Surface Proximity Effect, Imprint Memory of Ferroelectric Twins, and Tweed in the Paraelectric Phase of BaTiO3.

We have used energy-filtered photoemission electron microscopy (PEEM) at the photoemission threshold to carry out a microscopic scale characterization of the surface charge and domain structure of the (001) surface in BaTiO3. Signatures of ferroelectric and ferroelastic domains, and tweed, dominate the surface structure of BaTiO3 at room temperature. The surface ferroic signatures are maintained on heating to temperature (~550 K), well above the transition temperature (393 K). This surface proximity effect provides the mechanism for memory of the bulk ferroelectric domain arrangement up to 150 K above TC and thus can be considered as a robust fingerprint of the ferroelectric state near the surface. Self-reversal of polarization is observed for the tweed below TC and for the surface domains above TC. Annealing at higher temperature triggers the dynamic tweed which in turn allows a full reorganization of the ferroic domain configuration.

Biocatalysis with Escherichia coli-overexpressing cyclopentanone monooxygenase immobilized in polyvinyl alcohol gel.

UNLABELLED: This is the first reported study on the immobilization of living recombinant Escherichia coli cells that overexpress cyclopentanone monooxygenase in polyvinyl alcohol gel particles LentiKats®. Immobilized cells overexpressing cyclopentanone monooxygenase have been used as a model of biocatalyst for enantioselective Baeyer-Villiger biooxidation of rac-bicyclo[3.2.0]hept-2-en-6-one into regioisomeric lactones. This process is useful for the syntheses of cytostatic sarkomycin, several prostaglandins and other biologically active compounds. The original technique for qualitative analysis of enzyme expression within free cells and cells entrapped in LentiKats® using SDS-PAGE was developed and used for verification of optimal conditions for the induction of cyclopentanone monooxygenase. Here, we successfully performed six repeated batch Baeyer-Villiger biooxidations utilizing entrapped cells using 40% (w/v) polyvinyl alcohol gel particles in flasks with baffles. The latter conditions have been found to be the most appropriate achieving optimal oxygen transfer within LentiKats®. Moreover, immobilized cells retained their catalytic efficiency over six reaction cycles, while the catalytic efficiency of free cells decreased after three reaction cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: Immobilization in polyvinylalcohol gel particles is desirable technique with presumptive impact on industrial applications of recombinant whole-cell Baeyer-Villiger monooxygenases as biocatalysts for production of bioactive compounds and precursors of potentially new drugs. An original immobilization of cells E. coli with overproduced Baeyer-Villiger monooxygenase improved their stability in repetitive batch biooxidations as compared to free cells. Detected autoinduction of recombinant enzyme in pET22b+ plays significant role in application of immobilized cells as it may increase specific activity of cells in repetitive use under growing reaction conditions. Original technique for qualitative analysis of enzyme expression within immobilized cells was developed.

The direct magnetoelectric effect in ferroelectric-ferromagnetic epitaxial heterostructures.

Ferroelectric (FE) and ferromagnetic (FM) materials engineered in horizontal heterostructures allow interface-mediated magnetoelectric coupling. The so-called converse magnetoelectric effect (CME) has been already demonstrated by electric-field poling of the ferroelectric layers and subsequent modification of the magnetic state of adjacent ferromagnetic layers by strain effects and/or free-carrier density tuning. Here we focus on the direct magnetoelectric effect (DME) where the dielectric state of a ferroelectric thin film is modified by a magnetic field. Ferroelectric BaTiO3 (BTO) and ferromagnetic CoFe2O4 (CFO) oxide thin films have been used to create epitaxial FE/FM and FM/FE heterostructures on SrTiO3(001) substrates buffered with metallic SrRuO3. It will be shown that large ferroelectric polarization and DME can be obtained by appropriate selection of the stacking order of the FE and FM films and their relative thicknesses. The dielectric permittivity, at the structural transitions of BTO, is strongly modified (up to 36%) when measurements are performed under a magnetic field. Due to the insulating nature of the ferromagnetic layer and the concomitant absence of the electric-field effect, the observed DME effect solely results from the magnetostrictive response of CFO elastically coupled to the BTO layer. These findings show that appropriate architecture and materials selection allow overcoming substrate-induced clamping in multiferroic multi-layered films.

Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins.

A procedure for determination of apparent affinity constants K(D)(app) between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10(-7) to 9×10(-6)M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

Multiferroic phase transition near room temperature in BiFeO3 films.

In multiferroic BiFeO(3) thin films grown on highly mismatched LaAlO(3) substrates, we reveal the coexistence of two differently distorted polymorphs that leads to striking features in the temperature dependence of the structural and multiferroic properties. Notably, the highly distorted phase quasiconcomitantly presents an abrupt structural change, transforms from a standard to a nonconventional ferroelectric, and transitions from antiferromagnetic to paramagnetic at 360±20 K. These coupled ferroic transitions just above room temperature hold promises of giant piezoelectric, magnetoelectric, and piezomagnetic responses, with potential in many applications fields.

Low-symmetry phases in ferroelectric nanowires.

Ferroelectric nanostructures have recently attracted much attention due to the quest of miniaturizing devices and discovering novel phenomena. In particular, studies conducted on two-dimensional and zero-dimensional ferroelectrics have revealed original properties and their dependences on mechanical and electrical boundary conditions. Meanwhile, researches aimed at discovering and understanding properties of one-dimensional ferroelectric nanostructures are scarce. The determination of the structural phase and of the direction of the polarization in one-dimensional ferroelectrics is of technological importance, since, e.g., a low-symmetry phase in which the polarization lies away from a highly symmetric direction typically generates phenomenal dielectric and electromechanical responses. Here, we investigate the phase transition sequence of nanowires made of KNbO(3) and BaTiO(3) perovskites, by combining X-ray diffraction, Raman spectroscopy, and first-principles-based calculations. We provide evidence of a previously unreported ferroelectric ground state of monoclinic symmetry and the tuning of the polarization's direction by varying factors inherent to the nanoscale.

Degradation of high-molar-mass hyaluronan and characterization of fragments.

A sample of high-molar mass hyaluronan was oxidized by seven oxidative systems involving hydrogen peroxide, cupric chloride, ascorbic acid, and sodium hypochlorite in different concentrations and combinations. The process of the oxidative degradation of hyaluronan was monitored by rotational viscometry, while the fragments produced were investigated by size-exclusion chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and non-isothermal chemiluminometry. The results obtained imply that the degradation of hyaluronan by these oxidative systems, some of which resemble the chemical combinations present in vivo in the inflamed joint, proceeds predominantly via hydroxyl radicals. The hyaluronan fragmentation occurred randomly and produced species with rather narrow and unimodal distribution of molar mass. Oxidative degradation not only reduces the molecular size of hyaluronan but also modifies its component monosaccharides, generating polymer fragments that may have properties substantially different from those of the original macromolecule.

Degradation of high-molar-mass hyaluronan by an oxidative system comprising ascorbate, Cu(II), and hydrogen peroxide: inhibitory action of antiinflammatory drugs--naproxen and acetylsalicylic acid.

Changes in dynamic viscosity of the solutions of a high-molar-mass hyaluronan (HA) were monitored using a rotational viscometer. The degradative conditions generated in the HA solutions by a system comprising ascorbate plus Cu(II) plus H(2)O(2) were studied either in the presence or absence of a drug--naproxen or acetylsalicylic acid. Continual decrease of the dynamic viscosity of HA solution was indicative of the polymer degradation. Addition of the drug retarded/inhibited the HA degradation in a concentration-dependent manner. The characteristics of the fragmented polymers were investigated by FT-IR spectroscopy and by two different liquid chromatographic techniques, namely by size-exclusion chromatography equipped with a multi-angle light scattering photometric detector and by high-performance liquid chromatography connected on-line to a spectrofluorometer.

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression.

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.

Influence of mannan epitopes in glycoproteins--Concanavalin A interaction. Comparison of natural and synthetic glycosylated proteins.

Two natural glycoproteins/glycoenzymes, invertase and glucoamylase, and two neoglycoconjugates, synthetized from Saccharomyces cerevisiae mannan, bovine serum albumin and penicillin G acylase were tested for interaction with lectin Concanavalin A (Con A). The interaction of natural and synthetic glycoproteins with Con A was studied using three different experimental methods: (i). quantitative precipitation in solution (ii). sorption to Con A immobilized on bead cellulose; and (iii). kinetic measurement of the interaction by surface plasmon resonance. Prepared neoglycoproteins were further characterized: saccharide content, molecular weight, polydispersion, kinetic and equilibrium association constants with Con A were determined. It can be concluded that the used conjugation method proved to be able to produce neoglycoproteins with similar properties like natural glycoproteins, i.e. enzymatic activity (protein part) and lectin binding activity (mannan part) were preserved and the neoglycoconjugates interact with Con A similarly as natural mannan-type glycoproteins.

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.

Stability enhancement of Escherichia coli penicillin G acylase by glycosylation with yeast mannan.

Penicillin G acylase (PGA) from Escherichia coli was cross-linked with mannan dialdehydes. Conjugates were prepared with molecular masses varying from 140 to 580 kDa and containing from 18 to 50% (w/w) saccharides, the values depending on the reaction conditions (mannan/enzyme ratio), and by using mannans with different degrees of oxidation and weight-average molecular mass (M macro(w)). The pH- and thermo-stability of all preparations of glycosylated enzyme were improved remarkably, whereby the influence of the character of the linked mannan dialdehyde, its content, as well as the molecular mass of prepared glycoconjugates, on the stability of PGA, was evaluated. PGA glycosylated with the most oxidized mannan up to an M(w) of 490 kDa, containing 41% (w/w) saccharides, and retaining 90% of its original catalytic activity, showed the highest stability. The half-life of this PGA preparation increased significantly: 13-fold at pH 3, 7-fold at pH 10, and 3.5-fold at pH 8 (all at 37 degrees C), compared with the native enzyme. At higher temperatures (50 degrees C) even more significant stabilization was evident, a 16-fold increase in half-life, from 18 min to 289 min, at pH 8, being measured.

Study of interaction of mannan-BSA neoglycoconjugates with Concanavalin A.

Neoglycoconjugates prepared by synthesis of oxidized mannans from Saccharomyces cerevisiae with bovine serum albumin were studied for interaction with Concanavalin A. The mannan-bovine serum albumin neoglycoproteins, different in degree of mannan oxidation used for synthesis and its content in conjugates were prepared. The interaction of these glycoconjugates with Concanavalin A by using precipitation method was investigated. The conjugates prepared at high weight ratio mannan: protein (4:1) involved 47-65% of saccharides and formed by precipitation with Concanavalin A aggregates with low content of protein. The obtained results showed that conjugates with lower content of mannan (up to 30%) are more efficacious for their aggregation with Concanavalin A than the conjugates with high content of mannan.

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor.

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 µA/mM in phosphate buffer), low detection limit (0.8 µM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).

Novel glucose non-interference biosensor for lactose detection based on galactose oxidase-peroxidase with and without co-immobilised beta-galactosidase.

Two types of amperometric biosensors for lactose detection based either on co-immobilisation of two enzymes (galactose oxidase with peroxidase) or co-immobilisation of three enzymes (beta-galactosidase, galactose oxidase and peroxidase) were constructed. A graphite rod with pre-adsorbed ferrocene was used as a working electrode. The use of galactose oxidase instead of the frequently used glucose oxidase resulted in the construction of a glucose-non-interfering lactose sensor. Co-immobilisation of peroxidase with galactose oxidase allowed the effect of borate on the extension of the linear range and the effect of the working potential on galactose oxidase activation to be studied. The presence of beta-galactosidase greatly enhances the sensor's sensitivity, but its linear range is narrower than that of the sensor without beta-galactosidase. Addition of DEAE-dextran and inositol to the enzyme layer improved the half-life more than 16-fold compared with the sensor without stabilisers. A response time between 60 and 75 s (90% of the steady-state value) and a detection limit for lactose determination from 44 to 339 microM (signal-to-noise ratio = 3) were observed depending on the conditions. The precision of measurements of standard lactose solution for the trienzymatic and bienzymatic sensors was 2.19 and 2.02%, respectively. The precision of analysis of dairy products varied from 0.24 to 5.24%. Analyses of real samples showed good correlation with HPLC analysis; eight samples and 10 standard lactose solutions without pre-treatment were analysed in 1 h.

Use of bioluminometry for determination of active yeast biomass immobilized in ionotropic hydrogels.

The technique of bioluminometry was used to determine the biomass concentration of yeast cells immobilized in ionotropic hydrogel beads, including alginate, pectate, and kappa-carrageenan. The method uses determination of ATP extracted from viable cells, the concentration of which is then expressed as the active biomass concentration. Seven yeast strains divided into three categories (brewing, wine-making, and ethanol-producing yeasts) were tested, and different biomass concentrations were determined in all three immobilization materials. The described method is characterized by a good correlation (up to 99%) to classical dry biomass determination. The method is quicker, easier, and not so laborious, providing sufficient determination accuracy, and can be used for a rapid estimation of viable biomass in most biotechnological processes using immobilized living cells.

Calcium pectate gel beads for cell entrapment. 6. Morphology of stabilized and hardened calcium pectate gel beads with cells for immobilized biotechnology.

The structure of standard and stabilized calcium pectate gel (CPG) beads has been examined by scanning (SEM) and transmission (TEM) electron microscopy. A two-stage crosslinking procedure with polyethyleneimine (PEI) and glutaraldehyde (GA) led to the formation of a more compact layer on the bead surface. On the other hand, the stabilization procedure did not significantly change either gel bead interior or morphologic properties, vitality and biotransformation activity of immobilized bacterial cells (Nocardia tartaricans) against cis-epoxysuccinate as well as yeast cells (Trigonopsis variabilis) against cephalosporin C. The structure of these cells within the calcium pectate matrix remained unchanged. Moreover, the two-step chemical stabilization of CPG containing T. variabilis or N. tartaricans had a favourable effect on storage and operational stability at semi-continuous and continuous processing in stirred batch and packed-bed reactors. The most valuable effect of stabilization was the fact that the hardened CPG comprising the cells N. tartaricans resisted, for a long time (360 days and more), the destructive effects of the product (such strong sequestering reagent as L-(+)-tartaric acid) at high concentrations (up to 1 M). Non-hardened CPG was destroyed after 21 h. The reference materials, hardened and non-hardened calcium alginate gels (CAG), were destroyed over 3 h or 30 min, respectively.

Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins.

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Investigation of catalytic properties of immobilized enzymes and cells by flow microcalorimetry.

The investigation of catalytic properties of immobilized biocatalysts (IMB) is a time-consuming and not-always-simple procedure, requiring a simple and accurate method of enzyme-activity measurement. In comparison with generally-used techniques, flow microcalorimetry (FMC) has proven to be a very practical and versatile technique for direct monitoring of the course of enzyme reactions. The principal advantage of FMC is integration of the enzyme reaction and its monitoring in one step. This review summarizes the information needed for the complete kinetic or catalytic characterization of the IMB by FMC, without the requirement of any independent analytical method. The optimal experimental procedure is proposed. Examples of experimental studies on immobilized biocatalysts using the FMC are provided. The method is applicable to purified enzymes as well as to enzymes fixed in cells.

Stabilization of D-amino-acid oxidase from Trigonopsis variabilis by manganese dioxide.

Stabilization of immobilized D-amino-acid oxidase was achieved as follows. Yeast Trigonopsis variabilis producing D-amino-acid oxidase was used to deaminate cephalosporin C to glutaryl-7-aminocephalosporanic acid. Permeabilized cells were co-immobilized with manganese dioxide by entrapment in (poly)acrylamide gel so that hydrogen peroxide, liberated in the reaction, could be partially deactivated and both the enzyme and the substrate could be stabilized. Activity of entrapped cells was determined by HPLC and enzyme flow microcalorimetry. The process was evaluated in terms of activity, immobilization yield, storage stability and oxo-product formation by immobilized preparations. The storage stability of immobilized biocatalysts with MnO2 was nearly doubled and production of 2-oxoadipyl-7-aminocephalosporanic acid was 2-3-fold higher than by entrapped cells without MnO2. Glutaryl-7-aminocephalosporanic acid can be easily obtained from the resulting oxo-product by a non-enzymic reaction via externally added hydrogen peroxide.