Self-assembly of Aeropyrum pernix bacilliform virus 1 (APBV1) major capsid protein and its application as building blocks for nanomaterials.

Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications.

Quantitative Analysis of Collective Migration by Single-Cell Tracking Aimed at Understanding Cancer Metastasis.

Metastasis is a major complication of cancer treatments. Studies of the migratory behavior of cells are needed to investigate and control metastasis. Metastasis is based on the epithelial-mesenchymal transition, in which epithelial cells acquire mesenchymal properties and the ability to leave the population to invade other regions of the body. In collective migration, highly migratory "leader" cells are found at the front of the cell population, as well as cells that "follow" these leader cells. However, the interactions between these cells are not well understood. We examined the migration properties of leader-follower cells during collective migration at the single-cell level. Different mixed ratios of "leader" and "follower" cell populations were compared. Collective migration was quantitatively analyzed from two perspectives: cell migration within the colony and migration of the entire colony. Analysis of the effect of the cell mixing ratio on migration behavior showed that a small number of highly migratory cells enhanced some of the migratory properties of other cells. The results provide useful insights into the cellular interactions in collective cell migration of cancer cell invasion.

A high performance nanocomposite based bioanode for biofuel cell and biosensor application.

Herein, to improve the current density and sensitivity for biofuel cell and glucose sensing application, a bioanode based on redox polymer (PEI-Fc) binding polydopamine (PDA) coated MWCNTs (PEI-Fc/PDA/MWCNTs) nanocomposite and glucose oxidase (GOD) was fabricated. PDA/MWCNTs nanocomposite was prepared by spontaneous self-polymerization of dopamine on MWCNTs surface and the PEI-Fc/PDA/MWCNTs nanocomposite was prepared by a simple self-assembly method. The PEI-Fc/PDA/MWCNTs nanocomposite and the resulting bioanode were fully characterized. A maximum current density of 0.73 mA cm-2 at the resulting bioanode was obtained by linear sweep voltammetry (LSV) at the scan rate of 50 mV s-1 with 20 mM glucose concentration. Moreover, a linear range up to 4 mM, a high sensitivity of 57.2 µA mM-1 cm-2, a fast response time reaching 95% of the steady current (2 s) and a low limit of detection (0.024 mM) were achieved. The amperometric method demonstrated both the sensitivity and the stability of the bioanode for glucose-sensing was improved by the employed PDA layer. Finally, the biosensor was used for glucose detection in human serum samples showing good recoveries. This study proposed an excellent functional material prepared by a facile self-assembled method for applying in biofuel cells and second-generation biosensors.

Electrochemical characteristics of a gold nanoparticle-modified controlled enzyme-electrode contact junction electrode.

Biodevices in which biomolecules such as enzymes and antibodies are immobilized on the surface of electrode materials are capable of converting chemical energy into electrical energy, and are expected to contribute to solving energy problems and developing medical measurements especially as biobatteries and biosensors. Device performance depends on the interface formed between the biomolecule layer and electrode material, and the interface is required to simultaneously achieve a highly efficient enzymatic reaction and electron transfer. However, when enzymes were immobilized on a material surface, the enzymes undergoes a structural change due to the interaction between the enzyme and the electrode surface, making it difficult to maximize the function of the enzyme molecule on the material surface. In this study, we postulate that the structural change of the enzyme would be reduced and the electrochemical performance improved by making the contact area between the enzyme and the electrode extremely small and adsorbing it as a point. Therefore, we aimed to develop a high-power biodevice that retains enzyme structure and activity by interposing gold nanoparticles (AuNPs) between the enzyme and the electrode. The enzymatic and electrochemical properties of pyrroloquinoline quinone-dependent glucose dehydrogenase adsorbed on AuNPs of 5-40 nm diameter were investigated. We found that the characteristics differed among the particles, and the enzyme adsorbed on 20 nm AuNPs showed the best electrochemical characteristics.

One-Step Surface Immobilization of Protein A on Hydrogel Nanofibers by Core-Shell Electrospinning for Capturing Antibodies.

Nanofibers (NFs) are potential candidates as filter materials for affinity separation owing to their high liquid permeability based on their high porosity. Multiple and complex processes were conventionally performed to immobilize proteins for modifying NF surfaces. A simple method must be developed to immobilize proteins without impairing their biological activity. Herein, we succeeded in fabricating NFs with a core of cellulose acetate and a shell of hydrophilic polyvinyl alcohol immobilized with staphylococcal recombinant protein A by a one-step process based on core-shell electrospinning. A total of 12.9 mg/cm3 of antibody was captured in the fiber shell through high affinity with protein A immobilized in an aqueous environment of the hydrogel. The maximum adsorption site and dissociation constant evaluated by the Langmuir model were 87.8 µg and 1.37 µmol/L, respectively. The fiber sheet withstood triplicate use. Thus, our NF exhibited high potential as a material for membrane chromatography.

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection.

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (ß)), forming a heterohexameric structure (α3ß3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5-8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme's surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

Highly Efficient Multi-Step Oxidation Bioanode Using Microfluidic Channels.

With the rapid decline of fossil fuels, various types of biofuel cells (BFCs) are being developed as an alternative energy source. BFCs based on multi-enzyme cascade reactions are utilized to extract more electrons from substrates. Thus, more power density is obtained from a single molucule of substrate. In the present study, a bioanode that could extract six electrons from a single molecule of L-proline via a three-enzyme cascade reaction was developed and investigated for its possible use in BFCs. These enzymes were immobilized on the electrode to ensure highly efficient electron transfer. Then, oriented immobilization of enzymes was achieved using two types of self-assembled monolayers (SAMs). In addition, a microfluidic system was incorporated to achieve efficient electron transfer. The microfluidic system, in which the electrodes were arranged in a tooth-shaped comb, allowed for substrates to be supplied continuously to the cascade, which resulted in smooth electron transfer. Finally, we developed a high-performance bioanode which resulted in the accumulation of higher current density compared to that of a gold disc electrode (205.8 µA cm-2: approximately 187 times higher). This presents an opportunity for using the bioanode to develop high-performance BFCs in the future.

Formation of one-dimensional arrays of nanoparticles using segmented polyurethane nanofibers prepared by electrospinning.

The number density and the arrangement of metal nanoparticles in composite materials have a significant effect on their performance and hence their suitability for use in sensors and devices. Forming one-dimensional arrays of metal nanoparticles is one way of controlling their number density and arrangement in the devices. In this study, we fabricated one-dimensional arrays of gold nanoparticles by adsorbing them on linearly distributed hard segments present on the surfaces of segmented polyurethane nanofibers, which were produced by electrospinning under a stretching force. The length of the one-dimensional array was approximately 500 nm. Furthermore, the interparticle distance was almost constant at approximately 14 nm. Thus, the proposed method is suitable for fabricating one-dimensional arrays of metal nanoparticles with high precision.

Effects of multicopper oxidase orientation in multiwalled carbon nanotube biocathodes on direct electron transfer.

In this study, multicopper oxidase (MCO) was immobilized on multiwalled carbon nanotubes (MWCNTs) at two different orientations, and the electrochemical properties of the resulting cathodes were investigated. Using N- or C-terminal His-tagged MCO and MWCNTs, we constructed two types of cathodes. We assumed that the distance between the type 1 (T1)Cu of the C-terminal His-tagged MCO and the MWCNT surface was lesser than that between the T1Cu of the N-terminal His-tagged MCO and the MWCNT surface. In addition, in the C-terminal His-tagged MCO, T1Cu was expected to be closer to the MWCNT surface than the type 2/type 3 Cu site. The current density of the modified electrode with a C-terminal His-tagged MCO immobilized on an MWCNT surface was 1.3-fold higher than that of the electrode with an N-terminal His-tagged MCO immobilized on an MWCNT surface. In addition, the amount of H2 O2 produced by the N-terminal His-tagged MCO immobilized MWCNT modified electrodes was 2.3-fold higher than that produced by the C-terminal His-tagged MCO immobilized MWCNT electrodes. In direct electron transfer (DET)-type biocathodes, both the MCO orientation and the distance between the T1Cu of MCO and the electrode surface are important. The authors succeeded in constructing highly efficient DET-type electrodes.

Development of Biofuel Cell Using a Complex of Highly Oriented Immobilized His-Tagged Enzyme and Carbon Nanotube Surface Through a Pyrene Derivative.

For increasing the output of biofuel cells, increasing the cooperation between enzyme reaction and electron transfer on the electrode surface is essential. Highly oriented immobilization of enzymes onto a carbon nanotube (CNT) with a large specific surface area and excellent conductivity would increase the potential for their application as biosensors and biofuel cells, by utilizing the electron transfer between the electrode-molecular layer. In this study, we prepared a CNT-enzyme complex with highly oriented immobilization of enzyme onto the CNT surface. The complex showed excellent electrical characteristics, and could be used to develop biodevices that enable efficient electron transfer. Multi-walled carbon nanotubes (MWCNT) were dispersed by pyrene butyric acid N-hydroxysuccinimide ester, and then N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) and NiCl2 were added to modify the NTA-Ni2+ complex on the CNT surface. Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) was immobilized on the CNT surface through a genetically introduced His-tag. Formation of the MWCNT-enzyme complex was confirmed by monitoring the catalytic current electrochemically to indicate the enzymatic activity. PQQ-GDH was also immobilized onto a highly ordered pyrolytic graphite surface using a similar process, and the enzyme monolayer was visualized by atomic force microscopy to confirm its structural properties. A biofuel cell was constructed using the prepared CNT-enzyme complex and output evaluation was carried out. As a result, an output of 32 µW/cm² could be obtained without mediators.

Construction of a novel bioanode for amino acid powered fuel cells through an artificial enzyme cascade pathway.

OBJECTIVE: The construction of a novel bioanode based on L-proline oxidation using a cascade reaction pathway comprised of thermostable dehydrogenases. RESULTS: A novel multi-enzymatic cascade pathway, containing four kinds of dehydrogenases from thermophiles (dye-linked L-proline dehydrogenase, nicotinamide adenine dinucleotide (NAD)-dependent Δ1-pyrroline-5-carboxylate dehydrogenase, NAD-dependent L-glutamate dehydrogenase and dye-linked NADH dehydrogenase), was designed for the generation of six-electrons from one molecule of L-proline. The current density of the four-dehydrogenase-immobilized electrode, with a voltage of + 450 mV (relative to that of Ag/AgCl), was 226.8 µA/cm2 in the presence of 10 mM L-proline and 0.5 mM ferrocene carboxylate at 50 °C. This value was 4.2-fold higher than that of a similar electrode containing a single dehydrogenase. In addition, about 54% of the initial current in the multi-enzyme cascade bioanode was maintained even after 15 days. CONCLUSIONS: Efficient deep oxidation of L-proline by multiple-enzyme cascade reactions was achieved in our designed electrode. The multi-enzyme cascade bioanode, which was built using thermophilic dehydrogenases, showed high durability at room temperature. The long-term stability of the bioanode indicates that it shows great potential for applications as a long-lived enzymatic fuel cell.

Enzymological characteristics of a novel archaeal dye-linked D-lactate dehydrogenase showing loose binding of FAD.

A gene-encoding a dye-linked D-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity. To date, four types of Dye-DLDH have been identified in hyperthermophilic archaea (in Aeropyrum pernix, Sulfolobus tokodaii, Archaeoglobus fulgidus, and Candidatus Caldiarchaeum subterraneum). The amino acid sequence of T. tenax Dye-DLDH showed the highest similarity (45%) to A. pernix Dye-DLDH, but neither contained a known FAD-binding motif. Nonetheless, both homologs required FAD for enzymatic activity, suggesting that FAD binds loosely to the enzyme and is easily released unlike in other Dye-DLDHs. Our findings indicate that Dye-DLDHs from T. tenax and A. pernix are a novel type of Dye-DLDH characterized by loose binding of FAD.

A L-proline/O2 biofuel cell using L-proline dehydrogenase (LPDH) from Aeropyrum pernix.

To utilize amino acids from food waste as an energy source, L-proline/O2 biofuel cell was constructed using a stable enzyme from hyperthermophilic archaeon for long-term operation. On the anode, the electrocatalytic oxidation of L-proline by L-proline dehydrogenase from Aeropyrum pernix was observed in the presence of ferrocenecarboxylic acid as mediator. On the cathode, electrocatalytic oxygen reduction was detected. Ketjenblack modification of carbon cloth substrate increased the current density due to increased laccase loading and enhanced electron transfer reaction. The biofuel cell using these electrodes achieved a current density of 6.00 µA/cm2. We successfully constructed the first biofuel cell that generates power from L-proline.

Design of a multi-enzyme reaction on an electrode surface for an L-glutamate biofuel anode.

OBJECTIVES: To design and construct a novel bio-anode electrode based on the oxidation of glutamic acid to produce 2-oxoglutarate, generating two electrons from NADH. RESULTS: Efficient enzyme reaction and electron transfer were observed owing to immobilization of the two enzymes using a mixed self-assembled monolayer. The ratio of the immobilized enzymes was an important factor affecting the efficiency of the system; thus, we quantified the amounts of immobilized enzyme using a quartz crystal microbalance to further evaluate the electrochemical reaction. The electrochemical reaction proceeded efficiently when approximately equimolar amounts of the enzyme were on the electrode. The largest oxidation peak current increase (171 nA) was observed under these conditions. CONCLUSION: Efficient multi-enzyme reaction on the electrode surface has been achieved which is applicable for biofuel cell application.

Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors.

Dye-linked D-amino acid dehydrogenase from the thermophilic bacterium Rhodothermus marinus JCM9785: characteristics and role in trans-4-hydroxy-L-proline catabolism.

A gene from the thermophilic Gram-negative bacterium Rhodothermus marinus JCM9785, encoding a dye-linked D-amino acid dehydrogenase homologue, was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was a highly thermostable dye-linked D-amino acid dehydrogenase that retained more than 80% of its activity after incubation for 10 min at up to 70 °C. When enzyme-catalyzed dehydrogenation of several D-amino acids was carried out using 2,6-dichloroindophenol as the electron acceptor, D-phenylalanine was the most preferable substrate among the D-amino acids tested. Immediately upstream of the dye-linked D-amino acid dehydrogenase gene (dadh) was a gene encoding a 4-hydroxyproline 2-epimerase homologue (hypE). That gene was successfully expressed in E. coli, and the gene product exhibited strong 4-hydroxyproline 2-epimerase activity. Reverse transcription PCR and quantitative real-time PCR showed that the six genes containing the dadh and hypE genes were arranged in an operon and were required for catabolism of trans-4-hydroxy-L-proline in R. marinus. This is the first description of a dye-linked D-amino acid dehydrogenase (Dye-DADH) with broad substrate specificity involved in trans-4-hydroxy-L-proline catabolism.

Construction of a biocathode using the multicopper oxidase from the hyperthermophilic archaeon, Pyrobaculum aerophilum: towards a long-life biobattery.

OBJECTIVES: The life of biobatteries remains an issue due to loss of enzyme activity over time. In this study, we sought to develop a biobattery with a long life using a hyperthermophilic enzyme. RESULTS: We hypothesized that use of such hyperthermophilic enzymes would allow for the biofuel cells to have a long battery life. Using pyrroloquinoline quinone-glucose dehydrogenase and the multicopper oxidase from Pyrobaculum aerophilum, we constructed an anode and cathode. The maximum output was 11 µW at 0.2 V, and the stability of the both electrode was maintained at 70 % after 14 days. CONCLUSION: The biofuel cells that use hyperthermophilic enzymes may prolong their life.

Evaluation of chitosan-binding amino acid residues of chitosanase from Paenibacillus fukuinensis.

Chitosan oligosaccharides longer than a hexamer have higher bioactivity than polymer or shorter oligosaccharides, such as the monomer or dimer. In our previous work, we generated Paenibacillus fukuinensis chitosanase-displaying yeast using yeast cell surface displaying system and demonstrated the catalytic base. Here we investigated the specific function of putative four amino acid residues Trp159, Trp228, Tyr311, and Phe406 engaged in substrate binding. Using this system, we generated chitosanase mutants in which the four amino acid residues were substituted with Ala and the chitosanase activity assay and HPLC analysis were performed. Based on these results, we demonstrated that Trp159 and Phe406 were critical for hydrolyzing both polymer and oligosaccharide, and Trp228 and Tyr311 were especially important for binding to oligosaccharide, such as the chitosan-hexamer, not to the chitosan polymer. From the results, we suggested the possibility of the effective strategy for designing useful mutants that produce chitosan oligosaccharides holding higher bioactivity.

Atomic force microscopy visualization of hard segment alignment in stretched polyurethane nanofibers prepared by electrospinning.

Molecular-level orientation within nanofibers has been attracting attention as a tool for controlling and designing highly functional nanofibers. In this study, we used atomic force microscopy to visualize the phase separation between soft and hard segments on a polyurethane (PU) nanofiber surface prepared by electrospinning. Furthermore, the stretched nanofibers prepared with a high-speed rotating collector were found to have a different phase distribution in the phase-separated structure, with the hard segment domains aligned to the fiber axis. In contrast, unstretched PU nanofibers prepared without rotation were observed to have nonuniformly distributed segments. These results indicate that the application of an intense elongation force along the nanofiber axis with a rotating mandrel collector changed the distribution of segment alignments.

Selective miR-21 detection technology based on photocrosslinkable artificial nucleic acid-modified magnetic particles and hybridization chain reaction.

Recently, microRNA (miRNA) detection in blood has attracted attention as a new early detection technology for cancer. The extraction of target miRNA is a necessary preliminary step for detection; however, currently, most extraction methods extract all RNA from the blood, which limits the detection selectivity. Therefore, a method for the selective extraction and detection of target miRNA from blood is very important. In this study, we utilized photocrosslinkable artificial nucleic acids and the hybridization chain reaction (HCR) in an attempt to improve upon the current standard method RT-qPCR, which is hampered by problems with primer design and enzymatic amplification. By introducing photocrosslinkable artificial nucleic acids to oligonucleotide probes modified with magnetic particles with a sequence complementary to that of the target miRNA and irradiating them with light, covalent bonds were formed between the target miRNA and the oligonucleotide probes. These tight covalent bonds enabled the capture of miRNA in blood, and intensive washing ensured that only the target miRNA were extracted. After extraction, two types of DNA (H1 and H2) modified with fluorescent dyes were added and the fluorescence signals were amplified by the HCR in the presence of the target miRNA bound to the photocrosslinkable artificial nucleic acids, allowing for isothermal and enzyme-free miRNA detection. The novel method is suitable for selective miRNA detection in real blood samples. Because the reaction proceeds isothermally and no specialized equipment is used for washing, this detection technology is simple and selective and suitable for application to point-of-care technology using microfluidic devices.