Composite magnetic particles as carriers for laccase from Trametes versicolor

In this paper we report a study of laccase immobilisation on different kinds of carrier particles. The immobilisation of enzyme on the particle surface with respect to the immobilisation efficiency and the properties of the immobilised enzymes is discussed. The immobilisation of laccase on polystyrene particles bearing reactive beta-diketone groups is characterised by high efficiency, but grafting of the enzyme increases the stability of the colloidal system, which makes the separation/purification procedure difficult. Additionally, the extreme colloidal stability of the immobilisates hinders the application of such particles with immobilised enzymes in some applications where the recycling of the enzyme should be performed. It has been found that hybrid PS-AAEM particles equipped with maghemite show similar immobilisation efficiency to that of their analogues without maghemite and can additionally be manipulated in magnetic fields. The activity of the immobilised laccase is much higher in the pH region 5-7 and the temperature range 50-70 degrees C as compared with that of the free enzyme. Immobilised enzymes also exhibit much better storage stability.     

Structure and Properties of Polymer Biocomposite Materials

Results of studying the structure and properties of biocomposite materials are summarized. The materials in question include an enzyme (laccase, peroxidase), an ion- or electron-conducting polymer (Nafion, polymethylpyrrole), and a carbon substrate (compact, disperse). It is shown that the orientation of a large number of enzyme molecules in an enzyme/Nafion composite material on the substrate surface favors direct bioelectrocatalysis. During co-immobilization of an enzyme and polymethylpyrrole, conditions are realized under which the polymer takes part in the electron transfer between the active center of the enzyme and the surface of the electroconducting substrate. A fresh approach to constructing a biocomposite material is developed. The material is based on an extremely finely divided carbon material (colloidal graphite), which ensures a high specific activity of laccase immobilized on it. The size of colloidal-graphite particles is commensurate with that of the laccase molecule, owing to which the enzyme macromolecule is surrounded by carbon particles. As a result, practically all adsorbed enzyme molecules are electrochemically active and participate in direct bioelectrocatalysis.     

Adsorption, desorption and activities of acid phosphatase on various colloidal particles from an Ultisol

Adsorption, desorption and activity of acid phosphatase on various soil colloidal particles and pure clay minerals were studied. Higher adsorption amounts and low percentage of desorption of acid phosphatase were found on fine soil clays (<0.2 μm). Electrostatic force and ligand exchange are the major driving forces that are involved in the adsorption of enzymes on soil clays. More enzyme molecules were adsorbed on soil clays in the presence of organic components. However, enzymes on organic clays were more easily released. One-third of the enzyme on goethite was adsorbed via ligand exchange process. Some other interactions, such as van der Waals force, hydrophobic force and hydrogen bonding may be more important in the adsorption of enzyme on kaolinite and the enzyme in this system cannot be easily removed. Coarse clays (0.2–2 μm) and inorganic soil clays had higher affinities for enzyme molecules than fine clays and organic clays, respectively. The activity of enzyme bound on soil clays was inhibited and the thermal stability was increased in the presence of organic matter. Data obtained in this study are helpful for a better understanding of the interactions of enzymes with inorganic and organic constituents in soil and associated environments.     

胶体颗粒在聚电解质多层膜表面的可控组装

利用原子力显微镜和扫描电子显微镜研究了磺化聚苯乙烯胶体颗粒在由聚二甲基二烯丙基氯化铵和聚苯乙烯磺酸钠层状自组装而成的多层膜表面的组装.该组装受表面性质影响,通过对多层膜的最外层的组装条件或利用盐溶液对多层膜进行后处理可以控制胶体颗粒在膜表面的组装密度.     

PET/PC共混体系的酯交换反应对其高压结晶行为的影响

利用转矩流变仪、DSC、SEM及WAXD等表征手段研究了PET/PC共混体系的酯交换反应对其高压结晶行为的影响.SEM观察表明,PET和PC熔混时的酯交换反应有利于PET/PC体系在高压结晶时生成厚度较大的伸直链晶体,且可以促进其高压下酯交换反应的发生.楔形伸直链晶体和弯曲伸直链晶体的存在证明链滑移扩散和酯交换反应两种机制对体系中聚酯伸直链晶体的增厚有贡献.拟合分峰法和War-ren-Averbach傅里叶分析法的计算结果表明,随PET/PC体系熔混时酯交换反应程度的增加,高压结晶共混物的结晶度降低,PET的平均微晶尺寸增大,点阵畸变平均值则减小,而微晶尺寸分布变宽.提出了在共聚物组分都具备结晶能力时,结晶诱导化学反应和化学反应诱导结晶两种过程在一定条件下可同时发生的观点.     

Structure, Stability, and Activity of Adsorbed Enzymes

A proteolytic enzyme, α-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solution onto a hydrophobic Teflon surface and a hydrophilic silica surface. We investigated the influence of adsorption on the structure, the structure thermal stability and the activity of these enzymes. Probing the protein structure by circular dichroism spectroscopy indicates that Teflon promotes the formation of helical structure in α-chymotrypsin, but the reverse effect is found with cutinase. The perturbed protein structures on Teflon are remarkably stable, showing no heat-induced structural transitions up to 100°C, as monitored by differential scanning calorimetry. Contact with the hydrophilic silica surface leads to a loss in the helix content of both proteins. Differential scanning calorimetry points to a heterogeneous population of adsorbed protein molecules with respect to their conformational states. The fraction of the native-like conformation in the adsorbed layer increases with increasing coverage of the silica surface by the proteins. The specific enzymatic activity in the adsorbed state qualitatively correlates with the fraction of proteins in the native-like conformation.     

Adsorption orientations and interactions of methyl orange on negatively and positively charged colloidal silver particles

Both positively and negatively charged colloidal silver particles were prepared from chemical deoxidized methods. Then UV-visible absorption, fluorescence, and surface-enhanced Raman scattering of methyl orange adsorbed onto surfaces of these two kinds of particles were observed and compared with each other. The results indicate that dye molecules may adsorb onto these two kinds of silver surface in differing adsorption orientations with different interactions, which caused the different phenomena.     

Formation and structure of colloidal halos in two-dimensional suspensions of paramagnetic particles

Different Monte Carlo simulation approaches are used here to study the static structure induced by a spherical neutral substrate inserted in the midst of a two-dimensional suspension of paramagnetic particles. It is then observed that in some instances some of these particles are adsorbed to the surface of the substrate, forming colloidal halos. We investigate the necessary conditions for the formation of these halos and the dependence of the number of adsorbed particles on the relevant parameters of the system. The angular distribution of the adsorbed particles around the perimeter of the substrate is analyzed here too.     

Formation of a supramolecular gel between alpha-cyclodextrin and free and adsorbed PEO on the surface of colloidal silica: effect of temperature, solvent, and particle size

Aqueous solutions of alpha-cyclodextrin (alpha-CD) complex spontaneously with poly(ethylene oxide) (PEO), forming a supramolecular structure known as pseudopolyrotaxane. We have studied the formation of the complex obtained from the threading of alpha-CD onto PEO, both free in solution and adsorbed on colloidal silica. The kinetics of the reaction were studied by gravimetric methods and determined as a function of temperature and solvent composition for the PEO free in solution. PEO was then adsorbed on the surface of colloidal silica particles, and the monomers were displaced by systematically varying the degree of complexation, the concentration of particles, and the molecular weight of the polymer. The effect of the size of the silica particles on the yield of the reaction was also studied. With the adsorbed PEO, the complexation was found to be partial and to take place from the tails of the polymer. The formation of a gel network containing silica at high degrees of complexation was observed. Small-angle X-ray and neutron scattering experiments were performed to study the configuration of the polymeric chains and confirmed the partial desorption of the polymer from the surface of the silica upon complexation.     

Covalent attachment of trypsin on plasma polymerized allylamine

This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV–vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.     

Development of a colloidal lithography method for patterning nonplanar surfaces

A colloidal lithography method has been developed for patterning nonplanar surfaces. Hexagonal noncontiguously packed (HNCP) colloidal particles 127 nm-2.7 μm in diameter were first formed at the air-water interface and then adsorbed onto a substrate coated with a layer of polymer adhesive ～17 nm thick. The adhesive layer plays the critical role of securing the order of the particles against the destructive lateral capillary force generated by a thin film of water after the initial transfer of the particles from the air-water interface. The soft lithography method is robust and very simple to carry out. It is applicable to a variety of surface curvatures and for both inorganic and organic colloidal particles.     

Temperature Window for Encapsulation of an Enzyme into Thermally Shrunk,CaCO3 Templated Polyelectrolyte Multilayer Capsules

Encapsulation of enzymes allows to preserve their biological activities in various environmental conditions, such as exposure to elevated temperature or to proteases. This is particularly relevant for in vivo applications, where proteases represent a severe obstacle to maintaining the activity of enzymes. Polyelectrolyte multilayer capsules are suitable for enzyme encapsulation, where CaCO₃ particles and temperature-dependent capsule formation are the best templates and the most adequate method, respectively. In this work, these two areas are combined and, ALP (alkaline phosphatase), which is a robust and therapeutically relevant enzyme, is encapsulated into thermally shrunk polyelectrolyte multilayer (PDADMAC/PSS)₄ capsules templated on calcium carbonate particles (original average diameter: ≈3.5 µm). The activity of the encapsulated enzyme and the optimal temperature range for encapsulation are investigated. The enzymatic activity is almost four times higher upon encapsulation when the temperature range for encapsulation is situated just above the glass transition temperature (40 °C), while its optimal conditions are dictated, on the one hand, by the enzyme activity (better at lower temperatures) and, on the other hand, by the size and mechanical properties of capsules (better at higher temperatures).     

纳米二氧化硅包覆颜料黄的研究

采用静电自组装技术成功地将纳米二氧化硅粒子包覆在颜料黄的表面.研究结果表明,预吸附的聚电解质层数显著影响纳米二氧化硅的吸附量.随着包覆二氧化硅层数的增加,覆盖率逐渐增加,但包覆三层二氧化硅后,覆盖趋于平衡.吸附的纳米二氧化硅不仅可以提高颜料黄的亲水性,而且还能够散射紫外线,尤其是波长小于270nm的紫外线,提高了颜料黄的耐候性,同时又不影响颜料黄本身的颜色.     

Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.     

Adsorption and order formation of colloidal nanoparticles on a substrate: a Brownian dynamics study

Adsorption process and order formation of electrostatically stabilized colloidal particles with a radius of 50 nm onto a planar surface with countercharge are examined. We perform Brownian dynamics simulations with a new three-dimensional cell model, in which the particle-particle and particle-substrate interactions are modeled based on the DLVO theory. The simulations yield the following results: (1) a larger bulk concentration would be required for larger kappaa to reach order formation to compensate for the decrease in the bulk potential; (2) the phase transition from a disordered to an ordered structure of the adsorbed particles on the substrate is considered to be of the Kirkwood-Alder type of transition through the examination of the two-dimensional pressure of the adsorbed particles; (3) the adsorbed particles are found to form a hexagonally ordered array, only if what we call "one-directional average force" acting on an adsorbed particle exceeds a critical value, which is independent of the ionic strength, or the interaction potentials. The critical value of the one-directional average force is interpreted as the force needed to keep an ordered structure by localizing adsorbed particles at fixed positions. In addition, the critical force is used to develop a new model to estimate the surface coverage at the order-disorder transition and it is demonstrated that the new model gives better estimation than other models previously reported.     

Salt-regulated attraction and repulsion of spherical polyelectrolyte brushes towards polyelectrolyte multilayers

Adsorption of colloidal particles presents an interesting alternative to the modification of surfaces using covalent coupling or physisorption of molecules. However, to tailor the properties of these materials full control over the effective particle-substrate interactions is required. We present a systematic investigation of the adsorption of spherical polyelectrolyte brushes (SPB) onto polyelectrolyte multilayers (PEM). A brush layer grafted from colloidal particles allows the incorporation of various functional moieties as well as the precise adjustment of their adsorption behaviour. In the presence of oppositely charged surfaces the amount of adsorbed SPB monotonically increases with the ionic strength, whereas equally charged substrates efficiently prevent colloidal attachment below a threshold salt concentration. We found that the transition from the osmotic to the salted brush regime at approximately 100 mM coincided with a complete loss of substrate selectivity. In this regime of high ionic strength, attractive secondary interactions become dominant over electrosteric repulsion. Due to the soft polyelectrolyte corona a surface coverage exceeding the theoretical jamming limit could be realized. Both the adsorption kinetics and the resulting thin film morphologies are discussed. Our study opens avenues for the production of two-dimensional arrays and three-dimensional multilayered structures of SPB particles.     

Structure and function of enzymes adsorbed onto single-walled carbon nanotubes

We have examined the structure and function of two enzymes, alpha-chymotrypsin (CT) and soybean peroxidase (SBP), adsorbed onto single-walled carbon nanotubes (SWNTs). SBP retained up to 30% of its native activity upon adsorption, while the adsorbed CT retained only 1% of its native activity. Analysis of the secondary structure of the proteins via FT-IR spectroscopy revealed that both enzymes undergo structural changes upon adsorption, with substantial secondary structural perturbation observed for CT. Consistent with these results, AFM images of the adsorbed enzymes indicated that SBP retains its native three-dimensional shape while CT appears to unfold on the SWNT surface. This study represents the first in depth investigation of protein structure and function on carbon nanotubes, which is critical in designing optimal carbon nanotube-protein conjugates.     

Stability in Colloidal Mixtures Containing Particles with a Large Disparity in Size

An experimental approach, based on turbidity measurements, is proposed for studies of the stability in colloidal mixtures containing particles with large disparity in size. The main advantage of this approach is that it permits investigations even under conditions of comparable particle number concentrations of the two colloidal populations. Binary mixtures containing a poly(vinyl acetate) (PVAc) latex and a Ludox AS-40 silica sol were investigated. The silica particles were much smaller than the latex ones. The experimental stability factors were compared with the theoretical values computed on the basis of the Kihira-Ryde-Matijevic model (J. Chem. Soc., Faraday Trans. 88(16), 2379 (1992)) for interaction between spherical particles with unevenly distributed surface charges. All the experimental results support the idea that, even when both sols are negatively charged, the small silica particles are adsorbed onto the latex surface. Under these conditions, the heteroaggregates, which are composed of PVAc cores surrounded with silica particles, can be modeled as PVAc particles having "modified" surface characteristics (i.e., average Stern potential and varying extents of the surface charge segregation). Copyright 2001 Academic Press.     

Constructing Hybrid Protein Zymogens through Protective Dendritic Assembly

The modulation of protein uptake and activity in response to physiological changes forms an integral part of smart protein therapeutics. We describe herein the self‐assembly of a pH‐responsive dendrimer shell onto the surface of active enzymes (trypsin, papain, DNase I) as a supramolecular protecting group to form a hybrid dendrimer–enzyme complex. The attachment is based on the interaction between boronic acid and salicyl hydroxamate, thus allowing the macromolecular assembly to respond to changes in pH between 5.0 and 7.4 in a highly reversible fashion. Catalytic activity is efficiently blocked in the presence of the dendrimer shell but is quantitatively restored upon shell degradation under acidic conditions. Unlike the native proteases, the hybrid constructs are shown to be efficiently taken up by A549 cells and colocalized in the acidic compartments. The programmed intracellular release of the proteases induced cytotoxicity, thereby uncovering a new avenue for precision biotherapeutics.