A variety of positive/negative selection systems have been exploited as genome engineering tools and screening platforms for genetic switches. While numerous positive-selection systems are available, only a handful of negative-selection systems are useful for such applications. We previously reported a powerful negative-selection system using herpes simplex virus thymidine kinase (HsvTK) and the mutagenic nucleoside analog 6-(β-d-2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido [4,5-c][1,2] oxazin-7-one (dP). Upon addition of 1000 nM dP, cells expressing HsvTK quickly die, with unprecedented efficacy. However, this selection procedure elevates the spontaneous mutation rate of the host cells by 10-fold due to the mutagenic nature of dP. To decrease the operative concentration of dP required for negative selection, we systematically created the strains of Escherichia coli either by removing or overexpressing genes involved in DNA/RNA metabolism. We found that over-expression of NupC and NupG (nucleoside uptake-related inner membrane transporters), Tsx (outer membrane transporter), NdK (nucleotide kinase) sensitized E. coli cells to dP. Simultaneous overexpression of these three genes (ndk-nupC-tsx) significantly improved the dP-sensitivity of E. coli, lowering the necessary operative concentration of dP for negative selection by 10-fold. This enabled robust and selective elimination of strains harboring chromosomally-encoded hsvtk simply by adding as low as 100 nM dP, which causes only a modest increase in the spontaneous mutation frequency as compared to the cells without hsvtk.

Heterologous production of a useful carotenoid astaxanthin was achieved in a cyanobacterium Synechocystis sp. PCC 6803 with the aid of marine bacterial genes. Astaxanthin and its intermediates emerged at high levels, whereas β-carotene and zeaxanthin disappeared in the strain. Total carotenoid accumulation was nearly two fold compared with wild type. The astaxanthin-producing strain was capable of only growing heterotrophically, which was likely due to the absence of β-carotene. Further enhanced accumulation was pursued by gene overexpression for possible rate-limiting steps in the biosynthesis pathway.

Stringency (low leak) is one of the most important specifications required for genetic circuits and induction systems, but it is challenging to evolve without sacrificing the maximum output level. This problem also comes from the absence of truly tunable negative selection methods. This paper reports that stringently switching variants can sometimes emerge with surprising frequency upon mutations. We randomly mutated the previously generated leaky variants of LuxR, the quorum-sensing transcription activator from Vibrio fischeri, to restore the stringency. We found as much as 10-20% of the entire population exhibited significantly improved signal-to-noise ratios compared with their parents. This indicated that these mutants arose by the loss of folding capability by accumulating destabilizing mutations, not by introducing rare adaptive mutations, thereby becoming AHL-dependent folders. Only four rounds of mutagenesis and ON-state selection resulted in the domination of the entire population by the improved variants with low leak, without direct selection pressure for stringency. With this surprising frequency, conversion into the "ligand-addicted folders" should be one of the prevailing modes of evolving stringency both in the laboratory and in nature, and the workflow described here provides a rapid and versatile method of improving the signal-to-noise ratio of various genetic switches.

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

Carotenoids are structurally diverse pigments with various important biological functions. There has been a large interest in the search for novel carotenoid structures, since only a slight structural changes can result in a drastic difference in their biological functions. Carotenoid-modifying enzymes show remarkable substrate promiscuity, allowing rapid access to a vast set of novel carotenoids by combinatorial biosynthesis. We previously constructed a nonnatural carotenoid biosynthetic pathway in Escherichia coli that can produce C50 carotenoids having a longer chain than their natural C40 counterparts. In this study, a carotenoid 2,2'-hydroxylase (crtG) from Brevundimonas sp. SD212 was coexpressed together with our laboratory-engineered C50-zeaxanthin and C50-astaxanthin biosynthetic pathways. We identified six novel nonnatural C50-xanthophylls, namely, C50-nostoxanthin, C50-caloxanthin, C50-adonixanthin, C50-4-ketonostoxanthin, C50-2-hydroxyastaxanthin, and C50-2,2'-dihydroxyastaxanthin.

A weak acid cation exchange fiber used for large-scale antibody purification was prepared by radiation-induced graft polymerization of acrylic acid (AA) onto a commercially available nylon-6 fiber. The AA-grafted fiber was post-treated by conditioning in NaOH aqueous solution at 298 K followed by immersion in water at 353 K to increase the protein binding capacity of the cation exchange fiber. A 2 g/L lysozyme solution buffered at pH 6.0 flowed through a column charged with a treated or untreated AA-grafted fiber at a space velocity of 20 h(-1). The 10% dynamic binding capacity of the column charged with the former fiber of 200 mg/mL-column was 5.9-fold that of the column charged with the latter fiber, and 2.3-fold that (88 mg/mL-column) of a column charged with CM Sepharose (TM) Fast Flow (GE Healthcare).

Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C50 carotenoids by engineering a C30-carotenoid backbone synthase (CrtM) from Staphylococcus aureus. In the present work, we conducted a series of experiments to engineer C60 carotenoid pathways. Stepwise introduction of cavity-expanding mutations together with stabilizing mutations progressively shifted the product size specificity of CrtM toward efficient synthases for C60 carotenoids. By coexpressing these CrtM variants with hexaprenyl diphosphate synthase, we observed that C60-phytoene accumulated together with a small amount of C65-phytoene, which is the largest carotenoid biosynthesized to date. Although these carotenoids failed to serve as a substrate for carotene desaturases, the C25-half of the C55-phytoene was accepted by the variant of phytoene desaturase CrtI, leading to accumulation of the largest carotenoid-based pigments. Continuing effort should further expand the scope of carotenoids, which are promising components for various biological (light-harvesting, antioxidant, and communicating) and nonbiological (photovoltaic, photonic, and field-effect transistor) systems.

While the majority of the natural carotenoid pigments are based on 40-carbon (C40) skeleton, some carotenoids from bacteria have larger C50 skeleton, biosynthesized by attaching two isoprene units (C5) to both sides of the C40 carotenoid pigment lycopene. Subsequent cyclization reactions result in the production of C50 carotenoids with diverse and unique skeletal structures. To produce even larger nonnatural novel carotenoids with C50 + C5 + C5 = C60 skeletons, we systematically coexpressed natural C50 carotenoid biosynthetic enzymes (lycopene C5-elongases and C50-cyclases) from various bacterial sources together with the laboratory-engineered nonnatural C50-lycopene pathway in Escherichia coli. Among the tested enzymes, the elongases and cyclases from Micrococcus luteus exhibited significant activity toward C50-lycopene, and yielded the novel carotenoids C60-flavuxanthin and C60-sarcinaxanthin. Moreover, coexpression of M. luteus elongase with Corynebacterium cyclase resulted in the production of C60-sarcinaxanthin, C60-sarprenoxanthin, and C60-decaprenoxanthin.

Substrate tolerance of bacterial cyclases has been demonstrated in various contexts, but little is known about that of plant cyclases. Here, we tested two plant ε-cyclases to convert C50-lycopene, which we previously established by rounds of directed evolution. Unlike bacterial β-cyclases, two-end cyclase from lettuce exhibited complete specificity against this molecule, indicating that this enzyme has some mechanism that exerts size-specificity. Arabidopsis one-end cyclase At-y2 showed detectable activity to C50-lycopene. Interestingly, we found that it functions as a two-end cyclase in a C50 context. Based on this observation, a possible model for substrate discrimination of this enzyme is proposed.

The cellular activities of gymnosperms monoterpene synthases are largely compromised due to their requirement for manganese, which is deficient in microbial cells. Through site-saturation mutagenesis of the residue adjacent to metal-binding glutamate, we found that pinene synthase is highly mutable at this position yet drastically alter their metal binding preference, thereby quickly improving the cellular performance in heterologous hosts.

An efficient method for rare metal recovery from environmental water and urban mines is in high demand. Toward rapid and high-resolution rare metal ion separation, a novel bis(2-ethylhexyl) phosphate (HDEHP)-impregnated graft-type particle as a filler for a chromatography column is proposed. To achieve rapid and high-resolution separation, a convection-flow-aided elution mode is required. The combination of 35 μm non-porous particles and a polymer-brush-rich particle structure minimizes the distance from metal ion binding sites to the convection flow in the column, resulting in minimized diffusional mass transfer resistance and the convection-flow-aided elution mode. The HDEHP-impregnated graft-type non-porous-particle-packed cartridge developed in this study exhibited a higher separation performance for model rare metals, neodymium (III) and dysprosium (III) ions, and a narrower peak at a higher linear velocity, than those of previous HDEHP-impregnated fiber-packed and commercially available Lewatit® VP OC 1026-packed cartridges.

<p>For the removal of catechin from green-tea extracts, N-vinylpyrrolidone-grafted fiber was prepared by radiation-induced graft polymerization of N-vinylpyrrolidone (NVP) onto a commercially available 6-nylon fiber. The adsorption isotherm of the NVP fiber with a density of 3.9 mmol/g for catechin in green-tea extracts was well correlated by Langmuir-type equation. The maximum adsorption capacity and adsorption constant were 0.23 mmol/g and 13 L/mmol, respectively. Catechin adsorbed onto the NVP fiber was eluted completely with 0.1 M NaOH. In the second to fourth cycles of adsorption and elution, the amount of catechin adsorbed onto the NVP fiber remained almost constant.</p>

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GESM53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

A concentric pulse by motile cells of Escherichia coli (E. coli) propagates and the cells aggregate to form self-organized patterns.We summarize experimental and numerical results on the self-organized pattern formation of E. coli to elucidate some aspects of its mechanism. Our presentation includes experiments on E. coli patterns, as well as numerical simulations on the basis of a reaction-diffusionchemotaxis model.We find good agreement for one-dimensional propagating fronts in observation and simulation. However, corresponding results for two-dimensional circular bacterial clusters have still not been obtained.

Hydrous cerium oxide was impregnated onto a sufonic-acid-group-containing fiber (SS fiber) prepared by radiation-induced graft polymerization and subsequent chemical modifications. Cerium ions (Ce³⁺) were adsorbed onto SS fiber before the formation of hydrous cerium oxide (H-CeO) via precipitation by a reaction with a sodium hydroxide (NaOH) solution at various NaOH concentrations in the range from 0.001 to 0.1 mol L^&minus;1. The maximum percentage impregnation of H-CeO was 12 %. In the batch mode, neither SS fiber nor anion-exchange fiber removed any antimony ions, whereas H-CeO fiber captured antimony ions at a high rate.

<p>To raise the adsorption capacity of cation-exchange fiber for protein, two methods of pre-irradiation grafting using either a solution or an emulsion of glycidyl methacrylate (GMA) were compared. The epoxy group of poly-GMA grafted onto a commercially available 6-nylon fiber was converted into a sulfonic acid group by reaction with sodium sulfite. Lysozyme solution (pH 9.0) was passed through a bed charged with one of the cation-exchange fibers at a space velocity of 60 h⁻¹. At a degree of GMA grafting of 81–87%, the 10% dynamic binding capacity of the bed charged with the fiber prepared by the emulsion grafting was four-fold that of the bed prepared by the solution grafting. The graft chain formed near the periphery of the fiber by the emulsion grafting is considered to contribute to the rapid capture of protein.</p>

LuxR family transcriptional regulators are the core components of quorum sensing in Gram-negative bacteria and exert their effects through binding to the signaling molecules acyl-homoserine lactones (acyl-HSLs). The function of the LuxR homologs is remarkably plastic, and naturally occurring acyl-HSLs are structurally diverse. To investigate the molecular basis of the functional plasticity of Vibrio fischeri LuxR, we directed the evolution of LuxR toward three different specificities in the laboratory. We found an orthogonal pair of LuxR mutants specific either to 3-oxo-hexanoyl homoserine lactone or to 3-oxo-octanoyl homoserine lactone. Interestingly, the majority of the specificity changes did not arise from modulating the recognition event but rather from changing the efficiency of the transition from the inactive form to the active form upon signal binding. This finding explains how quorum sensing systems can rapidly diverge in nature and in the laboratory and how signal orthogonality and mutual inhibition frequently occur among closely related diverging systems.

Synthetic biologists are in need of genetic switches, or inducible sensor/promoter systems, that can be reliably integrated in multiple contexts. Using a liquid-based selection method, we systematically engineered the choline-inducible transcription factor BetI, yielding various choline-inducible and choline-repressive promoter systems with various input-output characteristics. In addition to having high stringency and a high maximum induction level, they underwent a graded and single-peaked response to choline. Taking advantage of these features, we demonstrated the utility of these systems for controlling the carotenoid biosynthetic pathway and for constructing two-input logic gates. Additionally, we demonstrated the rapidity, throughput, robustness, and cost-effectiveness of our selection method, which facilitates the conversion of natural genetic controlling systems into systems that are designed for various synthetic biology applications.

LuxR is the core component of Vibrio fischeri quorum sensing. It acts as the transcriptional activator by binding to its cognate signaling molecules 3-oxo-hexanoyl-homoserine lactone (3OC6HSL). Although several acyl-HSLs with 3-oxo groups are known to activate LuxR with similar efficiency, acyl-HSLs without 3-oxo groups are very weak inducers. We conducted a round of LuxR directed evolution to acquire LuxR mutants with higher signal sensitivity to octanoyl-homoserine lactone (C8HSL). All of the isolated mutants showed increased signal sensitivity to many other acyl-HSLs, including C8HSL, and some to the LuxR antagonist p-coumaroyl-HSL. The evolution of their ligand sensitivity proceeded through the stabilization of the signal-bound state, thereby elevating the effective concentration of LuxR at the ON-state.

Successful feeding of the substrate geranylpyrophosphate (GPP) to monoterpene synthase is critical to the efficient microbial production of monoterpenes. Overexpression of GPP synthases, metabolic channeling from GPP synthase to terpene synthases, and down-tuning of endogenous competitors have been successfully used to increase the production of monoterpene. Nevertheless, the production of monoterpenes has remained considerably lower than that of hemi-/sesqui-terpenoids. We tested whether it is effective to improve the cellular activity of monoterpene synthases. To this end, we developed a high-throughput screening system to monitor for elevated GPP consumption. Through a single round of mutagenesis and screening, we isolated a pinene synthase variant that outperformed the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibited drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenylpyrophosphate isomerase, and GPP synthase yielded 140 mg/L pinene in a flask culture.

At Tokyo Electric Power Company (TEPCO) Fukushima Daiichi nuclear power plant (NPP), water contaminated with radionuclides such as Cs-137 and Sr-90 has been stored in tanks and seawater-intake area. We have prepared cobalt-ferrocyanide-impregnated fibers via four steps: the grafting of an epoxy-group-containing monomer, the conversion of the epoxy group into positively charged groups, the binding of ferrocyanide ions ([Fe(CN)(6)](4-)), and the precipitation of cobalt ferrocyanide (Co-2[Fe(CN)(6)]) by contact with cobalt ions. However, the impregnation structure of cobalt ferrocyanide microparticles onto the fiber remains unclear. Here, we describe the impregnation structure from the results of rebinding [Fe(CN)(6)](4-) to the cobalt-ferrocyanide-impregnated fiber. The amount of [Fe(CN)(6)](4-) re-bound onto the fiber was found to decrease with increasing amount of Co-2[Fe(CN)(6)] initially impregnated. This suggests that the microparticles of cobalt ferrocyanide become entangled with the grafted polymer chains via multipoint electrostatic interactions.

For recovery of palladium (Pd) from a hydrochloric acid medium, dioctyl sulfide (DOS), a neutral extractant capable of specifically capturing Pd anionic species, was impregnated onto a hydrophobic ligand-containing polymer chain grafted onto 6-nylon fiber, using radiation-induced graft polymerization. The hydrophobic ligand was the dodecanethiol (C12S) moiety, which functions both in the impregnation of DOS via hydrophobic interaction and in the direct uptake of Pd species by a similar mechanism as DOS. The C12S group was immobilized at a density of 2.0 mmol/g through its reaction of C12S with the epoxy group of the poly-glycidyl methacrylate (GMA) chain grafted onto the nylon fiber, and DOS was impregnated onto the resulting fiber at a density of 0.40–1.1 mmol/g. For example, fiber impregnated with DOS at a density of 0.40 mmol/g possessed a saturation capacity of 0.70 mmol-Pd/g in 1 mol/L HCl. The molar binding ratios of Pd to impregnated DOS and immobilized C12S were calculated as 0.50 and 0.27, respectively. In addition, the DOS-impregnated fiber specifically captured Pd species from a mixture solution of Pd and platinum (Pt) species in 1–4 mol/L HCl.

By assembly and evolutionary engineering of T7-phage-based transcriptional switches made from endogenous components of the bet operon on the Escherichia coli chromosome, genetic switches inducible by choline, a safe and inexpensive compound, were constructed. The functional plasticity of the BetI repressor was revealed by rapid and high-frequency identification of functional variants with various properties, including those with high stringency, high maximum expression level, and reversed phenotypes, from a pool of BetI mutants. The plasmid expression of BetI mutants resulted in the choline-inducible (Bet-ON) or choline-repressible (Bet-OFF) switching of genes under the pT7/betO sequence at unprecedentedly high levels, while keeping the minimal leaky expression in uninduced conditions.

Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C50 backbones produced. We then further extend the C50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C50 carotenoids, including ∼ 90% C50-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.

Squalene is a precursor of thousands of bioactive triterpenoids and also has industrial value as a lubricant, health-promoting agent, and/or drop-in biofuel. To establish an efficient Escherichia coli-based system for squalene production, we tested two different squalene synthases and their mutants in combination with precursor pathways. By co-expressing a chimeric mevalonate pathway with human or Thermosynechococcus squalene synthase, E. coli accumulated squalene up to 230 mg/L or 55 mg/g-DCW in flask culture. We also determined that a significant truncation of squalene synthase at the C-terminus retains partial cellular activity. The squalene-producing strain described herein represents a convenient platform for gene discovery and the construction of the pathway toward natural and non-natural hopanoids/steroids.

Bis(2-ethylhexyl) phosphate (HDEHP) as an acidic extractant was impregnated onto a polymer chain grafted onto a 6-nylon fiber at a density of 0.43 mol/kg-product. Neodymium and dysprosium ions loaded onto the fiber packed into a bed were separated by elution chromatography using 0.2, 0.3, and 1.5 M hydrochloric acid as an eluent. At various space velocities and loaded amounts, the HDEHP-impregnated fiber-packed bed exhibited a higher peak height and a narrower half width because of shorter diffusional mass-transfer path of ions and larger external area per volume. The leakage of HDEHP impregnated was found to be negligible.

Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of 'selection escapees' can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.

The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3')-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C30 carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a "dehydrosqualene synthase." All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.

Most natural carotenoids have 40-carbon (C40) backbones, while some bacteria produce carotenoids with C30 backbones. Carotenoid backbone synthases, the enzyme that catalyze the first committed step in carotenoid biosynthesis, are known to be highly specific. Previously, using C30 backbone synthase (diapophytoene synthase, CrtM) from Staphylococcus aureus, we reported two size-shifting mutations, F26A and W38A, which confer C40 synthase activity at the cost of the original C30 synthase activity. In this study, we performed a directed evolution of the C40-specialist variant CrtMF26A in search of mutations that restore the original C30 synthase function. Examination of the resultant mutants, together with the site-directed mutagenesis study identified three new mutations (H12A, D27A and I240F) that affect the size specificity of this enzyme. After re-defining the reading frame, we obtained CrtM variants that are highly active in C30 and C40 carotenoid synthesis.

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C30 carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C30 carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a "squalene route" and a "lycopersene route" for C30 and C40 carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.

Potassium cobalt hexacyanoferrate compounds (KCo-HCFe's) were impregnated onto a 6-nylon fiber by radiation-induced graft polymerization and subsequent chemical modifications. First, dimethylaminoethyl methacrylate was graft-polymerized onto the nylon fiber. Second, hexacyanoferrate ions were bound to graft chains via an anion-exchange interaction. Third, KCo-HCFe's were formed on the nylon fiber via the precipitation reaction of hexacyanoferrate ions with cobalt ions in the presence of potassium chloride. The resulting KCo-HCFe-impregnated fiber had an impregnation percentage of the fiber for KCo-HCFe's of 7%. The cesium concentration in 10. ppm cesium chloride solution with the immersion of this fiber decreased to 0.6. ppm within 60. min at a ratio of liquid volume (10. mL) to fiber mass (0.1. g). The fiber was fabricated into a braid with a length of 100. cm and a diameter of 8. cm for practical use at sites contaminated with cesium. © 2013 Elsevier Ltd.

Ever-increasing repertories of RNA-based switching devices are enabling synthetic biologists to construct compact, self-standing, and easy-to-integrate regulatory circuits. However, it is rather rare that the existing RNA-based expression controllers happen to have the exact specification needed for particular applications from the beginning. Evolutionary design of is powerful strategy for quickly tuning functions/specification of genetic switches. Presented here are the steps required for rapid and efficient enrichment of genetic switches with desired specification using recently developed nucleoside kinase-based dual selection system. Here, the library of genetic switches, created by randomizing either the part or the entire sequence coding switching components, is subjected to OFF (negative) selection and ON (positive) selection in various conditions. The entire selection process is completed only by liquid handling, facilitating the parallel and continuous operations of multiple selection projects. This automation-liable platform for genetic selection of functional switches has potential applications for development of RNA-based biosensors, expression controllers, and their integrated forms (genetic circuits).

Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

Impregnation was used to affix bis(2-ethylhexyl) phosphate (HDEHP) to the dodecylamino group of a polymer chain grafted onto a 6-nylon fiber. The distribution coefficients of Nd and Dy ions on the fiber agreed well those for HDEHP in dodecane. HDEHP supported on the hydrophobic graft chain was found to behave similarly to HDEHP dissolved in organic solvent. Nd and Dy ions were successfully separated by elution chromatography with an HDEHP-impregnated fiber-packed bed, and leakage of HDEHP from the bed was found to be negligible during the chromatography.

Aliquat 336 was impregnated onto the polymer chain grafted onto a 2.0-mm-thick porous sheet with a porosity of 75% and a pore size of 1.2 mu m via the graft polymerization of glycidyl methacrylate and the subsequent reaction of the epoxy group with mercaptoundecanoic acid. In a 2:1 ethanol/4M NaOH (v/v) mixture, Aliquat 336 was impregnated at a density of 0.85mmol/g, which was comparable to that of conventional Aliquat-336-impregnated polymeric beads. The dynamic binding capacity for palladium was 0.60mmol/g when 100mg-Pd/L palladium chloride solution was forced to permeate at a space velocity of 3700h(-1.</SUP)

Glycidyl methacrylate (GMA) was graft-polymerized onto a porous sheet (75% porosity and 1.6-mu m average pore size). previously irradiated with an electron beam. The resultant grafted poly-GMA chain can be classified as a polymer brush. extending from the pore surface toward the pore interior and a polymer root invading the polymer matrix. The boundary crossing the pore/matrix interface can be detected in two independent ways: molar conversion of the epoxy group into a sulfonic. group providing a plateau in the molar conversion versus reaction time curve and molar conversion exhibiting a breakthrough point in the swelling ratio versus molar conversion curve. A higher irradiation dose was found to lead to a higher mole percentage of polymer brush in the graft chain. The dose range from 20 to 200 kGy corresponded to the mole percentage range of polymer brush from 6% to 15%.

Dimethylamino-gamma-butyric acid (DMGABA) as an ampholite was reacted with the epoxy group of the poly-glycidyl methacrylate chain grafted onto the pore surface of a porous hollow-fiber polyethylene membrane by radiation-induced graft polymerization. DMGABA was dissolved in a mixture of dioxane and water at various dioxane volume fractions, defined by dividing the dioxane volume by the total volume. The equilibrium binding capacity (EBC) of the DMGABA-immobilized porous hollow-fiber membrane for lysozyme was evaluated in the permeation mode. The EBC was varied from a 1/50-fold monolayer binding capacity to a 10-fold monolayer binding capacity by controlling the composition of the solvent used for DMGABA immobilization and the molar conversion of the epoxy group into the DMGABA group. (c) 2013 Elsevier Ltd. All rights reserved.

Potassium cobalt-hexacyanoferrate (KCoFC)-impregnated fibers were prepared by radiation-induced cografting of vinylbenzyltrimethylammonium chloride (VBTAC) and N-vinyl-2-pyrrolidone (NVP) and subsequent chemical modifications. A 10 mg-Cs/L cesium chloride solution with various sodium chloride concentrations up to 0.5 M was forced to flow through the KCoFC-fiber-packed bed to acquire a breakthrough curve. The dynamic binding capacity of the bed for cesium with 0.5 M sodium chloride was 30-fold higher than that without sodium chloride. The adsorption isotherm of the KCoFC fiber for cesium ions in seawater was correlated using the Langmuir equation with a saturation capacity of 20 mg-Cs/g-dry.

Removal of the major urinary protein, cauxin, a carboxylesterase, from cat urine is essential for distinguishing between physiological and abnormal proteinuria by a urine dipstick. We have previously developed a material for removing cauxin by using lens culinaris agglutinin (LCA) lectin which targets the N-linked oligosaccharides present in cauxin. To improve the affinity and specificity toward cauxin, we immobilized 1,1,1-trifluoro-3-(2-sulfanylethylsulfanyl) propane-2-one, an inhibitor of esterases, to a polymer chain grafted on to a porous hollow-fiber membrane by applying radiation-induced graft polymerization. Normal male urine was forced to permeate through the pores rimmed by the ligand-immobilized polymer chain. Cauxin could not be detected in the effluent from the membrane. The residence time of the urine across a membrane thickness of 1 mm was set at 7 s. The respective dynamic and equilibrium binding capacities of the membrane for cauxin were 2 and 3 mg/g. The developed cauxin-affinity membrane material was more effective for diagnosing cat kidney diseases than the LCA lectin tip.

An epoxy-group-containing vinyl monomer, i.e., glycidyl methacrylate, was graft-polymerized onto polyethylene particles with an average diameter of 35 mu m at various reaction temperatures within 278-333 K. The produced epoxy group was converted into a diethylamino group as an anion-exchange group. From the equilibrium binding capacity of the resultant particle-packed bed for bovine serum albumin (BSA) and the pressure loss required for a protein solution to flow through the bed, the formation of graft chains that were sufficiently long to hold BSA in multilayers and allow convective flow among them was predicted. The achieved ideal adsorption characteristics enabled a higher flow rate of the protein solution, which leads to a higher overall adsorption rate of the protein because of convective flow among the graft chains.

Dimethylaminoethyl methacrylate (DMAEMA) was graft-polymerized at a density of 1.7 mmol/g onto a 6-nylon fiber by radiation-induced graft polymerization. Urease was bound to the resultant anion-exchange fiber by electrostatic interaction. After crosslinking with transglutaminase, 80% of the bound urease was immobilized at a density of 41 mg/g of the fiber. Urea in water was quantitatively hydrolyzed during the permeation of a 0.20 mg/L urea solution through the urease-immobilized-fiber-packed bed with a diameter of 5.5 mm and a height of 27 mm in the residence time range of 12-180 s. The activity of immobilized urease did not deteriorate after repeated use of the fiber, i.e., during reaction and storage.

An epoxy-group-containing vinyl monomer, glycidyl methacrylate, was graft-polymerized onto an electron-beam-irradiated porous sheet to form a graft chain. Graft chains can be categorized according to the formation site into the polymer brush extending from the pore surface of a porous sheet and polymer root penetrating into the polymer matrix of the porous sheet. The polymer brush and root of the porous sheet govern its protein adsorptivity and liquid permeability. The mole percentages of the polymer brush and the root were determined from the dependence of the degree of swelling on the molar conversion of epoxy groups into ion-exchange groups with water as the solvent during functionalization. This method enables us to better understand the manner in which polymer chains are grafted onto porous polymers and helps us to design porous polymers for protein purification and enzyme immobilization.

Directed evolution is a well-established strategy to confer novel catalytic functions to the enzymes. Thanks to the relative ease of establishing color screening, carotenogenic enzymes can be rapidly evolved in the laboratory for novel functions. The combinatorial usages of the evolvants result in the creation of diverse set of novel, sometimes unnatural carotenoids. This chapter describes the directed evolution of diapophytoene (C(30) carotenoid) synthase CrtM to function in the foreign C(40) pathway, and the use of the CrtM variants thus obtained for the production of novel backbone structures.

This study investigates improvements in high-resolution elution chromatography of proteins loaded to a bed charged by way of cation-exchange polymer-brush-immobilized particles. A sulfonic acid group as a cation-exchange group was introduced into a polymer brush grafted onto a polyethylene particle with an average diameter of 35 mu m. A bed charged with the resulting cation-exchange polymer-brush-immobilized particles, SS bed, has a sulfonic acid group density of 0.36 mmol/mL-bed. A mixture of alpha-chymotripsinogen A (chy), cytochrome c (cyt), and lysozyme (lys) was loaded onto the SS bed. The subsequent linear gradient elution performance of the proteins adsorbed by the SS bed with 20 mM phosphate buffer (pH 6.0) and 1 M NaCl is compared with those by commercially available cation-exchange-bead-packed beds. The resolutions of the SS bed of both the chy/cyt and cyt/lys pairs at a linear velocity of 600 cm/h exceeded 1.9 at a loading amount of 0.8 mg/mL-bed, which was not achieved using the beds charged with SOURCE (R) 30S, POROS (R) 50HS, or Fractogel (R) EMD SO3- (s). Even at a loading amount of 12 mg/mL-bed, the resolutions of the SS bed for the chy/cyt and cyt/lys pairs at a linear velocity of 300 cm/h were 1.4 and 2.7, respectively.

An octadecylamino-group-introduced polymer chain grafted onto a porous sheet was impregnated with bis(2-ethylhexyl) hydrogen phosphate (HDEHP). A mixture of HDEHP and ethanol of various HDEHP concentrations was used for the impregnation. The porous sheet into which a C18H37NH group was introduced was immersed in HDEHP/ethanol solution before ethanol evaporation. The liquid permeability of a cartridge charged with the HDEHP-impregnated porous sheet in disk form prepared in 50 (v/v)% HDEHP/ethanol solution was 96% that of the starting-porous-disk-packed cartridge. The equilibrium binding capacity of the HDEHP-impregnated porous disk for yttrium ions was 0.32 mol per kg of the disk. In addition, the HDEHP-impregnated-porous-disc-packed cartridge was found to be applicable to the preconcentration of trace amounts of lanthanides in a multielement solution prior to their measurement by inductively coupled plasma mass spectrometry.

The first step toward elucidating the mutagenic effects of chemicals and pathways is to determine the specificity of the mutations generated spontaneously or in response to treatment with mutagens. We constructed a set of plasmid-encoded probes for the specific detection of each type of base substitution mutation. Using these probes, we were able to quickly determine both the mutation rate and the specificity of the mutations caused by different types of mutagens and mutagenic conditions. We also developed a PCR-based method to rapidly and robustly determine the mutation spectrum in response to various mutagenic samples in parallel. This system allows one to not only analyze the mutation specificity of various chemicals, but also to search for novel genetic elements that promote the specific mutation events.

By using electron-beam-induced graft polymerization, an epoxy-group-containing monomer, glycidyl methacrylate (GMA), was appended onto a 6-nylon fiber; subsequently, N-methylglucamine as a chelate-forming moiety was added to the epoxy group. The chelating group density of the resultant chelating fiber was 2.0 mmol/g, which was 74% of that of a commercially available chelating bead containing the same functionality. A 150 mg-B/L boron solution was forced to flow through the chelating-fiber-packed bed at the space velocity range from 10 to 100 h(-1), defined by dividing flow rate by bed volume (0.3 mL). At a space velocity of 20 h(-1), the dynamic binding capacity of the chelating-fiber-packed bed was 2.5-fold higher than that of the chelating-bead-packed bed.

The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecedented enrichment efficacy (>30,000-fold) of functional switches from non-functional ones in a single selection cycle. In addition, negative (OFF) selection was exceptionally stringent, allowing the rapid and efficient selection of non-leaky from leaky circuits.

A neutral extractant, tri-n-octylphosphine oxide (TOPO), was used to chemically modify a porous sheet, with a final density of 1.0 mmol/g. First, glycidyl methacrylate (GMA) was graft-polymerized onto a porous polyethylene sheet with an average pore diameter, porosity, and thickness of 1.2 mu m, 75%, and 2.0 mm, respectively. Second, an octadecane thiol group was introduced into the poly-GMA graft chain. Third. TOPO was deposited on the graft chain via a hydrophobic interaction. Bismuth chloride solution (BiCl3 in 0.15 M HNO3) was forced through the pores of the TOPO-modified porous sheet. The equilibrium binding capacity for bismuth was 0.19 mmol/g. Bismuth ions bound to TOPO were quantitatively eluted with 11 M HNO3. (C) 2010 Elsevier Ltd. All rights reserved.

We prepared nucleic-acid-base-immobilized porous membranes of a hollow-fiber form with pore size, porosity, and thickness of 0.2 mu m, 70%, and approximately 0.7 mm, respectively. Glycidyl methacrylate was graft-polymerized onto a polyethylene-made porous hollow-fiber membrane, followed by ring-opening of the epoxy group with the amino groups of adenine, guanine, and cytosine. The collection of palladium ions was achievable during the permeation of palladium chloride solution through the adenine-immobilized porous hollow-fiber membrane. The diffusional mass-transfer resistance of palladium ion to immobilized adenine was negligible because palladium ion was transported by permeative flow through the pores. The adenine-immobilized porous membrane with an immobilization density of 0.85 mol/kg of the membrane exhibited the highest molar binding ratio of palladium ion to immobilized adenine of 0.31 in 1 M hydrochloric acid. In addition, a quantitative elution with 4 M hydrochloric acid was experimentally demonstrated. (c) 2007 Published by Elsevier B.V.

A chelating porous sheet for use in solid-phase extraction was prepared by radiation-induced graft polymerization and subsequent chemical modifications. An epoxy-group-containing vinyl monomer was graft-polymerized onto a porous sheet made of polyethylene. The produced epoxy group of the graft chain was converted into an iminodiacetate group. The chelating porous sheet with a density of the iminodiacetate group of 2.1 mol/kg was cut into disks 13 mm in diameter to fit an empty cylindrical cartridge with a capacity of 6 mL. Breakthrough curves using the chelating-porous-disk-packed cartridge overlapped irrespective of the flow rate of the solution ranging up to 1500 mL/h because of negligible diffusional mass-transfer resistance of the copper ions to the iminodiacetate group of the graft chain.

Three kinds of ampholites, i.e., 3-aminopropionic acid (NH2C2H4COOH), (2-aminoethyl)-phosphonic acid (NH2C2H4PO3H2), and 2-aminoethane-1-sulfonic acid (NH2C2H4SO3H), were introduced into an epoxy group-containing polymer brush grafted onto a porous hollow-fiber membrane with a porosity of 70% and pore size of 0.36 mu m. The amphoteric group density of the hollow-fiber ranged from 0.50 to 0.72 mmol/g. Three kinds of proteins, i.e., lactoferrin (Lf), cytochrome c (Cyt c), and lysozyme (Ly), were captured by the amphoteric polymer brush during the permeation of the protein solution across the ampholite-immobilized porous hollow-fiber membrane. Multilayer binding of the protein to the amphoteric polymer brush, with a degree of multilayer binding of 3.3, 8.6, and 15 for Lf, Cyt c, and Ly, respectively, with the (2-aminoethyl)-phosphonic acid-immobilized porous hollow-fiber membrane, was demonstrated with a negligible diffusional mass-transfer resistance of the protein to the ampholite immobilized. The 2-aminoethane-1-sulfonic acid-immobilized porous hollow-fiber membrane exhibited the lowest initial flux of the protein solution, 0.41 m/h at a transmembrane pressure of 0.1 MPa and 298 K, and the highest equilibrium binding capacity of the protein, e.g., 130 mg/g for lysozyme. Extension and shrinkage of the amphoteric polymer brushes were observed during the binding and elution of the proteins.

As an extracurricular activity, 18 students in chemistry major tried to create four Escherichia coli 'robots': (1) an imaging system with swimmy bacteria
(2) a switchable aroma generator
(3) a balloon bacteria with a light-triggered inflator
and (4) an E. coli clock. None of the projects were completed, but a number of BioBricks were generated. They include an aroma synthesiser, a motility controller, and a size and shape controller. By assembling and tuning these BioBricks, we will be able to complete our robot manufacture. We belive these BioBricks would expand the toolbox in bacterial robotics. © 2007 The Institution of Engineering and Technology.

The mutation spectrum, together with mutation frequency, is decisive for the size and nature of genetic diversity. We constructed plasmid-encoded probes for specific detection of each and all of the six base substitutions. Using the set of the probes, we analyzed the mutation spectrumon the plasmids caused by different types of mutagens and mutator enzymes/alleles.

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products--those that could be made biosynthetically--remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these "evolved" pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed.

Using methods of laboratory evolution to force the C(30) carotenoid synthase CrtM to function as a C(40) synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C(45) and C(50) carotenoid backbones (mono- and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C(30) carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C(40) synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies.

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone. The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.

The C30 carotene synthase CrtM from Staphylococcus aureus and the C40 carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C40 and C30 biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function. Each displayed negligible ability to synthesize the natural carotenoid product of the other. After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C40 carotenoids. In contrast, we failed to find a single variant of CrtB with detectable C30 activity. Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C40 pathway. These mutants showed significant variation in performance in their original C30 pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity. We discovered that Phe 26 alone determines the specificity of CrtM. The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.

In this research, we synthesized a novel DNA-polymer conjugate and evaluated its application to an affinity precipitation separation of TATA-box binding protein (TBP), which is a representative general transcription factor. The conjugate was composed of two fractions. One was a double-stranded DNA modified by the grafting of poly(N-isopropylacrylamide) (PNIPAAm), which is known as a thermosensitive vinyl polymer. The other fraction is a native double-stranded DNA containing a specific base sequence (5'-TATAAA-3') called a TATA-box. These two fractions, which have EcoRI termini, were treated with T4 DNA ligase, and the block conjugate was obtained as a precipitate after two wash processes. When the resultant block conjugate was introduced into a sample solution containing TBP (0.26 microM) and bovine serum albumin (BSA) (0.39 microM), a rapid and selective precipitation separation of TBP under homogeneous conditions was achieved by controlling temperature. The purity of TBP in the precipitation fraction was estimated to be above 90%.

In this study we develop a sequence-specific precipitation separation system of oligonucleotide (ODN) using a conjugate between poly(N-isopropylacrylamide) (PNIPAM) and ODN. PNIPAM is known as a thermoresponsive polymer and dehydrates to precipitate above its phase transition temperature in an aqueous milieu. The principal advantage of this separation system using the conjugate is that the hybridization reaction between the conjugate and oligonucleotide is conducted in homogeneous solution. The conjugate was prepared by copolymerization between N-isopropylacrylamide and a vinyl-derivatized (dT)8. The obtained conjugate efficiently precipitated (dA)8 from solution when the solution contained more than 1.5 M NaCl. The conjugate containing 3 nmol of (dT)8 residue was able to precipitate 1.4 nmol of (dA)8, suggesting that the (dT)8 residue of the conjugate formed a triple helix with (dA)8. From an equimolar mixture of (dA)8 and its one point mutant, the conjugate selectively precipitated (dA)8: the highest selectivity was obtained for the isolation of (dA)8 from the mixture consisting of (dA)4dT(dA)3 and (dA)8. When the conjugate was applied for the precipitation of five oligo(dA)s having different chain lengths, the longer oligo(dA)s tended to be precipitated by the conjugate more efficiently than the shorter ones. The conjugate could be used repeatedly for precipitation of (dA)8 without showing any loss in precipitation efficiency. © 2001 John Wiley &amp
Sons, Inc.

Single-chain observation of giant DNAs grafted with poly(N-isopropylacrylamide) (PNIPAAM) was performed using fluorescence microscopy while changing the solution temperature. The PNIPAAM-grafted DNA was prepared through the intercalation of psoralen-terminated PNIPAAM. Individual DNAs grafted with PNIPAAM exhibit a sharp but continuous transition at around 34 °C, from an elongated coil to a collapsed compact state. It is found that the width of the transition is almost the same between the level of individual DNAs and the level of ensemble DNAs. The unique nature of this transition is discussed in comparison with the discrete nature of the folding transition in native double-strand DNAs. With the addition of spermidine, a trivalent amine, the conformation of individual T4DNAs changes in a markedly discrete manner, on the level of individual giant DNAs, whereas in the ensemble, the size of T4 DNAs changes rather mildly
i.e., the transition appears to occur over a temperature range of 30 degrees. Thus, it is confirmed that the cooperative transition on the DNAs grafted with PNIPAAM exhibits a sharper transition than the change in the ensemble average for the discrete phase transition in native DNAs induced by the polycation.

We have synthesized psoralen-terminated poly(N-isopropylacrylamide)(1) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)(2), both of which show temperature-responsive properties and DNA-binding ability. These polymers were grafted on plasmid double stranded DNA through a photochemical reaction of their terminal psoralen groups. The double stranded DNA grafted with the vinyl polymers turned out to lose the function as templates for RNA synthesis, while it showed characteristic solution property derived from the vinyl polymers. Ligation reaction between the polymer-grafted DNA and native DNA resulted in the AB-type block conjugate with the polymer-grafted DNA as A-segment and native DNA as B-segment. It was found that the block conjugate which have two different functional domains retains two functions independently; B-domain of the block conjugate worked as a template for RNA synthesis, while A-domain responded to temperature and made the conjugate precipitate. In this way, one can construct tailor-made and precisely-regulated structures with multiple functions at his own will by using recombinant DNA techniques.

Soluble conjugates between double stranded DNA and poly(N-isopropylacrylamide) (polyNIPAAm) was synthesized and applied to the selective separation of DNA fragments: the target fragments were connected to the DNA-polyNIPAAm conjugate with the aid of T4 DNA ligase, followed by temperature-induced precipitation. Target DNA was collected from the resultant precipitate in high purity.

Poly(N-isopropylacrylamide) having a terminal psoralen group was synthesized and covalently bound to plasmid pBR 322 DNA through a photochemical reaction. The resulting conjugate exhibited temperature-responsive precipitation due to the aggregation of poly(N-isopropylacrylamide) chains when heated above 31 °C. This system was used for the one-pot separation of restriction endonuclease EcoRI.

Polyacrylamide hydrogels comprising double-stranded DNA were synthesized and applied as the absorbent for DNA-binding mutagenic molecules. Vinyl groups were immobilized on salmon sperm DNA via photoreaction between DNA and a vinyl derivative of psoralen. The resulting DNA macromonomer was fed into the polymerization system of acrylamide and N,N'-methylenebisacrylamide to give a hybrid hydrogel between DNA and polyacrylamide. Adsorption functions of the DNA hydrogel were studied for a variety of DNA-binding drugs and dyes. Copyright (C) 1998 Elsevier Science B.V.

We synthesized poly(N-isopropylacrylamide) terminated with psoralen. Photo-induced reaction between DNA and the psoralen end group resulted in the grafting of poly(N-isopropylacrylamide) on double-stranded DNA. On the other hand, binding efficiency of DNA binding proteins such as restriction endonucleases to the DNA decreased significantly with increasing degree of polymer modification: At a certain level of polymer introduction, DNA was revealed to be practically 'jacketed'. Lithography was attempted to this grafting procedure. We found that pre-incubation of a restriction endonuclease EcoRI with DNA resulted in selective preservation of its binding site from polymer modification. The application of this technique would allow us facile methods to construct a precisely regulated nanoscale architecture.

We immobilized double-stranded DNA on poly(N-isopropylacrylamide), which is temperature-sensitive and gives precipitates from an aqueous solution when heated over 31°C. Photochemical conjugation with a vinyl derivative of psoralen was demonstrated to make the DNA polymerizable with a vinyl monomer. The radical copolymerization with N-isopropylacrylamide gave the temperature-responsive conjugate. The conjugate was shown to capture ethidium, a DNA-binding genotoxin, and precipitate with it when heated. About 95% of ethidium was separated from its highly diluted solution (3 ppm).

We have synthesized a vinyl monomer having a psoralen moiety, which can form a photoadduct with double-helical DNA. The monomer 1 was proved to have an ability to crosslink DNA double strands through a photochemical reaction when irradiated by UV light. The resulting DNA having vinyl groups was copolymerized with a comonomer such as acrylamide and N-isopropylacrylamide to give rise to a DNA—vinyl polymer conjugate. A conjugation based on covalent bondings was verified by using gel electrophoresis
the conjugation efficiency was found to be dependent upon concentration of the monomer which had been used in the antecedent photochemical reaction. This monomer will be a useful tool when anchoring double-helical DNA on polymeric materials for separating and sensing DNA-binding substances. © 1994, American Chemical Society. All rights reserved.

二次代謝経路（天然物経路）は生理活性と新規酵素の宝庫であり，リデザインも容易である。一方で，これを一次代謝に匹敵する馬力で高度に運転するためには，多くの新しい工学的努力を要する。本稿では，筆者たちのテルペノイド合成経路の進化工学の経験をもとに，天然物生合成経路の高効率運転を目指したリデザイン技術のあり方について議論する。

Tannic acid（TA）was immobilized onto a glycidyl methacrylate-grafted fiber. The TA-immobilized fiber（TA fiber）with a TA content of 25 ％ was applicable to the collection of vanadium（V）from the spring water of Mt. Fuji. The contact in a batch mode for 1 day of the 1 g TA fiber with 1 L spring water containing V at a concentration of 58 &mu;g-V/L led to collection of 70 ％. The amount of V adsorbed onto the TA fiber was quantitatively eluted with 0.5 M hydrochloric acid. In addition, the amount of V adsorbed remained constant after the third cycle of repeated adsorption and elution of V.

To raise the adsorption capacity of the adsorptive fiber for strontium ions in seawater, sodium titanium silicate was impregnated onto an anion-exchange fiber. Peroxotitanium complex anions were immobilized onto N, N-dimethylaminopropylacrylamide（DMAPAA）-grafted nylon-6 fiber, followed by a reaction with sodium orthosilicate. The resultant fiber exhibited a strontium removal percentage of 83 ％ and a selectivity of Sr to Ca of 4.3 in artificial seawater, which were 1.1- and two-fold, respectively, higher than those of the sodium-titanate-impregnated fiber.

<p>To remove catechin from green-tea extracts, N-vinylacetamide (NVAA) as an amide monomer was graft-polymerized onto a commercially available nylon-6 fiber. The resultant NVAA-grafted fiber with a degree of grafting of 79％ exhibited a saturation capacity for catechin of 0.78 mmol/g of the fiber, which is 3.7-fold higher than that of N-vinyl-2-pyrrolidone-grafted fiber. The catechin adsorbed onto the NVAA-grafted fiber was quantitatively eluted with 0.1 M NaOH and the fiber was repeatedly used for catechin adsorption.</p>

<p>Tannic acid (TA) was immobilized onto nylon-6 fiber to adsorb caffeine. Glycidyl methacrylate (GMA) was graft-polymerized to gamma-ray-irradiated nylon-6 fiber and subsequently tannic acid was coupled with the epoxy groups of poly-GMA graft chain. The TA-immobilized fiber with a content of tannic acid of 25% exhibited an equilibrium amount of adsorbed caffeine of 3.1×10⁻² mmol per g of the fiber at a concentration of 1 mM of caffeine solution. The amount of caffeine adsorbed was quantitatively eluted with 50°C hot water. After the eighth cycle of adsorption and elution, the amount of caffeine adsorbed onto the TA-immobilized fiber was retained without any deterioration.</p>

<p>To prepare a weakly acidic cation-exchange fiber capable of binding lysozyme dissolved in a high-concentration phosphate buffer, acrylic acid (AA) was graft-polymerized onto a nylon-6 fiber by radiation-induced graft polymerization. The dynamic binding capacity (DBC) of the bed charged with the AA-grafted fiber at a space velocity of 20 h⁻¹ was determined in a flow-through mode for lysozyme solutions buffered with 50–250 mM phosphate solution (pH 6.0). The DBC of the CM-Sepharose-FF-bead-packed column decreased from 88 to 9 mg/mL-bed with increasing buffer concentration. In contrast the AA-grafted-fiber-packed column exhibited a constant value of 200 mg/mL-bed independent of the buffer concentration from 50 to 200 mM.</p>

<p>To prepare adsorptive fiber capable of capturing strontium ions dissolved in seawater at high capacity, peroxotitanium complex (POTC) anions as precursor of sodium titanate were immobilized by repetitive immersion of dimethylaminoethyl methacrylate (DMAEMA)-grafted fiber in a POTC anion-containing solution. Through ten-times immobilization of POTC anions and their conversion into sodium titanate, impregnation percentage of the fiber amounted to 29％, which was approximately 1.6-fold higher than once immobilization and impregnation. A binding capacity of the resultant fiber in artificial seawater for strontium ions was raised up to 2.9 mg-Sr per gram of the fiber.</p>

N,N-Dimethylaminopropylacrylamide methyl chloride quaternary（DMAPAA-Q）was graft-polymerized onto a commercially available nylon fiber in various solvents by radiation-induced graft polymerization in order to prepare a strong basic anion-exchange fiber. Primary alcohol such as 1-butanol as a solvent enhanced the grafting of DMAPAA-Q. An anion-exchange fiber with a degree of grafting of 78 ％, i.e., an anion-exchange density of 2.1 mmol/g-dry, was obtainable. The anion-exchange fiber exhibited a chemical stability for immersion in 1 M NaOH for 3 h at 40 ℃.

<p>An anion-exchange fiber capable of rapidly capturing proteins was prepared by radiation-induced emulsion graft polymerization. Glycidyl methacrylate emulsified with a surfactant Tween 20 was graft-polymerized onto pre-irradiated polyethylene fiber. Subsequently, some of epoxy groups of the graft chain were reacted with diethylamine. The 2.0-cm high column charged with the resultant diethylamino-type anion-exchange fiber exhibited a dynamic binding capacity (DBC) for 76 mg of bovine serum albumin (BSA) per mL of the column at a space velocity (SV) of BSA solution of 60 h⁻¹. The DBC of the anion-exchange-fiber-packed column at an SV of 1200 h⁻¹, 33 mg-BSA/mL, was 35 fold higher than that of a commercially available anion-exchange bead (DEAE Sepharose Fast Flow)-packed column with an identical column height with the fiber-packed column. This advantage of the fiber over the bead will result from the localization of the graft chain at the periphery of the fiber.</p>

Sodium titanate was impregnated onto a 6-nylon fiber by means of radiation-induced graft polymerization of dimethylaminopropyl acrylamide （DMAPAA） and subsequent chemical modifications. Peroxotitanium complex anions were bound to the DMAPAA-grafted fiber, followed by a reaction with sodium hydroxide to form sodium titanate as precipitate on the fiber. A quantitative elution of sodium titanate impregnated onto the fiber with 1 M nitric acid revealed that the molar ratio of Na to Ti was 0.76, which was almost consistent with that of sodium titanate （Na₄Ti₅O₁₂） produced in a homogeneous system. During the immersion of sodium titanate-impregnated fiber in a mixture of 0.5 M NaCl and 0.05 M SrCl₂, a molar ion-exchange ratio of Sr with Na was determined as being 0.55 by a quantitative elution of Sr-containing precipitate on the fiber.

For the removal of boron in liquid, N-methylglucamine （NMG） as a chelating moiety was immobilized to the poly-glycidyl methacrylate chain grafted onto a rayon fiber irradiated by means of electron beam. The density of the NMG immobilized was 1.9 mmol/g of the product. The resultant fiber was compared to a 6-nylon-based chelating fiber with an NMG density of 1.9 mmol/g that was derived from an electron-beam-irradiated 6-nylon fiber. The equilibrium binding capacity agreed with both fibers at 15 mg-B/g. The dynamic binding capacities of the rayon -and 6-nylon-based chelating fibers for boron were 9.4 and 4.5 mg-B/g, respectively, determined from respective breakthrough curves at a space velocity of 40 h^－1 of 150 mg-B/L solution flowing through a 10 mm-high fiber-packed bed.

An iminodiacetate-type chelating fiber（IDA fiber）was prepared by means of radiation-induced graft polymerization of an epoxy-group-containing monomer, glycidyl methacrylate, onto a commercially available 6-nylon fiber and subsequent conversion of the epoxy group into an iminodiacetate group capable of removing radioactive strontium ions from seawater. Adsorption isotherms of the IDA fiber for three kinds of alkaline earth-metal group ions, i.e., Sr^2＋, Ca^2＋, and Mg^2＋, in artificial seawater were found to be well described by ternary-component Langmuir adsorption isotherms. We proposed a strontium removal technique for the immersion of the fiber with a divided amount rather than an undivided amount into seawater in a closed area. The reduction in the amount of fiber required for an identical removal percentage of strontium was demonstrated in a batch mode. Experimental adsorption-equilibrium data as a function of the number of divided immersions agreed well with the curves calculated from the ternary-component Langmuir adsorption isotherms and mass-balance equations for each alkaline earth-metal ion.

The meltdown of three reactors of the TEPCO Fukushima Daiichi nuclear power station (NPS) caused by the Great East Japan Earthquake on March 11th 2011 resulted in the emission of radionuclides such as cesium-137 and strontium-90 to the environment. For example, radioactive cesium exceeding the legal discharge limit (90 Bq/L, 2×10^<-13> M) was detected in the seawater of the seawater-intake area of the NPS at the end of September 2014. Adsorbents with a high selectivity for cesium ions over other alkali metal ions such as sodium and potassium ions are required for cesium removal from seawater because sodium and potassium ions dissolve respectively at much higher concentrations of 5×10^<-1> and 1×10^<-2> M than cesium ions (2×10^<-9> M). In addition, the simple operations of the immersion in seawater and the recovery of the adsorbents from seawater are desirable at decontamination sites. We prepared a cobalt-ferrocyanide-impregnated fiber capable of specifically capturing cesium ions in seawater by radiation-induced graft polymerization and chemical modifications. First, a commercially available 6-nylon fiber was irradiated with γ-rays. Second, an epoxy-group-containing vinyl monomer, glycidyl methacrylate, was graft-polymerized onto the γ-rayirradiated nylon fiber. Third, the epoxy ring of the grafted polymer chain was reacted with triethylenediamine to obtain an anion-exchange fiber. Fourth, ferrocyanide ions, [Fe(CN)_6]^<4->, were bound to the anion-exchange group of the polymer chains. Finally, the ferrocyanide-ion-bound-fiber was placed in contact with cobalt chloride to precipitate insoluble cobalt ferrocyanide onto the polymer chains. Insoluble cobalt ferrocyanide was immobilized at the periphery of the fiber. However, the impregnation structure remains unclear. Here, we clarified the structure of insoluble cobalt ferrocyanide impregnated onto the polymer chain grafted onto the fiber to ensure the chemical and physical stability of the adsorptive fiber in various contaminated waters. The adsorption rate and capacity of the fiber for cesium ions were compared with those of a zeolite as a conventional adsorbent.

The Great East Japan Earthquake and the tsunami that followed caused the meltdown of three reactors of the TEPCO Fukushima Daiichi nuclear power station (NPS), resulting in the emission of radionuclides such as cesium-137 and strontium-90 to the environment. Radioactive strontium was detected in seawater and groundwater at concentrations of 1.8 × 10^2 and 5.5 × 10^5 Bq/L, respectively, on October 7th 2014. Nonradioactive strontium dissolves at a concentration of 8 mg/L in seawater. No adsorbent can distinguish radioactive strontium from nonradioactive strontium; therefore, the adsorbent must collect both ions which coexist with other alkaline-earth metal ions such as magnesium and calcium ions. Inorganic compounds and chelate-forming resins are candidate adsorbents for strontium removal. However, It is difficult to use these adsorbents to process a large volume of water contaminated with radionuclides because of their granule and bead forms. We have prepared two kinds of adsorptive fiber by radiation-induced graft polymerization and subsequent chemical modifications: (1) sodium-titanate-impregnated fiber (ST fiber) and (2) iminodiacetate-group-immobilized fiber (IDA fiber). The preparation scheme of the ST fiber consisted of four steps. First, a commercially available 6-nylon fiber was irradiated with γ-rays to produce radicals. Second, sodium styrene sulfate was graft-polymerized onto the irradiated fiber. Third, a titanium species [Ti(OH)_2^<2+>]was bound to the sulfonic acid group of the grafted polymer chain. Finally, the titanium species was converted into sodium titanate with sodium hydroxide, and the resulting precipitate was impregnated onto the fiber. On the other hand, the IDA fiber was prepared as follows. An epoxy-group-containing vinyl monomer, glycidyl methacrylate, was graft-polymerized onto a previously γ-ray-irradiated 6-nylon fiber. Subsequently, the epoxy group was converted into an iminodiacetate group as a chelate-forming group by a reaction with disodium iminodiacetate. The former and latter fibers are applicable to strontium removal from seawater and groundwater, respectively.

Three kinds of diamines, i.e., ethylene diamine (EDA), diethylene diamine (DEDA), and triethylene diamine (TEDA), were immobilized to the poly(glycidyl methacrylate) chain grafted onto a porous hollow–fiber membrane. The resultant anion–exchange porous hollow fibers were evaluated as ion–exchangers for protein binding in a permeation mode. The DEDA– and TEDA–immobilized porous hollow fibers exhibited higher flux for a buffer (pH 8.0) and lower binding capacity for bovine serum albumin than the EDA–immobilized porous hollow fiber. This will be due to that DEDA and TEDA crosslink between neighboring epoxy groups of the graft chain.

To enhance the removal rate of ruthenium from water, nucleic-acid base-immobilized fibers as adsorbent were prepared by means of radiation-induced graft polymerization of glycidyl methacrylate and the subsequent addition of nucleic-acid bases. The density of adenine immobilized onto 6-nylon fiber was 1.2 mmol/g. Ther removal rate of ruthenium increased with an increase in sodium chloride concentration in the ruthenium solution. The removal rate was represented by second-order reaction with respect to ruthenium concentration. The dependence of the initial removal rate of ruthenium from the solution on the temperature demonstrated that the activation energy of overall adsorption of ruthenium onto adenine-immobilized fiber was 45 kJ/mol, irrespective of the sodium chloride concentration ranging from 0.025 to 0.5 M.

In order to remove strontium from seawater, sodium titanate was impregnated onto a commercially available 6-nylon fiber by means of radiation-induced graft polymerization and subsequent chemical modifications. First, dimethyaminopropyl acrylamide as an originally anion-exchange-group-containing vinyl monomer was graft-polymerized onto the electron-beam-irradiated nylon fiber, followed by binding of a peroxo complex of titanium anions to the anion-exchange group of the graft chain. Then, bound titanium species were converted into insoluble sodium titanate through a reaction with sodium hydroxide. The equilibrium binding capacity of the sodium-titanate-impregnated fiber for strontium in seawater was calculated as 1.7 mg/g-Sr of the fiber from Langmuir adsorption isotherm.

Insoluble cobalt ferrocyanide（Co-FC）microparticles were impregnated onto a polymer chain grafted onto the surface of a 6-nylon fiber by means of radiation-induced graft polymerization and subsequent precipitation. The resultant Co-FC-impregnated fiber was immersed in either nonradioactive or radioactive cesium（Cs）solution in seawater in an initial Cs concentration range from 60 to 5.0×10^－4 g-Cs/m³．The adsorption isotherm correlated well with a Langmuir-type equation. In addition, the mass-transfer capacity coefficient was determined by fitting the experimental data of the rate of Cs adsorption onto the Co-FC-impregnated fiber to theoretical adsorption curves based on the Cs concentration difference between the bulk and the interface in seawater as a driving force of the overall adsorption rate. Decontamination factors（DFs）as functions of fiber weight and the contact time required for the removal of cesium ions from the contaminated seawater in a closed area were estimated using the above-mentioned values.

Sodium titanate was impregnated onto a commercially available 6-nylon fiber by means of radiation-induced graft polymerization of dimethyaminopropyl acrylamide（DMAPAA）and subsequent chemical modifications. A peroxo complex of titanium anions was bound onto the DMAPAA-grafted fiber before the bound titanium species was converted to sodium titanate through precipitation with sodium hydroxide. Impregnation percentage of sodium titanate of the fiber was constant at 20 ％ in the range of sodium hydroxide concentration in a mixture of methanol and water at a volume fraction of methanol of 80 ％ of 0.001 to 1 M, whereas the removal percentage of strontium from seawater leveled off at 80 ％ above a sodium hydroxide concentration in water of 0.1 M. Determination of adsorption isotherms in seawater demonstrates that the sodium-titanate-impregnated fiber with an impregnation percentage of 10 ％ exhibited 2.6-fold higher amount of strontium adsorbed in seawater per g of sodium titanate（8.8 mg-Sr/g）than a commercially available granular adsorbent for strontium, SrTreat^&reg;（3.4 mg-Sr/g）.

Catalase or palladium was immobilized to the polymer chain grafted onto a 6-nylon fiber to decompose hydroperoxide(H₂O₂) in ultrapure water. Catalase was adsorbed to a dimethylamino group of poly-dimethylaminoethylmethacrylate chain, followed by crosslinking with transglutaminase. Palladium complex ions were bound to the anion-exchange group and subsequent reduction with hydrazine. The density of immobilized catalase and impregnated palladium were 2.2×10^－4 and 0.54mmol/g, respectively. The catalase-immobilized fiber quantitatively decomposed H₂O₂ in water up to a space velocity through the fiber‐packed bed of 400h^－1. In contrast, almost 100% decomposition of H₂O₂ in water was attainable using the palladium-impregnated fiber above a space velocity of 3000h^－1.

A porous hollow–fiber membrane containing an anion–exchange graft chain is used as an adsorber for the removalof undesirable proteins. The adsorption and elution of the proteins are followed by the regeneration of the mem-brane. The volume of a buffer solution used for the regeneration of the anion–exchange porous hollow–fiber mem-brane was reduced by controlling the anion–exchange group distribution along the graft chain. First, glycidylmethacrylate(GMA)was graft-polymerized onto an electron–beam–irradiated porous hollow–fiber membrane.Second, diethylamino group was introduced into the epoxy group of the graft chain with water as a poor solvent forthe poly GMA chain. In a permeation mode, the membrane was regenerated with 1.0 M sodium hydroxide beforebeing rinsed with a phosphate buffer (pH 7.0). When the anion–exchange group was introduced preferentially intothe polymer brush, the buffer solution usage required to rinse the membrane to pH 7.2 was reduced by 40% com-pared to when the anion–exchange group was introduced uniformly along the graft chain. The binding capacity ofBSA to the polymer brush remained constant irrespective of the anion-exchange group distribution along the graft chain.

In a conventional desalination process, with the progression of desalination, i.e., decreasing the concentration of ions, the electrical resistance of the electrodialyzer increases. Here, ion-exchange groups were appended onto the currently-used spacer of the electrodialyzer to enhance ion transport and reduce the electrical resistance of the electrodialyzer. By means of radiation-induced graft polymerization, glycidyl methacrylate was graft-polymerized onto a polyolefin-made spacer with a thickness of 0.8 mm and an aperture ratio of 72 ％, and the produced epoxy group was converted into a sulfonic acid group. The electrical resistance of the sulfonic-acid type ion-exchange spacer with a salt-splitting capacity of 1.4 mmol/g was reduced in the 1.0 mmol/LNaCl concentration by 41 ％ compared to that of the starting spacer.

Anion-exchange fiber was prepared by means of radiation-induced graft polymerization of glycidyl methacrylate onto 6-nylon fiber, followed by a reaction with triethylenediamine (TEDA). Ferrocyanide ions ([Fe (CN)₆]^4－) were bound to the anion-exchange fiber with potassium ferrocyanide solution before the fiber was immersed in cobalt chloride solution. The resultant insoluble cobalt ferrocyanide microcrystals were immobilized by the TEDA-introduced polymer chain grafted onto the 6-nylon fiber. The impregnation percentage of insoluble cobalt ferrocyanide onto the fiber amounted to 12％, which was approximately two-fold higher than that of previous cesium-adsorptive fiber. The higher percentage impregnation of the fiber resulted in higher adsorption capacity for cesium ions in seawater.

Titanium hydroxide (TiO₂・xH₂O) was impregnated by repeated reaction of titanium ionic species-bound cation-exchange fiber with sodium hydroxide (NaOH) solution. The amount of titanium hydroxide deposited increased with an increase in the repeated number of deposition. The TiO₂・xH₂O-impregnated fiber was treated with NaOH in a mixture of methanol and H₂O to convert titanium hydroxide into sodium titanate (Na_xTi_yO_z). After the ninth repetition, an the impregnation percentage calculated from the mass ratio of sodium titanate to the cation-exchange fiber amounted to 110 ％. The amount of strontium adsorbed onto the fiber prepared by the ninth repetition rose to 0.86 mg-Sr per g of the fiber, which was almost equivalent to that of a commercially available strontium adsorbent. The selectivity of Sr over Mg increased with the repetition number of titanium hydroxide deposited onto the fiber, whereas that of Sr over Ca remained constant irrespective of it.

Hydrous titanium oxide was impregnated onto a 6-nylon fiber by immersing titanium-ion-bound cation-exchange fiber into sodium hydroxide solution with various concentrations ranging from 0.1 to 1.0 M. First, Ti (OH)₂^2＋ as an ionic species of titanium was bound to a sulfonic acid group of the poly-sodium styrene sulfonate (SSS) grafted onto the nylon-fiber by means of radiation-induced graft polymerization. The maximum amount of titanium ions bound throughout the entire SSS-grafted fiber was 1.3 mmol/g. By precipitation of Ti (OH)₂^2＋ eluted by sodium ions of NaOH solution with hydroxide ions, hydrous titanium oxide was formed in the periphery of the fiber. Bound Ti (OH)₂^2＋ was quantitatively converted into the precipitate of hydrous titanium oxide while a negligible leakage of titanium was detected in the NaOH solution. The impregnation percentage of the resultant fiber defined by the content of hydrous titanium oxide in the product fiber was 14 ％.

For the removal of radioactive strontium dissolved in seawater in the harbor near the Fukushima Daiichi Nuclear Power Plant, two kinds of sodium-titanate (ST) -impregnated fiber were prepared by means of radiation-induced graft polymerization and subsequent chemical modifications: one was ST-impregnated fiber, or SSS-ST fiber, originating from cation-exchange fiber that was prepared by grafting sodium styrene sulfonate (SSS) onto a 6-nylon fiber, and the other was DMAEMA-ST fiber originating from anion-exchange fiber prepared by grafting dimethylaminoethyl methacrylate (DMAEMA) onto 6-nylon fiber. In a batch adsorption mode, these ST-impregnated fibers exhibited a higher removal rate of strontium in seawater than commercially available ST adsorbent (SrTreat) in a granulous form. At a mass ratio of seawater to fiber of 100, the percentages for the removal of strontium were 86 ％ and 83 ％ for the SSS-ST and DMAEMA fibers, respectively. From the viewpoint of endurance against alkaline conditions in the preparation scheme, the SSS-ST fiber was judged to be feasible for practical use.

Cobalt or nickel ferrocyanide was impregnated into an anion-exchange polymer chain grafted onto a commercially available 6-nylon fiber. First, ferrocyanide ions (Fe (CN)₆^4－) were bound to the anion-exchange fiber. Second, the fiber was made to reach with cobalt or nickel ions to form insoluble cobalt or nickel ferrocyanide via precipitation. For comparison, a commercially available anion-exchange bead was used as a support for the impregnation of insoluble cobalt or nickel ferrocyanide. The contents of metal ferrocyanides impregnated onto the fiber were approximately half those impregnated onto the bead. Adsorption isotherms of insoluble cobalt or nickel ferrocyanide-impregnated fiber and bead for cesium ions in seawater correlated well with a Langmuir-type isotherm. The saturation capacity for cesium ions in seawater per gram of insoluble metal ferrocyanides was almost identical between the fiber and the bead.

Cation-exchange polymer-brush-immobilized particles were prepared using polyethylene particles with an average diameter of 60 &mu;m as a starting material for electron-beam-induced graft polymerization. Typical proteins, i.e., chymotrypsinogen A, cytochrome C, and lysozyme, were loaded onto a cation-exchange polymer brush immobilized particle (cation-exchange particle)-packed bed followed by gradient elution with a mixture of phosphate buffer (pH 6.0) and 1 M NaCl. Resolution performance was compared between the cation-exchange particle and commercially available cation-exchange bead (SOURCE 30S, GE Healthcare Co.) using the respective packed heights of the bed under an identical bed pressure loss, i.e., 1.5 and 2.5 cm. Even at higher linear velocity and total protein load, the cation-exchange-particle-packed bed exhibited a higher resolution for both chymotrypsinogen A/cytochrome C and cytochrome C/lysozyme combinations than the SOURCE-30S-packed bed.<br>

We prepared two kinds of adsorptive fibers for the removal of strontium from seawater by means of radiation-induced graft polymerization and subsequent chemical modifications: iminodiacetate-group-containing chelating fiber and sodium-titanate-impregnated fiber. Evaluation of the removal of strontium from seawater in a batch mode demonstrated that these adsorptive fibers showed a higher adsorption rate than conventional chelating beads and sodium titanium oxide granules. From the determination of concentration factors of strontium, calcium, and magnesium in seawater, the sodium-titanate-impregnated fiber was found to exhibit a higher affinity for strontium over calcium and magnesium in seawater than the chelating fiber. At a mass ratio of seawater to sodium-titanate-impregnated fiber of 100, the concentration factors of strontium, calcium, and magnesium in seawater were 600, 190, and 18 mL/g, respectively.

We proposed a method for purifying proteins based on an interaction between dually-immobilized affinity ligands and dually-tagged proteins. Streptavidin (SA) and nickel (Ni) ions as two kinds of ligands were immobilized to the polymer brush grafted onto a porous hollow-fiber membrane. Whereas, green fluorescent protein (GFP) containing both streptavidin binding peptide (SBP) and poly histidine tag (His-tag) as two kinds of tags was genetically produced. The resultant dually-tagged protein solution was permeated through the pores of the dual-affinity-ligands- immobilized porous hollow-fiber membrane. The dual-affinity ligands and dually-tagged proteins were found to fit on the polymer chain grafted onto the pore surface at a binding efficiency 2%.

&nbsp;&nbsp;For the removal of boron from aqueous solutions, N-methylglucamine (NMG) was immobilized onto the poly(glycidyl methacrylate) chains grafted onto a trunk nylon fiber with a diameter 25 &mu;m. A binary mixture of water/organic solvent was used as solvent for the NMG immobilization to epoxy groups in the grafted poly(glycidyl methacrylate) chains. Organic solvents used were 1,4-dioxane, isopropanol, N,N-dimethylacetamide, tetrahydrofuran, and dimethyl sulfoxide. Among tested organic solvents, 1,4-dioxane and isopropanol gave the NMG immobilization yields higher than the other three solvents did, when the volume fraction of each organic solution was 0.8 except for tetrahydrofuran (0.7) at the NMG concentration of 0.2 M. The 0.2 M NMG solutions prepared by water/1,4-dioxane and water/isopropanol binary mixed solvents mentioned above gave molar NMG immobilization yields of 74 and 68% at reaction temperature of 80 and 60&deg;C, respectively. One of nylon-based chelating fibers containing 1.9 mmol of NMG/g exhibited the equilibrium binding capacity of 12 mg-B/g corresponding to the molar boron to NMG binding ratio of 0.66.<br>

&nbsp;&nbsp;An iminodiacetate (IDA) group was introduced into the crosslinked polymer chain grafted onto a porous sheet. An epoxy-group-containing monomer, glycidyl methacrylate, and a crosslinker, ethyleneglycoledimethacrylate, were cografted to the porous sheet with various concentrations of crosslinker of 1.0 to 6.0 mol% in a monomer solution, followed by epoxy ring opening with disodium iminodiacetate. A 500 mg/L copper chloride solution was permeated through the resultant chelating porous sheet at a residence time of 10 sec. The dynamic binding capacity of the crosslinked chelating porous sheet with an IDA group density of 1.7 mmol/g for copper ions was 1.7-fold higher than that of a noncrosslinked chelating porous sheet. This improvement results from the restriction of the axial diffusion along the sheet thickness induced by the gradient of the amount of copper ions adsorbed by the crosslinked graft chain along the porous sheet thickness. The dynamic binding capacity of the crosslinked chelating porous sheet decreased with increasing space velocity. This indicates that the time required for copper ions to diffuse in the radial direction of the pores is not negligible compared to the residence time of the copper chloride solution across the porous sheet.<br>

Glycidyl methacrylate (GMA) and divinyl benzene (DVB) were cografted onto a high-density polyethylene (HDPE) film with a thickness of 35 &mu;m by electron-beam-induced graft polymerization. First, one surface of the resultant GMA/DVB-cografted film reacted with trimethylammonium chloride to convert the epoxy group of the cograft chain into trimethylammonium group capable of selectively permeating mono-valent cations. Second, the remaining epoxy group across the entire film was converted into a sulfonic acid group by a reaction with sodium 3-mercapto-1-propanesulfonate. Electrodialysis of a mixture of 0.5 mol/L NaCl and 0.05 mol/L MgCl₂ using a pair of the resultant mono-valent cation selective cation-exchange membrane and a commercially available anion-exchange membrane exhibited a permselectivity coefficient of 0.56.

Cation-exchange membranes containing a sulfonic acid group were prepared by electron-beam-induced graft polymerization of sodium styrene sulfonate (SSS) onto a nylon-6 film with a thickness of 25 &mu;m. The lamella sizes and lamella-to-lamella intervals of the resultant cation-exchange membranes (SSS membranes) were evaluated by X-ray diffraction (XRD) analysis and small-angle neutron scattering (SANS), respectively. With increasing degrees of grafting, the lamella size decreased, whereas the lamella-to-lamella interval increased. This can be explained by that the poly-SSS chain grafted to the periphery of the lamella of nylon 6 partially destroys the lamella and invades the amorphous domain among the lamella. The SSS membrane with a degree of grafting of 150% exhibited a similar performance in the electrodialysis of 0.5 M sodium chloride as a current cation-exchange membrane and possessed the lamella sizes and lamella-to-lamella intervals of 7.6 and 13 nm, respectively.

Bromo-butyl styrene (BBS) was graft-polymerized onto a high-density polyethylene film with a thickness of 35 &mu;m, previously irradiated with an electron beam. The BBS-grafted film was converted into a strongly basic anionexchange membrane (BBS-TMA membrane) by reacting with trimethylamine hydrochloride. The thickness and resistance of the BBS-TMA membrane with a BBS grafting degree of 140% and a TMA group density of 2.2 mol/kg were 50 &mu;m and 0.57 &Omega;cm2, respectively, in 0.50 M sodium chloride at 298 K. After 42-day immersion of this membrane in 5.0 M NaOH at 353 K, the transport number of the BBS-TMA membrane decreased by 1.6% to 0.925 from its initial value of 0.94.

Gallium (Ga) ions as an affinity ligand were immobilized to the polymer chain grafted onto a porous hollow-fiber membrane to specifically capture phospho-compounds. An epoxy-group-containing vinyl monomer, glycidyl methacrylate, was graft-polymerized to an electron-beam-irradiated porous hollow-fiber membrane. The partial conversion of the epoxy group into an iminodiacetate group was effective in increasing the molar conversion in the subsequent introduction of a nitrilotriacetate group capable of forming a complex with Ga ions. The amount of Ga ions immobilized was 0.55 mmol per gram of the membrane. The overall rate of adsorption of phosphotyrosine to the Ga-ion-immobilized porous hollow-fiber membrane increased with an increase in the rate of permeation of phosphotyrosine solution through the pores because of the negligible diffusional mass-transfer resistance of phosphotyrosine to the affinity ligand.

N, N–dimethyl–γ–aminobutyric acid (DMGABA) as a carboxybetaine was introduced into the polymer chain grafted onto the pore surface of a polyethylene-made porous hollow-fiber membrane. The DMGABA–immobilized porous hollow-fiber membrane (DMGABA fiber) was prepared by the radiation–induced graft polymerization of glycidyl methacrylate (GMA) and the subsequent addition of DMGABA to the epoxy group of the grafted poly–GMA chain. The dose of the electron beam, the degree of GMA grafting (dg), and the molar conversion of the epoxy group into the DMGABA group were optimized to minimize the amount of protein adsorbed. As a result, a dose of 200 kGy, a dg of 140%, and a molar conversion above 3% were selected from the viewpoints of the protein binding capacity of the DMGABA fiber. The binding capacity of the DMGABA fiber for lysozyme in carbonate buffer (pH 9.0) was reduced to less than 10% that of the original polyethylene membrane. The lysozyme binding capacity of the DMGABA fiber in Britton–Robinson universal buffer (pH 7.0) was 17 ng per cm2 of the pore surface.

Glycidyl methacrylate (GMA), an epoxy-group-containing monomer, was graft-polymerized onto a high-density polyethylene (HDPE) film with a thickness of 35&mu;m. Divinyl benzene (DVB) was co-grafted as a cross-linker. Toluene used as a solvent of a mixture of GMA and DVB provided a uniform density of the grafted polymer chain across the membrane. Subsequently, the produced epoxy group was converted into anion and cation-exchange groups by a reaction with trimethylammonium chloride and sodium sulfite, respectively. The resultant anion- and cation- exchange membranes exhibited 0.55 and 0.34&Omega;cm², respectively, which were lower membrane resistances with ion-exchange capacity comparable to commercially available ion-exchange membranes. The highest brine concentration of 3.7mol/L for electrodialysis of 0.50mol/L sodium chloride as a model seawater was attained using the ion-exchange membranes prepared from GMA/DVB-co-grafted membranes with a DVB mole percentage of 1.0mol%.

A novel preparation scheme for size-exclusion polymer chain, capable of adsorbing low–molecular–mass targetswhile retarding proteins, was suggested. An epoxy–group–containing vinyl monomer, glycidyl methacrylate, waspolymerized onto a polyethylene–made porous hollow–fiber membrane by radiation–induced graft polymerization.Water was added to the epoxy group of the graft chain, followed by the reaction of the remaining epoxy group withtrimethylamine. Phosphate ionic species were adsorbed to the resultant anion–exchange porous hollow–fiber mem-brane, whereas bovine serum albumin (BSA)was not bound to the membrane. This remarkable difference in thebinding characteristics was explained by a distribution of the functional groups along the graft chain: a shrunken con-formation in the upper part of the graft chain exclusively containing the diol group retards BSA and accepts phos-phate ionic species. In contrast, a swollen conformation in the lower part along the graft chain exclusively containingthe trimethylammonium group captures the phosphate ionic species invading the upper part. The successive addi-tion of the reactantsin the introduction of the modification of the epoxy group of the graft chain provided a size exclu-sion structure for the graft chain.

An immobilized metal affinity porous membrane of a hollow-fiber form was applied to the purification of geneticallyengineered histidine (His)–tagged fusion protein. An iminodiacetate (IDA)group (–N(CH2COOH)2)was introducedinto the poly–glycidyl methacrylate chain grafted onto a polyethylene-made porous hollow–fiber membrane.Subsequently, nickel ions were bound to the IDA group before the permeation of a His–tagged green fluorescent pro-tein (GFP)solution through the porous membrane. The resultant immobilized nickel affinity porous membrane(immobilized Ni membrane)had a ligand density of 0.36 mol/kg and a phosphate buffer flux of 0.4 m/h at a perme-ation pressure of 0.1 MPa and 298 K. His–tagged GFP adsorbed to the immobilized Ni membrane was eluted by per-meating a 0.5 M imidazole solution through the porous membrane. From an SDS–PAGE analysis, the purity of theprotein was found to be improved from 35 to 97%.

Cation- and anion-exchange membranes were prepared by electron-beam-induced graft polymerization of an epoxy-group-containing vinyl monomer, glycidyl methacrylate, onto trunk polymeric films and subsequent introduction of sulfonic acid and trimethylammonium groups into the epoxy group, respectively. Nine kinds of commercially available polymeric films were used as a trunk polymeric film: low-density and high-density polyethylene (LDPE and HDPE), ultra-high-molecular-weight polyethylene (UHMW-PE), polyacrylonitrile (PAN), polyamide (NY), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), copolymer of ethylene and tetrafluoroethylene (ETFE), and copolymer of tetrafluoroethylene and perfluoroalkoethylene (PFA). HDPE- and NY-made films were selected as favorable trunk polymeric films according to the grafting rate of GMA and the strength of the resultant GMA-grafted membranes. Small angle X-ray scattering (SAXS) analysis demonstrated that the distance between the crystalline regions of HDPE increased from 25 to 49 and 38 nm, accompanied by the graft polymerization of GMA and introduction of sulfonic acid and trimethylammonium groups, respectively.

Anion- and cation-exchange membranes were prepared by means of electron-beam-induced graft polymerization of vinyl benzyltrimethylammonium chloride (VBTAC) and sodium styrene sulfonate (SSS), respectively, onto a nylon-6-made film. VBTAC- and SSS-grafted membranes prepared for a reaction time of 48 and 8h in methanol and water as a solvent of VBTAC and SSS had membrane resistances of 3.2 and 2.2&Omega;cm², respectively, for 0.5mol/L sodium chloride. These membrane resistances were to 20% higher and 8% lower than those of commercially available ion-exchange membranes, ASA and CSO, supplied by AGC Engineering Co. Electrodialysis of 0.5mol/L sodium chloride with the resultant ion-exchange membranes was performed at a current capacity of 30mA/cm² (25&deg;C). The chloride ion concentration in brine for the electrodialysis with a pair of the VBTAC-grafted membrane and CSO were 3.8mol/L, which was 95% that for electrodialysis with a pair of ASA and CSO; whereas a pair of ASA and the SSS-grafted membrane exhibited 105% chloride ion concentration in brine compared to a pair of ASA and CSO.

An immobilized metal affinity porous sheet was prepared by radiation-induced graft polymerization and subsequent chemical modifications. A 2-mm thick porous sheet, made of polyethylene, with a pore size and porosity of 1.3&mu;m and 75%, respectively, was irradiated with electron beam. An epoxy-group-containing vinyl monomer was graft-polymerized onto the irradiated porous sheet, followed by the conversion of the epoxy group into an iminodiacetate group. The resultant chelating porous sheet was cut into disks 13 mm in diameter suitable for the packing into the cylindrical cartridge. Nickel ions were immobilized before the permeation of a histidine (His) -tagged fusion protein solution through the porous sheet. Purification of His-tagged fusion green fluorescent proteins was demonstrated in a permeation mode using the nickel-ion-immobilized metal affinity porous sheet.

The recent developments of affinity techniques concerning single- and double-stranded DNA are reviewed. Based on the opinion that the immobilization of DNA is essential for the analytical and preparative use of the ligand function of DNAs, the two items are extensively discussed concerning the choice of conjugation chemistry and supports. Finally, a variety of applications, mainly for sensing and purification, are discussed.

The ultimate purpose of this study is to reconstitute the protein-localization system via reconstitution of yeast prenylation enzymes Ram-1/-2 system in Escherichia coli. We succeeded in establishing the colorimetric screening method to indirectly visualize the activity of protein prenylation systems. Also established was the selection platform for protein prenylation by way of the monitoring of expression level of reporter genes under the control of transcription factors with prenylation tags. We also succeeded in the improvement of the selection system by elevating the efficacy of incorporating reagent called dP, the hyper-mutagenic nucleoside analogues, into genomic DNA thereby reducing the necessary concentration of dP added to the selection media. This way, we could have established the robust and reproducible selection system for improving protein prenylation systems in heterologous hosts.

我々が開発したテルペン活性のスクリーニング法では、基質消費活性が高いほど、つまり細胞活性が高いほど「より白い」コロニを形成する。「白さ」を指標として、テルペン酵素の細胞活性をハイスループットに見積もることができる。これを用いて，以下２つの方向で実験を行った。
(1) タバコ由来5-epi-aristolochene（防カビ剤の前駆体）合成酵素(TEAS)，イチイ由来のタキサジエン（抗がん剤タキソールの前駆体）合成酵素（TaxS）を変異PCR法によってランダム変異 (～2変異/gene/世代)を導入し，ライブラリ化（多様化）した。つぎに，それぞれを黄色ブドウ球菌由来のC30カロテノイド合成経路とともにひとつの大腸菌内に発現させた。活性の無い酵素変異体の導入された大腸菌は，色素由来の黄色いコロニを与えたが，活性をもつ変異体を導入した細胞は，白っぽいコロニを与える。白いコロニから得た配列をサンプリングして解読したところ，ストップコドンやフレームシフト，そして重篤な構造破壊／機能低下をもたらす変異は完全に除去されていた。こうして，世界で初めて，テルペン酵素としての機能におけるMutability mapが描けることができるようになった。
(2) ゲラニオール酵素とTEASの２つの酵素について，ランダム変異→活性スクリーニング活性変異体を調べたところ，親よりも白いコロニーを幾つも得た。これらを解析したところ，細胞活性は有意に向上していたが，おもに異種発現性の向上によって説明できるものが殆どであった。