selection or high-throughput screening, to identify the rare mutants that approach the target characteristics, followed by an iterative process of building fitness into the resulting variants. [85,86]. Recent findings have indicated that the quest for achieving low protein loads of industrially important enzymes can be achieved through modelling synergy studies that allows the construction of defined enzyme mixtures and hold the key for many biomass conversion based process [87].

The authors would like to express their gratitude to Rhodes University for granting A.B. a Rhodes University Postdoctoral Fellowship.

8. Future prospects The enzymatic machinery of H. orenii, with special reference to extracellular enzymes, offers tremendous biotechnological potential. H. orenii is an anerobe, and because it has enzymes required for gluconeogenesis and fermentation to ethanol and acetate, it may be an attractive strain for the conversion of starch into bioethanol. Only a few enzymes from this organism have been

References [1] Mesbah NM, Wiegel J. Natronovirga wadinatrunensis gen. nov., sp. nov. and Natranaerobius trueperi sp. nov., two halophilic, alkalithermophilic microorganisms from soda lakes of the Wadi An Natrun, Egypt. Int J Syst Ev Microbiol 2009;59:2042–8. [2] Gomes J, Steiner W. The biocatalytic potential of extremophiles and extremozymes. Food Technol Biotechnol 2004;42(4):223–35. [3] Litchfield CD, Gillevet PM. Microbial diversity and complexity in hypersaline environments: a preliminary assessment. J Indust Microbiol Biotechnol 2002;28:48–55. [4] Setati ME. Diversity and industrial potential of hydrolase producing producing halophilic/halotolerant eubacteria. African J Biotechnol 2010;9(11):1555–60. [5] Ventosa A, Nieto JJ, Oren A. Biology of aerobic moderately halophilic bacteria. Microbiol Mol Biol Rev 1998;62:504–44. [6] Rainey FA, Zhilina TN, Boulygina ES, Stackebrandt E, et al. The taxonomic study of the fermentative halophilic bacteria: description of Haloanaerobiales ord. nov., Halobacteroidaceae fam. nov., Orenia gen. nov. and further taxonomic rearrangements at the genus and species level. Anaerobe 1995;1:185–99. [7] Imhoff JF. True marine and halophilic anoxygenic phototrophic bacteria. Arch Microbiol 2001;176:243–54. [8] Oren A. The prokaryotes; an evolving electronic resource for the microbiological community. New York: Springer-Verlag; 2000. [9] Mesbah N, Wiegel J. Halophilic Thermophiles A Novel Group of Extremophiles. In: Satyanarayana T, editor. Microbial diversity: current perspectives and potential applications. New Delhi: I.K. International Publishing House Private Limited; 2005. p. 91–118. [10] Maki M, Leung TK, Qin W. The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci 2009;5(5):500–16. [11] Kumar L, Awasthi G, Singh B. Extremophiles A novel source of industrially important enzymes. Biotechnology 2011;10(2):121–35.

168

A. Bhattacharya, B.I. Pletschke / Enzyme and Microbial Technology 55 (2014) 159–169

[12] Cayol JL, Ollivier B, Patel BKC, Prensier G, Guezennec J, Garcia JL. Isolation and characterization of Halothermothrix orenii gen.nov., sp. nov., a halophilic, thermophilic, fermentative, strictly anaerobic bacterium. Int J Syst Bacteriol 1994;44(3):534–40. [13] Mavromatis K, Ivanova N, Anderson I, Lykidis A, Hooper SD, et al. Genome analysis of anaerobic thermohalophilic bacterium Halothermothrix orenii. PLoS ONE 2009;4(1):e4192. [14] Sivakumar N, Li N, Tang JW, Patel BKC, Swaminathan K. Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at polyextreme condition. FEBS Lett 2006;580:2646–52. [15] Delgado-Garcia M, Valdivia-Urdiales B, Aguilar-Gonzalvez CN, et al. Halophilic hydrolases as a new tool for the biotechnological industries. J Sci Food Agri 2012;92:2575–80. [16] Karan R, Capes MD, DasSharma S. Function and biotechnology of extremophilic enzymes in low water activity. Aquatic Biosys 2012;8:4. [17] Mijts BN, Patel BK. Random sequence analysis of genomic DNA of an anaerobic, thermophilic, halophilic bacterium, Halothermothrix orenii. Extremophiles 2001;5:61–9. [18] Hickey DA, Singer GAG. Genomic and proteomic adaptation to growth at high temperature. Genome Biol 2004;5:117. [19] Lin TY, Timasheff SN. On the role of surface tension in the stabilization of globular proteins. Protein Sci 1996;5:372–81. [20] Mijts BN, Patel BK. Cloning, sequencing and expression of an a-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme. Microbiology 2002;148:2343–9. [21] Tan T, Mijts BN, Swaminathan K, Patel BKC, Divne C. Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch. J Mol Biol 2008;378:850–68. [22] Rao MB, Tanksale AM, Gathe MS, Deshpande VV. Molecular and biotechnological aspects of microbial proteases. Micrbiol Mol Biol Rev 1998;62(3):597–635. [23] Gupta R, Gigras P, Mohapatran H, Goswami KV, Chauhan B. Microbial ␣-amylase: a biotechnological perspective. Process Biochem 2003;8: 1599–616. [24] Kim TJ, Park WS, Choi SK, Yoon YJ, Park KH, Kim JW. Molecular cloning and characterization of a thermostable pullulanase from a Thermus Strain IM6501. Food Science Biotechnol 2009;3:188–94. [25] van der Marrel MJEC, van der Veen B, Uitdehaag JCM, Leemhuis H, Dijkhuizen L. Properties and pplications of starch-converting enzymes of the ␣-amylase Family. J Biotechnol 2002;94:137–55. [26] Tanimoto T, Omatsu M, Ikuta A, Yuki N, Murakami H, Nakano H, Kitahata S. Synthesis of novel heterobranched ␤-cyclodextrins having ␤-D-Nacetylglucosaminyl-maltotriose on the side chain. Biosci Biotechnol Biochem 2005;69(4):732–9. [27] Duan KJ, Sheu DC, Lin CT. Transglucosylation of a fungal alphaglucosidase. The enzyme properties and correlation of isomaltooligosaccharide production. Ann NY Acad Sci 1995;750:325–8. [28] Mala S, Dvorakova H, Hrabal R, Kralova B. Towards regioselective synthesis of oligosaccharides by use of alpha-glucosidases with different substrate specificity. Carbohydr Res 1999;322:209–18. [29] Takenaka F, Uchiyama H. Synthesis of alpha-D-glucosylglycerol by alphaglucosidase and some of its characteristics. Biosci Biotechnol Biochem 2000;64:1821–6. [30] Jiang Z, Wei Y, Li D, Chai P, Kusakabe I. High-level production, purification and characterization of a thermostable ␤-mannanase from the newly isolated Bacillus subtilis WY34. Carbohydr Polym 2006;66:88–96. [31] Moreira LRS, Filho EXF. An overview of mannan structure and mannandegrading enzyme systems. Appl Microbiol Biotechnol 2008;79: 165–78. [32] Vlahopoulos S, Gritzapis AD, Perez SA, Cacoullos N, Papamichail M, Baxevanis CN. Mannose addition by yeast Pichia Pastoris on recombinant HER-2 protein inhibits recognition by the monoclonal antibody herceptin. Vaccine 2009;27(34):4704–8. [33] Kori LD, Hofmann A, Patel BKC. Expression, purification, and preliminary crystallographic analysis of recombinant ␤-glucosidase (BglA) from the halothermophile Halothermothrix orenii. Acta Cryst F 2011;67:111–3. [34] Zhang YH, Lynd LR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: non-complexed cellulase systems. Biotechnol Bioeng 2004;88:797–824. [35] Belancic A, Gunata Z, Vallier MJ, Agosin E. ␤-glucosidase from grape native yeast Debaryomyces vanrijiae: Purification, characterization, and its effect on monoterpene content of a Muscat grape juice. J Agric Food Chem 2003;51:1453–9. [36] Zagrobelny M, Bak S, Moller BL. Cyanogenesis in plants and arthropods. Phytochemistry 2008;69:1457–68. [37] Park TH, Choi KW, Park CS, Lee SB, Kang HY, et al. Substrate specificity, and transglycosylation catalysed by a thermostable ␤-glucosidase from marine hyperthermophile Thermotoga neapolitana. Appl Microbiol Biotechnol 2007;69:411–22. [38] Yabuki M, Kasai Y, Ando A, Fujii T. Rapid method of converting fungal cells into protoplast with a high regeneration frequency. Exp Mycol 1984;8: 386–90. [39] Sakuda S, Nishinato Y, Ohi M, Watanabe M, Takayama S, et al. Effects of demethylallosamidine, a potent yeast chitinase inhibitor, on the cell division of yeast. Agric Biol Chem 1990;54:1333–5.

[40] Dai DH, Hu WL, Li W. Purification and characterization of a novel extracellular chitinase from thermophilic Bacillus sp. Hu1. African J Biotech 2011;10(13):2476–85. [41] Purwani EY, Maggy TS, Yaya R, Jae KH, Yu RP. Characteristics of thermostable chitinase enzymes from the indonesian Bacillus sp.13.26. Enzyme Microbial Technol 2004;35:147–53. [42] Marrakchi M, Dzyadevych SV, Lagarde F, Martelet C, Jaffrezic-Renault N. Conductometric biosensor based on glucose oxidase and beta-galactosidase for specific lactose determination in milk. Material Sci Eng C 2008;28: 872–5. [43] Panesar R, Panesar PS, Singh RS, Kennedy JF, Bera MB. Production of lactose-hydrolyzed milk using ethanol permeabilized yeast cells. Food Chem 2007;101:786–90. [44] Kobayashi T, Liu D, Ogawa H, Miwa Y, Nagasaka T, et al. Removal of blood group A/B antigen in organs by ex vivo and in vivo administration of endo␤-galactosidase (ABase) for ABO-incompatible transplantation. Transplant Immunol 2009;20:132–8. [45] Li J, Robertson DE, Short JM, Wang PG. Chemical and enzymatic synthesis of glycoconjugates 4. Control of regioselectivity in high yielding synthesis of (␤D-fucopyranosyl)-O-D-xylopyranosyl disaccharides using a CLONENZYMETM thermophilic glycosidase. Tetrahedron Lett 1998;39:8963–6. [46] De la Cruz J, Pintor-Toro JA, Benitez T, Llobell A, Romero LC. A novel endo-ˇ-1,3glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol 1995;177:6937–45. [47] Blattel V, Larisika M, Pfeiffer P, Novak C, et al. ␤-1,3-Glucanase from Delftia tsuruhatensis strain MV01 and its potential application in vinification. Appl Environ Microbiol 2011;77(3):11–20. [48] Chua TK, Seetharaman J, Patel BKC, et al. Crystal structure of a fructokinase homolog from Halothermothrix orenii. J Struct Biol 2010;171:397–401. [49] Kori LD, Hofman A, Patel BKC. Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from thermohalophile Halothermothrix orenii. Acta Cryst 2012;68(Pt2):240–3. [50] Barker SA. Pure fructose syrups. Process Biochem 1976;11:20–5. [51] Bhosale SH, Rao MB, Deshpande VV. Molecular industrial aspects of glucose isomerase. Microbiological Rev 1996;60(2):280–300. [52] Wiseman A. Handbook of enzyme biotechnology. Chichester, United Kingdom: Ellis Horwood Ltd; 1975. [53] Shallom D, Belakhov V, Solomon D, Gilead-Gropper S, Baasov T, et al. The identification of the acid-base catalyst of ␣-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51glycoside hydrolase. FEBS Lett 2002;514:163–7. [54] Rahman SAKM, Kato K, Kawai S, Takamizawa K. Substrate specificity of the ␣-L-arabinofuranosidase from Rhizomucor pusillus HHT-1. Carbohydr Res 2003;338:1469–76. [55] Saha BC. ␣-L-Arabinofuranosidases: biochemistry, molecular biology and application in biotechnology. Biotech Adv 2000;18:403–23. [56] Spagna G, Andreani F, Salatelli E, Romagnoli D, Casarini D, Pifferi PG. Immobilization of the glycosidases: ␣-L-arabinofuranosidase and ␣-Dglucopyranosidase from Aspergillus niger on a chitosan derivative to increase the aroma of wine. Part II. Enzyme Microb Technol 1998;23:413–21. [57] Romboust FM, Voragen AGJ, Searle-van Leenwen MF, Geraeds CCJM, et al. The arabinanases of Asperigillus niger–purification and characterization of two ␣-L-arabinofuranosidase and an endo-1,5-␣-L-arabinanase. Carbohydr Polym 1998;9:25–47. [58] Hespell RB, O’Bryan P. Purification and characterization of an ␣-Larabinofuranosidase from Butyrivibrio fibrisolvens GS113. Appl Environ Microbiol 1992;58:1082–8. [59] Biskup SE, Intert F, Holthnujzen JH, Stengele M, Schultz G. Glycosically bound volatiles-A review 1986–1991. Flav Fragr J 1993;8:61–80. [60] Takao M, Akiyama K, Sakai T. Purification and characterization of thermostable endo-1,5-␣-L-Arabinase from a strain of Bacillus thermodenitrificans. Appl Environ Microbiol 2002;68:1639–46. [61] Bae EA, Park SY, Kim DH. Constitutive ␣-glucosidase hydrolysing ginsenoside Rb1 and Rb2 from human intestinal bacteria. Biol Pharm Bull 2000;23: 1481–5. [62] Bezalel L, Shoham Y, Rosenberg E. Characterization and delignification activity of a thermostable ␣-L-arabinofuranosidase from Bacillus stearothermophilus. Appl Microbiol Biotechnol 1993;40:57–62. [63] Sørensen HR, Pedersen S, Viksø-Nielsen A, Meyer AS. Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue. Enzyme Microb Technol 2005;36:773–84. [64] Rémond C, Plantier-Royon R, Aubry N, Maes E, et al. Synthesis of pentosecontaining disaccharides using a thermostable ␣-L-arabinofuranosidase. Carbohydr Res 2004;339:2019–25. [65] Kroutil W, Mang H, Edegger K, Faber K. Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols. Curr Opin Chem Biol 2004;8:120–6. [66] Schlag S, Suresh B, Yoneyama M. SRI Consulting CEH Report–Hydrogen. 2007. URL http://www.sriconsulting.com/CEH/Public/Reports/743.5000/ [67] Krupp M, Widmann R. Biohydrogen production by dark fermentation: Experiences of continuous operation in large lab scale. Int J Hydrogen Energy 2009;34(10):4509–16. [68] Sharma A, Bhattacharya A, Pujari R, Shrivastava A. Characterization of carbonic anhydrase from diversified genus for biomimetic carbon-dioxide sequestration. Indian J Microbiol 2008;48:365–71.

A. Bhattacharya, B.I. Pletschke / Enzyme and Microbial Technology 55 (2014) 159–169 [69] Sharma A, Bhattacharya A. Enhanced biomimetic sequestration of CO2 into CaCO3 using purified carbonic anhydrase from indigenous bacterial strains. J Mol Catal B: Enzym 2010;67:122–8. [70] Sharma A, Bhattacharya A, Shrivastava A. Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials. Enzym Microb Technol 2011;48:416–26. [71] Petrou CE, Pappis PC. Biofuels: a survey on pros and cons. Energy Fuel 2009;23:1055–66. [72] Subhadra B, Edwards M. An integrated renewable energy park approach for algal biofuel production in United States. Energy Policy 2010;38:4897–902. [73] John PR, Anisha GS, Nampoothri KM, Pandey A. Micro and macroalgal biomass: a renewable source for bioethanol. Bioresour Technol 2011;102:186–93. [74] Nigam PS, Singh A. Production of liquid biofuels from renewable sources. Prog Energy Combust Sci 2011;37:52–68. [75] Matsumoto M, Yokouchi H, Suzuki N, Ohata H, Matsunaga T. Saccharification of marine microalgae using marine bacteria for ethano production. Appl Biochem Biotechnol 2003;105–108:247–54. [76] Rubin EM. Genomics of cellulosic biofuels. Nature 2008;454:841–5. [77] Rahman RN, Baharum SN, Salleh AB, Basri M. S5 lipase: an organic solvent tolerant enzyme. J Microbiol 2006;44:583–90. [78] Torres S, Castro GR. Non-aqueous biocatalysis in homogenous solvent system. Food Technol Biotechnol 2004;42:271–7. [79] Gupta MN, Roy I. Enzymes in organic media, forms, functions and applications. Eur J Biochem 2004;13:2575–83. [80] Karbalaei-Haidari H, Ziaee A-A, Amoozegar M. Purification and biochemical characterization of a protease secreted by the Salinivibrio sp. strain AF-2004 and its behaviour in organic solvents. Extremophiles 2007;11: 237–43. [81] Fukushima T, Mizuki T, Echigo A, Inoue A, Usami R. Organic solvent tolerance of halophilic ␣-amylase from a haloarcheon, Haloarcula sp. strain S-1. Extremophiles 2005;9:85–9. [82] Shafiei M, Ziaee Abed-Ali Mohammad AA. Purification and characterization of an organic-solvent-tolerant halophilic ␣-amylase from the moderately halophilic Nesterenkonia sp. Strain F. J Ind Microbiol Biotechnol 2011;38:275–81. [83] Arnold FH, Georgiou G. Directed enzyme evolution: screening and selection methods. Methods in Molecular Biology Series: Humana Press; 2003. [84] Robertson DE, Noel JP. Methods in enzymology: protein engineering, vol. 388. Elsevier; 2004. [85] Hoppe C, Schomburg D. Prediction of protein thermostability with a direction- and distance-dependent knowledge-based potential. Protein Sci 2005;14:2682–92.

169

[86] Korkegian A, Black ME, Baker D, Stoddard BL. Computational thermostabilization of an enzyme. Science 2005;308:857–60. [87] van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—factors affecting enzymes, conversion and synergy. Biotechnol Adv 2012;30:1458–80. [88] Hirsch P, Hoffman B. Dichotomicrobium thermohalophilum, gen. nov., spec. nov., budding prosthecate bacteria from the solar lake (Sinai) and some related strains. Syst Appl Microbiol 1989;11:291–301. [89] Antón J, Oren A, Benlloch S, Rodríguez-Valera F, Amann R, Roselló-Mora R. Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. Int J Syst Ev Microbiol 2002;52: 485–91. [90] Liaw HJ, Mah RA. Isolation and characterization of Haloanaerobacter chitinovorans gen. nov., sp. nov., a halophilic, anaerobic, chitinolytic bacterium from a solar saltern. Appl Env Micro 1992;58:260–6. [91] Mouné S, Manac’h N, Hirschler A, Caumette P, Willison JC, Matheron R. Haloanaerobacter salinarius sp. nov., a novel halophilic fermentative bacterium that reduces glycine-betaine to trimethylamine with hydrogen or serine as electron donors; emendation of the genus Haloanaerobacter. Int J Syst Ev Microbiol 1999;49:103–12. [92] Zhilina TN, Zavarzin GA, Bulygina ES, Kevbrin VV, Osipov GA, Chumakov KM. Ecology, physiology and taxonomy studies on a new taxon of Haloanaerobiaceae, Haloincola saccharolytica gen. nov., sp. nov. Syst Appl Microbiol 1992;15:275–84. [93] Imhoff JF, Suling J. The phylogenetic relationship among Ectothiorhodospiracaea: a reevaluation of their taxonomy on the basis of 16S rDNA analysies. Arch Microbiol 1996;165:106–13. [94] Raymond JC, Sistrom WR. Ectothiorhodospira halophila, a new species of the genus Ectothiorhodospira. Archiv fur Mikrobiologie 1969;69:121–6. [95] Bowers KJ, Mesbah NM, Wiegel J. Natranaerobius ‘grantii’ and Natranaerobius ‘jonesii’, spp. nov., two anaerobic halophilic alkaliphiles isolated from the Kenyan-Tanzanian Rift [abstract]. ASM General Meeting 2008 Boston, MA, I-007. [96] Mesbah NM, Hedrick DB, Peacock AD, Rohde M, Wiegel J. Natranaerobius thermophilus gen. nov., sp. nov., a halophilic, alkalithermophilic bacterium from soda lakes of the Wadi An Natrun, Egypt, and proposal of Natranaerobiaceae fam. nov. and Natranaerobiales ord. nov. Int J Syst Ev Micro 2007;57: 2507–12. [97] Bowers KJ, Meshbah NH, Wiegel J. Biodiversity of poly-extremophilic Bacteria: Does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico-chemical boundary for life? Saline Syst 2009;5:9.