The determination of the stability constant for the iron(II) complex of the biochelator pyridine-2,6-bis(monothiocarboxylic acid)

Pyridine-2,6-bis(monothiocarboxylic acid), also known as pyridine-2,6-dithiocarboxylic acid (pdtc), is a unique and powerful metal chelator produced by Pseudomonas stutzeri and Pseudomonas putida. The actual physiological roles of pdtc in these pseudomonads are not known with certainty, though it is likely that the compound acts as a siderophore, an antibiotic, or both. The stability constant of Fe^III(pdtc)₂ ^2- was determined in previous work to be 10^33.36. Here we determined that the stability constant of Fe^II(pdtc)₂ ^2- is 10¹². We determined this stability constant through potentiometric and spectrophotometric measurements of a ligand-ligand competition study using 2,6-pyridine dicarboxylic acid as the competitor for iron. Comparing the stability constant for Fe^II(pdtc)₂ ^2- to the constant for Fe^III(pdtc)₂ ^2- shows that the stability constant of Fe^II(pdtc)₂ ^2- is approximately 21 orders of magnitude smaller. This represents a very significant decrease in the binding strength of pdtc toward iron. Thus, if the host cell produces pdtc as a siderophore for sequestering Fe(III), it is likely that a second metabolite or a membrane protein of the host cell is used for reduction of the chelated iron at or near the cell membrane in order to facilitate its release from pdtc for cellular use.     

Immobilized cell reactors in mineralization of dicarboxylic acid solid waste

Dicarboxylic acid solid waste containing phthalic acid, malic acid, quinone, saturated and unsaturated dicarboxylic esters etc., are discharged in huge quantities during the crackdown of benzene over the catalyst vanadium at temperatures greater than 500 °C in a dicarboxylic acid manufacturing industry. Concern over the biological effects of these compounds underlines the necessity to treat this solid waste. The role of yeast Saccharomyces cerevisiae and anaerobic mixed bacterial cultures immobilized in activated carbon, in sequential two stage anoxic reactors, were investigated for the degradation of dicarboxylic acid solid waste (DASW). In the first stage, DASW was dissolved in water to yield a concentration of 0.5% w/v and was treated in yeast Saccharomyces cerevisiae immobilized reactor at an optimum residence time of 24 h. The yeast fermented samples were further treated in an upflow anaerobic reactor containing mixed culture immobilized in activated carbon at an Hydraulic Retention Time (HRT) of 0.2076 days at an hydraulic flow rate of 14.6×10⁻³ m³/day and Chemical Oxygen Demand (COD) loading rate of 4.3 kg/m³/day. The intermediates that were formed during the yeast fermentation and the anaerobic degradation of DASW were characterized by HPLC, proton NMR, C¹³ NMR and mass spectrometry.     

Quantitation of the energetic state in mitochondria and submitochondrial vesicles with 8-anilino-1-naphthalene sulfonic acid

Some of the apparently anomalous findings made with the fluorescent probe 8-anilino-1-naphthalene sulfonic acid (ANS) have been reinvestigated using rat liver mitochondria. The results have been found compatible with current views on energy conservation.The direction of fluorescence and proton flux changes under different conditions have been delineated. The relation of these results to consideration of membrane polarity and organization is discussed.The reliability of ANS fluorescence changes in determining the level of energization of mitochondria and submitochondrial preparations is discussed.Abbreviations used ANS 8-anilino-1-naphthalene sulfonic acid - F _E and H⁺ _E O₂ dependent change in fluorescence and H⁺ in mitochondria and SMP - SMP submitochondrial preparation     

Fermentative degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by a defined coculture of strictly anaerobic bacteria

Degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) under strictly anaerobic conditions was studied in enrichment cultures from marine and freshwater sediments. In all cases, dipicolinic acid was completely degraded. From an enrichment culture from a marine sediment, a defined coculture of two bacteria was isolated. The dipicolinic acid-fermenting bacterium was a Gram-negative, non-sporeforming strictly anaerobic short rod which utilized dipicolinic acid as sole source of carbon, energy, and nitrogen, and fermented it to acetate, propionate, ammonia, and 2CO₂. No other substrate was fermented. This bacterium could be cultivated only in coculture with another Gram-negative, non-sporeforming rod from the same enrichment culture which oxidized acetate to CO₂ with fumarate, malate, or elemental sulfur as electron acceptor, similar to Desulfuromonas acetoxidans. Since this metabolic activity is not important in substrate degradation by the coculture, the basis of the dependence of the dipicolinic acid-degrading bacterium on the sulfur reducer may be sought in the assimilatory metabolism.     

Development,validation and characterization of an analytical method for the quantification of hydrolysable urinary metabolites and plasma protein adducts of 2,4- and 2,6-toluene diisocyanate, 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate

Occupational exposure to diisocyanates within the plastic industry causes irritation and disorders in the airway. The aim of this study was to develop, validate and characterize a method for the determination of 2,4-toluenediamine (2,4-TDA), 2,6-toluenediamine (2,6-TDA), 1,5-diaminonaphthalene (1,5-NDA) and 4,4′-methylenedianiline (4,4′-MDA) in hydrolysed urine and plasma, and to study the correlation between the plasma and urinary levels of these potential biomarkers of 2,4-toluene diisocyanate (2,4-TDI), 2,6-toluene diisocyanate (2,6-TDI), 1,5-naphthalene diisocyanate (1,5-NDI) and 4,4′-methylenediphenyl diisocyanate (4,4′-MDI), respectively. Samples were hydrolysed with 0.3 M NaOH at 100°C for 24 h. The diamines were extracted, derivatized with pentafluoropropionic acid anhydride, and quantified by selected ion monitoring on gas chromatography-mass spectrometry. The repeatability and reproducibility of the method were 7-18% and 7-19%, respectively. Dialysis experiments showed that the metabolites of 2,4-TDI, 2,6-TDI, 1,5-NDI and 4,4′-MDI in plasma were exclusively protein adducts. No free diamines were found in urine, indicating that all diisocyanate-related metabolites were in a conjugated form. For each diisocyanate-related biomarker, there were strongly significant correlations (p<0.001) between individual levels of metabolites in plasma and urine, with Spearman's rank correlation coefficient (r_s) values of 0.74-0.90. The methods presented here will be valuable for the development of biological monitoring methods for diisocyanates.     

Oxidation of syringic acid by extracellular peroxidase of white-rot fungus,Pleurotus ostreatus

The oxidative degradation of syringic acid by the extracellular peroxidase ofPleurotus ostreatus was studied. Three products formed in the oxidation of syringic acid by the peroxidase in the presence of O₂ and H₂O₂ were identified as 2,6-dimethoxyphenol, 2,6-dimethoxy-1,4-dihydroxybenzene, and 2,6-dimethoxy-1,4-benzoquinone. A free radical was detected as the reaction intermediate of the extracellular peroxidase-catalyzed oxidation of acetosyringone. These results can be explained by mechanisms involving the production of a phenoxy radical and subsequent decarboxylation. This is the first time that 2,6-dimethoxyphenol has been identified in extracellular peroxidase-catalyzed reactions.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

The Escherichia coli SLC26 homologue YchM (DauA) is a C4‐dicarboxylic acid transporter

The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM‐dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C₄‐dicarboxylic acids and that its substrate specificity differs from the well‐characterized succinate transporter, DctA. Accordingly ychM was re‐designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C₄‐dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

代谢工程改造大肠杆菌合成丙二酸

丙二酸是一种重要的有机二元羧酸,其应用价值遍及化工、医药、食品等领域。本文以大肠杆菌为底盘细胞,过表达了ppc、aspC、panD、pa0132、yneI和pyc基因,成功构建了丙二酸合成重组菌株大肠杆菌BL21(TPP)。该菌株在摇瓶发酵条件下,丙二酸产量达到0.61 g/L。在5 L发酵罐水平,采用间歇补料的方式丙二酸的积累量达3.32 g/L。本研究应用了融合蛋白技术,将ppc和aspC、pa0132和yneI分别进行融合表达,构建了工程菌BL21(SCR)。在摇瓶发酵水平,该菌株丙二酸的积累量达到了0.83 g/L,较出发菌株BL21(TPP)提高了36%。在5 L发酵罐中,工程菌BL21(SCR)的丙二酸产量最高达5.61 g/L,较出发菌株BL21(TPP)提高了69%。本研究实现了丙二酸在大肠杆菌中的生物合成,为构建丙二酸合成的细胞工厂提供了理论依据和技术基础,同时也对其他二元羧酸的生物合成具有启发和指导意义。     

Metal binding by pyridine-2,6-bis(monothiocarboxylic acid), a biochelator produced by Pseudomonas stutzeri and Pseudomonas putida

Pyridine-2,6-bis(monothiocarboxylic acid) (pdtc),a natural metal chelator produced by Pseudomonas stutzeri and Pseudomonas putidathat promotes the degradation of carbon tetrachloride, was synthesized and studiedby potentiometric and spectrophotometric techniques. The first two stepwise protonationconstants (pK) for successive proton addition to pdtc were found to be 5.48 and2.58. The third stepwise protonation constant was estimated to be 1.3. The stability (affinity)constants for iron(III), nickel(II), and cobalt(III) were determined by potentiometric orspectrophotometric titration. The results show that pdtc has strong affinity for Fe(III)and comparable affinities for various other metals. The stability constants (log K) are 33.93 for Co(pdtc)₂ ^1-; 33.36 for Fe(pdtc)₂ ^1-; and 33.28 for Ni(pdtc)₂ ^2-. These protonationconstants and high affinity constants show that over a physiological pH range theferric pdtc complex has one of the highest effective stability constants for ironbinding among known bacterial chelators.     

Catabolism of 2,6-dinitrophenol by Alcaligenes eutrophus JMP 134 and JMP 222

Both Alcaligenes eutrophus JMP 134 and its plasmid-free derivative Alcaligenes eutrophus JMP 222 utilize 2,6-dinitrophenol as sole source of carbon, energy, and nitrogen. In the presence of ammonia resting cells of these strains release two mol of nitrite per mol of 2,6-dinitrophenol. Alcaligenes eutrophus JMP 222-1D, a mutant of strain JMP 222 obtained by transposon (Tn5) mutagenesis, is able to use 2,6-dinitrophenol as nitrogen source but not as source of carbon and energy. Resting cells of this mutant liberate only one mol of nitrite per mol of 2,6-dinitrophenol. A single metabolite was detected by high-pressure liquid chromatography and identified as 2-hydroxy-5-nitropenta-2,4-dienoic acid from the mass spectrum, the ¹H-, and ¹³C-NMR spectra. Strain JMP 222-1S, a spontaneous mutant of strain JMP 222-1D, accumulates 4-nitropyrogallol which was identified as the initial metabolite of 2,6-dinitrophenol degradation.Non-standard abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,6-DNP 2,6-dinitrophenol - HNMA 2-hydroxy-5-nitromuconic acid - HNPA 2-hydroxy-5-nitropenta-2,4-dienoic acid - NB nutrient broth - NMR nuclear magnetic resonance - NPG 4-nitropyrogallol - O.D. optical density - t_R retention time - UV/Vis ultraviolet/visible     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Metabolic integration in locust flight: the effect of octopamine on fructose 2,6-bisphosphate content of flight muscle in vivo

The biogenic amine octopamine was injected into the haemolymph of 20-days old male locusts,Locusta migratoria, and the content of fructose 2,6-bisphosphate, a potent activator of glycolysis, was measured in the flight muscle after various time. Octopamine brought about a transient increase in fructose 2,6-bisphosphate. After the injection of 10 l of 10 mmol·l^-1 d, l-octopamine fructose 2,6-bisphosphate was increased by 61% within 2 min. Ten minutes after the injection fructose 2,6-bisphosphate was increased to 6.71±0.89 nmol·g^-1 flight muscle, almost 300% over the control value. Flight caused fructose 2,6-bisphosphate in flight muscle to decrease, but this decrease was counteracted by octopamine injected into the haemolymph of flying locusts. Octopamine and fructose 2,6-bisphosphate may act as signals to stimulate the oxidation of carbohydrate and to integrate muscle performance and metabolism. This mechanism appears particularly significant in the initial stage of flight when carbohydrates are the main fuel.Abbreviations F2,6P₂ fructose 2,6-bisphosphate - F6P fructose 6-phosphate - PFK1 6-phosphofructokinase (EC 2.7.1.11) - P _i inorganic phosphate - PP _i -PFK pyrophosphate dependent fructose 6-phosphate phosphotransferase (EC 2.7.1.90)     

Synthesis of (R)-2-phenylpropanoic acid from its racemate through an isomerase-involving reaction by Nocardia diaphanozonaria

(R)-2-Phenylpropanoic acid was synthesized from the racemic acid through an isomerization reaction involving resting cells of Nocardia diaphanozonaria JCM3208. The isomerization activity of the cells was enhanced 25-fold by adding 5.5 mM racemic 2-phenylpropanoic acid to the culture medium. When 5 mM racemic 2-phenylpropanoic acid was included in the reaction mixture (4 ml) containing resting cells (100 mg dry cell wt) in 25 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0) at 30 °C for 8 h, 4.56 mM (R)-2-phenylpropanoic acid (95.8% e.e.) was formed with a 91% molar conversion yield.     

代谢工程改造大肠杆菌合成己二酸

己二酸是一种具有重要应用价值的二元羧酸,是合成尼龙-66的关键前体。目前,生物法生产己二酸存在生产周期长、生产效率低的问题。本研究选择一株野生型高产琥珀酸菌株大肠杆菌(Escherichia coli) FMME N-2为底盘细胞,首先通过引入逆己二酸降解途径的关键酶,成功构建了可合成0.34 g/L己二酸的E. coli JL00菌株;接着,对合成路径限速酶进行表达优化,使E. coli JL01菌株在摇瓶发酵条件下产量达到0.87 g/L;随后,通过敲除sucD基因、过表达acs基因和突变lpd基因的组合策略平衡己二酸合成前体的供应,优化菌株E. coli JL12己二酸产量进一步提升至1.51 g/L;最后,在5 L发酵罐上对己二酸发酵工艺进行优化。工程菌株经72 h分批补料发酵,己二酸的产量达到22.3 g/L,转化率为0.25 g/g,生产强度为0.31 g/(L·h),具备了一定的应用潜力。本研究可为包括己二酸在内的多种二元羧酸细胞工厂的构建提供理论依据和技术基础。     

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO₂ using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO₂ to carboxylic acids.     

Promotion of Flowering in Sagittaria pygmaea Miq. by 2,6-Diisopropylphenoxyacetic Acid

The flowering of Sagittaria pygmaea Miq. was promoted by 2,6-diisopropylphenoxyacetic acid, as well as by gibberellic acid (GA₃). Uniconazole canceled the promotive effect of the phenoxy-acetic acid, while prohexadione shortened the period required for flowering. Endogenous GAs seem to play an important role in the flowering of S. pygmaea, and 2,6-diisopropylphenoxyacetic acid might affect GA biosynthesis or metabolism.     

Syntheses and crystal structures of dimethyltin (IV) derivatives with 2,6-pyridinedicarboxylic acid

A novel cyclic dimethyltin complex [Me₂Sn(2,6-pdc)]₃ (1) (2,6-pdc = 2,6-pyridinedicarboxylate) was synthesized by the reaction of dimethyltin (IV) dichloride and 2,6-pyridinedicarboxylate acid in methanol under solvothermal conditions (150 °C). However, under room temperature (25 °C), we obtained a ladder complex [Me₂Sn(2,6-pdc)]₂(MeOH)₂ (2). Characterization of complexes 1 and 2 was achieved using elemental analysis, IR, ¹H, ¹³C and ¹¹⁹Sn NMR spectra and X-ray diffraction. X-ray data of 1 revealed that it was an unusual cyclic complex with a discrete cyclotrinuclear unit, in which the 12-membered cyclic cavity is almost completely planar. X-ray data of 2 showed that it was a ladder complex, in which a crystallizing methanol molecule is found in each formula unit.     

Production of acetic acid by Clostridium thermoaceticum in electrodialysis culture using a fermenter equipped with an electrodialyser

Electrodialysis culture of Clostridium thermoaceticum increased the yield of acetate by its continuous removal. In normal batch cultures without pH control the yield was 4.2 g acetic acid/800 ml, while in pH-controlled culture it was 16.8 g/800 ml. Although electrodialysis cultures gave almost the same yield (15.4 g/800 ml) as that in pH-controlled cultures, sparging CO₂ into the broth in electrodialysis culture increased the amount of acetic acid to 22.3 g/800 ml. CO₂ sparging into normal cultures with or without pH control did not significantly increase the amount of acetate produced but yields, in terms of amounts of glucose consumed, were higher than without sparging. The theoretical yield was almost obtained in pH-controlled, electrodialysis cultures with CO₂ sparging.The authors are with the Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, Japan