Investigation of structural effects and behaviour of pseudomonas aeruginosa amidase encapsulated in reserved micelles

The acetohydroxamic acid synthesis reaction was studied using whole cells, cell-free extract and purified amidase from the strains of Pseudomonas aeruginosa L10 and A13 entrapped in a reverse micelles system composed of cationic surfactant tetradecyltrimethyl ammonium bromide. The specific activity of amidase, yield of synthesis and storage stability were determined for the reversed micellar system as well as for free amidase in conventional buffer medium. The results have revealed that amidase solutions in the reverse micelles system exhibited a substantial increase in specific activity, yield of synthesis and storage stability. In fact, whole cells from P. aeruginosa L10 and AI3 in reverse micellar medium revealed an increase in specific activity of 9.3- and 13.9-fold, respectively, relatively to the buffer medium. Yields of approximately 92% and 66% of acetohydroxamic acid synthesis were obtained for encapsulated cell free extract from P. aeruginosa L10 and A13, respectively. On the other hand, the half-life values obtained for the amidase solutions encapsulated in reverse micelles were overall higher than that obtained for the free amidase solution in buffer medium. Half-life values obtained for encapsulated purified amidase from P. aeruginosa strain L10 and encapsulated cell-free extract from P. aeruginosa strain AI3 were of 17.0 and 26.0 days, respectively. As far as the different sources biocatalyst are concerned, the data presented in this work has revealed that the best results, in both storage stability and biocatalytic efficiency, were obtained when encapsulated cell-free extract from P. aeruginosa strain AI3 at 14/0 of 10 were used. Conformational changes occurring upon encapsulation of both strains enzymes in reverse micelles of TAB in heptane/octanol were additionally identified by FTIR spectroscopy which clarified the biocatalysts performances.

Substrate interaction with recombinant amidase from Pseudomonas aeruginosa during biocatalysis

The interaction of a variety of substrates with Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain, was investigated using difference FTIR spectroscopy. The amides used as substrates showed an increase in hydrogen bonding upon association in multimers, which was not seen with esters. Evidence for an overall reduction or weakening of hydrogen bonding while amide and ester substrates are interacting with the enzyme is presented. The results describe a spectroscopic approach for analysis of substrate-amidase interaction and in situ monitoring of the hydrolysis and transferase reaction when amides or esters are used as substrates.

Inhibition of Aspergillus fumigatus and its biofilm by Pseudomonas aeruginosa is dependent on the source, phenotype and growth conditions of the bacterium

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

Pseudomonas aeruginosa amidase: Aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonasaeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of solubleamidase, relatively to other proteins, was obtained in the condition at 37 degrees C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all tested growth conditions. Addition of ethanol at 25 degrees C in the culture medium improved amidase yield, which quantitatively aggregated in a biologically active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37 degrees C by Furier transform infrared spectroscopy (FTIR) analysis demonstrated a lower content of intermolecular interactions, which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.

Environmental and track perturbations on multiple pantograph interaction with catenaries in high-speed trains

The top velocity of high-speed trains is generally limited by the ability to supply the proper amount of energy through the pantograph-catenary interface. The deterioration of this interaction can lead to the loss of contact, which interrupts the energy supply and originates arcing between the pantograph and the catenary, or to excessive contact forces that promote wear between the contacting elements. Another important issue is assessing on how the front pantograph influences the dynamic performance of the rear one in trainsets with two pantographs. In this work, the track and environmental conditions influence on the pantograph-catenary is addressed, with particular emphasis in the multiple pantograph operations. These studies are performed for high speed trains running at 300 km/h with relation to the separation between pantographs. Such studies contribute to identify the service conditions and the external factors influencing the contact quality on the overhead system. (C) 2013 Elsevier Ltd. All rights reserved.

Surface morphology and phase transformations of femtosecond laser-processed saphire

The morphological and structural modifications induced in sapphire by surface treatment with femtosecond laser radiation were studied. Single-crystal sapphire wafers cut parallel to the (0 1 2) planes were treated with 560 fs, 1030 nm wavelength laser radiation using wide ranges of pulse energy and repetition rate. Self-ordered periodic structures with an average spatial periodicity of similar to 300 nm were observed for fluences slightly higher than the ablation threshold. For higher fluences the interaction was more disruptive and extensive fracture, exfoliation, and ejection of ablation debris occurred. Four types of particles were found in the ablation debris: (a) spherical nanoparticles about 50 nm in diameter; (b) composite particles between 150 and 400 nm in size; (c) rounded resolidified particles about 100-500 nm in size; and (d) angular particles presenting a lamellar structure and deformation twins. The study of those particles by selected area electron diffraction showed that the spherical nanoparticles and the composite particles are amorphous, while the resolidified droplets and the angular particles, present a crystalline a-alumina structure, the same of the original material. Taking into consideration the existing ablation theories, it is proposed that the spherical nanoparticles are directly emitted from the surface in the ablation plume, while resolidified droplets are emitted as a result of the ablation process, in the liquid phase, in the low intensity regime, and by exfoliation, in the high intensity regime. Nanoparticle clusters are formed by nanoparticle coalescence in the cooling ablation plume. (C) 2013 Elsevier B.V. All rights reserved.

Production of hydroxamic acids by immobilized Pseudomonas aeruginosa cells Kinetic analysis in reverse micelles

Intact cells from Pseudomonas aeruginosa strain L10 containing amidase were used as biocatalysts both free and immobilized in a reverse micellar system. The apparent kinetic constants for the transamidation reaction in hydroxamic acids synthesis, were determined using substrates such as aliphatic, amino acid and aromatic amides and esters, in both media. In reverse micelles, K-m values decreased 2-7 fold relatively to the free biocatalyst using as substrates acetamide, acrylamide, propionamide and glycinamide ethyl ester. We have concluded that overall the affinity of the biocatalyst to each substrate increases when reactions are performed in the reversed micellar system as opposed to the buffer system. The immobilized biocatalyst in general, exhibits higher stability and faster rates of reactions at lower substrates concentration relatively to the free form, which is advantageous. Additionally, the immobilization revealed to be suitable for obtaining the highest yields of hydroxamic acids derivatives, in some cases higher than 80%. (C) 2013 Elsevier B.V. All rights reserved.

The effect of metal transfer stability (spattering) on fume generation, morphology and composition in short-circuit MAG welding

The main objective of this work was to evaluate the hypothesis that the greater transfer stability leads also to less volume of fumes. Using an Ar + 25%CO2 blend as shielding gas and maintaining constant the average current, wire feed speed and welding speed, bead-on-plate welds were carried out with plain carbon steel solid wire. The welding voltage was scanned to progressively vary the transfer stability. Using two conditions of low stability and one with high stability, fume generation was evaluated by means of the AWS F1.2:2006 standard. The influence of these conditions on fume morphology and composition was also verified. A condition with greater transfer stability does not generate less fume quantity, despite the fact that this condition produces fewer spatters. Other factors such as short-circuit current, arcing time, droplet diameters and arc length are the likely governing factors, but in an interrelated way. Metal transfer stability does not influence either the composition or the size/morphology of fume particulates. (c) 2014 Elsevier B.V. All rights reserved.

Fresh-state properties of self-compacting mortar and concrete with combined use of limestone filler and fly ash

This paper presents the results of a study on the behaviour of self-compacting concrete (SCC) in the fresh and hardened states, produced with binary and ternary mixes of fly ash (FA) and limestone filler (LF), using the method proposed by Nepomuceno. His method determines the SCC composition parameters in the mortar phase (self-compacting mortar - SCM) easily and efficiently, whilst guaranteeing the SCC properties in both the fresh and hardened states. For this, 11 SCMs were studied: one with cement (C) only; three with FA at 30%, 60% and 70% C substitution; three with LF at 30%, 60% and 70% C substitution; four with FA + LF in combinations of 10-20%, 20-10%, 20-40% and 40-20% C substitution. Once the composition of these mortars was defined, 18 SCC mixes were produced: 14 binary SCC mixes were produced with the seven binary mortar mixes, and four ternary SCC mixes were produced with the four ternary mortar mixes. In addition to the methodology proposed by Nepomuceno, the combined use of FA and LF in ternary mixtures was tested. The results confirmed that the method could yield SCC with adequate properties in both the fresh and hardened states. It was also possible to determine the SCC composition parameters in the mortar phase (self-compacting mortar - SCM) that will guarantee the SCC properties in both the fresh and hardened states, as confirmed through the optimized behaviour of the SCC in the fresh state and the promising results in the hardened state (compressive strength). The potential demonstrated by the joint use of LF and FA through the synergetic interaction of both additions is emphasized.