The antifungal mechanisms of plant volatile compound 1-octanol against Aspergillus flavus growth.

The exploitation of active ingredients from plant volatile organic compounds as natural gaseous fungicides shows remarkable potential for controlling fungal decay in postharvest agroproducts. Although 1-octanol is a common component of cereal volatiles, its antifungal potency against spoilage fungi in postharvest grains remains unclear. In this study, we studied the effectiveness of 1-octanol against Aspergillus flavus growth in postharvest grains and its mechanisms of action. 1-Octanol vapor and liquid contact dose-dependently inhibited A. flavus spore germination and mycelial growth at a low concentration. The simulated storage experiment demonstrated that 300 µL/L of 1-octanol vapor completely controlled A. flavus growth in wheat, corn, and paddy grains with 20% moisture content. 1-Octanol treatment irreversibly damaged the conidial and mycelial morphology of A. flavus and caused electrolyte leakage due to reduced plasma membrane integrity. It induced apoptosis along with morphological abnormalities, phosphatidylserine externalization, mitochondrial membrane potential depolarization, intracellular reactive oxygen species accumulation, and DNA fragmentation in A. flavus cells. Metabolomic analysis revealed that 1-octanol treatment disrupted the biosynthesis of unsaturated fatty acids, ATP-binding cassette transporters, amino acid metabolism, and glycerophospholipid metabolism. This study demonstrated the promising application potential of 1-octanol as a biofumigant for preventing fungal spoilage of postharvest cereal grains. KEY POINTS: • (1) 1-Octanol inhibits Aspergillus flavus growth in the vapor phase and liquid contact; • (2) 1-Octanol damages membrane integrity and induces apoptosis of A. flavus; • (3) Metabolomic changes in A. flavus mycelia were analyzed after 1-octanol treatment.

Improved Bioproduction of 1-Octanol Using Engineered Synechocystis sp. PCC 6803.

1-Octanol has gained interest as a chemical precursor for both high and low value commodities including fuel, solvents, surfactants, and fragrances. By harnessing the power from sunlight and CO2 as carbon source, cyanobacteria has recently been engineered for renewable production of 1-octanol. The productivity, however, remained low. In the present work, we report efforts to further improve the 1-octanol productivity. Different N-terminal truncations were evaluated on three thioesterases from different plant species, resulting in several candidate thioesterases with improved activity and selectivity toward octanoyl-ACP. The structure/function trials suggest that current knowledge and/or state-of-the art computational tools are insufficient to determine the most appropriate cleavage site for thioesterases in Synechocystis. Additionally, by tuning the inducer concentration and light intensity, we further improved the 1-octanol productivity, reaching up to 35% (w/w) carbon partitioning and a titer of 526 ± 5 mg/L 1-octanol in 12 days. Long-term cultivation experiments demonstrated that the improved strain can be stably maintained for at least 30 days and/or over ten times serial dilution. Surprisingly, the improved strain was genetically stable in contrast to earlier strains having lower productivity (and hence a reduced chance of reaching toxic product concentrations). Altogether, improved enzymes and environmental conditions (e.g., inducer concentration and light intensity) substantially increased the 1-octanol productivity. When cultured under continuous conditions, the bioproduction system reached an accumulative titer of >3.5 g/L 1-octanol over close to 180 days.

Production of 1-octanol in Escherichia coli by a high flux thioesterase route.

1-octanol is a valuable molecule in the chemical industry, where it is used as a plasticizer, as a precursor in the production of linear low-density polyethylene (LLDPE), and as a growth inhibitor of tobacco plant suckers. Due to the low availability of eight-carbon acyl chains in natural lipid feedstocks and the selectivity challenges in petrochemical routes to medium-chain fatty alcohols,1-octanol sells for the highest price among the fatty alcohol products. As an alternative, metabolic engineers have pursued sustainable 1-octanol production via engineered microbes. Here, we report demonstration of gram per liter titers in the model bacterium Escherichia coli via the development of a pathway composed of a thioesterase, an acyl-CoA synthetase, and an acyl-CoA reductase. In addition, the impact of deleting fermentative pathways was explored E. coli K12 MG1655 strain for production of octanoic acid, a key octanol precursor. In order to overcome metabolic flux barriers, bioprospecting experiments were performed to identify acyl-CoA synthetases with high activity towards octanoic acid and acyl-CoA reductases with high activity to produce 1-octanol from octanoyl-CoA. Titration of expression of key pathway enzymes was performed and a strain with the full pathway integrated on the chromosome was created. The final strain produced 1-octanol at 1.3 g/L titer and a >90% C8 specificity from glycerol. In addition to the metabolic engineering efforts, this work addressed some of the technical challenges that arise when quantifying 1-octanol produced from cultures grown under fully aerobic conditions where evaporation and stripping are prevalent.

Bioderivatization as a concept for renewable production of chemicals that are toxic or poorly soluble in the liquid phase.

Bio-based production technologies may complement or replace petroleum-based production of chemicals, but they face a number of technical challenges, including product toxicity and/or water insolubility. Plants and microorganisms naturally biosynthesize chemicals that often are converted into derivatives with reduced toxicity or enhanced solubility. Inspired by this principle, we propose a bioderivatization strategy for biotechnological chemicals production, defined as purposeful biochemical derivatization of intended target molecules. As proof of principle, the effects of hydrophobic (e.g., esterification) and hydrophilic (e.g., glycosylation) bioderivatization strategies on the biosynthesis of a relatively toxic and poorly soluble chemical, 1-octanol, were evaluated in Escherichia coli and Synechocystis sp. PCC 6803. The 1-octanol pathway was first optimized to reach product titers at which the host displayed symptoms of toxicity. Solvent overlay used to capture volatile products partially masked product toxicity. Regardless of whether solvent overlay was used, most strains with bioderivatization had a higher molar product titer and product yield, as well as improved cellular growth and glucose consumption, compared with strains without bioderivatization. The positive effect on bioproduction was observed with both the hydrophobic and hydrophilic strategies. Interestingly, in several combinations of genotype/induction strength, bioderivatization had a positive effect on productivity without any apparent effect on growth. We attribute this to enhanced product solubility in the aqueous or solvent fraction of the bioreactor liquid phase (depending on the derivative and medium used), with consequent enhanced product removal. Overall, under most conditions, a benefit of bioproduction was observed, and the bioderivatization strategy could be considered for other similar chemicals as well.

Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts.

BACKGROUND: The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. RESULTS: Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. CONCLUSIONS: This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.

1-Octanol, a self-inhibitor of spore germination in Penicillium camemberti.

Penicillium camemberti is a technologically relevant fungus used to manufacture mold-ripened cheeses. This fungal species produces many volatile organic compounds (VOCs) including ammonia, methyl-ketones, alcohols and esters. Although it is now well known that VOCs can act as signaling molecules, nothing is known about their involvement in P. camemberti lifecycle. In this study, spore germination was shown to be self-regulated by quorum sensing in P. camemberti. This phenomenon, also called "crowding effect", is population-dependent (i.e. observed at high population densities). After determining the volatile nature of the compounds involved in this process, 1-octanol was identified as the main compound produced at high-spore density using GC-MS. Its inhibitory effect was confirmed in vitro and 3 mM 1-octanol totally inhibited spore germination while 100 µM only transiently inhibited spore germination. This is the first time that self-inhibition of spore germination is demonstrated in P. camemberti. The obtained results provide interesting perspectives for better control of mold-ripened cheese processes.

H2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O2-tolerant hydrogenase and a P450 monooxygenase.

An in vivo biotransformation system is presented that affords the hydroxylation of n-octane to 1-octanol on the basis of NADH-dependent CYP153A monooxygenase and NAD(+)-reducing hydrogenase heterologously synthesized in a bacterial host. The hydrogenase sustains H2-driven NADH cofactor regeneration even in the presence of O2, the co-substrate of monooxygenase.

Indexes of polar interactions between ionizable drugs and membrane phospholipids measured by IAM-HPLC: their relationships with data of Blood-Brain Barrier passage.

Phospholipid affinity indexes logkw(IAM) for 21 structurally non-related basic, acidic, ampholytic, and neutral drugs were measured by HPLC on two different phospholipid stationary phases (Immobilized Artificial Membrane - IAM). The differences between the experimental values and those expected for neutral isolipophilic compounds Δ/Δ(')logkw(IAM) were assumed as a measure of polar and electrostatic forces involved in the interactions drug/membrane. Blood Brain Barrier (BBB) permeation data (logBB) weakly related with n-octanol lipophilicity values of the neutral forms (logP(N)), but only after the exclusion of two analytes. No relationship was observed with the lipophilicity of the mixture of charged and neutral forms (logD(7.4)) and very poor relationships were also found with logkw(IAM) values. In contrast, a good linear inverse relationship was found between log BB and Δ/Δ(')logkw(IAM) values. This relationship was also observed on an enlarged set of 42 data points obtained by assembling the drugs considered with 21 drugs examined in our previous works (acids and bases). The results suggest that Δ/Δ(')logkw(IAM) parameter, as a sole descriptor, is effective in evaluating the capability of a compound to cross the BBB, indicating that the polar/electrostatic interactions, which are the additional interactions described by IAM, are of prime importance in predicting BBB passage.

Odor-taste learning assays in Drosophila larvae.

The Drosophila larva is an emerging model for studies in behavioral neurogenetics because of its simplicity in terms of cell number. Despite this simplicity, basic features of neuronal organization and key behavior faculties are shared with adult flies and with mammals. Here, we describe a pavlovian-type learning assay in fruit fly larvae. A group of larvae is sequentially exposed to specific odors in the presence or the absence of sugar, and then tested to determine whether they prefer the odor previously experienced with the reward. The protocol uses a two-group, reciprocal training design: One group of Drosophila larvae is exposed to n-amyl acetate (AM) with a sugar reward (+), then subsequently exposed to 1-octanol (OCT) with no reward (denoted AM+/OCT). The other group receives the reciprocal training (AM/OCT+). The two groups of larvae are then tested for their choices between AM and OCT. Relatively higher preferences for AM after AM+/OCT training than after AM/OCT+ training reflect associative learning and are quantified by the learning index (LI). This method offers a robust, simple, cheap, and reasonably quick test for learning ability (an aversive version is available as well, using either high-concentration salt or quinine as punishment). With the concerted efforts of the Drosophila research community, we anticipate it will allow us to unravel the full circuitry underlying odor-taste learning on a single-cell level.

Engineered catalytic biofilms for continuous large scale production of n-octanol and (S)-styrene oxide.

This study evaluates the technical feasibility of biofilm-based biotransformations at an industrial scale by theoretically designing a process employing membrane fiber modules as being used in the chemical industry and compares the respective process parameters to classical stirred-tank studies. To our knowledge, catalytic biofilm processes for fine chemicals production have so far not been reported on a technical scale. As model reactions, we applied the previously studied asymmetric styrene epoxidation employing Pseudomonas sp. strain VLB120ΔC biofilms and the here-described selective alkane hydroxylation. Using the non-heme iron containing alkane hydroxylase system (AlkBGT) from P. putida Gpo1 in the recombinant P. putida PpS81 pBT10 biofilm, we were able to continuously produce 1-octanol from octane with a maximal productivity of 1.3 g L ⁻¹(aq) day⁻¹ in a single tube micro reactor. For a possible industrial application, a cylindrical membrane fiber module packed with 84,000 polypropylene fibers is proposed. Based on the here presented calculations, 59 membrane fiber modules (of 0.9 m diameter and 2 m length) would be feasible to realize a production process of 1,000 tons/year for styrene oxide. Moreover, the product yield on carbon can at least be doubled and over 400-fold less biomass waste would be generated compared to classical stirred-tank reactor processes. For the octanol process, instead, further intensification in biological activity and/or surface membrane enlargement is required to reach production scale. By taking into consideration challenges such as biomass growth control and maintaining a constant biological activity, this study shows that a biofilm process at an industrial scale for the production of fine chemicals is a sustainable alternative in terms of product yield and biomass waste production.

Complete degradation of di-n-octyl phthalate by Gordonia sp. strain Dop5.

The present study describes the assimilation of di-n-octyl phthalate by an aerobic bacterium, isolated from municipal waste-contaminated soil sample utilizing di-n-octyl phthalate as the sole source of carbon and energy. The isolate was identified as Gordonia sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic and spectrometric analyses revealed a complete di-n-octyl assimilation pathway. In the degradation process, mono-n-octyl phthalate, phthalic acid, protocatechuic acid and 1-octanol were identified as the degradation products of di-n-octyl phthalate. Furthermore, phthalic acid was metabolized via protocatechuic acid involving protocatechuate 3,4-dioxygenase while 1-octanol was metabolized by NAD(+)-dependent dehydrogenases to 1-octanoic acid, which was subsequently degraded via ß-oxidation, ultimately, leading to tricarboxylic acid cycle intermediates. Apart from phthalic acid and 1-octanol metabolizing pathway enzymes, two esterases, di-n-octyl phthalate hydrolase and mono-n-octyl phthalate hydrolase involved in di-n-octyl phthalate degradation were found to be inducible in nature. This is the first report on the metabolic pathway involved in the complete degradation of di-n-octyl phthalate by a single bacterial isolate, which is also capable of efficiently degrading other phthalate esters of environmental concern having either shorter or longer alkyl chains.

Characterization of three propane-inducible oxygenases in Mycobacterium sp. strain ENV421.

AIMS: Mycobacterium sp. strain ENV421 has the ability to cometabolize a variety of chemicals following growth on propane as a sole source of carbon and energy. In this study, we used genetic and biochemical approaches to identify and characterize multiple propane-inducible oxygenase genes in ENV421. METHODS AND RESULTS: Gene clusters encoding a CYP153-type cytochrome P450 oxygenase (P450), an AlkB-type alkane monooxygenase (AlkB) and a soluble diiron monooxygenase were identified and cloned using degenerate PCR primers. Reverse transcriptase PCR showed that all three gene clusters were induced by propane. Substrate specificity studies revealed that despite the fact that ENV421 does not grow on medium length alkanes, cloned versions of both the AlkB and P450 were capable of octane oxidation, forming n-octanol. Additionally, the P450 oxygenase had the ability to oxidize indole, medium-to-long-chain alkylbenzenes and a variety of para-substituted methylalkylbenzenes. Successful cloning and expression of the diiron monooxygenase was not achieved, so its substrate specificity could not be determined. CONCLUSIONS: Three types of short-to-medium-chain alkane oxygenases were induced by propane in ENV421, even though the cloned AlkB and P450 oxygenases did not oxidize propane. Curiously, they both oxidized octane, which is not a growth substrate for ENV421. Furthermore, the P450, typically operating as terminal alkane hydroxylase, exhibited interesting regio- and stereoselectivity, catalysing linear alkanes, alkylbenzenes and indole. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first example of a propane-inducible P450 with a broad substrate specificity, including linear alkanes, alkylbenzenes and a multiring compound. The induction of three distinct oxygenase classes by propane is also an interesting finding because it might explain why propane serves as an effective stimulant that promotes the biodegradation of a various environmental contaminants.

Efficient synthesis of octyl-ß-d-galactopyranoside by Bacillus spore-displayed ß-galactosidase using an amphiphilic 1,2-dimethoxyethane co-solvent.

For enzymatic synthesis of octyl-ß-d-galactopyranoside (octyl-gal) from lactose and n-octanol, Escherichia coli ß-galactosidase (ß-Gal) was expressed and displayed on the surfaces of Bacillus subtilis spores. The spore-displayed ß-Gal was found to be stable when an amphiphilic 1,2-dimethoxyethane (DME) was used as a co-solvent; the transgalactosylation efficiency and octyl-gal conversion were optimal at 50% (v/v) DME. In addition, the product was maximally obtained from 100mM lactose in a phosphate buffer/n-octanol/DME (25/25/50, v/v) mixture. By increasing the agitation speed and the amount of spores displaying ß-Gal, a yield of 33.7 mM octyl-gal was obtained over 24h in a batch mode, which is much higher than in other octyl-gal bioconversion processes, such as those involving lipid-coating, reverse micelles, or whole cells. On the other hand, intermittent addition of spore-displayed ß-Gal and/or lactose in the reaction medium had no effect on the octyl-gal yield. The synthesized octyl-gal was hydrolyzed by the spore-displayed ß-Gal, and a high concentration of octyl-gal competitively inhibited the enzymes (K(i) value of 10.8mM). In summary, we demonstrate that octyl-gal synthesis by spore-displayed ß-Gal in non-aqueous medium can be significantly improved with the use of DME as a co-solvent.

Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli.

The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C(5)-C(12) alkanes to primary alcohols both in the wild-type organism by growth on C(5)-C(12) alkanes as sole carbon source and in vitro. Despite this, successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme for the production of primary alcohols and carboxylic acids is feasible and in fact potentially more promising than n-octane oxidation due to lower product and substrate toxicity. Yields are reported of 1-dodecanol of up to 2 g/L(organic) and dodecanoic acid up to 19.7 g/L(organic) in a 2 L stirred tank reactor with 1L aqueous phase and 200 mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9 g/L(organic)/h (0.35 g/L(total)/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5 µmol/min/g(dcw)) than on n-octane (21 µmol/min/g(dcw)); similar to the 5-fold difference observed between wild-type growth rates using the two respective alkanes as sole carbon source. Despite this, both total volumetric rate and final yield exceeded n-octane oxidation by 3.5-fold under the same conditions, due to the lower toxicity of n-dodecane and its oxidation products to E. coli compared to the 8-carbon equivalents. Substrate access limitations and the overoxidation of 1-dodecanol to dodecanoic acid were identified as the most important limitations to be addressed.

Magnaporthe oryzae cell wall hydrolysate induces ROS and fungistatic VOCs in rice cell cultures.

Plants react to microbial attack with a number of defense mechanisms, including the synthesis of volatile organic compounds (VOCs) and the production of reactive oxygen species (ROS). These responses are triggered by elicitors derived from either the cell surface of pathogens or the incomplete hydrolysis of the plant cell wall. Here we show the response of rice (Oryza sativa L., cv Gigante Vercelli) cell cultures following treatment with cell wall hydrolysates prepared from the rice blast Magnaporthe oryzae. Elicitation prompted the production of several plant VOCs, which were analyzed by stir bar sorptive extraction from both the liquid and head-space phase (SBSE and HSSE, respectively) and gas chromatography coupled to mass spectrometry (GC-MS) analysis. VOCs included alkanes, alkenes and long-chain alcohols as well as cinnamyl alcohol, myristicin, a sesquiterpene alcohol (caryolan-1-ol), 1-butanamide and 2-pentylfuran. The major released compounds, 1-octanol and 1-decanol, were found to induce ROS production in both elicited and non-elicited rice cells and showed fungistatic activity against the pathogen M. oryzae. The possible role of induced VOCs and ROS production in the plant-pathogen interaction is discussed.

Influence of pyridine versus piperidine ligands on the chemical, DNA binding and cytotoxic properties of light activated trans,trans,trans-[Pt(N3)2(OH)2(NH3)(L)].

The photocytotoxicity and photobiochemical properties of the new complex trans,trans,trans-[Pt(N(3))(2)(OH)(2)(NH(3))(piperidine)] (5) are compared with its analogue containing the less basic and less lipophilic ligand pyridine (4). The log P (n-octanol/water) values were of -1.16 and -1.84 for the piperidine and pyridine complexes, respectively, confirmed that piperidine increases the hydrophobicity of the complex. Density Functional Theory (DFT) and time-dependent density functional theory (TDDFT) calculations indicate that 5 has accessible singlet and triplet states which can promote ligand dissociation when populated by both UVA and visible white light. When activated by UVA or white light, both compounds showed similar cytotoxic potencies in various human cancer cell lines although their selectivity was different. The time needed to reach similar antiproliferative activity was noticeably decreased by introducing the piperidine ligand. Neither compound showed cross-resistance in three oxoplatin-resistant cell lines. Furthermore, both compounds showed similar anticlonogenic activity when activated by UVA radiation. Interactions of the light-activated complexes with DNA showed similar kinetics and levels of DNA platination and similar levels of DNA interstrand cross-linking (ca. 5%). Also the ability to unwind double stranded DNA were comparable for the piperidine analogue (24°, respectively), while the piperidine complex showed higher potency in changing the conformation of DNA, as measured in an ethidium bromide binding assay. These results indicate that the nature of the heterocyclic nitrogen ligand can have subtle influences on both the phototoxicity and photobiochemistry of this class of photochemotherapeutic agents.

A transmembrane amino acid in the GABAA receptor ß2 subunit critical for the actions of alcohols and anesthetics.

Alcohols and inhaled anesthetics enhance the function of GABA(A) receptors containing α, ß, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the ß subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA(A) receptor ß(2) subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant α(1)ß(2)(N265C)γ(2S) receptors [but not α(1)ß(2)(I264C)γ(2S)], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the ß(2) subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant α(1)ß(2)(N265C)γ(2S) receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.

Heterologous expression of the alcohol dehydrogenase (adhI) gene from Geobacillus thermoglucosidasius strain M10EXG.

A thermostable alcohol dehydrogenase (ADH-I) isolated from the potential thermophilic ethanologen Geobacillus thermoglucosidasius strain M10EXG has been characterised. Inverse PCR showed that the gene (adhI) was localised with 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3 hexuloisomerase (PHI) on its genome. The deduced peptide sequence of the 1020-bp M10EXG adhI, which corresponds to 340 amino acids, shows 96% and 89% similarity to ADH-hT and ADH-T from Geobacillus stearothermophilus strains LLD-R and NCA 1503, respectively. Over-expression of M10EXG ADH-I in Escherichia coli DH5alpha (pNF303) was confirmed using an ADH activity assay and SDS-PAGE analysis. The specific ADH activity in the extract from this recombinant strain was 9.7(+/-0.3) U mg(-1) protein, compared to 0.1(+/-0.01) U mg(-1) protein in the control strain. The recombinant E. coli showed enzymatic activity towards ethanol, 1-butanol, 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol and 2-propanol, but not methanol. In silico analysis, including phylogenetic reconstruction and protein modeling, confirmed that the thermostable enzyme from G. thermoglucosidasius is likely to belong to the NAD-Zn-dependent family of alcohol dehydrogenases.

Activated sludge inhibition by chemical stressors--a comprehensive study.

The effects of shock loads of 1-chloro-2,4-dinitrobenzene (CDNB); cadmium; 1-octanol; 2,4-dinitrophenol (DNP); weakly complexed cyanide; pH 5, 9, and 11; and high ammonia levels on activated sludge biomass growth, respiration rate, flocculation, chemical oxygen demand removal, dewaterability, and settleability were studied. For all chemical shocks, except ammonia and pH, concentrations that caused 15, 25, and 50% respiration inhibition were used to provide a single pulse shock to sequencing batch reactor systems containing a nitrifying or non-nitrifying biomass. Cadmium and pH 11 shocks were most detrimental to all processes, followed by CDNB. The DNP and cyanide primarily affected respiration, while pH 5, pH 9, octanol, and ammonia did not affect the treatment process to a significant extent. A chemical source-process effect matrix is provided, which we believe will aid in the development of methods that prevent and/or attenuate the effects of toxic shock loads on activated sludge systems.

Solution equilibria of copper(II) complexation with N,N'-(2,2'-azanediylbis(ethane-2,1-diyl))dipicolinamide: a bio-distribution and dermal absorption study.

The protonation equilibria of a pentadentate ligand, N,N'-(2,2'-azanediylbis(ethane-2,1-diyl))dipicolinamide ([H(2)(5555)-N]) and the complexation of this ligand with Cu(II) Ca(II), Zn(II) and Ni(II) have been studied by pH-potentiometry, (1)H NMR spectroscopy and UV-vis spectrophotometry. (1)H NMR detected the protonation of the pyridyl groups and formation of Cu[H(2)(5555)-N]H species at low pH, while amide group deprotonation at higher pH resulted in the formation of Cu[H(2)(5555)-N]H(-1) and Cu[H(2)(5555)-N]H(-2) species in solution. Potentiometric detection of protonated species was limited by the acidic nature of the pyridyl nitrogen donors. From UV-vis spectroscopy it is suggested that the amide nitrogens are coordinated. This conclusion is supported by Molecular Mechanics calculations. Water-octanol partition coefficients for the Cu(II)-[H(2)(5555)-N] system indicated that although the Cu[H(2)(5555)-N]H(-1) species is largely hydrophilic, approximately 54% of the complex goes into the organic phase. This percentage is able to promote dermal absorption of copper with a calculated penetration rate of 1.92x10(-1)cmh(-1). This was confirmed by dermal absorption studies which illustrate the role of hydrophobicity in promoting percutaneous drug administration.