Halophilic Enzymes Research Articles

Covalent Immobilization of Alcohol Dehydrogenase (ADH2) from Haloferax volcanii: How to Maximize Activity and Optimize Performance of Halophilic Enzymes

Alcohol dehydrogenase from halophilic archaeon Haloferax volcanii (HvADH2) was successfully covalently immobilized on metal-derivatized epoxy Sepabeads. The immobilization conditions were optimized by investigating several parameters that affect the halophilic enzyme-support interaction. The highest immobilization efficiency (100%) and retention activity (60%) were achieved after 48h of incubation of the enzyme with Ni-epoxy Sepabeads support in 100mM Tris-HCl buffer, pH 8, containing 3M KCl at 5°C. No significant stabilization was observed after blocking the unreacted epoxy groups with commonly used hydrophilic agents. A significant increase in the stability of the immobilized enzyme was achieved by blocking the unreacted epoxy groups with ethylamine. The immobilization process increased the enzyme stability, thermal activity, and organic solvents tolerance when compared to its soluble counterpart, indicating that the immobilization enhances the structural and conformational stability. One step purification-immobilization of this enzyme has been carried out on metal chelate-epoxy Sepabeads, as an efficient method to obtain immobilized biocatalyst directly from bacterial extracts.

Halophilic enzymes: characteristics, structural adaptation and potential applications for biocatalysis

Halophilic enzymes: characteristics, structural adaptation and potential applications for biocatalysis

Characterization of a d-Stereoselective Aminopeptidase (DamA) Exhibiting Aminolytic Activity and Halophilicity from Aspergillus oryzae

β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide (pNA)], the enzyme preferred D-Leu-pNA and D-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward D-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0-11.0. DamA also exhibited aminolytic activity, producing D-Leu-D-Leu-NH2 from D-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from D-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a D-amino acid at the N-terminus as well as physiologically active peptides.

Draft Genome Sequence of the Moderately Halophilic Bacterium Marinobacter lipolyticus Strain SM19.

Marinobacter lipolyticus strain SM19, isolated from saline soil in Spain, is a moderately halophilic bacterium belonging to the class Gammaproteobacteria. Here, we report the draft genome sequence of this strain, which consists of a 4.0-Mb chromosome and which is able to produce the halophilic enzyme lipase LipBL.

Henryk (Heini) Eisenberg (1921–2009): From polymer chemistry to halophilic enzymes to chromatin and back

Henryk (Heini) Eisenberg (1921–2009): From polymer chemistry to halophilic enzymes to chromatin and back

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii

The effect of various organic solvents on the catalytic activity, stability and substrate specificity of alchohol dehydrogenase from Haloferax volcanii (HvADH2) was evaluated. The HvADH2 showed remarkable stability and catalysed the reaction in aqueous-organic medium containing dimethyl sulfoxide (DMSO) and methanol (MeOH). Tetrahydrofuran and acetonitrile were also investigated and adversely affected the stability of the enzyme. High concentration of salt, essential to maintain the enzymatic activity and structural integrity of the halophilic enzyme under standard conditions may be partially replaced by DMSO and MeOH. The presence of organic solvents did not induce gross changes in substrate specificity. DMSO offered a protective effect for the stability of the enzyme at nonoptimal pHs such as 6 and 10. Salt and solvent effects on the HvADH2 conformation and folding were examined through fluorescence spectroscopy. The fluorescence findings were consistent with the activity and stability results and corroborated the denaturing properties of some solvents. The intrinsic tolerance of this enzyme to organic solvent makes it highly attractive to industry.

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98% pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70°C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

Halophilic hydrolases as a new tool for the biotechnological industries

Halophilic micro-organisms are able to survive in high salt concentrations because they have developed diverse biochemical, structural and physiological modifications, allowing the catalytic synthesis of proteins with interesting physicochemical and structural properties. The main characteristic of halophilic enzymes that allows them to be considered as a novel alternative for use in the biotechnological industries is their polyextremophilicity, i.e. they have the capacity to be thermostable, tolerate a wide range of pH, withstand denaturation and tolerate high salt concentrations. However, there have been relatively few studies on halophilic enzymes, with some being based on their isolation and others on their characterisation. These enzymes are scarcely researched because attention has been focused on other extremophile micro-organisms. Only a few industrial applications of halophilic enzymes, principally in the fermented food, textile, pharmaceutical and leather industries, have been reported. However, it is important to investigate applications of these enzymes in more biotechnological processes at both the chemical and the molecular level. This review discusses the modifications of these enzymes, their industrial applications and research perspectives in different biotechnological areas.

Expression and one-step purification of a β-galactosidase by fusion with elastin-like polypetides

Thermally triggered reversible phase transition of elastin-like polypeptide (ELP) allows for a simple, economical and scalable procedure of protein purification. This technique is especially useful for purifying salt-requiring enzymes such as halophilic enzymes which require high salt concentration to keep natural structure and activity. In this study, a highly hydrophilic/acidic β-galactosidase cloned from halotolerant Planococcus sp.L4 was used as a target protein to apply ELP tags for purification. A high-level expression of β-galactosidase tagged with 80 repeats of Val-Pro-Gly-Val-Gly pentapeptide (galactosidase-ELP[V5-80]) was achieved in Escherichia coli BLR(DE3) at 21°C for 24h, accounting for around 50% of the total protein. The enzyme activity of the fusion by optimized protocol should be reached as much as 3 folds of that by rapid IPTG-induction, implying that measures to avoid possible errors during protein expression can be helpful for keeping bioactivities. The optimal condition for precipitating ELP-tagged protein was performed with a simple, rapid and sensitive method by examining the activity of supernatant after the first-round hot spin. The fusion protein aggregated effectively at 37°C with 1.5M ammonium sulfate and yielded highly pure protein with a recovery higher than 90% by one cycle. These results suggested that inverse transition cycling (ITC) process provides a potential for the large-scale purification of halophilic β-galactosidase.

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3–20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Halophilic enzyme activation induced by salts

Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3–6 M. To remain folded and active, their constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K+ triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl.

Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance

Some ionic liquids (ILs) have been shown to be very effective solvents for biomass pretreatment. It is known that some ILs can have a strong inhibitory effect on fungal cellulases, making the digestion of cellulose inefficient in the presence of ILs. The identification of IL-tolerant enzymes that could be produced as a cellulase cocktail would reduce the costs and water use requirements of the IL pretreatment process. Due to their adaptation to high salinity environments, halophilic enzymes are hypothesized to be good candidates for screening and identifying IL-resistant cellulases. Using a genome-based approach, we have identified and characterized a halophilic cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis. Hu-CBH1 is present in a gene cluster containing multiple putative cellulolytic enzymes. Sequence and theoretical structure analysis indicate that Hu-CBH1 is highly enriched with negatively charged acidic amino acids on the surface, which may form a solvation shell that may stabilize the enzyme, through interaction with salt ions and/or water molecules. Hu-CBH1 is a heat tolerant haloalkaliphilic cellulase and is active in salt concentrations up to 5 M NaCl. In high salt buffer, Hu-CBH1 can tolerate alkali (pH 11.5) conditions and, more importantly, is tolerant to high levels (20% w/w) of ILs, including 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Interestingly, the tolerances to heat, alkali and ILs are found to be salt-dependent, suggesting that the enzyme is stabilized by the presence of salt. Our results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes.

Overexpression and purification of halophilic proteins in Haloferax volcanii

Halophilic enzymes function optimally at high salt concentrations and are active at low water availability. Such conditions are encountered at elevated concentrations of solutes such as salts and sugars, and at high concentrations of organic solvents. However, expression in heterologous hosts such as Escherichia coli can cause problems, since halophilic proteins typically misfold and aggregate in conditions of low ionic strength. We have harnessed the sophisticated genetic tools available for the haloarchaeon Haloferax volcanii, to develop a system for the overexpression and purification of halophilic proteins under native conditions.

Catalytic and thermodynamic characterization of protease from Halobacterium sp. SP1(1)

Osmolytes KCl, glycerol, mannitol, trehalose, sucrose, betaine, proline and Na-glutamate at different concentrations (5–30%) were investigated as effective solutes for retaining the activity of Halobacterium sp. SP1(1) protease in the absence of NaCl. Maximum activity was observed in the presence of 30% Na-glutamate. Kinetic and thermodynamic parameters for casein hydrolysis revealed that the protease was equally efficient in the presence of Na-glutamate as in NaCl. The enzyme was active over a broader range of temperature (20–80 °C) and was highly stable even at 80 °C with Na-glutamate. Thermodynamic parameters (ΔH*, ΔS*, ∆G*) for irreversible inactivation of protease at different temperatures (20–80 °C) were determined in the presence of Na-glutamate and NaCl. The efficiency of these osmolytes for thermal stability of protease was 30% (1.6 M) Na-glutamate >4 M (∼25%) NaCl >2 M (∼10%), suggesting that the effect exerted by the osmolyte depends not only on its chemical nature but also on its concentration. Na-glutamate was thus found to play an important role in thermal stabilization of enzyme substituting for NaCl. Moreover, substitution of NaCl by Na-glutamate may increase the applicability of halophilic enzymes in biotechnology and industry, which is otherwise limited to high NaCl concentrations.

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. Beta-lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several "difficult-to-express" proteins as a BLA fusion protein and verified biological activities of human interleukin 1alpha and human neutrophil alpha-defensin, HNP-1.

Behaviour in different denaturant agents and structural characterization by fluorescence techniques of Haloferax mediterranei D-2-hydroxyacid dehydrogenase

The halophilic enzyme D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei is found to be an oligomeric enzyme composed of two identical subunits. Fluorescence spectra of native and denatured protein and effect of denaturants such as urea and guanidine hydrochloride on enzyme activity of halophilic D-2-hydroxyacid dehydrogenase have been analysed. Native D2-HDH shows the maximum emission at 340nm. The denaturation process caused an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum from 340 to 350nm. When urea was used as denaturant agent the enzyme required long incubation times, higher to 24h, to unfold. Fluorescence quenching by KI and acrylamide was also carried out; showing that the tryptophan residues are mainly located near the enzyme surface.

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an alpha-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50 degrees C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5-3 M NaCl.

Genomics of deep‐sea and sub‐seafloor microbes

Over two‐thirds of the surface of the earth is covered by oceans, which have an average depth of about 3800 m. As each drop of ocean water contains > 105 cells, the > 1030 microbial cells in the ocean represent the largest reservoir of microbes on earth (Whitman et al., 1998). Communities of bacteria, archaea, protists and unicellular fungi account for most of the oceanic biomass and metabolism. Marine microbes are known to play an essential role in the global cycling of nitrogen, carbon, oxygen, phosphorous, iron, sulfur and trace elements (Karl, 2007).

Intrinsic halotolerance of the psychrophilic α-amylase from Pseudoalteromonas haloplanktis

The halotolerance of a cold adapted alpha-amylase from the psychrophilic bacterium Pseudoalteromonas haloplanktis (AHA) was investigated. AHA exhibited hydrolytic activity over a broad range of NaCl concentrations (0.01-4.5 M). AHA showed 28% increased activity in 0.5-2.0 M NaCl compared to that in 0.01 M NaCl. In contrast, the corresponding mesophilic (Bacillus amyloliquefaciens) and thermostable (B. licheniformis) alpha-amylases showed a 39 and 46% decrease in activity respectively. Even at 4.5 M NaCl, 80% of the initial activity was detected for AHA, whereas the mesophilic and thermostable enzymes were inactive. Besides an unaltered fluorescence emission and secondary structure, a 10 degrees C positive shift in the temperature optimum, a stabilization factor of >5 for thermal inactivation and a Delta T(m) of 8.3 degrees C for the secondary structure melting were estimated in 2.7 M NaCl. The higher activation energy, half-life time and T(m) indicated reduced conformational dynamics and increased rigidity in the presence of higher NaCl concentrations. A comparison with the sequences of other halophilic alpha-amylases revealed that AHA also contains higher proportion of small hydrophobic residues and acidic residues resulting in a higher negative surface potential. Thus, with some compromise in cold activity, psychrophilic adaptation has also manifested halotolerance to AHA that is comparable to the halophilic enzymes.