No-salt Conditions Research Articles

High NaCl concentrations induce the resistance to thermal denaturation of an extremely halotolerant (salt-activated) β-mannanase from Bacillus velezensis H1.

β-mannanase catalyzes the hydrolysis of mannans β-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant β-mannanases are already reported, existence of a high halotolerant β-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant β-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48h incubation under optimal conditions of 50°C and pH 5.5. The enzyme's salt activation nature was revealed when its maximum activity was obtained in the presence of 4M NaCl,it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (Tm) increased steadily with increasing NaCl concentrations reaching 75.5°C in the presence of 2.5M NaCl. The Km and Vmax values were 5.63mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13mg/mL of reducing sugars from olive mill wastes (OMW)after 24h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications.

Fungal endophytes from salt-adapted plants confer salt tolerance and promote growth in wheat (Triticum aestivum L.) at early seedling stage.

With increasing human global population, increased yield under saline conditions is a desirable trait for major food crops. Use of endophytes, isolated from halophytic hosts, seems to be an exciting approach for conferring salt tolerance to a salt-sensitive crop. Therefore, in the current study, fungal endophytes were isolated from halophytic plants' roots and their ability to withstand in vitro salt stress was evaluated. The fungal endophytes could withstand up to 1M NaCl concentrations and this tolerance was independent of their host or tissue source. When inoculated on salt-sensitive wheat seeds/seedlings, several of the endophytes showed a positive impact on germination and biomass-related parameters upon salt stress, both in vitro and under glasshouse conditions. One of the isolates from dicot plants (identified as Microsphaeropsis arundinis) could successfully colonize wheat and promote its growth under salt and no-salt conditions. Amongst the fungal isolates that are known to be natural endophytes of wheat, Chaetomium globosum was the best performing isolate and has previously been reported to be an effective biocontrol agent. Based on the results of our preliminary study, we suggest that these fungal endophytes could prove beneficial for enhancing the salt stress tolerance of wheat crop.

Facile induction and stabilization of intramolecular antiparallel G-quadruplex of d(TTAGGG)n on interaction with triazine-2-imidazole ethyl amine compound and its Cu(II), Zn(II) complexes under no-salt conditions

Facile induction and stabilization of intramolecular antiparallel G-quadruplex of d(TTAGGG)n on interaction with triazine-2-imidazole ethyl amine compound and its Cu(II), Zn(II) complexes under no-salt conditions

Trizbenzim, Cu-Trizbenzim and Zn-Trizbenzim as G-Quadruplex Inducing and Stabilizing Compounds on Human Telomeric Sequence and their Anticancer Properties.

The benzimidazole and their derivatives have rich biological relevance with respect to available natural amino acids and their role in protein folding and quaternary conformations. Thus the ligand trizbenzim and their Cu(II) and Zn(II) metal complexes were prepared to induce G-quadruplex conformation even under no-salt conditions with remarkable anticancer activities. The ligand N,N',N''-Tris-(1H-benzoimidazol-2-ylmethyl)-[1,3,5]triazine-2,4,6-triamine ( trizbenzim) and its Cu and Zn complexes (Cu-trizbenzim, Zn-trizbenzim) were synthesized and characterized by IR, NMR, and MALDI-TOF techniques. The pure ligand and its complexes interacted with human telomere DNA sequence d(TTAGGG), HTelo8and HTelo20and the interactions were followed by circular dichroism spectroscopy, FID assay, and molecular docking techniques. The compounds were tested for anticancer activity towards selected cell lines. All the three compounds stabilized the HTelo8 and HTelo20 in parallel and antiparallel G-quadruplex conformations with salt conditions. Under no-salt conditions, the compounds induce and stabilize the G-quadruplex conformation in antiparallel topology selectively. The pure ligand, Cu-trizbenzim, and Zn-trizbenzim were involved in partial or classical intercalation and some backbone interactions on the strand. The FID assay using thiazole orange intercalator supports the proposed intercalation mode of binding for the three compounds, especially for the pure ligand and the Cu-complex. The MOE docking experiments using X-ray and NMR derived G-quadruplex models with the title compounds extensively support the G-quadruplex induction and stabilization of the telomere sequence by these compounds. The guanines bases involved in the G-tetrad formation interact well with the triazine and the benzimidazole part of the ligand through strong π-π interactions. The primary mode of binding is described as end stacking and intercalation of the compounds to the G-quadruplex structures. The Cu-trizbenzim exhibited more anticancer property in comparison to the pure ligand and the Zntrizbenzim complex. The IC50 values were in the nanomolar range from 50 to 150nM in concentration. This novel self-induction of G-quadruplex is novel without the presence of alkali metal ions.

Effect of arbuscular mycorrhizal symbiosis on ion homeostasis and salt tolerance-related gene expression in halophyte Suaeda salsa under salt treatments

Halophytes can remove large quantities of salts from saline soils, so their importance in ecology has received increasing attention. Preliminary studies have shown that arbuscular mycorrhizal (AM) fungi can improve the salt tolerance of halophytes. However, few studies have focused on the molecular mechanisms and effects of AM fungi in halophytes under different salt conditions. A pot experiment was carried out to investigate the effects of Funneliformis mosseae inoculation on growth, nutrient uptake, ion homeostasis and the expression of salt tolerance-related genes in Suaeda salsa under 0, 100, 200 and 400 mM NaCl. The results showed that F. mosseae promoted the growth of S. salsa and increased the shoot Ca2+ and Mg2+ concentrations under no-salt condition and high-salt condition. In addition, AM fungi increased the K+ concentration and maintained a high K+/Na+ ratio at 400 mM NaCl, while AM fungi decreased the K+ concentration and reduced the K+/Na+ ratio at 0 mM NaCl. AM fungi downregulated the expression of SsNHX1 in shoots and the expression of SsSOS1 in roots at 400 mM NaCl. These effects may decrease the compartmentation of Na+ into leaf vacuoles and restrict Na+ transport from roots to shoots, leading to an increase in root Na+ concentration. AM symbiosis upregulated the expression of SsSOS1 in shoots and downregulated the expression of SsSOS1 and SsNHX1 in roots at 100 mM NaCl. However, regulation of the genes (SsNHX1, SsSOS, SsVHA-B and SsPIP) was not significantly different with AM symbiosis at 0 mM or 200 mM NaCl. The results revealed that AM symbiosis might induce diverse modulation strategies in S. salsa, depending on external Na+ concentrations. These findings suggest that AM fungi may play significant ecological roles in the phytoremediation of salinized ecosystems.

A Salt-Signaling Network Involving Ethylene, Extracellular ATP, Hydrogen Peroxide, and Calcium Mediates K+/Na+ Homeostasis in Arabidopsis.

This work aimed at investigating the interactive effects of salt-signaling molecules, i.e., ethylene, extracellular ATP (eATP), H2O2, and cytosolic Ca2+ ([Ca2+]cyt), on the regulation of K+/Na+ homeostasis in Arabidopsis thaliana. The presence of eATP shortened Col-0 hypocotyl length under no-salt conditions. Moreover, eATP decreased relative electrolyte leakage and lengthened root length significantly in salt-treated Col-0 plants but had no obvious effects on the ethylene-insensitive mutants etr1-1 and ein3-1eil1-1. Steady-state ionic flux kinetics showed that exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) and eATP-Na2 (an eATP donor) significantly increased Na+ extrusion and suppressed K+ loss during short-term NaCl treatment. Moreover, ACC remarkably raised the fluorescence intensity of salt-elicited H2O2 and cytosolic Ca2+. Our qPCR data revealed that during 12 h of NaCl stress, application of ACC increased the expression of AtSOS1 and AtAHA1, which encode the plasma membrane (PM) Na+/H+ antiporters (SOS1) and H+-ATPase (H+ pumps), respectively. In addition, eATP markedly increased the transcription of AtEIN3, AtEIL1, and AtETR1, and ACC treatment of Col-0 roots under NaCl stress conditions caused upregulation of AtRbohF and AtSOS2/3, which directly contribute to the H2O2 and Ca2+ signaling pathways, respectively. Briefly, ethylene was triggered by eATP, a novel upstream signaling component, which then activated and strengthened the H2O2 and Ca2+ signaling pathways to maintain K+/Na+ homeostasis under salinity.

Responses of Spinach to Salinity and Nutrient Deficiency in Growth, Physiology, and Nutritional Value

Salinity and nutrient-depleted soil are major constraints to crop production, especially for vegetable crops. The effects of salinity and nutrient deficiency on spinach (Spinacia oleracea L.) were evaluated in sand cultures under greenhouse conditions. Plants were watered every day with Hoagland nutrition solution, deprived of nitrogen (N), phosphorous (P), or potassium (K) for nutrient deficiency, either with or without 20/10 mm sodium chloride (NaCl)/calcium chloride (CaCl2) for salinity treatment. Salinity significantly decreased shoot fresh weight (FW) and dry weight (DW), leaf relative water content (RWC), and specific leaf area (SLA) relative to controls after 4 weeks of treatment and increased chlorophyll content, maximum photochemical efficiency (Fv/Fm), and photochemical yield [Y(II)]. Nitrogen deficiency greatly reduced shoot FW and DW, SLA, and chlorophyll content, regardless of salt treatment. Y(II) and Fv/Fm were reduced by N deficiency and salinity treatment. Phosphorous and K deficiencies, similarly, decreased shoot FW and DW irrespective of salinity treatment and increased chlorophyll content without salt stress. Phosphorous deficiency increased Y(II) under control and Fv/Fm under both control and salt treatment. Salinity and nutrient deficiency also affected the nutritional value of spinach. Salt stress increased carotenoid and flavonoid contents, and reducing power in full-strength Hoagland solution, and decreased leaf ferrous ion chelating ability (FICA). Nutrient deficiency increased reducing power regardless of salinity treatment. Nitrogen deficiency increased anthocyanin and total phenolic contents, decreased carotenoids and flavonoids regardless of salinity treatment, and increased antioxidant capacity under no-salt conditions. Phosphorous deficiency increased carotenoid and flavonoid contents under no-salt condition and enhanced total phenolic content and reduced FICA and amino acid content under salt stress. Potassium deficiency increased total phenolic, carotenoid, and flavonoid contents and antioxidant capacity under non-salt condition, but decreased FICA regardless of salinity treatment. These results suggest that spinach nutritional value could be improved with only moderately or slightly reduced yield through cultural practices that impose either low fertilizer levels or slight salt stress.

Phenol degradation by halophilic fungal isolate JS4 and evaluation of its tolerance of heavy metals.

Phenol is one of the most common pollutants in many kinds of industrial wastewater, some of which are in high salinity, resulting in more difficulties of biodegradation. In this work, a halophilic strain capable of utilizing phenol as sole source of carbon and energy in both hypersaline and no-salt media was isolated and identified as genus Debaryomyces. The optimization of environmental parameters including phenol concentration, pH, dissolved oxygen as well as salinity was carried out and tolerance of heavy metals by the strain was evaluated. The strain Debaryomyces sp. was able to grow in culture when initial phenol concentration, pH, agitation and salinity were at wide ranges (0-1200 mg L(-1), 4.0-10.0, 50-200 rpm, 0 %-15 %, respectively). High removal efficiency was hardly affected in the presence of 5 mM of Zn (II) and Mn (II). Under optimal conditions (pH 6.0, 200 rpm, 1 % of salinity without heavy metals), 500 mg L(-1) of phenol could be completely degraded within 32 h. The high removal efficiency of phenol by the strain with significant variations of process parameters might contribute to the bioremediation of phenol-polluted environments under hypersaline or no-salt conditions.

Association mapping of soybean seed germination under salt stress.

Soil salinity is a serious threat to agriculture sustainability worldwide. Seed germination is a critical phase that ensures the successful establishment and productivity of soybeans in saline soils. However, little information is available regarding soybean salt tolerance at the germination stage. The objective of this study was to identify the genetic mechanisms of soybean seed germination under salt stress. One natural population consisting of 191 soybean landraces was used in this study. Soybean seeds produced in four environments were used to evaluate the salt tolerance at their germination stage. Using 1142 single-nucleotide polymorphisms (SNPs), the molecular markers associated with salt tolerance were detected by genome-wide association analysis. Eight SNP-trait associations and 13 suggestive SNP-trait associations were identified using a mixed linear model and the TASSEL 4.0 software. Eight SNPs or suggestive SNPs were co-associated with two salt tolerance indices, namely (1) the ratio of the germination index under salt conditions to the germination index under no-salt conditions (ST-GI) and (2) the ratio of the germination rate under salt conditions to the germination rate under no-salt conditions (ST-GR). One SNP (BARC-021347-04042) was significantly associated with these two traits (ST-GI and ST-GR). In addition, nine possible candidate genes were located in or near the genetic region where the above markers were mapped. Of these, five genes, Glyma08g12400.1, Glyma08g09730.1, Glyma18g47140.1, Glyma09g00460.1, and Glyma09g00490.3, were verified in response to salt stress at the germination stage. The SNPs detected could facilitate a better understanding of the genetic basis of soybean salt tolerance at the germination stage, and the marker BARC-021347-04042 could contribute to future breeding for soybean salt tolerance by marker-assisted selection.

고검화도 폴리비닐알콜 필름에서의 이색성 유기염료의 염착 특성

Abstract: Dyeing of a poly(vinyl alcohol) (PVA) film with a high degree hydrolysis (>98%) using Direct Black 22, adichroic organic dye, was investigated under various conditions (dye concentration, salt (Na 2 SO 4 ) concentration, andtemperature). This process could correctly be analyzed using the Freundlich isotherm ( C film =K F C soln1/n ) based on the poremodel. The Freundlich exponent 1/n appeared to be constant over a broad range of salt concentrations, but the Freun-dlich constant K F assumed the maximum value for [Na 2 SO 4 ]=~0.010 M. However, this value was 10 times greater thanthat under the no-salt conditions. Analysis of the adsorption kinetics revealed that only the early stage of dyeing was fol-lowed second-order, with an activation energy of 50.2 kJ/mol. However, gradual deviation from the second-orderoccurred with an increase in the dyeing time. These results indicated that the surfaces of the inner pores in the PVA filmare dyed through multilayer adsorption, which is accompanied by complicated kinetics.Keywords: poly(vinyl alcohol), dichroic dye, dyeing, Freundlich's isotherm, pore model, the second-order kinetics

Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions

Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Performance was comparable to that observed using conventional HIC conditions with high salt.

Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations.

Explicit solvent molecular dynamics simulations have been used to complement preceding experimental and computational studies of folding of guanine quadruplexes (G-DNA). We initiate early stages of unfolding of several G-DNAs by simulating them under no-salt conditions and then try to fold them back using standard excess salt simulations. There is a significant difference between G-DNAs with all-anti parallel stranded stems and those with stems containing mixtures of syn and anti guanosines. The most natural rearrangement for all-anti stems is a vertical mutual slippage of the strands. This leads to stems with reduced numbers of tetrads during unfolding and a reduction of strand slippage during refolding. The presence of syn nucleotides prevents mutual strand slippage; therefore, the antiparallel and hybrid quadruplexes initiate unfolding via separation of the individual strands. The simulations confirm the capability of G-DNA molecules to adopt numerous stable locally and globally misfolded structures. The key point for a proper individual folding attempt appears to be correct prior distribution of syn and anti nucleotides in all four G-strands. The results suggest that at the level of individual molecules, G-DNA folding is an extremely multi-pathway process that is slowed by numerous misfolding arrangements stabilized on highly variable timescales.

Giant Unilamellar Vesicles Electroformed from Native Membranes and Organic Lipid Mixtures under Physiological Conditions

In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this “cell size” model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low- or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mechanical properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amount of salt present in the solution.

Non-Surface Activity and Micellization of Ionic Amphiphilic Diblock Copolymers in Water. Hydrophobic Chain Length Dependence and Salt Effect on Surface Activity and the Critical Micelle Concentration

We reported previously (Macromolecules 2003, 36, 5321; Langmuir, 2004, 20, 7412) that amphiphilic diblock copolymers having polyelectrolytes as a hydrophilic segment show almost no surface activity but form micelles in water. In this study, to further investigate this curious and novel phenomenon in surface and interface science, we synthesized another water-soluble ionic amphiphilic diblock copolymer poly(hydrogenated isoprene)-b-sodium poly(styrenesulfonate) PIp-h2-b-PSSNa by living anionic polymerization. Several diblock copolymers with different hydrophobic chain lengths were synthesized and the adsorption behavior at the air/water interface was investigated using surface tension measurement and X-ray reflectivity. A dye-solubilization experiment was carried out to detect the micelle formation. We found that the polymers used in this study also formed micelles above a certain polymer concentration (cmc) without adsorption at the air-water interface under a no-salt condition. Hence, we further confirmed that this phenomenon is universal for amphiphilic ionic block copolymer although it is hard to believe from current surface and interface science. For polymers with long hydrophobic chains (more than three times in length to hydrophilic chain), and at a high salt concentration, a slight adsorption of polymer was observed at the air-water interface. Long hydrophobic chain polymers showed behavior "normal" for low molecular weight ionic surfactants with increasing salt concentration. Hence, the origin of this curious phenomenon might be the macroionic nature of the hydrophilic part. Dynamic light scattering analysis revealed that the hydrodynamic radius of the block copolymer micelle was not largely affected by the addition of salt. The hydrophobic chain length-cmc relationship was found to be unusual; some kind of transition point was found. Furthermore, very interestingly, the cmc of the block copolymer did not decrease with the increase in salt concentration, which is in clear contrast to the fact that cmc of usual ionic small surfactants decreases with increasing salt concentration (Corrin-Harkins law). These behaviors are thought to be the special, but universal, characteristics of ionic amphiphilic diblock copolymers, and the key factor is thought to be a balance between the repulsive force from the water surface by the image charge effect and the hydrophobic adsorption.

Effect of Addition of Calcium Chloride on the Liquid−Liquid Equilibria of the Water + Propionic Acid + 1-Butanol System at 303.15 K

The influence of the addition of CaCl 2 to the water + propionic acid + 1-butanol ternary system was studied at (303.15 ± 0.05) K. The quaternary system was represented projecting four sectional quaternary planes corresponding to 4.3%, 9.3%, 15.1%, and 20.3% CaCl 2 in aqueous solution onto the face of the tetrahedron containing the ternary system water + propionic acid + 1-butanol. Therefore, all compositions are given on a salt-free basis for the solubility curves and for tie-line data. The addition of salt always enhances significantly the distribution coefficients and selectivities, while the region of heterogeneity increases as compared to the no-salt condition. Band type curves were obtained for solubility curves above 4.3% CaCl 2 , and no salt precipitation was observed. The experimental results show that CaCl 2 is not distributed in the same proportion between the phases at equilibrium and that 1-butanol appears to be a good solvent for extraction purposes. A comparison between the influence of CaCl 2 and NaCl on this ternary system is also made using these experimental results with earlier data. A high value of the Sestchenow's constant was obtained by a linear regression method, which is an indication that a great salting-out effect is produced by the addition of CaCl 2 , which is more important than for NaCl.

Liquid-liquid equilibria for the system water + propionic acid + 1-butanol at 303.2 K. Effect of addition of sodium chloride

Solubility and tie-line data were obtained for the ternary system: water + propionic acid + 1-butanol at 303.2 K. The ‘salting-out’ effect was studied by using sodium chloride (NaCl) at three concentrations within its solubility limits. The effect of the addition of this salt on the liquid-liquid equilibrium data of the ternary system has been investigated at the same temperature, determining independently both the solubility curve and the tie lines. In all cases, the addition of salt enhances significantly the distribution coefficients and selectivities, while the region of heterogeneity increases as compared to the no-salt condition. Tie-line data were correlated by the method of Othmer and Tobias [R.E. Othmer, P.E. Tobias, Ind. Eng. Chem. 34 (1942) 690–700.] and their parameters were evaluated. From the experimental results, it is evident that NaCl is not distributed in the same proportion between the phases at equilibrium, and that 1-butanol is a good solvent for extraction purposes.

Measurement and prediction of liquid—liquid equilibria of ethyl acetate—methanol—water in the presence of dissolved inorganic salts

The effect of sodium chloride, calcium chloride and zinc chloride at concentrations 5–20% by weight on the liquid—liquid equilibrium (LLE) data of the ternary system ethyl acetate(S)—methanol(C)—water(A) has been investigated experimentally at 30 °C. The region of heterogeneity is significantly increased with the addition of sodium and calcium chloride salts. A change in the direction of tie-line was observed for 10% sodium chloride, 20% calcium chloride and saturated zinc chloride exhibiting thereby a tendency to form solutropism for this system, which is non-solutropic under a no-salt condition. The selectivity values are also altered in comparison with that of a no-salt condition. The tie-line data have been correlated by the Eisen—Joffe equation. The solubility envelopes for the salt systems have been predicted using the NRTL model, from a knowledge of the binary vapor—liquid equilibria (VLE) determined experimentally in our laboratory employing the iso-activity criterion.