Halotolerant Enzymes Research Articles

Novel Approach of Tackling Wax Deposition Problems in Pipeline Using Enzymatic Degradation Process: Challenges and Potential Solutions

Anthropogenic activities have led to hydrocarbon spills, and while traditional bioremediation methods are costly and time-consuming, recent research has focused on engineered enzymes for managing pollutant. The potential of enzymes for resolving wax flow problems in the petroleum industry remains unexplored. This paper offers a comprehensive review of the current state of research activities related to the bioremediation of petroleum-polluted sites and the biodegradation of specific petroleum hydrocarbons. The assayed enzymes that took part in the degradation were discussed in detail. Lipase, laccase, alkane hydroxylase, alcohol dehydrogenase, esterase, AlkB homologs and cytochrome P450 monooxygenase are among the enzymes responsible for the degradation of more than 50% of the hydrocarbons in contaminated soil and wastewater and found to be active on carbon C8 to C40. The possible biodegradation mechanism of petroleum hydrocarbons was also elucidated. The enzymes’ primary metabolic pathways include terminal, subterminal, and ω-oxidation. Next, given the successful evidence of the hydrocarbon treatment efficiency, the authors analyzed the opportunity for the enzymatic degradation approach if it were to be applied to a different scenario: managing wax deposition in petroleum-production lines. With properties such as high transformation efficiency and high specificity, enzymes can be utilized for the treatment of viscous heavy oil for transportability, evidenced by the 20 to 99% removal of hydrocarbons. The challenges associated with the new approach are also discussed. The production cost of enzymes, the characteristics of hydrocarbons and the operating conditions of the production line may affect the biocatalysis reaction to some extent. However, the challenges can be overcome by the usage of extremophilic enzymes. The combination of technological advancement and deployment strategies such as the immobilization of a consortium of highly thermophilic and halotolerant enzymes is suggested. Recovering and reusing enzymes offers an excellent strategy to improve the economics of the technology. This paper provides insights into the opportunity for the enzymatic degradation approach to be expanded for wax deposition problems in pipelines.

Bioprospecting of Culturable Halophilic Bacteria Isolated from Mediterranean Solar Saltern for Extracellular Halotolerant Enzymes

Bioprospecting of Culturable Halophilic Bacteria Isolated from Mediterranean Solar Saltern for Extracellular Halotolerant Enzymes

Rational design of a novel halotolerant ATP regeneration system for biocatalytic CTP production

AbstractBACKGROUNDAcetyl phosphate/acetate kinase system is a cost‐saving and efficient ATP regeneration system. However, it suffers from accumulation of salts during catalytic reactions. It inhibits the activity of acetate kinase (ACK), a crucial enzyme of this process, and hence decreases the rate of product accumulation and yield of CTP.RESULTSA new halotolerant ACK enzyme based ATP regeneration platform was established in this research by applying the concept of rational design to optimize its building blocks. Selection of residues for rational design followed several rules. First, the less conserved residue positions on the protein surface were considered. Second, avoiding significant residue interactions, suchas salt bridges. Third, following the feature of nature occurring halotolerant enzymes, the basic and neutral surface residues were replaced with acidic ones. Our mutants represent high catalytic activities and high IC50 value (inhibit activity by 50%) at high salt concentrations. Also, when the halotolerant ACK was applied to catalyze CTP production, the maximum titer was 213.5 ± 1.6 mM (which is 12.3‐fold higher than that of the traditional process), the productivity was 13.3 ± 0.1 mM L−1 h−1, and the molar yield to 5′‐cytidylate monophosphate (CMP) and utilization efficiency of energy were 92.4% and 72.7%, respectively.CONCLUSIONThis strategy can be applied to modify other enzymes in the industry and gives insight into resolving salt accumulation during biocatalytic processes. In addition, the halotolerant Adenosine Triphosphate (ATP) regeneration system built in this work can be applied to the synthesis of several other high energy compounds. © 2023 Society of Chemical Industry (SCI).

Catalytic and Spectroscopic Properties of the Halotolerant Soluble Methane Monooxygenase Reductase from Methylomonas methanica MC09.

The soluble methane monooxygenase receives electrons from NADH via its reductase MmoC for oxidation of methane, which is itself an attractive C1 building block for a future bioeconomy. Herein, we present biochemical and spectroscopic insights into the reductase from the marine methanotroph Methylomonas methanica MC09. The presence of a flavin adenine dinucleotide (FAD) and [2Fe2S] cluster as its prosthetic group were revealed by reconstitution experiments, iron determination and electron paramagnetic resonance spectroscopy. As a true halotolerant enzyme, MmoC still showed 50 % of its specific activity at 2 M NaCl. We show that MmoC produces only trace amounts of superoxide, but mainly hydrogen peroxide during uncoupled turnover reactions. The characterization of a highly active reductase is an important step for future biotechnological applications of a halotolerant sMMO.

Characterization of a Solvent-Tolerant Amidohydrolase Involved in Natural Product Heterocycle Formation

Heterocycles are important building blocks in pharmaceutical drugs and their enzymatic synthesis is attracting increasing interest. In recent years, various enzymes of the amidohydrolase superfamily were reported to catalyze heterocycle-forming condensation reactions. One of these enzymes, MxcM, is biochemically and kinetically characterized in this study. MxcM generates an imidazoline moiety in the biosynthesis of the natural product pseudochelin A, which features potent anti-inflammatory properties. The enzyme shows maximal activity at 50 °C and pH 10 as well as a kcat/Km value of 22,932 s−1 M−1 at its temperature optimum. Experimental data suggest that the activity of MxcM does not depend on a catalytic metal ion, which is uncommon among amidohydrolases. MxcM is highly active in diverse organic solvents and concentrated salt solutions. Furthermore, we show that MxcM is also capable to introduce imidazoline rings into derivatives of its natural substrate myxochelin B. Overall, MxcM is a solvent-stable, halotolerant enzyme with promising biochemical and kinetic properties and, in future, might become a valuable biocatalyst for the manufacturing of pharmaceutical drugs.

Crude oil and pyrene degradation by halotolerant fungi Embellisia sp. KJ59 and Alternaria sp. KJ66 isolated from saline soils

ABSTRACT In recent years, investigating halophilic and halotolerant fungi enzymes has confirmed their significant potential in the biodegradation of crude oil. The aim of the current study was to evaluate the biodegradation of crude oil by halotolerant fungal Embellisia sp. KJ59 and Alternaria sp. KJ66. The fungal strains were isolated on Czapek Dox Agar (CDA), potato dextrose agar (PDA), and malt extract agar (MEA) containing 10% NaCl. Seven strains that could grow in salinity, ranging from 0 to 17% were selected. Out of the seven isolates, isolates KJ59 and KJ66 were selected as superior strains because they were more efficient in the biodegradation of crude oil on Minimal Salt Medium containing 1% crude oil and different salinity. The isolates were identified as Embellisia sp. and Alternaria sp. based on its nucleotide sequence of internal transcribed spacer gene. They could biodegrade petroleum, ranging from 81.3 to 95.1% in NaCl concentrations of 0, 2.5, and 5% on PDB over a period of 14 days. Pyrene removal by these strains was over 88.3% in saline and non-saline MSM containing 100 mg L−1 pyrene. Finally, Embellisia sp. KJ59 and Alternaria KJ66 were introduced fungal strains for crude oil biodegradation as the sole or supplementary carbon source in the presence of different salt concentrations. Furthermore, they are potential fungal strains for bioremediation of crude oil in non-saline and contaminated saline environments.

A Novel Carboxylesterase Derived from a Compost Metagenome Exhibiting High Stability and Activity towards High Salinity.

Halotolerant lipolytic enzymes have gained growing interest, due to potential applications under harsh conditions, such as hypersalinity and presence of organic solvents. In this study, a lipolytic gene, est56, encoding 287 amino acids was identified by functional screening of a compost metagenome. Subsequently, the gene was heterologously expressed, and the recombinant protein (Est56) was purified and characterized. Est56 is a mesophilic (Topt 50 °C) and moderate alkaliphilic (pHopt 8) enzyme, showing high thermostability at 30 and 40 °C. Strikingly, Est56 is halotolerant as it exhibited high activity and stability in the presence of up to 4 M NaCl or KCl. Est56 also displayed enhanced stability against high temperatures (50 and 60 °C) and urea (2, 4, and 6 M) in the presence of NaCl. In addition, the recently reported halotolerant lipolytic enzymes were summarized. Phylogenetic analysis grouped these enzymes into 13 lipolytic protein families. The majority (45%) including Est56 belonged to family IV. To explore the haloadaptation of halotolerant enzymes, the amino acid composition between halotolerant and halophilic enzymes was statistically compared. The most distinctive feature of halophilic from non-halophilic enzymes are the higher content of acidic residues (Asp and Glu), and a lower content of lysine, aliphatic hydrophobic (Leu, Met and Ile) and polar (Asn) residues. The amino acid composition and 3-D structure analysis suggested that the high content of acidic residues (Asp and Glu, 12.2%) and low content of lysine residues (0.7%), as well as the excess of surface-exposed acidic residues might be responsible for the haloadaptation of Est56.

Biochemical and Taxonomic Characterization of Novel Haloarchaeal Strains and Purification of the Recombinant Halotolerant α-Amylase Discovered in the Isolate.

Haloarchaea are salt-loving archaea and potential source of industrially relevant halotolerant enzymes. In the present study, three reddish-pink, extremely halophilic archaeal strains, namely wsp1 (wsp-water sample Pondicherry), wsp3, and wsp4, were isolated from the Indian Solar saltern. The phylogenetic analysis based on 16S rRNA gene sequences suggests that both wsp3 and wsp4 strains belong to Halogeometricum borinquense while wsp1 is closely related to Haloferax volcanii species. The comparative genomics revealed an open pangenome for both genera investigated here. Whole-genome sequence analysis revealed that these isolates have multiple copies of industrially/biotechnologically important unique genes and enzymes. Among these unique enzymes, for recombinant expression and purification, we selected four putative α-amylases identified in these three isolates. We successfully purified functional halotolerant recombinant Amy2, from wsp1 using pelB signal sequence-based secretion strategy using Escherichia coli as an expression host. This method may prove useful to produce functional haloarchaeal secretory recombinant proteins suitable for commercial or research applications. Biochemical analysis of Amy2 suggests the halotolerant nature of the enzyme having maximum enzymatic activity observed at 1 M NaCl. We also report the isolation and characterization of carotenoids purified from these isolates. This study highlights the presence of several industrially important enzymes in the haloarchaeal strains which may potentially have improved features like stability and salt tolerance suitable for industrial applications.

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Isolation and Characterization of a Novel Cold-Active, Halotolerant Endoxylanase from Echinicola rosea sp. Nov. JL3085T.

We cloned a xylanase gene (xynT) from marine bacterium Echinicola rosea sp. nov. JL3085T and recombinantly expressed it in Escherichia coli BL21. This gene encoded a polypeptide with 379 amino acid residues and a molecular weight of ~43 kDa. Its amino acid sequence shared 45.3% similarity with an endoxylanase from Cellvibrio mixtus that belongs to glycoside hydrolases family 10 (GH10). The XynT showed maximum activity at 40 °C and pH 7.0, and a maximum velocity of 62 μmoL min−1 mg−1. The XynT retained its maximum activity by more than 69%, 51%, and 26% at 10 °C, 5 °C, and 0 °C, respectively. It also exhibited the highest activity of 135% in the presence of 4 M NaCl and retained 76% of its activity after 24 h incubation with 4 M NaCl. This novel xylanase, XynT, is a cold-active and halotolerant enzyme that may have promising applications in drug, food, feed, and bioremediation industries.

The crystal structure of the tetrameric DABA-aminotransferase EctB, a rate-limiting enzyme in the ectoine biosynthesis pathway.

l-2,4-diaminobutyric acid (DABA) aminotransferases can catalyze the formation of amines at the distal ω-position of substrates, and is the intial andrate-limiting enzyme in the biosynthesis pathway of the cytoprotecting molecule (S)-2-methyl-1,4,5,6-tetrahydro-4-pyrimidine carboxylic acid (ectoine). Although there is an industrial interest in the biosynthesis of ectoine, the DABA aminotransferases remain poorly characterized. Herein, we present the crystal structure of EctB (2.45Å), a DABA aminotransferase from Chromohalobactersalexigens DSM 3043, a well-studied organism with respect to osmoadaptation by ectoine biosynthesis. We investigate the enzyme's oligomeric state to show that EctB from C.salexigens is a tetramer of two functional dimers, and suggest conserved recognition sites for dimerization that also includes the characteristic gating loop that helps shape the active site of the neighboring monomer. Although ω-transaminases are known to have two binding pockets to accommodate for their dual substrate specificity, we herein provide the first description of two binding pockets in the active site that may account for the catalytic character of DABA aminotransferases. Furthermore, our biochemical data reveal that the EctB enzyme from C.salexigens is a thermostable, halotolerant enzyme with a broad pH tolerance which may be linked to its tetrameric state. Put together, this study creates a solid foundation for a deeper structural understanding of DABA aminotransferases and opening up for future downstream studies of EctB's catalytic character and its redesign as a better catalyst for ectoine biosynthesis. In summary, we believe that the EctB enzyme from C.salexigens can serve as a benchmark enzyme for characterization of DABA aminotransferases. DATABASE: Structural data are available in PDB database under the accession number 6RL5.

Study on a Novel Cold-Active and Halotolerant Monoacylglycerol Lipase Widespread in Marine Bacteria Reveals a New Group of Bacterial Monoacylglycerol Lipases Containing Unusual C(A/S)HSMG Catalytic Motifs

Monoacylglycerol lipases (MGLs) are present in all domains of life. However, reports on bacterial MGLs are still limited. Until now, reported bacterial MGLs are all thermophilic/mesophilic enzymes from warm terrestrial environments or deep-sea hydrothermal vent, and none of them originates from marine environments vastly subject to low temperature, high salts, and oligotrophy. Here, we characterized a novel MGL, GnMgl, from the marine cold-adapted and halophilic bacterium Glaciecola nitratireducens FR1064T. GnMgl shares quite low sequence similarities with characterized MGLs (lower than 31%). GnMgl and most of its bacterial homologs harbor a catalytic Ser residue located in the conserved C(A/S)HSMG motif rather than in the typical GxSxG motif reported on other MGLs, suggesting that GnMgl-like enzymes might be different from reported MGLs in catalysis. Phylogenetic analysis suggested that GnMgl and its bacterial homologs are clustered as a separate group in the monoglyceridelipase_lysophospholipase family of the Hydrolase_4 superfamily. Recombinant GnMgl has no lysophospholipase activity but could hydrolyze saturated (C12:0-C16:0) and unsaturated (C18:1 and C18:2) MGs and short-chain triacylglycerols, displaying distinct substrate selectivity from those of reported bacterial MGLs. The substrate preference of GnMgl, predicted to be a membrane protein, correlates to the most abundant fatty acids within the strain FR1064T, suggesting the role of GnMgl in the lipid catabolism in this marine bacterium. In addition, different from known bacterial MGLs that are all thermostable enzymes, GnMgl is a cold-adapted enzyme, with the maximum activity at 30°C and retaining 30% activity at 0°C. GnMgl is also a halotolerant enzyme with full activity in 3.5M NaCl. The cold-adapted and salt-tolerant characteristics of GnMgl may help its source strain FR1064T adapt to the cold and saline marine environment. Moreover, homologs to GnMgl are found to be abundant in various marine bacteria, implying their important physiological role in these marine bacteria. Our results on GnMgl shed light on marine MGLs.

A Halotolerant Endo-1,4-β-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification.

The use of non-potable water (such as seawater) is an attractive alternative for water intensive processes such as biomass pretreatment and saccharification steps in the production of biochemicals and biofuels. Identification and application of halotolerant enzymes compatible with high-salt conditions may reduce the energy needed for non-potable water treatment and decrease waste treatment costs. Here we present the biochemical properties of a halotolerant endo-1,4-β-xylanase produced by Aspergillus clavatus in submerged fermentation, using paper sludge (XPS) and sugarcane bagasse (XSCB), and its potential application in the hydrolysis of agroindustrial residues. The peptide mass fingerprint and amino acid sequencing of the XPS and XSCB enzymes showed primary structure similarities with an endo-1,4-β-xylanase from Aspergillus clavatus (XYNA_ASPCL). Both enzyme preparations presented good thermal stability at 50°C and were stable over a wide range of pH and Vmax up to 2450U/mg for XPS. XPS and XSCB were almost fully stable even after 24h of incubation in the presence of up to 3M NaCl, and their activity were not affected by 500mM NaCl. Both enzyme preparations were capable of hydrolyzing paper sludge and sugarcane bagasse to release reducing sugars. These characteristics make this xylanase attractive to be used in the hydrolysis of biomass, particularly with brackish water or seawater.

Haloadaptation: insights from comparative modeling studies between halotolerant and non-halotolerant dehalogenases

Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma

Saccharification of lignocellulosic biomass using seawater and halotolerant cellulase with potential application in second-generation bioethanol production

Global water scarcity at an alarming stage has triggered the interest of many environmentalists and global researchers. Use of freshwater in biomass-based industries would result in depletion of a precious natural resource, which is not sustainable in the long term. Thus, water management technologies are critical to the successful operation of an ethanol plant. Utilization of seawater-based systems and halotolerant enzymes can be a breakthrough in this context. The present study involves marine bacterial strains Bacillus oceanisediminis, Brevibacterium halotolerans, and Psychrobacter celer capable of producing halotolerant cellulases, isolated from Gopalpur, Odisha. The crude enzyme extracts and direct bacterial cultures were independently utilized for saccharification of pretreated rice straw, and the treated rice straw was characterized for the production of reducing sugars using high-performance liquid chromatography (HPLC). The possible bond breakage and formation during saccharification of cellulose was assessed using attenuated total reflectance with Fourier transform infrared (ATR-FTIR) spectroscopy. The relative fraction and size of crystallites in cellulose was evaluated by X-ray diffraction (XRD) study. The biomass saccharified using the crude cellulase from B. oceanisedimins was utilized for the production of bioethanol in freshwater and seawater-based media using Saccharomyces cerevisiae NCIM 3570 and Candida shehatae NCIM 3500. The maximum fermentation efficiency (45.74%) was recorded for saccharified rice straw in freshwater using a consortium of immobilized yeasts. The highest fermentation efficiency (36.69%) was recorded in the seawater system by immobilized S. cerevisiae.

Isolation and screening of extracellular anticancer enzymes from halophilic and halotolerant bacteria from different saline environments in Iran.

It was confirmed that several enzymes have anti-cancer activity. The enzymes L-asparaginase, L-glutaminase, and L-arginase were chosen according to amino acids starvation in cancer cells and screened in halophilic and halotolerant bacteria, given probably less immunological reactions of halophilic or halotolerant enzymes in patients. Out of 110 halophilic and halotolerant strains, isolated from different saline environments in Iran and screened, some could produce a variety of anticancer enzymes. A total of 29, 4, and 2 strains produced L-asparaginase, L-glutaminase, and L-arginase, respectively. According to the phenotypic characteristics and partial 16S rRNA gene sequence analysis, the positive strains-strains with the ability to produce these anticancer enzymes-were identified as the members of the genera: Bacillus, Dietzia, Halobacillus, Rhodococcus, Paenibacillus and Planococcus as Gram-positive bacteria and Pseudomonas, Marinobacter, Halomonas, Idiomarina, Vibrio and Stappia as Gram-negative bacteria. The production of anticancer enzymes was mostly observed in the rod-shaped Gram-negative isolates, particularly in the members of the genera Halomonas and Marinobacter. Most of the enzymes were produced in the stationary phase of growth and the maximum enzyme activity was experienced in strain GBPx3 (Vibrio sp.) for L-asparaginase at 1.0IU/ml, strain R2S25 (Rhodococcus sp.) for L-glutaminase at 0.6IU/ml and strain GAAy3 (Planococcus sp.) for L-arginase at 3.1IU/ml. The optimum temperature and pH for L-asparaginase and L-glutaminase activities in selected strains were similar to the physiological conditions of human body and the enzymes could tolerate NaCl up to 7.5% concentration.

Amide hydrolyzing potential of amidase from halotolerant bacterium Brevibacterium sp. IIIMB2706

Till date, amidases from halophiles and halotolerant micro-organisms have not been much explored. In the present study, Brevibacterium sp. IIIMB2706 strain was isolated from salt fields of Gujarat, India, using propionitrile as a nitrogen source in the mineral base media and explored for its amidase activity. Amidase from Brevibacterium sp. IIIMB2706 exhibited substrate affinity towards isobutyramide, propionamide and butyramide. The optimum temperature and pH required for its maximum activity was 45 °C and 7.0, respectively. Effect of salt concentration on amidase activity was also studied and maximum activity was observed in presence of 50 g L−1 NaCl with significant activity up to 200 g L−1 NaCl which justifies its halotolerant nature. Various organic solvents compatibility profile showed that the enzyme was highly active in presence of 10% methyl alcohol. Henceforth, halotolerant enzymes may find application in industrial processes where substrate requires organic solvents for solubilization.

Isolation and characterization of denitrifying halophilic bacteria from Bahr Al-Milh Salt Lake, Karbala, Iraq

Objectives: Nitrogen fixation is considered as a significant biological process, which is important in agricultural and environmental implications. Herein we reported denitrifying bacteria from Bahr Al-Milh Salt Lake, Karbala, Iraq, using molecular and phenotypical characteristics. Material and Methods: Samples were collected from eastern parts of the Bahr Al-Milh. The strains were grown in different saline concentrations of nutrient broth (2.5% to 22.5%). Nitrate and Nitrite reduction activities were assessed for all the isolates. Molecular analysis was performed by BLAST alignment and MEGA7 software. The 16S rRNA sequences of newly found strains were submitted in the GenBank database. Results: Two hundreds and eighteen strains were isolated, 76.6% of which were nitrate reductase and 25.5% nitrate-nitrite reductase positive strains. 68% slightly and 32% moderately halophilic bacteria were found. Isolates with nitrite reduction activity belonged to five genera including Bacillus, Halobacillus, Virgibacillus, Oceanobacillus and Idiomarina. The isolates with the ability of producing nitrate-nitrite reductase consisted of bacteria in genera Halomonas and Halobacillus. Conclusions: Apart from industrial and biotechnological applications, the present information might be useful in order to fertilize saline soil for agricultural aims. The isolated strains could be considered as a source of halotolerant enzymes in agriculture and environmental implications in hypersaline areas.

Characterization and mutation analysis of a halotolerant serine protease from a new isolate of Bacillus subtilis.

A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential. Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions. Changing the pI of a bacterial serine protease may be an effective strategy to improve the enzyme's halotolerance.

Salt Adaptation and Evolutionary Implication of a Nah-related PAHs Dioxygenase cloned from a Halophilic Phenanthrene Degrading Consortium

Polycyclic aromatic hydrocarbons (PAHs) pollutions often occur in marine and other saline environment, largely due to anthropogenic activities. However, study of the PAHs-degradation genotypes in halophiles is limited, compared with the mesophilic terrestrial PAHs degraders. In this study, a bacterial consortium (CY-1) was enriched from saline soil contaminated with crude oil using phenanthrene as the sole carbon source at 10% salinity. CY-1 was dominated by the moderate halophilic Marinobacter species, and its dominant PAHs ring-hydroxylating dioxygenase (RHD) genotypes shared high identity to the classic nah-related RHDs found in the mesophilic species. Further cloning of a 5.6-kb gene cluster from CY-1 unveiled the existence of a new type of PAHs degradation gene cluster (hpah), which most probably evolves from the nah-related gene clusters. Expression of the RHD in this gene cluster in E. coli lead to the discovery of its prominent salt-tolerant properties compared with two RHDs from mesophiles. As a common structural feature shared by all halophilic and halotolerant enzymes, higher abundance of acidic amino acids was also found on the surface of this RHD than its closest nah-related alleles. These results suggest evolution towards saline adaptation occurred after horizontal transfer of this hpah gene cluster into the halophiles.