Addition Of Divalent Metal Cations Research Articles

Biodegradation of the strobilurin fungicide pyraclostrobin by Burkholderia sp. Pyr-1: Characteristics, degradation pathway, water remediation, and toxicity assessment

Pyraclostrobin, a widely used fungicide, poses significant risks to both the environment and human health. However, research on the microbial degradation process of pyraclostrobin was scarce. Here, a pyraclostrobin-degrading strain, identified as Burkholderia sp. Pyr-1, was isolated from activated sludge. Pyraclostrobin was efficiently degraded by strain Pyr-1, and completely eliminated within 6 d in the presence of glucose. Additionally, pyraclostrobin degradation was significantly enhanced by the addition of divalent metal cations (Mn2+ and Cu2+). The degradation pathway involving ether bond and N–O bond cleavage was proposed by metabolite identification. The sodium alginate-immobilized strain Pyr-1 had a higher pyraclostrobin removal rate from contaminated lake water than the free cells. Moreover, the toxicity evaluation demonstrated that the metabolite 1-(4-chlorophenyl)-1H-pyrazol-3-ol significantly more effectively inhibited Chlorella ellipsoidea than pyraclostrobin, while its degradation products by strain Pyr-1 alleviated the growth inhibition of C. ellipsoidea, which confirmed that the low-toxic metabolites were generated from pyraclostrobin by strain Pyr-1. The study provides a potential strain Pyr-1 for the bioremediation in pyraclostrobin-contaminated aquatic environments.

Ferrous ion Enhanced Lipopeptide Antibiotic Iturin A Production from Bacillus amyloliquefaciens B128

ABSTRACT The effect of different carbon sources, initial pH, and addition of divalent metal cations to the shaker flask cultivation medium was observed with the aim of improving iturin A production from Bacillus amyloliquefaciens B128. Among different C-sources tested, soft sugar enhanced iturin A production to 68.78 mg/L, which is 5 fold higher than nutrient broth cultivation concentration. This concentration further enhanced to 71.34 mg/L, after 6 days cultivation by the application of starting cultivation pH at 6.64. Furthermore, when this medium was supplemented with 0.2 mM of iron, iturin A concentration increased to 92.78 mg/L, after 5 days with no pH control cultivation. Interestingly, with the stating pH at 6.64 and 0.2 mM of ferrous sulfate, the highest iturin A production of 121.28 mg/L was obtained. Thus, the present study provides useful information for enhancing iturin A production in cultivation.

Fluorescent probe sensor based on (R)-(-)-4-phenyl-2-oxazolidone for effective detection of divalent cations.

Significant progress attained in sensor science in recent years has resulted in the development of highly efficient fluorescence probes for sensing metal ions. Fluorescent molecular probes based on (R)-(-)-4-phenyl-2-oxazolidone are reported here. Fluorescence studies indicated that the molecular probe could be used successfully to sense divalent metal cations such as Cu2+ , Co2+ , Pb2+ , and Zn2+ . The addition of divalent metal cations to the molecular probe produced a specific interaction pattern under UV-visible and fluorescence spectroscopy. These molecules could detect metal cations using fluorescence quenching. Stern-Volmer plots were used to determine quenching rate coefficients, which were calculated to be 2 × 101 , 1.06 × 103 and 7.39 × 102 M-1 s-1 for copper, cobalt, and zinc respectively. Calculation of limit of detection for heavy metal cations revealed that the reported molecular probes improved the limit of detection compared with available standard data. Limit of quantitation values were also well within the permissible range. The frontier energy gap of highest occupied molecular orbital to the lowest unoccupied molecular orbital was evaluated using the density functional theory approach and Gaussian 09 W software, which complemented the coordination of azetidinones with divalent metal ions.

Purification and Characterization of Cellulase from Obligate Halophilic Aspergillus flavus (TISTR 3637) and Its Prospects for Bioethanol Production.

A cellulase from the extreme obligate halophilic fungus, Aspergillus flavus, isolated from a man-made solar saltern in Phetchaburi, Thailand, was purified by ammonium sulfate precipitation and using Sephadex G-100 gel filtration column chromatography. The cellulase was found to be approximately 55kDa by SDS-PAGE. Using CMC as a substrate, the specific activity of the cellulase was 62.9units (U)mg-1 with Vmax and Km values of 37.87molmin-1mg-1 and 3.02mgmL-1, respectively. Characterization of the enzyme revealed it to be an extremozyme, having an optimum activity at pH 10, 60°C, and 200gL-1 of NaCl. The enzyme activity was not significantly altered by the addition of divalent metal cations at 2mM and neither did ß-mercaptoethanol, while EDTA was found strongly inhibiting the cellulase. Compared with commercial cellulase, the purified cellulase from A. flavus was more active in the extremity of conditions, especially at pH 10, 60°C, and 150gL-1 NaCl, whereas the commercial cellulase had a very low activity.

Pcal_0970: an extremely thermostable L-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity.

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100°C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4mmol/L and Vmax of 355 ± 13μmolmin-1mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6kJmol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150min at 100°C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.

Impact of cadmium and phosphate ions on the hematite nanorings formation

It has been suggested that the addition of divalent metal cations during the hydrothermal synthesis of hematite (α-Fe2O3) in the presence of phosphate ions has a strong impact on the hematite morphology, crystal growth and dissolution (Gotić et al., J. Mol. Struct. 993 (2011) 167). In this work, the same system was modified with divalent cadmium ions and the impacts of cadmium and phosphate ions on the hematite nanorings formation have been studied. It has been found that formed hematite and akaganeite (β-FeOOH) precipitates did not contain any trace of cadmium impurities. On the contrary, the modification with divalent cadmium cations changes the PO43−/Fe3+ molar ratio in the system. Upon the addition of cadmium ions the PO43−/Fe3+ molar ratio changes from 0.036 in a pure system to 0.051 in the cadmium modified system. The relative increase of phosphate concentration at early stage of precipitation induced the change of spindle to spherical morphology and formation of the poorly crystallised akaganeite-like phase. The appearance of akaganeite-like phase directly correlated with the delay in the crystallisation of hematite. Besides, the relative increase of phosphate concentration has the prevailing effect for the change of spindle to pseudosphere and nanotube to nanoring particle morphologies.

Innate cation sensitivity in a semiconducting polymer

Water-gated organic thin film transistors (OTFTs) using the hole transporting semiconducting polymer, poly(2,5-bis(3-hexadecylthiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT), show an innate response of their threshold voltage to the addition of divalent metal cations to the gating water, without deliberately introducing an ion-sensitive component. A similar threshold response is shown for several divalent cations, but is absent for monovalent cations. Response is absent for transistors using the inorganic semiconductor ZnO, or the similar organic semiconductor poly(3-hexylthiophene) (rrP3HT), instead of PBTTT. We assign innate cation sensitivity to residues of the organometallic Pd(0) complex used as catalyst in PBTTT synthesis which bears strong resemblance to typical metal chelating agents. Organometallic Pd(0) residues are absent from ZnO, and also from rrP3HT which is polymerised with a different type of catalyst. However, when Pd(0) complex is deliberately added to rrP3HT casting solutions, resulting OTFTs also display threshold response to a divalent cation.

X-ray Crystal Structure of Divalent Metal-Activated β-xylosidase, RS223BX.

We report the X-ray crystal structure of a glycoside hydrolase family 43 β-xylosidase, RS223BX, which is strongly activated by the addition of divalent metal cations. The 2.69Å structure reveals that the Ca(2+) cation is located at the back of the active-site pocket. The Ca(2+) is held in the active site by the carboxylate of D85, an "extra" acid residue in comparison to other GH43 active sites. The Ca(2+) is in close contact with a histidine imidazole, which in turn is in contact with the catalytic base (D15) thus providing a mechanism for stabilizing the carboxylate anion of the base and achieve metal activation. The active-site pocket is mirrored by an "inactive-site" pocket of unknown function that resides on the opposite side of the monomer.

Activation of a GH43 β-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity

RS223-BX of glycoside hydrolase family 43 is a β-d-xylosidase that is strongly activated (kcat/Km as much as 116-fold) by the addition of divalent metal cations, Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+. Slow activation by Mg2+ was demonstrated (kon 0.013s−1mM−1, koff 0.008s−1) at pH 7.0 and 25°C. koff and kon values are independent of Mg2+ concentration, but koff and kon are slower in the presence of increasing levels of substrate 4-nitrophenyl-β-d-xylopyranoside. The kinetics strongly suggest that M2+ binds to the enzyme rapidly, forming E M2+, followed by slow isomerization to the activated enzyme, E∗ M2+. Moderately high values of kcat (7–30s−1) were found for M2+-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25°C. Certain M2+-activated RS223-BX exhibit the highest reported values of kcat/Km of any β-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn2+, 190s−1mM−1), xylotriose (Ca2+, 150s−1mM−1) and xylotetraose (Ca2+, 260s−1mM−1). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations.

Hydrothermal synthesis of α-Fe 2O 3 nanorings with the help of divalent metal cations, Mn 2+, Cu 2+, Zn 2+ and Ni 2+

The morphologies of α-Fe 2O 3 spindles and nanotubes was modified by the addition of divalent metal cations M 2+ (M = Mn, Cu, Zn, Ni). Divalent metal cations gradually modify the spindle to pseudosphere and the nanotube to nanoring particle morphologies. At a higher concentration of added divalent cations the nanodisc morphology was obtained. The SEM/EDS and TEM/EDX analyses showed that precipitates contained Fe, O and P elements, but none of the precipitates contained divalent metal cations. The XRD patterns of each investigated sample fitted well to pure hematite (α-Fe 2O 3) and there were no other phases observed. The Mössbauer and XRD lines broadened with the addition of divalent metal cations. The mean crystallite sizes were calculated using the Scherrer equation from XRD line broadening of 104 and 110 lines of hematite. With the addition of a Mn 2+ cation the mean crystallite size gradually increased in the a-axis direction. The relative high value of the mean crystallite size in the a-axis direction was found for nanorings modified by a Cu 2+ cation. This high crystal distortion in the a-axis direction upon adding Cu 2+ was explained by a strong Jahn–Teller effect of Cu 2+ that has a tetragonally distorted coordination sphere in an octahedral coordination. Thus, quite opposite to a pure system, the modification by divalent metal cations induced the preferential growth of α-Fe 2O 3 crystals in the a-axis direction. It was suggested that divalent metal cations introduced the defects into the planes parallel to the basal plane (0 0 1) and thus changed the type of hydroxyl groups on hematite planes, which as a consequence switched the preferential growth of the α-Fe 2O 3 along the c-axis to the growth along the a-axis.

The M17 Leucine Aminopeptidase of the Malaria Parasite Plasmodium falciparum: Importance of Active Site Metal Ions in the Binding of Substrates and Inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

The N-Acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis Is a Zinc Metalloenzyme

The de-N-acetylation of N-acetyl-D-glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei, the causative agent of African sleeping sickness, and GlcNAc-PI de-N-acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de-N-acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de-N-acetylase with divalent metal cations restores activity in the order Zn(2+) > Cu(2+) > Ni(2+) > Co(2+) > Mg(2+). Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de-N-acetylase as a zinc metalloenzyme will facilitate the rational design of anti-protozoan parasite drugs.

Separation of organic acids by capillary zone electrophoresis in buffers containing divalent metal cations

The addition of divalent metal cations to the background electrolyte influences the migration time of organic acids that are involved in complexation equilibria with the metals. This effect derives from a change in charge distribution and represent a means of manipulation of selectivity. In the present investigation, we evaluated the effects produced on the separation of a test mixture, composed of aromatic and aliphatic acids, by a series of divalent cations added to the background electrolyte. The use of a coated capillary allowed us to exclude the influence of wall effects on the separation.

Characterization and comparison of synthetic immobile and mobile Holliday junctions.

Eight synthetic Holliday junction (HJ) oligonucleotides containing an immobile or a mobile junction were characterized by gel electrophoresis, ultraviolet absorption and circular dichroism (CD) spectroscopy. Four 24-mer deoxyribonucleotides formed stable immobile and mobile HJs in 0.1 M NaCl at 5 muM strand concentration at room temperature. However, the immobile HJ constructed from four 18-mers was less stable, and four 12-mers did not form the HJ structure under the conditions used. A comparison of the melting profiles of the HJs with those of the duplexes corresponding to the arms of four-way junctions indicated that the thermal stability of the HJ was similar to that of the individual arm and the cooperativity of the melting behavior of the HJ was relatively higher than that of the individual arm duplex. The Tms of the mobile HJs containing 4, 6, 8, and 10 base-pair homologous cores at junctions were essentially identical with that of the immobile HJ of the same size. There is a tendency that the HJ containing a larger homologous core region becomes more resistant to thermal denaturation. The addition of divalent metal cations, Mg2+ and Ca2+, to the solutions of the HJs raised their melting temperatures. The difference found for the CD spectra of the HJs which differ only in the arrangement of the HJ depended primarily upon the DNA sequence flanking the junction. The RuvC protein binds to the immobile and mobile HJs, regardless of the presence and the size of the homologous core at the junction.

Interaction of Na+ and Mg2+ ions in acetylcholine receptor channels of frog skeletal muscle changes in character with an increase in agonist concentration.

Measurements of agonist-induced single-channel currents of nicotinic acetylcholine receptor channels (nAChR) have shown that addition of divalent metal cations (Ca2+, Mg2+, Ba2+) in millimolar amounts causes a decrease of 40% or more in currents carried by monovalent cations (Na+, Cs+). Conventional voltage-clamp measurements were made at frog (Rana pipiens) sartorius endplates to determine if this occlusive interaction between mono- and divalent cations is preserved in macroscopic currents produced by higher concentrations of nAChR agonists, as would be expected on the basis of simple summation of unitary currents. Single ion nAChR currents of Na+ (INa+ ) and Mg2+(IMg2+ ) and the composite current when both cations were present (INa+, Mg2+ ) were measured in high-K+, low ionic strength bathing media during local superfusion with various concentrations of carbamylcholine. It was found that, at lower test carbamylcholine levels of 27 and 54 microM, peak values of INa+, Mg2+ were 40% and 53% of the respective INa+ , in agreement with the effects observed from single-channel measurements. At all higher carbamylcholine levels, however, peak INa+, Mg2+ exceeded INa+ and increased instead with the sum of INa+ and IMg2+ as if there was an independent movement of these ions through the channels rather than the occlusive interaction found at lower carbamylcholine levels. This suggests that with exposure to increasing agonist concentrations above those commonly used in single-channel measurements there are changes in the open state of nAChR affecting cation selectivity possibly in the narrow pore region of the channels.

5′-Nucleotidase from bovine brain cortex: effect of solubilization on enzyme kinetics and modulation

The kinetic characteristics and the EDTA inhibition of microsomal 5?-nucleotidase from bovine brain cortex were studied and compared with the properties of the enzyme solubilized with Lubrol WX. The K(m) value after enzyme solubilization was not significantly different from that of the membrane-bound enzyme. Likewise, di- and trinucleotides performed a similar competitive inhibition of the two forms of the enzyme. In contrast, divalent cations inhibited the intact microsomal enzyme activity at the same concentrations in which they increased the soluble-enzyme activity. The solubilization of microsomal 5?-nucleotidase did not change the progressive and irreversible character of the EDTA inhibition, but the mechanism of the irreversible inhibition was different. The addition of divalent metal cations did not affect the irreversibility of either inhibition, even though the effect on the residual activities was different. The Arrhenius plot of the 5?-nucleotidase activity in intact microsomal fraction exhibited a well-defined break at 31 +/- 0.1 degrees C, whereas that of the solubilized enzyme was a straight line. It is concluded then that microsomal 5?-nucleotidase from bovine brain cortex does not require the membrane environment to express its activity, although the influence of this lipidic environment was evident in the differences observed in the enzyme activity modulation by EDTA, cations and temperature.

Selectivity to K+ and Na+ of protoplast fractions isolated from different regions of Aspergillus nidulans hyphae.

The selectivity to K+ and Na+ of protoplast samples representing cytoplasm isolated from different regions of the hyphal filament of Aspergillus nidulans was investigated. Concentrations of both ions contained in successive protoplast fractions were measured. During lytic digestion, protoplasts were released first from apical regions and subsequently from progressively older regions of hyphae. A low K+/Na+ ratio was found in protoplasts containing primarily apical cytoplasm and a high K+/Na+ ratio was found in protoplasts originating from older regions of hyphae. The ratios were the same whether MgSO4 or mannitol was used as stabilizer. Absolute concentrations of both ions were higher in protoplasts of apical origin. Protoplasts stabilized in mannitol lost more ions than those stabilized in MgSO4 over an 8 h incubation period. Na+ losses were higher from apical protoplasts whereas K+ losses were higher from protoplasts liberated from older regions of hyphae. The addition of divalent metal cations (1.5 mM-Mn2+ or Mg2+) reduced losses of Na+ from protoplasts but did not affect loss of K+. Data obtained using protoplast samples were related to those obtained for intact mycelium. Absolute losses of both ions from mycelium were lower than for protoplasts but when compared on a protein basis the data suggested that protoplasts possess properties similar to those of intact mycelium in terms of K+ and Na+ selectivity.

Protein-RNA interactions in belladonna mottle virus investigated by laser Raman spectroscopy.

Raman difference spectroscopy of the belladonna mottle virus (BDMV) and its separated RNA and protein components indicates that molecular interaction occurs between the single-stranded RNA genome and capsid subunits. The molecular interactions that stabilize the virion at pH 5.0 are altered or eliminated at pH 8.0, even though release of the RNA from the capsid is prevented by the addition of divalent metal cations (Ca2+). From the perturbations that occur to Raman lines of cytosine and adenine rings of the encapsidated RNA molecule between pH values of 5.0 and 8.0, it is concluded that cytosines are protonated in significant numbers at the conditions which maintain the native virus structure and that the stacking of adenines is altered by changes in pH. The degree of protonation of RNA bases can be reduced by elevation of the pH to 8.0 for encapsidated RNA or by release of the RNA from the capsid at pH 5.0. Although the protein groups that interact with the viral RNA cannot be identified unambiguously from the Raman spectra, it is apparent that the molecular environments of aromatic amino acid side chains are altered with the same changes in pH (from 5.0 to 8.0) that perturb the cytosine and adenine ring structures. No significant change in secondary structures of the capsid subunit can be detected with changes in pH or with RNA release. On the other hand, the characteristic Raman lines of the phosphate groups of packaged RNA differ from those of naked RNA at all pH values examined, most likely as a result of specific electrostatic binding of divalent cations to RNA phosphates within the virus shell.

Spectroscopic sensitivity of linear -benzoadenine nucleotides to divalent metal counterions, side chain conformations, micelles, and enzymes

From pK(a) data for lin-benzoadenosine 5'-mono-, 5'-di-, and 5'-triphosphates, which are fluorescent "stretched-out" analogues of adenine nucleotides, it was possible to designate the cases of interaction of phosphate with the heteroaromatic moiety. The addition of divalent metal cations or quaternary ammonium micelles diminishes the direct intramolecular interaction between the phosphate(s) and base and consequently brings the pK(a) values close to that of lin-benzoadenosine. Fluorescence spectroscopy was used to investigate the interaction of lin-benzoadenine nucleotides with Mg(2+), Mn(2+), and Co(2+). The association constants for the formation of such complexes were obtained from measurements of steady-state fluorescence quenching. Phase and modulation measurements of the fluorescence lifetimes of lin-benzoadenine nucleotides as a function of Co(2+) concentration permitted determination of the static component of the quenching due to intramolecular complex formation. The association constants of the lin-benzoadenine nucleotides with all of the divalent metal ions studied were greater than those observed for the corresponding adenine nucleotides and were in the order: lin-benzo-ATP > lin-benzo-ADP > lin-benzo-AMP. Fourier transform (1)H NMR of lin-benzo-ATP in the presence of Co(2+) showed broadening of the aromatic proton signals, the 2-H signal (corresponding to the 8-H in ATP) being the most affected. Models are proposed to explain the phosphate-base interaction, the influence of metal ions on base protonation, and the intramo- the intramolecular quenching observed in the complexes due to paramagnetic ion (Co(2+), Mn(2+)) and base interaction.