Kiyomi Matsuda

Water purification through bioconversion of phenol compounds by tyrosinase and chemical adsorption by chitosan beads.

Enzymatic removal of various phenol compounds from artificial wastewater was undertaken by the combined use of mushroom tyrosinase (EC 1.14.18.1) and chitosan beads as function of pH value, temperature, tyrosinase dose, and hydrogen peroxide‐to‐substrate ratio. Chitosan film incubated in a p‐crersol+tyrosinase mixture had the main peaks at 400–470 nm assigned to chemically adsorbed quinone derivatives, which increased over the immersion time. These results indicate that removal of phenol compounds is caused by their tyrosinase‐catalyzed oxidation to the corresponding quinone derivatives and the subsequent chemical adsorption on the chitosan film. The optimum conditions for quinone adsorption were determined to be pH 7 and 45 °C for p‐cresol. Some alkyl‐substituted phenol compounds were removed by adsorption of quinone derivatives enzymatically generated on the chitosan beads, and the % removal for p‐cresol, 4‐ethylphenol, 4‐n‐propylphenol, 4‐n‐butylphenol, and p‐chlorophenol went up to 93%. In addition, 4‐tert‐butylphenol underwent tyrosinase‐catalyzed oxidation in the presence of hydrogen peroxide. This procedure was applicable to removal of chlorophenols and alkyl‐substituted phenols.

Removal of p-Alkylphenols from Aqueous Solutions by Combined Use of Mushroom Tyrosinase and Chitosan Beads

Enzymatic removal of p-alkylphenols from aqueous solutions was investigated through the two-step approach, the quinone conversion of p-alkylphenols with mushroom tyrosinase (EC 1.14.18.1) and the subsequent adsorption of quinone derivatives enzymatically generated on chitosan beads at pH 7.0 and 45 °C as the optimum conditions. This technique is quite effective for removal of various p-alkylphenols from an aqueous solution. The % removal values of 97–100% were obtained for p-n-alkylphenols with carbon chain lengths of 5 to 9. In addition, removal of other p-alkylphenols was enhanced by increasing either the tyrosinase concentration or the amount of added chitosan beads, and their % removal values reached >93 except for 4-tert-pentylphenol. This technique was also applicable to remove 4-n-octylphenol (4NOP) and 4-n-nonylphenol (4NNP) as suspected endocrine disrupting chemicals. The reaction of quinone derivatives enzymatically generated with the chitosan’s amino groups was confirmed by the appearance of peaks for UV–visible spectrum measurements of the chitosan films incubated in the p-alkylphenol and tyrosinase mixture solutions. In addition, 4-tert-pentylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide.

Determination of optimum process parameters for peroxidase‐catalysed treatment of bisphenol A and application to the removal of bisphenol derivatives

Systematic investigations were carried out to determine the optimum process parameters such as the hydrogen peroxide (H2O2) concentration, concentration and molar mass of poly(ethylene glycol) (PEG) as an additive, pH value, temperature and enzyme dose for treatment of bisphenol A (BPA) with horseradish peroxidase (HRP). The HRP‐catalysed treatment of BPA was effectively enhanced by adding PEG, and BPA was completely converted into phenoxy radicals by HRP dose of 0.10 U/cm3. The optimum conditions for HRP‐catalysed treatment of BPA at 0.3 mM was determined to be 0.3 mM for H2O2 and 0.10 mg/cm3 for PEG with a molar mass of 1.0 × 104 in a pH 6.0 buffer at 30 °C. Different kinds of bisphenol derivatives were completely or effectively treated by HRP under the optimum conditions determined for treatment of BPA, although the HRP dose was further increased as necessary for some of them. The aggregation of water‐insoluble oligomers generated by the enzymatic radicalization and radical coupling reaction was enhanced by decreasing the pH values to 4.0 with HCl after the enzymatic treatment, and BPA and bisphenol derivatives were removed from aqueous solutions by filtering out the oligomer precipitates.

Design and Characterization of Endosomal‐pH‐Responsive Coiled Coils for Constructing an Artificial Membrane Fusion System

A weakly acidic pH-responsive polypeptide is believed to have the potential for an endosome escape function in a polypeptide-triggered delivery system. For constructing a membrane fusion device with pH-responsiveness, we have designed novel polypeptides that are capable of forming an α2 coiled coil structure. Circular dichroism spectroscopy reveals that a polypeptide, AP-LZ(EH5), with a Glu and His salt-bridge pair at a staggered position in the hydrophobic core forms a stable coiled coil structure only at endosomal pH values (pH 5.0 to 5.5). On the basis of their endosomal-pH responsiveness, a boronic acid/polypeptide conjugate (BA-H5-St) was also designed as a pilot molecule to construct a pH-responsive, one-way membrane fusion system with a sugarlike compound (phosphatidylinositol: PI)-containing liposome as a target. Membrane fusion behavior was characterized by lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays. These studies reveal that membrane fusion is clearly observed when the pH of the experimental system is changed from 7.4 (physiological condition) to 5.0 (endosomal condition).

Influence of Position of Substituent Groups on Removal of Chlorophenols and Cresols by Horseradish Peroxidase and Determination of Optimum Conditions

Enzymatic treatment of o-, m-, and p-chlorophenols and o-, m-, and p-cresols from artificial wastewater was undertaken through the enzymatic conversion into the corresponding phenoxy radicals with horseradish peroxidase (HRP) and nonenzymatic radical coupling reaction. The concentration of chlorophenols and cresols decreased sharply over the reaction time and water-insoluble oligomer precipitates were generated. The optimum conditions were determined to be the H2O2 concentration of 2.5 mM and poly(ethylene glycol) with molecular mass of 1.0×104 (10K-PEG) of 0.10 mg/cm3 at 30 °C for treatment of p-chlorophenol at 2.5 mM. The optimum pH values depended on the relative position of a chlorine atom for chlorophenols and on a methyl group for cresols. Concentrations of HRP and 10K-PEG were increased to 1.0 U/cm3 and 1.0 mg/cm3 respectively to treat m-chlorophenol highly. For o-chlorophenol, a decrease in the pH value to 3.0 after the enzymatic treatment led to the enhancement of the aggregation of oligomer precipitates. The % residual value for o-cresol effectively decreased by absorbing water-soluble intermediates on the chitosan films. These results indicate that chlorophenols and cresols were removed to a great degree by this technique, although the detailed procedure depended on the position of substituent groups of chlorophenols and cresols.

Construction of a pH-Responsive Artificial Membrane Fusion System by Using Designed Coiled-Coil Polypeptides

In many viruses, pH-responsive coiled-coil domains in the specific fusion proteins play important roles in membrane fusion and the infection of viruses into host cells. To investigate the relationship between the conformational change of the coiled coil and the fusion process, we have introduced a de novo designed polypeptide as a model system of the coiled-coil domain. This system enables the systematic study of the dynamics of pH-responsive coiled-coil polypeptide-membrane interactions. First, we designed and synthesized pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptides. Then the relationship between the pH-induced conformational change of the polypeptide and the membranes interactive properties was studied by physicochemical methods. Structural changes in the designed polypeptides were examined by means of circular dichroism measurements. And finally, the behavior of the membrane fusion was investigated by leakage of liposomal contents, turbidity analysis, dynamic light scattering, and lipid mixing experiments. Our data show that coiled-coil formation under acidic pH conditions enhances polypeptide-induced membrane fusion. The results in this study demonstrate that an artificial membrane fusion system can be constructed on a molecular level by the use of a pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptide.

Target-selective vesicle fusion system with pH-selectivity and responsiveness

The present paper reports on induction of target-selective liposomal vesicle fusion triggered by molecular recognition on a vesicle surface. Phosphatidylinositol (PI) having a sugar-like cyclic cys-diol structure was selected as a recognition target. Since diol sugars are abundant on cell surfaces, vesicle fusion systems based on the recognition of diol functionalities can be relevant for liposome-based drug delivery. Here, we design and synthesize a novel phenylboronic acid derivative with a tertiary amine group adjacent to the boron atom as a pilot molecule toward PI at a physiological condition. The pilot vesicle, or EggPC liposome containing this phenylboronic acid derivative causes selective membrane fusion toward a target liposomal vesicle containing sugar-like cyclic cys-diol structure at a physiological condition. From lipid mixing and inner-leaflet mixing assays, we demonstrate that the fusion event activated by inter-vesicular complex formation occurs rapidly. Furthermore, we also construct the target selective fusion system working only at the endosomal pH by the use of weakly acidic lipid, 1,2-dipalmitoyl-sn-glycero-3-succinate (DPGS) containing the pilot vesicle. The lipid mixing and inner-leaflet mixing assays make it clear that the vesicle fusion proceeds over pH range 5.0–5.5, whose range is upper than the pKa of the boronic acid moiety on cys-diol complexation and lower than the pKa of the carboxyl group of DPGS.

Tuning the enzymatic hydrolysis of biodegradable polyesters and its application to surface patterning

Biodegradable polyester surfaces with controlled enzymatic degradability were fabricated by simple UV–ozone and UV treatments. The surface modification was applied for the development of an enzymatic lithographic technique to produce a patterned soft interface with various architecture designs.

Target-Selective One-Way Membrane Fusion System Based on a pH-Responsive Coiled Coil Assembly at the Interface of Liposomal Vesicles

The coiled coil trimer structure is a common motif observed in membrane fusion processes of specific fusion proteins such as the hemagglutinin glycoprotein. The HA2 subunit in the hemagglutinin changes its conformation or geometry to be favorable to membrane fusion in response to endosomal weakly acidic pH. This pH responsiveness is indispensable to an artificial polypeptide-triggered delivery system as well as the membrane fusion reaction in biology. In this study, we have constructed an AAB-type coiled coil heteroassembled system that is sensitive to weakly acidic pH. The heterotrimer is formed from two kinds of polypeptides containing an Ala or a Trp residue at a hydrophobic a position, and it was observed that the Glu residue at the other a position induced an acidic pH-dependent conformational change. On the basis of this pH-responsive coiled coil heteroassembled system, a boronic acid coupled working polypeptide for the combination of an intervesicular complex with a sugarlike compound on the surface of the target liposome, and a supporting polypeptide for the construction of a pH-responsive heterotrimer with the working polypeptide were designed and synthesized. The process of membrane fusion was characterized by lipid-mixing, inner-leaflet lipid-mixing, and content-mixing assays. The target selective vesicle fusion is clearly observed at a weakly acidic pH, where the working polypeptides form a heterotrimeric coiled coil with the supporting polypeptides in a 1:2 binding stoichiometry and the surfaces between pilot and target vesicles come into close proximity to each other.

Design, Construction, and Characterization of High-Performance Membrane Fusion Devices with Target-Selectivity

Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system.