Novel biodegradation system for bisphenol A using laccase-immobilized hollow fiber membranes.

Radiation-induced graft polymerization was applied to prepare membranes for multilayer immobilization of laccase, which has biodegradation ability for bisphenol A (BPA). Glycidyl methacrylate (GMA) was grafted onto porous polyethylene membranes as the monomer of polymer brushes, and aminoethanol (AE) was introduced to the grafted GMA membrane, creating unfolded polymer brushes that serve as a good support for multilayer immobilization of laccase. The objectives of this study were as follows: adjustment of space velocity (SV) for optimum performance; enhancement of stability in organic media through moisture retention; biodegradation of BPA at continuous operation; and investigation of the effects of redox mediators. Laccase and membrane activities were increased at higher SVs as a result of stronger substrate transport. The 1.85% moisture retention as a result of high-density AE containing polymer brushes demonstrated the improved stability of immobilized laccase over free laccase in methanol-containing solutions. BPA was removed with an activity of 0.11 mol/h/kg-membrane. The effects of three major laccase mediators on BPA oxidation was studied, and only 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was shown to increase the oxidation of BPA to 100% at low SVs. Improved stability of laccase and high removal rates in the continuous biodegradation of BPA were achieved by the presented method.

Structural analysis of a calix[4]arene-based Platonic Micelle.

We have recently introduced the concept of "Platonic micelles", the preference of spherical micelles to specific aggregation numbers mostly coinciding with the number of faces of platonic solids. This effect was observed on bulky, mostly calix[4]arene-based surfactant systems with small aggregation numbers. The preferred aggregation numbers result in better sphere coverage, highliting the packing and the "protection" of hydrophobic cores from the aqueous solvent as the most important factor for this preference. In the present study we further explore the interactions that drive the packing of the highly charged PACaL3 surfactant into highly symmetrical hexameric micelles. We performed a series of molecular dynamics simulations that yielded a large set of structures and an ensemble in good agreement with the experimental Small Angle X-ray Scattering data was selected. The geometry and the rigidity of the calix[4]arene group with proper tail length and headgroup volume are the driving forces for the high symmetry and monodispersity of the micelle. The charge of the headgroups is mainly responsible for inhibiting the formation of higher order structures. Sodium, shown to be important for the stability of the micelle, is not directly interacting with the micelle implying that the calix[4]arene ring is a C2ν symmetry conformation.

Platonic Micelles: Monodisperse Micelles with Discrete Aggregation Numbers Corresponding to Regular Polyhedra.

The concept of micelles was first proposed in 1913 by McBain and has rationalized numerous experimental results of the self-aggregation of surfactants. It is generally agreed that the aggregation number (Nagg) for spherical micelles has no exact value and a certain distribution. However, our studies of calix[4]arene surfactants showed that they were monodisperse with a defined Nagg whose values are chosen from 6, 8, 12, 20, and 32. Interestingly, some of these numbers coincide with the face numbers of Platonic solids, thus we named them "Platonic micelles". The preferred Nagg values were explained in relation to the mathematical Tammes problem: how to obtain the best coverage of a sphere surface with multiple identical circles. The coverage ratio D(N) can be calculated and produces maxima at N = 6, 12, 20, and 32, coinciding with the observed Nagg values. We presume that this "Platonic nature" may hold for any spherical micelles when Nagg is sufficiently small.

Polypod-Shaped DNAs: Small-Angle X-ray Scattering and Immunostimulatory Activity.

We explored in detail the relationship between the structure in aqueous solution and immunostimulatory activity of polypod-shaped DNAs, called polypodnas. The polypodnas were constructed using 3-6 oligodeoxynucleotides (ODNs) to obtain tri-, tetra-, penta-, and hexapodna, each of which had 3, 4, 5, and 6 arms made of double-stranded DNA, respectively. A highly potent immunostimulatory CpG sequence was included into each of the polypodnas. Synchrotron X-ray scattering analysis showed that the double-stranded DNA arms of all of the polypodnas adopted a B-form DNA conformation. The analysis also suggested that some nucleotides in the central parts of pentapodna and hexapodna did not form base pairs, whereas those of tripodna and tetrapodna all formed base pairs. This difference would occur because of an increase in steric hindrance and electrical repulsion with increasing number of arms. The pentapodna and hexapodna induced a large amount of tumor necrosis factor α-release from macrophage-like cells compared with the tripodna and tetrapodna, suggesting that the partly loosened DNA in polypodna with many arms is advantageous for exposing the immunostimulatory sequences of the polypodna.

Enhancement of immobilized lipase activity by design of polymer brushes on a hollow fiber membrane.

A polymer brush possessing aminoethanol (AE) functional groups for lipase immobilization was grafted onto a hollow fiber membrane by radiation-induced graft polymerization. Almost the AE groups-grafted polymer brushes unfold through positive charge repulsion between the AE groups, enabling multi-layer immobilization of lipase. The hydroxyl groups in AE can also retain water molecules around hydrophilic part of the lipase. In this study, we controlled the length and density of the polymer brushes consisting of the glycidyl methacrylate (GMA) by changing the concentration of GMA monomer during radiation-induced graft polymerization. Immobilized lipase showed the highest activity on the grafted membrane when 5 wt% of glycidyl methacrylate as monomer for the radiation-induced graft polymerization was used. Consequently high efficiency esterification (approximately 1600 mmol/h/g-membrane) was achieved in five-layer lipase on AE polymer brush than that in monolayer lipase on the polymer brush possessing only hydroxyl groups. Moreover, the polymer brush possessing AE functional groups for lipase immobilization maintained high activity on the reuse for several times.

Production and removal of superoxide anion radical by artificial metalloenzymes and redox-active metals.

Generation of reactive oxygen species is useful for various medical, engineering and agricultural purposes. These include clinical modulation of immunological mechanism, enhanced degradation of organic compounds released to the environments, removal of microorganisms for the hygienic purpose, and agricultural pest control; both directly acting against pathogenic microorganisms and indirectly via stimulation of plant defense mechanism represented by systemic acquired resistance and hypersensitive response. By aiming to develop a novel classes of artificial redox-active biocatalysts involved in production and/or removal of superoxide anion radicals, recent attempts for understanding and modification of natural catalytic proteins and functional DNA sequences of mammalian and plant origins are covered in this review article.

X-ray scattering from immunostimulatory tetrapod-shaped DNA in aqueous solution to explore its biological activity-conformation relationship.

We carried out synchrotron X-ray scattering experiments from four DNA supermolecules designed to form tetrapod shapes; these supermolecules had different sequences but identical numbers of total base pairs, and each contained an immunostimulatory CpG motif. We confirmed that the supermolecules did indeed form the expected tetrapod shape. The sample that had the largest radius of gyration (Rg) induced the most cytokine secretion from cultured immune cells. Structural analysis in combination with a rigid tetrapod model and an atomic scale DNA model revealed that the larger Rg can be ascribed to dissociation of the DNA double strands in the central connecting portion of the DNA tetrapod. This finding suggests that the biological activity is related to the ease with which single DNA strands can be formed.

Synthesis and characterization of a calix[4]arene amphiphilie bearing cysteine and uniform Au nanoparticle formation templated by its four cysteine moieties.

A novel calix[4]arene amphiphilic molecule, denoted by CCaL3, was synthesized and found to form a spherical micelle consisting of 12 molecules at low pH in aqueous solution. Furthermore, uniform Au nanoparticles with 2.0 nm in diameter were synthesized in aqueous solution on the template consisting of the four cysteines of the upper rim of CCaL3. Asymmetric field flow fractionation coupled with light scattering showed that there was no dispersity in the CCaL3 micellar aggregation number. When AuCl4(-) ions were added into the CCaL3 micelle solution, induced circular dichroism (ICD) appeared, indicating appearance of the structural chirality of the CCaL3/AuCl4(-) complex. A combination of electron microscopy and small-angle X-ray scattering showed that helically coiled bilayer sheets were formed upon addition of AuCl4(-). Subsequent reduction with the amine of cysteine moieties led to uniform Au nanoparticles formation with 2.0 nm in diameter on the micellar plate surface. The nanoparticle size was almost equal to the size of cavity constructed by the four cysteines on the calix[4]arene upper rim, indicating that the growth of Au nanoparticles was spatially controlled by the host-guest interaction between the cysteines and Au.

A bimolecular micelle constructed from amphiphilic pillar[5]arene molecules.

An amphiphilic macrocycle based on pillar[5]arene with polar lysine head groups spontaneously self-assembles into a bimolecular micelle in water. This self-assembled structure was characterized by small angle X-ray scattering (SAXS), field flow fluctuation coupled with multi-angle light scattering (FFF-MALS) and atomic force microscopy (AFM). The self-assembly of amphiphilic pillar[5]arene into dimeric spherical micelles represents a new molecular architecture for micelle formation.

ß-1,3-D-glucan schizophyllan/poly(dA) triple-helical complex in dilute solution.

A certain length of poly(deoxyadenylic acid) (dA(X)) can form a novel complex with ß-1,3-D-glucan schizophyllan (SPG) with a stoichiometric composition of one dA binding two main chain glucoses. We measured dilute solution properties for the complex with light and small-angle X-ray scattering as well as intrinsic viscosity and found that the complex behaves as a semiflexible rod without branching or cross-linking. We analyzed the data with the wormlike cylinder model, and the chain dimensions and the persistence length for the complexes were consistently determined. The chain flexibility was reduced to almost 25% upon complexation for dA/SPG and to 15% for S-dA/SPG, where S-dA denotes the phosphorothioated DNA analogue. The changes in the molar mass per unit length and the diameter indicated that the helix was elongated or stretched along the axis direction upon the complexation.

Molecular dynamics studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution.

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.

DNA binding of tilorone: 1H NMR and calorimetric studies of the intercalation.

The fluorene derivative tilorone has received great attention as a DNA intercalator and has been widely recognized as an inducer of interferon. The biological activity of tilorone is known to be related to its binding mode with DNA; however, few structural and thermodynamic studies have elaborated on this issue. This paper presents two-dimensional (2-D) NMR and isothermal titration calorimetry (ITC) for the tilorone/DNA complex, coupled with circular dichroism (CD) spectroscopy and viscosity measurements. NMR investigation suggests that tilorone binds to DNA through intercalation, showing greater affinity for insertion between AT base pairs than between CG pairs. CD spectral changes were observed for T/B (tilorone/DNA base pair molar ratio) ratios greater than the stoichiometric ratio generally expected for intercalators (i.e., T/B = 0.5, according to the neighbor-exclusion principle). However, there was a clear plateau in the CD intensity between T/B < 0.35 and T/B > 0.45. From comparison with NMR and other measurements, we postulate that CD changes below the plateau should be related to the intercalation and the latter to electrostatic interactions and nonspecific bindings. ITC data showed that DeltaH < -TDeltaS < 0, which indicated that tilorone/DNA binding is enthalpy controlled. The magnitude of Kb (the binding constant) was of the same order as that of ethidium bromide. The stoichiometric number, obtained from ITC, CD, and UV data, implied a relatively smaller value (0.28-0.35) than that of the neighbor-exclusion principle. This is because side chains located in the groove disrupt further intercalation to the adjacent sites.