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.

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.

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.

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.

Lectin-glycoenzyme column chromatography monitored by enzyme flow microcalorimetry.

A method based on the flow microcalorimetric determination of catalytic activity of immobilized enzyme in a so-called enzyme thermistor was used to monitor the process of lectin affinity chromatography of invertase on Concanavalin A-bead cellulose. The strong biospecific interaction between Concanavalin A and invertase was employed to determine the bound enzyme and this principle was used for the investigation of an alternative direct method for monitoring the lectin affinity chromatography of glycoenzymes. The results obtained by flow microcalorimetry showed that the catalytic activity of invertase immobilized on Concanavalin A-bead cellulose can be compared directly with the thermometric value delta Tmax. The validity of the method was also confirmed by the enzyme thermistor post-column method, which is based on the determination of the product from the immobilized invertase enzymatic reaction. The adsorption and desorption in the chromatography column were examined by flow microcalorimetry in small samples withdrawn from the column. Attention has been given to the operating parameters and the storage stability of the affinity sorbent. The binding ability of the affinity matrix decreased with the number of consecutive chromatographic runs, although its storage stability was satisfactory.

Stepwise immobilization of proteins via their glycosylation.

Glycosyl derivatives of bovine serum albumin in which the glycosyl residue is represented by mono- or disaccharide can be, after periodate oxidation, coupled to polyhydrazides having a macroporous matrix (cross-linked polyacrylamide, bead cellulose). The amount of the linked neoglycoprotein depends not only on the physical structure of the matrix but also on the degree of its substitution with hydrazide groups and on the type and concentration of glycosyl residue in the neoglycoprotein. A high degree of substitution as well as the presence of the D-galactosyl unit both play a positive role. Owing to the fact that the glucosyl unit in disaccharide residues (cellobiosyl, lactosyl) also contributes positively to spacer properties, in the monolactosyl derivative of albumin exhibits good binding properties towards macroporous polyhydrazides. While the high sugar-containing conjugates of glycosyl derivatives of albumin with polyhydrazides are stable for two weeks at pH 6-9, the conjugates of the monolactosyl derivative of albumin can only be stored at pH 7.5. The binding site of albumin immobilization is considered.

The glycosylated enzyme-binding assay for the study of the interaction of free and immobilized lectins with carbohydrates.

The glycosylated enzymes (invertase and glucose oxidase) were used as the competitive markers for a simple and rapid determination of the lectin-saccharide interactions. The method, based on the formation of the conjugate of an appropriate glycoenzyme with the specific carbohydrate-binding lectins and the inhibition of the conjugate formation with a monosaccharide, was described. This method was used to estimate the relative carbohydrate specificity of Concanavalin A for monosaccharides derived from D-mannose. The inhibition effect of the saccharides on the formation of Concanavalin A-glycosylated enzyme precipitate was compared with their influence on the enzyme sorption on conjugate Concanavalin A-bead cellulose support. The amount of the interacting enzyme was estimated either indirectly from its concentration in a supernatant that was determined spectrophotometrically (Con A was in a free or immobilized form) or directly in the immobilized form linked to Con A-sorbent using the flow microcalorimetric method. The results obtained, using different methods, agreed in general.

Simple estimation of carrier binding capacity using sorption kinetics curve-fitting.

Kinetic curves of the sorption of biological-like compounds and proteins onto insoluble carriers were determined either by linearisation via double reciprocal transformation or by non-linear fitting using the following equation: B = (1/K1) X [t/(t + K2/K1)]. Both these procedures provided non-significant differences in the values of parameters of sorption kinetics, namely of equilibrium sorption (BM), sorption half-time (t1/2) and initial sorption rate (vo). Moreover, these methods proved to be sufficient to provide a precise description of the kinetics of different types of sorption, such as chemical, physical, ionic, biospecific and non-specific sorption. Both computing procedures make it possible (i) to calculate the parameters BM, t1/2 and vo even in instances where they cannot be established experimentally, and (ii) to replace the graphic estimation with computation. The assumption that all types of sorption mentioned above will be kinetically controlled in a uniform way proved to be reasonable.

Application of adsorption kinetics for estimation of dissociation constants.

Substances such as drugs, as well as special ligands with expressive biospecific properties, all with different affinities, interact with proteins which can be characterized by dissociation constants. The method for estimation of the dissociation constant on the basis of adsorption kinetics was verified for two typical cases: adsorption of lactate dehydrogenase onto bead cellulose derivatized by reactive dyes C.I.2. or C.I.19, and adsorption of different drugs (neuroleptics and local anesthetics) onto calmodulin immobilized on agarose gel. The real equilibrium values obtained by using the complete time-concentration model of adsorption were fitted according to the respective adsorption isotherms by non-linear regression.

Reactions of cellulose isothiocyanates with thiol and amino compounds.

A cellulose isothiocyanate has been prepared by treatment of cellulose with 2,4-di-isocyanatotoluene followed by hydrolysis and reaction of the resulting amine with thiophosgene. The cellulose isothiocyanate was characterized by its binding capacity with respect to [14C]-glycine, [131 I]-human serum albumin, and 2-mercaptoethanol. An analytical method for binding capacity, based on reaction with [35 S]-alpha-toluenethiol, was developed. Because of the aromatic character of the NCS group of the cellulose isothiocyanate, the covalently bonded thiol can be quantitatively liberated.

Biochemical engineering of biocatalysts immobilized on cellulosic materials.

Complete design of the optimum immobilized biocatalyst seems to still be a matter of the future. To be successful, it would require numerical determination of all significant parameters at each enzyme engineering phase, that is at the design of the carriers, immobilized biocatalysts and immobilized reactors. Future research trends should follow this strategy. For processing, cellulosic materials have been considered carriers that fulfill requests to an example model: they represent a unique family of carriers that cover a broad variety of physical and chemical properties, immobilizing techniques, and immobilized reactors as well. The reason for writing this review article was to test the reliability of such a processing and subsequently, to confront theoretical considerations with practical applications of biocatalysts immobilized on cellulose materials.

Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system.

The possibility of using the enzyme thermistor (ET) for the direct determination of kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different preparations of invertase conjugated to bead celluloses. Two different ET columns packed with IME were operated in the mode of a differential enzyme reactor (short length, low substrate conversion). Kinetic parameters of the above IME reactor were computed by a nonlinear curve-fitting procedure. The obtained kinetic parameters were superverified by means of an independent differential reactor (DR) system. This system utilized an indirect postcolumn analytical method based on determination of glucose concentration in the stirred reservoir. Best agreement between the data acquired by direct (ET) and indirect (DR) methods was obtained if the ET column was operated at flow rates within the range of 1.0-1.5 ml min-1 using invertase-cellulose chlorotriazine conjugate. Influence of heat loss and flow nonideality is discussed. The proposed ET method offers a rapid, convenient, and general approach to determination of kinetic constants of IME preparations by omitting postcolumn analytical methods.

Direct determination of the cephalosporin transforming activity of immobilized cells with use of an enzyme thermistor. 1. Verification of the mathematical model.

A simple method for the direct measurement of the catalytic properties of immobilized cells in the flow minicalorimeter, the enzyme thermistor (ET), is presented. A Trigonopsis variabilis strain with cephalosporin C-transforming activity was used as the model system. The yeast cells were immobilized either by crosslinking with a homobifunctional reagent or by entrapment in gels. The actual activity of the immobilized cells used in the ET was estimated by means of a stirred-batch reactor measurement in conjunction with HPLC analysis of substrate and products. Similar results were also obtained using D-amino acid oxidase (EC 1.4.3.3) isolated from T. variabilis cells and immobilized by gel entrapment. This calibration procedure was found to be appropriate for all biocatalyst systems used. The thermometric signal was proportional to the amount of biocatalyst immobilized in the ET minicolumn. It was shown that the rate of reaction catalyzed by T. variabilis entrapped in calcium pectate gel was limited by internal diffusion to an extent depending on the cell concentration in the biocatalyst particle. This approach offers a direct method for studying the kinetic properties of immobilized cells.

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.

Metabolic characteristics of bacterial cells entrapped in beaded calcium alginate and/or pectate gels.

A mixture of heterotrophic bacteria and collection strains of Escherichia coli and Pseudomonas fluorescens were immobilized in calcium alginate or pectate gels. Comparison of respiratory activity, substrate uptake and biosynthetic capacity of immobilized cells showed that both types of carriers permit a prolonged preservation of metabolic activity but the transfer of substances through the gel is faster in the pectate. Morphological changes include some intracellular structures, partial shrinkage of the plasma membrane of immobilized cells, and transformation of a rod-like cell shape to an oval one.

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.

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.