Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H₂O₂-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Pre-steady state kinetic characterization of human peroxiredoxin 5: taking advantage of Trp84 fluorescence increase upon oxidation

Human peroxiredoxin 5 (PRDX5) catalyzes different peroxides reduction by enzymatic substitution mechanisms. Enzyme oxidation caused an increase in Trp84 fluorescence, allowing performing pre-steady state kinetic measurements. The technique was validated by comparing with data available from the literature or obtained herein by alternative approaches. PRDX5 reacted with organic hydroperoxides with rate constants in the 10⁶-10⁷ M⁻¹ s⁻¹ range, similar to peroxynitrite-mediated PRDX5 oxidation, whereas its reaction with hydrogen peroxide was slower (10⁵ M⁻¹ s⁻¹). The method allowed determining the kinetics of intramolecular disulfide formation as well as thioredoxin 2-mediated reduction. The reactivities of PRDXs with peroxides were surprisingly high considering thiol pK_a, indicating that other protein determinants are involved in PRDXs specialization. The order of reactivities between PRDX5 towards oxidizing substrates differ from other PRDXs studied, pointing to a selective action of PRDXs with respect to peroxide detoxification, helping to rationalize the multiple enzyme isoforms present even in the same cellular compartment.     

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H₂O₂, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 10⁴ M^− 1 s^− 1, 30 M^− 1 s^− 1, 3.4 × 10³ M^− 1 s^− 1, and 8.6 × 10³ M^− 1 s^− 1, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Short term effects of hypoxia and bioturbation on solute fluxes, denitrification and buffering capacity in a shallow dystrophic pond

The effects of short term hypoxia on bioturbation activity and inherent solute fluxes are scarcely investigated even if increasing number of coastal areas are subjected to transient oxygen deficits. In this work dark fluxes of oxygen (O₂), dissolved inorganic carbon (TCO₂) and nutrients across the sediment-water interface, as well as rates of denitrification (isotope pairing), were measured in intact sediment cores collected from the dystrophic pond of Sali e Pauli (Sardinia, Italy). Sediments were incubated at 100, 70, 40 and 10% of O₂ saturation in the overlying water, with both natural benthic communities, dominated by the polychaete Polydora ciliata (11.100 ± 2.500  ind. m^− 2), and after the addition of individuals of the deep-burrower polychaete Hediste diversicolor. Below an uppermost oxic layer of ~ 1 mm, sediments were highly reduced, with up to 6 mM of S²⁻ in the 5 mm layer. Flux of S²⁻ and O₂ calculated from pore water gradients were 8.61 ± 1.12 and − 2.27 ± 0.56 mmol m^− 2 h^− 1, respectively. However, sediment oxygen demand (SOD) calculated from core incubation was − 10.52 ± 0.33 mmol m^− 2 h^− 1, suggesting a major contribution of P. ciliata to O₂-mediated sulphide oxidation. P. ciliata also strongly stimulated NH₄⁺ and PO₄³⁻ fluxes, with rates ~ 15 and ~ 30 folds higher, respectively, than those estimated from pore water gradients. P. ciliata activity was significantly reduced at 10% O₂ saturation, coupled to decreased rates of solutes transfer. The addition of H. diversicolor further stimulated SOD, NH₄⁺ efflux and SiO₂ mobilisation. Similarly to P. ciliata, the degree of stimulation of SOD and NH₄⁺ flux by H. diversicolor depended on the level of oxygen saturation. TCO₂ regeneration, respiratory quotients, PO₄³⁻ fluxes and denitrification of added ¹⁵NO₃⁻ were not affected by the addition of H. diversicolor, but depended upon the O₂ levels in the water column. Denitrification rates supported by water column ¹⁴NO₃⁻ and sedimentary nitrification were both negligible (< 0.5 µmol m^− 2 h^− 1). They were not significantly affected by oxygen saturation nor by bioturbation, probably due to the limited availability of NO₃⁻ in the water column (< 3 µM) and O₂ in the sediments. This study demonstrates for the first time the integrated short term effect of transient hypoxia and bioturbation on solute fluxes across the sediment-water interface within a simplified lagoonal benthic community.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k_cat′ for desulfination of l-cysteine sulfinate increased to 0.5 s^− 1 (from 0.05 s^− 1 in wild-type enzyme), whereas k_cat′ for transamination of the same substrate was reduced from 510 s^− 1 to 0.05 s^− 1. Similarly, k_cat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s^− 1 to 0.07 s^− 1, whereas k_cat′ for transamination was reduced from 530 s^− 1 to 0.13 s^− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots

Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1′E)-12-(1′-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid. Its identification was confirmed by the data of ultraviolet spectroscopy, mass spectra, ¹H NMR, COSY, catalytic hydrogenation. The isomeric divinyl ether (8E,1′E,3′Z)-12-(1′,3′-nonadienyloxy)-8-nonenoic (colneleic) acid was detected as a minor product. Incubations with linoleic acid hydroperoxides revealed that 13-hydroperoxide was a preferential substrate, while the 9-hydroperoxide was utilized with lesser efficiency.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Effect of substituent dtp to optical properties of heterobimetallic M/Ag/S nest-shaped clusters (M = Mo, W)

Clusters [MoS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (1), [WS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (2) and [WOS₃Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (3) were synthesized by the reaction of (NH₄)₂MoS₄/(NH₄)₂WS₄, (NH₄)₂WOS₃ with Ag[S₂P(OPrⁱ)₂]. Their structures have been characterized by X-ray diffraction. The clusters consist of a distorted tetrahedral MS₄ (or MOS₃) (M = Mo, W) with three Ag atoms and three sulfur atom bridges (Fig. 1), and resemble roughly that of cubane-like clusters. The nonlinear optical (NLO) properties were studied with an 8 ns pulsed laser at 532 nm. Its optical response to the incident light exhibits good optical absorptive and refractive effects, with α₂ = 1.56 × 10⁻¹⁰ m W⁻¹, n₂ = 3.87 × 10⁻¹⁷ m² W⁻¹ for cluster 1; α₂ = 1.33 × 10⁻¹⁰ m W⁻¹, n₂ = 6.52 × 10⁻¹⁷ m² W⁻¹for cluster 2; and α₂ = 2.54 × 10⁻¹⁰ m W⁻¹, n₂ = 4.07 × 10⁻¹⁷ m² W⁻¹ for cluster 3 for a 1.56 × 10⁻⁴ mol dm⁻³ CH₂Cl₂ solution.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Lipid hydroperoxide-induced and hemoglobin-enhanced oxidative damage to colon cancer cells

Epidemiological studies have indicated that Western diets are related to an increase in a series of malignancies. Among the compounds that are credited for this toxic effect are heme and lipid peroxides. We evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxides (LAOOH) on a series of toxicological endpoints, such as cytotoxicity, redox status, lipid peroxidation, and DNA damage. We demonstrated that the preincubation of SW480 cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, malonaldehyde formation, and DNA fragmentation and that these effects were related to the peroxide group and the heme present in Hb. Furthermore, Hb and LAOOH alone exerted a toxic effect on the endpoints assayed only at concentrations higher than 100 μM. We were also able to show that SW480 cells presented a higher level of the modified DNA bases 8-oxo-7,8-dihydro-2′-deoxyguanosine and 1,N²-etheno-2′-deoxyguanosine compared to the control. Furthermore, incubations with Hb led to an increase in intracellular iron levels, and this high level of iron correlated with DNA oxidation, as measured as EndoIII- and Fpg-sensitive sites. Thus, Hb from either red meat or bowel bleeding could act as an enhancer of fatty acid hydroperoxide genotoxicity, which contributes to the accumulation of DNA lesions in colon cancer cells.     

Bicarbonate-catalyzed hydrogen peroxide oxidation of cysteine and related thiols

The effect of bicarbonate on the rates of the H₂O₂ oxidation of cysteine, gluthathione, and N-acetylcysteine to the corresponding disulfides was investigated. The relative oxidation rates at pH 8 for the different thiols are inversely related to the pK_a values of the thiol groups, and the reactive nucleophiles are identified as the thiolate anions or their kinetic equivalents. The second-order rate constants at 25 °C for the reaction of the thiolate anions with hydrogen peroxide are 17 ± 2 M⁻¹ s⁻¹ for all three substrates. In the presence of bicarbonate (>25 mM), the observed rate of thiolate oxidation is increased by a factor of two or more, and the catalysis is proposed to be associated with the formation of peroxymonocarbonate from the equilibrium reaction of hydrogen peroxide with bicarbonate (via CO₂). The calculated second-order rate constants for the direct reaction of the three thiolate anions with peroxymonocarbonate fall within the range of 900-2000 M⁻¹ s⁻¹. Further oxidation of disulfides by peroxymonocarbonate results in the formation of thiosulfonate and sulfonate products. These results strongly suggest that peroxymonocarbonate should be considered as a reactive oxygen species in aerobic metabolism with relevance in thiol oxidations.     

Development of cesium phosphotungstate salt and chitosan composite membrane for direct methanol fuel cells

A novel composite membrane has been developed by doping cesium phosphotungstate salt (Cs_xH_3−xPW₁₂O₄₀ (0 ≤ x ≤3), Cs_x-PTA) into chitosan (CTS/Cs_x-PTA) for application in direct methanol fuel cells (DMFCs). Uniform distribution of Cs_x-PTA nanoparticles has been achieved in the chitosan matrix. The proton conductivity of the composite membrane is significantly affected by the Cs_x-PTA content in the composite membrane as well as the Cs substitution in PTA. The highest proton conductivity for the CTS/Cs_x-PTA membranes was obtained with x = 2 and Cs₂-PTA content of 5 wt%. The value is 6 × 10⁻³ S cm⁻¹ and 1.75 × 10⁻² S cm⁻¹ at 298 K and 353 K, respectively. The methanol permeability of CTS/Cs₂-PTA membrane is about 5.6 × 10⁻⁷, 90% lower than that of Nafion-212 membrane. The highest selectivity factor (φ) was obtained on CTS/Cs₂-PTA-5 wt% composite membrane, 1.1 × 10⁴/S cm⁻³ s. The present study indicates the promising potential of CTS/Cs_x-PTA composite membrane as alternative proton exchange membranes in direct methanol fuel cells.     

A simple bacterial transformation method using magnesium- and calcium-aminoclays

An efficient and user-friendly bacterial transformation method by simple spreading cells with aminoclays was demonstrated. Compared to the reported transformation approaches using DNA adsorption or wrapping onto (in)organic fibers, the spontaneously generated clay-coated DNA suprastructures by mixing DNA with aminoclay resulted in transformants in both Gram-negative (Escherichia coli) and Gram-positive cells (Streptococcus mutans). Notably, the wild type S. mutans showed comparable transformation efficiency to that of the E. coli host for recombinant DNA cloning. This is a potentially promising result because other trials such as heat-shock, electroporation, and treatment with sepiolite for introducing DNA into the wild type S. mutans failed. Under defined conditions, the transformation efficiency of E. coli XL1-Blue and S. mutans exhibited ~ 2 × 10⁵ and ~ 6 × 10³ CFU/μg of plasmid DNA using magnesium-aminoclay. In contrast, transformation efficiency was higher in S. mutans than that in E. coli XL1-Blue for calcium-aminoclay. It was also confirmed that each plasmid transformed into E. coli and S. mutans was stably maintained and that they expressed the inserted gene encoding the green fluorescent protein during prolonged growth of up to 80 generations.     

Biosensor based on the biocatalysis of microperoxidase-11 in nanocomposite material of multiwalled carbon nanotubes/room temperature ionic liquid for amperometric determination of hydrogen peroxide

A novel nanocomposite material of multiwalled carbon nanotubes (MWCNTs) and room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) was explored and used to construct a novel microperoxidase-11 (MP-11) biosensor for the determination of hydrogen peroxide (H₂O₂). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to characterize the performance of the biosensor. Under the optimized experimental conditions, H₂O₂ could be detected in a linear calibration range of 0.5 to 7.0 × 10⁻⁷ mol L⁻¹ with a correlation coefficient of 0.9949 (n = 9) and a detection limit of 3.8 × 10⁻⁹ mol L⁻¹ at 3σ. The modified electrodes displayed excellent electrochemical response, high sensitivity, long-term stability, and good bioactivity and selectivity.