Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding a-L-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0-5.0) and an optimum temperature of 70 degrees C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 degrees C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic. (C) 2012 Elsevier Ltd. All rights reserved.

Immobilization of a recombinant endo-1,5-arabinanase secreted by Aspergillus nidulans strain A773

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 degrees C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 degrees C. The half-life of glyoxyl 4% derivative at 60 degrees C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose. (C) 2012 Elsevier B.V. All rights reserved.

Tailoring Molecular Architectures with Cobalt Tetrasulfonated Phthalocyanine: Immobilization in Layer-by-Layer Films and Sensing Applications

In the field of organic thin films, manipulation at the nanoscale can be obtained by immobilization of different materials on platforms designed to enhance a specific property via the layer-by-layer technique. In this paper we describe the fabrication of nanostructured films containing cobalt tetrasulfonated phthalocyanine (CoTsPc) obtained through the layer-by-layer architecture and assembled with linear poly(allylamine hydrochloride) (PAH) and poly(amidoamine) dendrimer (PAMAM) polyelectrolytes. Film growth was monitored by UV-vis spectroscopy following the Q band of CoTsPc and revealed a linear growth for both systems. Fourier transform infrared (FTIR) spectroscopy showed that the driving force keeping the structure of the films was achieved upon interactions of CoTsPc sulfonic groups with protonated amine groups present in the positive polyelectrolyte. A comprehensive SPR investigation on film growth reproduced the deposition process dynamically and provided an estimation of the thicknesses of the layers. Both FTIR and SPR techniques suggested a preferential orientation of the Pc ring parallel to the substrate. The electrical conductivity of the PAH films deposited on interdigitated electrodes was found to be very sensitive to water vapor. These results point to the development of a phthalocyanine-based humidity sensor obtained from a simple thin film deposition technique, whose ability to tailor molecular organization was crucial to achieve high sensitivity.

Galactomannan thin films as supports for the immobilization of Concanavalin A and/or dengue viruses

The immobilization of the glucose/mannose-binding lectin from Concanavalia ensiformis seeds (ConA) onto a monolayer made of a galactomannan extracted from Leucaena leucocephala seeds (GML), which was adsorbed onto - amino-terminated surfaces, was investigated by means of ellipsometry and atomic force microscopy. The mean thickness of GML monolayer, which polysaccharide consists of linear 1 -> 4-linked beta-D-mannopyranosil units partially substituted at C-6 by alpha-D-galactopyranosyl units, amounted to (1.5 +/- 0.2) nm. ConA molecules adsorbed onto GML surfaces forming (2.0 +/- 0.5) nm thick layers. However, in the presence of mannose the adsorption failed, indicating that ConA binding sites were blocked by mannose and were no longer available for mannose units present in the GML backbone. The GML film was also used as support for the adsorption of three serotypes of dengue virus particles (DENV-1, DENV-2 and DENV-3), where DENV-2 formed the thickest film (4 +/- 2) nm. The adsorbed layer of DENV-2 onto ConA-covered GML surfaces presented mean thickness values similar to that determined for DENV-2 onto bare GML surfaces. The addition of free mannose units prevented DENV-2 adsorption onto ConA-covered GML films by similar to 50%, suggesting competition between virus and mannose for ConA binding sites. This finding suggests that if ConA is also adsorbed to GML surface and its binding site is blocked by free mannose, virus particles are able to recognized GML mannose unities substituted by galactose. interactions between polysaccharides thin films, proteins, and viruses are of great relevance since they can provide basis for the development of biotechnological devices. These results indicate that GML is a potential polysaccharide for biomaterials development, as those could involve interactions between ConA in immune system and viruses. (C) 2011 Elsevier B.V. All rights reserved.

Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy

The research interest of this study is to investigate surface immobilization strategies for proteins and other biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique. The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with other functional molecules make this protein a prime candidate for supramolecular architectures. The recrystallization behavior on gold or on the secondary cell wall polymer (SCWP) was recorded by SPR. The optical thicknesses and surface densities for different protein layers were calculated. In DNA hybridization tests performed in order to discriminate different mismatches, recombinant S-layer-streptavidin fusion protein matrices showed their potential for new microarrays. Moreover, SCWPs coated gold chips, covered with a controlled and oriented assembly of S-layer fusion proteins, represent an even more sensitive fluorescence testing platform. Additionally, S-layer fusion proteins as the matrix for LHCII immobilization strongly demonstrate superiority over routine approaches, proving the possibility of utilizing them as a new strategy for biomolecular coupling. In the study of the SPFS hCG immunoassay, the biophysical and immunological characteristics of this glycoprotein hormone were presented first. After the investigation of the effect of the biotin thiol dilution on the coupling efficiently, the interfacial binding model including the appropriate binary SAM structure and the versatile streptavidin-biotin interaction was chosen as the basic supramolecular architecture for the fabrication of a SPFS-based immunoassay. Next, the affinity characteristics between different antibodies and hCG were measured via an equilibrium binding analysis, which is the first example for the titration of such a high affinity interaction by SPFS. The results agree very well with the constants derived from the literature. Finally, a sandwich assay and a competitive assay were selected as templates for SPFS-based hCG detection, and an excellent LOD of 0.15 mIU/ml was attained via the “one step” sandwich method. Such high sensitivity not only fulfills clinical requirements, but is also better than most other biosensors. Fully understanding how LHCII complexes transfer the sunlight energy directionally and efficiently to the reaction center is potentially useful for constructing biomimetic devices as solar cells. After the introduction of the structural and the spectroscopic features of LHCII, different surface immobilization strategies of LHCII were summarized next. Among them the strategy based on the His-tag and the immobilized metal (ion) affinity chromatography (IMAC) technique were of great interest and resulted in different kinds of home-fabricated His-tag chelating chips. Their substantial protein coupling capacity, maintenance of high biological activity and a remarkably repeatable binding ability on the same chip after regeneration was demonstrated. Moreover, different parameters related to the stability of surface coupled reconstituted complexes, including sucrose, detergent, lipid, oligomerization, temperature and circulation rate, were evaluated in order to standardize the most effective immobilization conditions. In addition, partial lipid bilayers obtained from LHCII contained proteo-liposomes fusion on the surface were observed by the QCM technique. Finally, the inter-complex energy transfer between neighboring LHCIIs on a gold protected silver surface by excitation with a blue laser (λ = 473nm) was recorded for the first time, and the factors influencing the energy transfer efficiency were evaluated.

High frequency acoustics in colloid-based meso- and nanostructures by spontaneous Brillouin light scattering

Materials that can mold the flow of elastic waves of certain energy in certain directions are called phononic materials. The present thesis deals essentially with such phononic systems, which are structured in the mesoscale (<1 µm), and with their individual components. Such systems show interesting phononic properties in the hypersonic region, i.e., at frequencies in the GHz range. It is shown that colloidal systems are excellent model systems for the realization of such phononic materials. Therefore, different structures and particle architectures are investigated by Brillouin light scattering, the inelastic scattering of light by phonons.rnThe experimental part of this work is divided into three chapters: Chapter 4 is concerned with the localized mechanical waves in the individual spherical colloidal particles, i.e., with their resonance- or eigenvibrations. The investigation of these vibrations with regard to the environment of the particles, their chemical composition, and the influence of temperature on nanoscopically structured colloids allows novel insights into the physical properties of colloids at small length scales. Furthermore, some general questions concerning light scattering on such systems, in dispute so far, are convincingly addressed.rnChapter 5 is a study of the traveling of mechanical waves in colloidal systems, consisting of ordered and disordered colloids in liquid or elastic matrix. Such systems show acoustic band gaps, which can be explained geometrically (Bragg gap) or by the interaction of the acoustic band with the eigenvibrations of the individual spheres (hybridization gap).rnWhile the latter has no analogue in photonics, the presence of strong phonon scatterers, when a large elastic mismatch between the composite components exists, can largely impact phonon propagation in analogy to strong multiple light scattering systems. The former is exemplified in silica based phononic structures that opens the door to new ways of sound propagation manipulation.rnChapter 6 describes the first measurement of the elastic moduli in newly fabricated by physical vapor deposition so-called ‘stable organic glasses’. rnIn brief, this thesis explores novel phenomena in colloid-based hypersonic phononic structures, utilizing a versatile microfabrication technique along with different colloid architectures provided by material science, and applying a non-destructive optical experimental tool to record dispersion diagrams.rn

Immobilization and characterization of recombinant esterase enzyme on chitosan nanoparticles

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).

Goal-directed colloid administration improves the microcirculation of healthy and perianastomotic colon

BACKGROUND: The aim of this study was to compare the effects of goal-directed colloid fluid therapy with goal-directed crystalloid and restricted crystalloid fluid therapy on healthy and perianastomotic colon tissue in a pig model of colon anastomosis surgery. METHODS: Pigs (n = 27, 9 per group) were anesthetized and mechanically ventilated. A hand-sewn colon anastomosis was performed. The animals were subsequently randomized to one of the following treatments: R-RL group, 3 ml x kg(-1) x h(-1) Ringer lactate (RL); GD-RL group, 3 ml x kg(-1) x h(-1) RL + bolus 250 ml of RL; GD-C group, 3 ml x kg(-1) x h(-1) RL + bolus 250 ml of hydroxyethyl starch (HES 6%, 130/0.4). A fluid bolus was administered when mixed venous oxygen saturation dropped below 60%. Intestinal tissue oxygen tension and microcirculatory blood flow were measured continuously. RESULTS: After 4 h of treatment, tissue oxygen tension in healthy colon increased to 150 +/- 31% in group GD-C versus 123 +/- 40% in group GD-RL versus 94 +/- 23% in group R-RL (percent of postoperative baseline values, mean +/- SD; P < 0.01). Similarly perianastomotic tissue oxygen tension increased to 245 +/- 93% in the GD-C group versus 147 +/- 58% in the GD-RL group and 116 +/- 22% in the R-RL group (P < 0.01). Microcirculatory flow was higher in group GD-C in healthy colon. CONCLUSIONS: Goal-directed colloid fluid therapy significantly increased microcirculatory blood flow and tissue oxygen tension in healthy and injured colon compared to goal-directed or restricted crystalloid fluid therapy.

STUDIES INTO THE MOLECULAR MECHANISMS REGULATING MUSCULAR ATROPHY RESULTING FROM HINDLIMB IMMOBILIZATION IN THE RAT (PROTEIN, SYNTHESIS, RNA)

The purpose of the work performed in this dissertation was to examine some of the possible regulatory mechanisms involved in the initiation of muscular atrophy during periods of decreased muscle utilization resulting from hindlimb immobilization in the rat. A 37% decrease in the rate of total muscle protein synthesis which has been observed to occur in the first 6 h of immobilization contributes significantly to the observed loss of protein during immobilization.^ The rates of cytochrome c and actin synthesis were determined in adult rat red vastus lateralis and gastrocnemius muscles, respectively, by the constant infusion and incorporation of ('3)H-tyrosine into protein. The fractional synthesis rates of both actin and cytochrome c were significantly decreased (P < 0.05) in the 6th h of hindlimb immobilization.^ RHA was extracted from adult rat gastrocnemius muscle by modification of the phenol: chloroform: SDS extraction procedures commonly used for preparation of RNA for hybridization analysis from other mammalian tissues. RNA content of rat gastrocnemius muscle, as determined by this method of extraction and its subsequent quantification by UV absorbance and orcinol assay, was significantly greater than the RNA content previously determined for adult rat gastrocnemius by other commonly employed methods.^ RNA extracted by this method from gastrocnemius muscles of control and 6h immobilized rats was subjected to "dot blot" hybridization to ('32)P-labelled probe from plasmid p749, containing a cDNA sequence complementary to (alpha)-actin mRNA and from rat skeletal muscle. (alpha)-Actin specific mRNA content as estimated by this procedure is not significantly decreased in rat gastrocnemius following 6h or hindlimb immobilization. However, (alpha)-actin specific mRNA content is significantly decreased (P < 0.05) in adult rat gastrocnemius (alpha)-actin specific mRNA is not decreased in adult rat gastrocnemius muscle following 6h of immobilization, a time when actin synthesis is significantly decreased, it is concluded that a change in (alpha)-actin specific mRNA content is not the initiating event responsible for the early decrease in actin synthesis observed in the 6th h of immobilization. ^

Selection and nuclear immobilization of exportable RNAs

The intracellular distribution of RNAs depends on interactions of cis-acting nuclear export elements or nuclear retention elements with trans-acting nuclear transport or retention factors. To learn about the relationship between export and retention, we isolated RNAs that are exported from nuclei of Xenopus laevis oocytes even when most RNA export is blocked by an inhibitor of Ran-dependent nucleocytoplasmic transport, the Matrix protein of vesicular stomatitis virus. Export of the selected RNAs is saturable and specific. When present in chimeric RNAs, the selected sequences acted like nuclear export elements in promoting efficient export of RNAs that otherwise are not exported; the pathway used for export of these chimeric RNAs is that used for the selected RNAs alone. However, these chimeric RNAs, unlike the selected RNAs, were not exported in the presence of Matrix protein; thus, the nonselected sequences can cause retention of the selected RNA sequences under conditions of impaired nucleocytoplasmic transport. We propose that most RNAs are transiently immobilized in the nucleus and that release of these RNAs is an essential and early step in export. Release correlates with functional Ran-dependent transport, and the lack of export of chimeric RNAs may result from interference with the Ran system.

Fluid resuscitation with colloid or crystalloid solutions in critically ill patients: a systematic review of randomised trials

Objective: To determine the effect on mortality of resuscitation with colloid solutions compared with resuscitation with crystalloids.

Immobilization of oligodeoxyribonucleotides with multiple anchors to microchips

Facile modification of oligodeoxyribonucleotides is required for efficient immobilization to a pre-activated glass surface. This report presents an oligodeoxyribonucleotide which contains a hairpin stem–loop structure with multiple phosphorothioate moieties in the loop. These moieties are used to anchor the oligo to glass slides that are pre-activated with bromoacetamidopropylsilane. The efficiency of the attachment reaction was improved by increasing the number of phosphorothioates in the loop, as shown in the remarkable enhancement of template hybridization and single base extension through catalysis by DNA polymerase. The loop and stem presumably serve as lateral spacers between neighboring oligodeoxyribonucleotides and as a linker arm between the glass surface and the single-stranded sequence of interest. The oligodeoxyribonucleotides of this hairpin stem–loop architecture with multiple phosphorothioate moieties have broad application in DNA chip-based gene analysis.