Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

L-Glutaminase Synthesis by Marine Halomonas meridiana Isolated from the Red Sea and Its Efficiency against Colorectal Cancer Cell Lines.

L-glutaminase is an important anticancer agent that is used extensively worldwide by depriving cancer cells of L-glutamine. The marine bacterium, Halomonas meridian was isolated from the Red Sea and selected as the more active L-glutaminase-producing bacteria. L-glutaminase fermentation was optimized at 36 h, pH 8.0, 37 °C, and 3.0% NaCl, using glucose at 1.5% and soybean meal at 2%. The purified enzyme showed a specific activity of 36.08 U/mg, and the molecular weight was found to be 57 kDa by the SDS-PAGE analysis. The enzyme was highly active at pH 8.0 and 37 °C. The kinetics' parameters of Km and Vmax were 12.2 × 10-6 M and 121.95 µmol/mL/min, respectively, which reflects a higher affinity for its substrate. The anticancer efficiency of the enzyme showed significant toxic activity toward colorectal adenocarcinoma cells; LS 174 T (IC50 7.0 µg/mL) and HCT 116 (IC50 13.2 µg/mL). A higher incidence of cell death was observed with early apoptosis in HCT 116 than in LS 174 T, whereas late apoptosis was observed in LS 174 T more than in HCT 116. Also, the L-glutaminase induction nuclear fragmentation in HCT 116 was more than that in the LS 174T cells. This is the first report on Halomonas meridiana as an L-glutaminase producer that is used as an anti-colorectal cancer agent.

Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis.

3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldDHb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L-1 of 3HP with a yield and productivity of 0.93 g g-1 1,3-propanediol and 2.4 g L-1 h-1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32-45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP.

Identification, characterization, and immobilization of a novel YbfF esterase from Halomonas elongata.

The YbfF esterase family, which has a bifurcated binding pocket for diverse ligands, could serve as excellent biocatalysts in industrial and biotechnological applications. Here, the identification, characterization, and immobilization of a novel YbfF esterase (YbfFHalomonas elongata) from Halomonas elongata DSM 2581 is reported. Biochemical characterization of YbfF was carried out using activity staining, chromatographic analysis, kinetic analysis, activity assay, acetic acid release, and pH-indicator-based hydrolysis. YbfFH.elongata displayed broad substrate specificity, including that for p-nitrophenyl esters, glucose pentaacetate, tert-butyl acetate, and ß-lactam-containing compounds, with high efficiency. Based on a homology model of YbfFH.elongata, Trp237 in the substrate-binding pocket, a critical residue for catalytic activity and substrate specificity was identified and characterized. Furthermore, crosslinked enzyme aggregates and nanoflower formation were explored to enhance the chemical stability and recyclability of YbfFH.elongata. The present study is the first report of a YbfF esterase from extremophiles, and explains its protein stability, catalytic activity, substrate specificities and diversities, kinetics, functional residues, amyloid formation, and immobilization.

Salt tolerance of nitrate reductase in Halomonas sp. B01.

A systematic study on the lack of dissimilatory nitrate reductase (NAR) properties in Halomonas strains had been reported so far. The effects of different factors on Halomonas sp. B01 NAR activity were investigated. The salt tolerance of NAR was characterized. The denitrification process under high salt conditions was reported. Halomonas sp. B01 expressed membrane-bound NAR under induced culture by nitrate. The optimum pH of the enzyme reaction system was 8, and the optimum temperature was 30 °C. The mRNA expression abundance of narH in NAR encoding gene was highest in the 60 g/L NaCl inducing matrix. The NaCl concentration of optimum growth and induction of NAR were both 60 g/L. The ectoine added to the NAR vitro enzyme reaction system could maintain NAR activity under high NaCl concentration. In the range of 0-60 g/L NaCl, the NAR activity was stable at 17.7 (± 0.3) U/mg. The denitrification was performed by Halomonas sp. B01 at 60 g/L NaCl, and the denitrification rate reached 97.1% at 24 h. This study reveals for the first time the NAR properties of Halomonas strains, which provides a theoretical and technical basis for the nitrogen removal of high-salt nitrogenous wastewater using this strain.

Purification, biochemical characterization and DNA protection against oxidative damage of a novel recombinant superoxide dismutase from psychrophilic bacterium Halomonas sp. ANT108.

A novel superoxide dismutase (referred hereafter to as HsSOD) from the psychrophilic bacterium Halomonas sp. ANT108 was purified and characterized. Escherichia coli (E. coli) was selected as the expression host. After recombinant HsSOD (rHsSOD) was purified, the specific activity was determined to be 213.47 U/mg with a purification ratio of approximately 3.61-fold. SDS-PAGE results demonstrated that rHsSOD has the molecular weight of 31.3 kDa, and type identification revealed that it belongs to Cu/Zn SOD. The optimum activity of rHsSOD was at 35 °C and 28% of its maximum activity remained at 0 °C. Further enzymatic assays indicated that rHsSOD exhibited thermal instability with a half-life of 20 min at 60 °C. Moreover, Cu2+ and Zn2+ significantly promoted rHsSOD activity. The values of Km and Vmax were 0.33 mM and 476.19 U/mg, respectively. Interestingly, rHsSOD could avoid DNA strand breakage formed by metal-catalyzed oxidation, demonstrating its antioxidant capacity. To summarize, the results suggested that rHsSOD has relatively high catalytic efficiency and oxidation resistance at low temperatures.

Engineering Halomonas bluephagenesis for L-Threonine production.

Halophilic Halomonas bluephagenesis (H. bluephagenesis), a chassis for cost-effective Next Generation Industrial Biotechnology (NGIB), was for the first time engineered to successfully produce L-threonine, one of the aspartic family amino acids (AFAAs). Five exogenous genes including thrA*BC, lysC* and rhtC encoding homoserine dehydrogenase mutant at G433R, homoserine kinase, L-threonine synthase, aspartokinase mutant at T344M, S345L and T352I, and export transporter of threonine, respectively, were grouped into two expression modules for transcriptional tuning on plasmid- and chromosome-based systems in H. bluephagenesis, respectively, after pathway tuning debugging. Combined with deletion of import transporter or/and L-threonine dehydrogenase encoded by sstT or/and thd, respectively, the resulting recombinant H. bluephagenesis TDHR3-42-p226 produced 7.5 g/L and 33 g/L L-threonine when grown under open unsterile conditions in shake flasks and in a 7 L bioreactor, respectively. Engineering H. bluephagenesis demonstrates strong potential for production of diverse metabolic chemicals.

Enhanced tolerance to inhibitors of Escherichia coli by heterologous expression of cyclopropane-fatty acid-acyl-phospholipid synthase (cfa) from Halomonas socia.

Bacteria have evolved a defense system to resist external stressors, such as heat, pH, and salt, so as to facilitate survival in changing or harsh environments. However, the specific mechanisms by which bacteria respond to such environmental changes are not completely elucidated. Here, we used halotolerant bacteria as a model to understand the mechanism conferring high tolerance to NaCl. We screened for genes related to halotolerance in Halomonas socia, which can provide guidance for practical application. Phospholipid fatty acid analysis showed that H. socia cultured under high osmotic pressure produced a high portion of cyclopropane fatty acid derivatives, encoded by the cyclopropane-fatty acid-acyl phospholipid synthase gene (cfa). Therefore, H. socia cfa was cloned and introduced into Escherichia coli for expression. The cfa-overexpressing E. coli strain showed better growth, compared with the control strain under normal cultivation condition as well as under osmotic pressure (> 3% salinity). Moreover, the cfa-overexpressing E. coli strain showed 1.58-, 1.78-, 3.3-, and 2.19-fold higher growth than the control strain in the presence of the inhibitors furfural, 4-hydroxybenzaldehyde, vanillin, and acetate from lignocellulosic biomass pretreatment, respectively. From a practical application perspective, cfa was co-expressed in E. coli with the polyhydroxyalkanoate (PHA) synthetic operon of Ralstonia eutropha using synthetic and biosugar media, resulting in a 1.5-fold higher in PHA production than that of the control strain. Overall, this study demonstrates the potential of the cfa gene to boost cell growth and production even in heterologous strains under stress conditions.

Transaminase-Catalyzed Continuous Synthesis of Biogenic Aldehydes.

The physiological role of biogenic aldehydes, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL), has been associated with cardiovascular and neurodegenerative disorders. The availability of these substrates is limited and robust synthetic methodologies would greatly facilitate further biological studies. Herein, a transaminase-mediated single-step process in continuous mode, which leads to excellent product yields (90-95 %), is reported. Coimmobilization of the pyridoxal phosphate (PLP) cofactor eliminated the need for exogenous addition of this reagent without affecting the longevity of the system, delivering a truly self-sufficient process.

Recombinant production and characterization of a novel esterase from a hypersaline lake, Acigöl, by metagenomic approach.

The aim of this study was to isolate a novel esterase from a hypersaline lake by sequence-based metagenomics. The metagenomic DNA was isolated from the enriched hypersaline lake sediment. Degenerate primers targeting the conserved regions of lipolytic enzymes of halophilic microorganisms were used for polymerase chain reaction (PCR) and a whole gene was identified by genome walking. The gene was composed of 783 bp, which corresponds to 260 amino acids with a molecular weight of 28.2 kDa. The deduced amino acid sequence best matched with the esterase from Halomonas gudaonensis with an identity of 91%. Recombinantly expressed enzyme exhibited maximum activity towards pNP-hexanoate with a kcat value of 12.30 s-1. The optimum pH and temperature of the enzyme were found as 9 and 30 °C, respectively. The effects of NaCl, solvents, metal ions, detergents and enzyme inhibitors were also studied. In conclusion, a novel enzyme, named as hypersaline lake "Acigöl" esterase (hAGEst), was identified by sequence-based metagenomics. The high expression level, the ability to maintain activity at cold temperatures and tolerance to DMSO and metal ions are the most outstanding properties of the hAGEst.

Ectoine hydroxylase displays selective trans-3-hydroxylation activity towards L-proline.

L-Hydroxyproline (Hyp) is a valuable intermediate for the synthesis of pharmaceuticals; consequently, a practical process for its production has been in high demand. To date, industrial processes have been developed by using L-Pro hydroxylases. However, a process for the synthesis of trans-3-Hyp has not yet been established, because of the lack of highly selective enzymes that can convert L-Pro to trans-3-Hyp. The present study was designed to develop a biocatalytic trans-3-Hyp production process. We speculated that ectoine hydroxylase (EctD), which is involved in the hydroxylation of the known compatible solute ectoine, may possess the ability to hydroxylate L-Pro, since the structures of ectoine and 5-hydroxyectoine resemble those of L-Pro and trans-3-Hyp, respectively. Consequently, we discovered that ectoine hydroxylases from Halomonas elongata, as well as some actinobacteria, catalyzed L-Pro hydroxylation to form trans-3-Hyp. Of these, ectoine hydroxylase from Streptomyces cattleya also utilized 3,4-dehydro-L-Pro, 2-methyl-L-Pro, and L-pipecolic acid as substrates. In the whole-cell bioconversion of L-Pro into trans-3-Hyp using Escherichia coli expressing the ectD gene from S. cattleya, only 12.4 mM trans-3-Hyp was produced from 30 mM L-Pro, suggesting a rapid depletion of 2-oxoglutarate, an essential component of enzyme activity as a cosubstrate, in the host. Therefore, the endogenous 2-oxoglutarate dehydrogenase gene was deleted. Using this deletion mutant as the host, trans-3-Hyp production was enhanced up to 26.8 mM from 30 mM L-Pro, with minimal loss of 2-oxoglutarate. This finding is not only beneficial for trans-3-Hyp production, but also for other E. coli bioconversion processes involving 2-oxoglutarate-utilizing enzymes.

Exploring useful fermentation strategies for the production of hydroxyectoine with a halophilic strain, Halomonas salina BCRC 17875.

Hydroxyectoine, an ectoine derivative, is the most common compatible solute in halophilic microorganisms for resisting harsh environments. Compatible solutes can be utilized in fields such as cosmetics, medicine, and biochemistry. Moderately halophilic microorganisms produce much less hydroxyectoine as compared with ectoine. In this study, we first evaluate the effect of medium formulation (i.e., yeast extract (YE) medium and high yeast extract (HYE) medium) on hydroxyectoine production. In addition, an investigation of hydroxyectoine production by Halomonas salina under optimal conditions for vital factors (i.e., iron and α-ketoglutarate) and hydroxylase activity was also carried out. As a result, hydroxyectoine production was obviously elevated (0.9 g/L to 1.8 g/L) when the HYE medium was utilized. Furthermore, hydroxyectoine production further increased to 2.4 g/L when both the α-ketoglutarate and iron factors were added to the HYE medium in the early stationary phase. In addition, we found that ectoine hydroxylase activity increased more when a combination of iron and α-ketoglutarate was used than when either was used alone. The results showed that the alteration of iron and α-ketoglutarate clearly stimulated the expression of ectoine hydroxylase, which in turn affected hydroxyectoine synthesis. This study also showed that hydroxyectoine production was further raised from 2.4 g/L to 2.9 g/L when 50 mM of α-ketoglutarate and 1 mM of iron were added to the HYE medium. Ultimately, the experimental results showed using the optimal conditions further elevated the hydroxyectoine production yield to 2.90 g/L, which was over 3-fold higher than the best results obtained from the original medium.

The role of the acyl-CoA thioesterase "YciA" in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli.

Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

Isolation, cloning and characterization of an azoreductase and the effect of salinity on its expression in a halophilic bacterium.

Understanding the molecular mechanisms of azo dye decolorization is important for the development of effective bioremediation for textile-colored wastewater. A halophilic bacterium Halomonas sp. strain GT was isolated, which could degrade the azo dye Acid Brilliant Scarlet GR at 10% NaCl. The complete genome sequence of this strain was obtained using the PacBio RS II platform. Genome annotation revealed that four proteins are related to decolorization of azo dyes, such as azoreductase, laccases, benzene 1,2-dioxygenase, and catechol 1,2-dioxygenase. The putative azoreductase gene of Halomonas sp. strain GT responsible for the decolorization of azo dye in high salt environment was isolated. Phylogenetic tree analysis showed that the azoG (azoreductase gene of Halomonas sp. strain GT) and its homologs constituted a new branch of the NADH depending azoreductases, with all the homologous sequence of the protein from halophilic bacteria. At high NaCl concentrations, azoreductase gene expression and azoreductase activity were restrained in Halomonas sp. strain GT, which resulted in low a decolorization rate.

Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site-directed mutagenesis.

Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X-ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three-dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik-Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half-life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild-type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase.

Exploring a broad spectrum nitrilase from moderately halophilic bacterium Halomonas sp. IIIMB2797 isolated from saline lake.

Nitrile hydrolyzing moderate halophilic bacterium Halomonas sp. IIIMB2797 was isolated from Sambhar Lake, India. Maximum cell biomass and nitrilase production were observed at 60 g L-1 NaCl in the production media which confirms its moderate halophilic nature. Nitrilase of Halomonas sp. IIIMB2797 proved to be inducible in nature as maximum activity was observed when valeronitrile was added in the production media. Whole cells of Halomonas sp. IIIMB2797 exhibited broad substrate affinity towards aromatic and aliphatic nitriles. Optimum pH and temperature for nitrilase activity was observed at 7.0 and 45 °C, respectively. Effect of salinity on nitrilase activity was also studied and maximum activity was observed in presence of 50 g L-1 NaCl in 0.1 M phosphate buffer of pH 7.0. The interesting feature of the study is that whole cells of Halomonas sp. IIIMB2797 exhibited higher nitrilase activities in presence of organic solvents which may be useful in biotransformation of nitriles to corresponding carboxylic acids for industrial applications.

Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope.

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering.

New Findings on Aromatic Compounds' Degradation and Their Metabolic Pathways, the Biosurfactant Production and Motility of the Halophilic Bacterium Halomonas sp. KHS3.

The study of the aromatic compounds' degrading ability by halophilic bacteria became an interesting research topic, because of the increasing use of halophiles in bioremediation of saline habitats and effluents. In this work, we focused on the study of aromatic compounds' degradation potential of Halomonas sp. KHS3, a moderately halophilic bacterium isolated from hydrocarbon-contaminated seawater of the Mar del Plata harbour. We demonstrated that H. sp. KHS3 is able to grow using different monoaromatic (salicylic acid, benzoic acid, 4-hydroxybenzoic acid, phthalate) and polyaromatic (naphthalene, fluorene, and phenanthrene) substrates. The ability to degrade benzoic acid and 4-hydroxybenzoic acid was analytically corroborated, and Monod kinetic parameters and yield coefficients for degradation were estimated. Strategies that may enhance substrate bioavailability such as surfactant production and chemotactic responses toward aromatic compounds were confirmed. Genomic sequence analysis of this strain allowed us to identify several genes putatively related to the metabolism of aromatic compounds, being the catechol and protocatechuate branches of ß-ketoadipate pathway completely represented. These features suggest that the broad-spectrum xenobiotic degrader H. sp. KHS3 could be employed as a useful biotechnological tool for the cleanup of aromatic compounds-polluted saline habitats or effluents.

Cloning and characterization of Halomonas elongata L-asparaginase, a promising chemotherapeutic agent.

L-asparaginase has been used in the treatment of patients with acute lymphoblastic leukemia (ALL) for more than 30 years. Rapid clearance of the enzyme from blood stream and its L-glutaminase-dependent neurotoxicity has led to searching for new L-asparaginases with more desirable properties. In the present study, L-asparaginase coding gene of Halomonas elongata was isolated, expressed in Escherichia coli, purified, and characterized. The purified protein was found to have a molecular mass of 39.5 kDa and 1000-folds more activity towards L-asparagine than L-glutamine. Enzyme-specific activity towards L-asparagine was determined to be 1510 U/mg, which is among the highest reported values for microbial L-asparaginases. K m , Vmax, and k cat values were 5.6 mM, 2.2 µmol/min, and 1.96 × 103 1/S, respectively. Optimum temperature was found to be 37 °C while the enzyme showed maximum activity at a wide pH range (from 6 to 9). Enzyme half-life in the presence of human serum at 37 °C was 90 min which is three times higher when compared with reported values for E. coli L-asparaginase. Enzyme showed cytotoxic effects against Jurkat and U937 cell lines with an IC50 of 2 and 1 U/ml, respectively. Also, no toxic effects on human erythrocytes and Chinese hamster ovary cell lines were detected, and just minor inhibitory effects on human umbilical vein endothelial cells were observed. This is the first report describing the therapeutic potentials of a recombinant L-asparaginase isolated from a halophilic bacterium as an anticancer agent.

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2.

Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na+ and Li+ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na+ and Li+ expulsion, as well as complementary growth following exposure to 300 mM Na+ and 100 mM Li+ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile.IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu38, in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.