Earticle

Polylactic acid (PLA) is an increasingly attractive polymer that can be used in many industries. It is currently synthesized by two-step processes composed of fermentative production of lactic acid followed by rather complicated chemical polymerization step. In this lecture, I will report on the results of combined metabolic and enzyme engineering for the establishment of new pathways in E. coli for the biosynthesis of PLA and lactate containing polyester copolymers. [This work was supported by the Korean Systems Biology Research Project from the Ministry of Education, Science and Technology. Further supports by LG Chem and WCU (R32-2008-000-10142-0) program are appreciated.]

Repeat proteins have recently been of great interest as the templates for the development of protein scaffolds with wide applications in biotechnology and biomedical fields due to their unique structural and biophysical features. We here present the design of a binding scaffold based on repeat proteins by module engineering. A template scaffold is first constructed by joining appropriate numbers of repeat modules between the N- and C-capping motifs. The N-terminal domain of the template scaffold is then redesigned based on the internalin B cap by computational method. Using this approach, we developed a new binding scaffolds based on variable lymphocyte receptors (VLRs) composed of leucine-rich repeat (LRR) modules. The newly designed scaffold, termed ‘epebody’ showed a high-level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering and general applicability of the Repebody was demonstrated by rationally designing molecular binders for two target proteins (myeloid differentiation protein-2 and hen egg lysozyme). The crystal structures of the designed molecular binders revealed rigid backbone structures and unique interaction interfaces for the respective targets. The Repebody scaffold is well suited to create target-specific molecular binders by rational and molecular evolution approaches. The combined results demonstrate that our approach can be effectively used for the development of new binding scaffolds based on repeat proteins

Detection of lymph node (LN) metastasis by magnetic resonance imaging (MRI) has obtained clinical significance for treating cancer patients. LN metastasis often happens through regional lymphatic system, leading to distal tumor formation invluding lungs, liver and bones. Successful imaging of small and microscopic LN metastasis provides the helpful information in deciding the therapeutic option of cancer. Mannan is a water-soluble polysaccharide having high content of D-mannose residues which can be recognized by mannose receptors on activated macrophages and dendritic cells. Mannan-coated superparamagnetic iron oxide nanoparticles (Mannan-SPION) were developed to be specifically delivered to macrophages in lymph node by receptor-mediated endocytosis. Mannan-SPION was proven to be suitable for MR imaging due to small size, excellent stability in ferrofluid, and low cytotoxicity. In addition, mannan-SPION exhibited enhanced targeted delivery efficiency to macrophages in lymph nodes in vivo compared with PVA-SPION. Especially, LN enhancement of Mannan-SPION on MRI was dramatically increased at the later stage after intravenous injection compared with PVA-SPION control, indicative of the potential to successfully detect micrometastasis in LN.

Principally, chemical and physical methods have been used for immobilizing bioactive materials. However, there are some of drawbacks with those methods. For example, not only chemical method may produce potential toxic by-product and, the cost is high but also in case of physical method shows low efficiency of immobilizing bioactive material and it is difficult to control the best condition of reaction. To solve these problems, recently, immobilizing bioactive materials by photo reaction has been researched widely. The advantages of photo-immobilizing are 1) high selectivity of chemical reactions or processes under mild conditions (ambient temperture of also much below), 2) typically no need for added catalysts or special solvents, 3) spatially addressable effects (2D and 3D structuring possible), 4) applicable to very small and (relatively) large scales and 5) simple procedures. To use for photo-immobilization, various natural polymers, such as gelatin, chitosan, hyaluronic acid are reacted by irradiation to UV or visible light. They could be applied for medical area widely. For example, coating agent for bioinnert devices such as stent and implant, anti-adhesive agent, wound dressing and bio-adhesive.

In this study, the effect of pH of chitosan solution and chitosan-fucoidan mass ratio was investigated on the turbidity and aggregation pattern of suspension, the yield of dried mass, the extent of electrostatic interaction, the element analysis of dried mass, the particle size distribution and the morphology of polyelectrolyte complex prepared by polyelectrolyte complexation between chitosan and fucoidan.. With regard to the interaction of chitosan and fucoidan, the amino groups of chitosan electrostatically bind to the sulfate groups of fucoidan to form chitosan-fucoidan complex. It was observed that the turbidity and the yield of chitosan-fucoidan complex formation are dependent on the pH of chitosan solution as well as chitosan-fucoidan mass ratio. As the pH of chitosan solution increased, the effect of chitosan-fucoidan mass ratio becomes greater on the turbidity and the yield of chitosan-fucoidan complex. The chitosan-fucoidan ratio of 1:0.6, 1:0.8 and 1:1 at pH 6 led to the formation of big visible aggregates. However the lower ratio at this pH had no visible aggregation in the prepared dispersion. The result of FT-IR confirmes the formation of chitosan-fucoidan complex carrying the amino group from chitosan and the sulfate group from fucoidan. In the result of FT-IR the involvement of amide groups in the interaction is also evident, which may be exerted through hydrogen bonding interaction. The prepared chitosan-fucoidan complex nanoparticles were observed by scanning electron microscope and the mean size of prepared nanoparticle size distribution ranges between 350nm-900nm. The nanopartices tend to grow as the pH of chitosan increased up to 3.69, after which they become smaller. This pattern of growth is prominent as the mass ratio of chitosan and fucoidan decrease.

Recently, a great deal of interest has been developed to isolate novel bioactive compounds from natural resources due to their numerous health beneficial effects. Among them, the polysaccharides are widely distributed in the nature and their molecular structures have caused to their various biological activities. Chitosan is a naturally abundant β-1,4-linked copolymer of N-acetyl-2-amino-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-Dglucopyranose units and is a naturally occurring, biodegradable, non-allergenic deacetylated derivative of chitin, found abundantly in nature. Even though chitosan is known to have important functional activities, poor solubility makes them difficult to use in the food and biomedicinal applications. Unlike chitosan, its hydrolyzed products as chitosan oligosaccharides (COS) are readily soluble in water due to their shorter chain lengths and free amino groups in D-glucosamine units. It has been reported that novel COS derivatives can be used as physiologically bioactive materials since they possess various health beneficial biological activities, such as antioxidative, antitumor, antimicrobial, anti-hypertensive, antidiabetic, calcium absorption enhancing hypocholesterolemic activities and immune enhancing effects. Therefore, it is needful to prepare various types of COS derivatives, such as N-acetyl COS, Amino ethyl-COS (AE-COS), Dimethyl amino ethyl-COS (DMAE-COS) and sulfated COS with different degrees of deacetylation and molecular weights. Furthermore, the determination of their uninvestigated biological activities such as enzyme inhibitory (β-secretase, MMP, AchE), intra-cellular free radical scavenging, and anti-HIV-1 activites are useful to expand the health of human beings. The use of these novel COS derivatives as nutraceuticals, functional foods, pharmaceuticals and cosmeceuticals are promissing.

Bacterial cellulose (BC), produced by bacteria of genus Gluconoacetobacter is a pure biopolymer and exhibits higher degree of polymerization and crystallinity, high water holding capacity, and better adsorption of liquids compared to plant cellulose. The micro fibril of the BC form a high reticulated structure which is stabilized by inter and intra molecular hydrogen bonds. The inspiring mechanical properties and reinforcing potentials, presence of hydrogen binding sites, abundance, and biodegradable nature of BC nanocrystals make them perfect candidate for the processing of polymer nanocomposites. The composites of BC and chitosan (BC-Ch) have been synthesized by treating BC sheets with Ch solution. In this presentation, the strategy for the higher productivity of BC production is introduced. The purpose of the preparation of BC-Ch composite is introduced and the improved physico-chemical propertied will be reported.

During the past decade, aromatic dioxygenases isolated from various bacterial strains have increasingly attracted research interests primarily due to the potential application as biocatalysts for regioselective and enantioselective synthesis of vicinal cis-dihydrodiols (1). As a representative example, the toluene dioxygenase from Pseudomonas putida 39D has been extensively used to produce synthons for chemoenzymatic synthesis (2). Rhodococcus sp. strain DK17 can metabolize various benzene derivatives including o-xylene, toluene, ethylbenzene, and indan by degrading them through a common pathway initiated by a common aromatic oxygenase (3-6). The DK17 oxygenase enzyme possesses the unique ability to perform distinct regioselective hydroxylations that depend on the position and the size of the substituent groups on the aromatic ring. For example, the enzyme catalyzes dioxygenation on the aromatic ring of o-xylene to produce the o-xylene cis-3,4-dihydrodiol form of dihydrodiol or on ethylbenzene to produce the two dihydrodiols, ethylbenzene cis-2,3- and cis-3,4-dihydrodiol. The same enzyme has also been reported to oxidize m-xylene into 3-methylbenzylalcohol and 2,4-dimethylphenol at a ratio of 9:1. This presentation reports on the structure-based engineering and the potential application of the aromatic oxygenase in industrial biocatalysis.

For sustainable low carbon, recycling economy in future, marine biomass has potential as resources for bio-based energy, plastics, chemicals, and so on. We have been developing the biorefinery system to utilizing carbohydrates derived from brown algae to produce organic acids with high efficiency and high productivity. Alginates, laminaran and mannitol are most abundant carbohydrates of Laminaria japonica, but they are not easy-fermentable saccharides for most microorganisms even after pretreated chemically and/or physically, which contributes to low efficiency and availability of microbial transformation of brown algae. To achieve a high performance biorefinery system using brown algae, 1) we, from the gut of marine animals, screened and identified microorganisms which can digest polysaccharides of L. japonica to monosaccharides and simultaneously ferment them into organic acids; and 2) also, as the platform technology of bio-based chemical production, we have developed the continuous process of improved efficiency and yield of the organic acids-production via biotransformation of brown algae. Further research on metabolic flux analysis and metabolic engineering of the targeted microorganism will improve the technology of biotranformation of brown algae as resources for renewable biomaterials.

A systematic approach to determine the optimal operation strategy for nitrogen and phosphorus removal of sequencing batch reactors (SBR) has been developed and applied to successfully to a pilot-scale SBR. Activated Sludge Model No. 2d (ASM2d) is employed to model the SBR. Then, based on a survey of the relevant literature and a preliminary model-based analysis of the system, the following degrees of freedoms were identified and used for the SBR optimization: oxygen set-point in the aerobic phase (SO) and the lengths of anaerobic (TAN), aerobic (TA), and feeding (TF) phases. A grid of scenarios is formulated as fullfactorial experimental design to simulate the effect of the key degrees of freedom in the SBR system. Based on the effluent quality and robustness index of the best scenarios, the SBR operation under the condition (TF = 60 min, TAN = 140 min, TA = 240 min, SO = 2.0 mg O2/L) appeared to be the best scenario to provide effluent quality below discharge standards accompanied with good system stability. Under this scenario, the existing SBR performance for phosphorus removal could be improved by 93%.

Precious metals are widely being used in various industries because of their specific physical and chemical properties. Conventional methods for the recovery of low concentrations of the dissolved precious metal ions from solution phases include solvent extraction, chemical precipitation and ion exchange. These methods have significant disadvantages, including incomplete metal recovery, high capital costs, high reagent and energy requirements, and other waste products that require disposal. Such disadvantages, along with the increasing demand for new and economic processes for the recovery of metal ions from industrial effluents, have resulted in the investigation of alternative separation technologies. In this talk, sorption-based recovery of precious metals is introduced and the required specification of sorbents is discussed. Especially, strategies for design of powerful sorbents are suggested with some successful application cases.

Recently, an therapeutic angiogenesis based on human adipose-derived stem cells (hASCs) in an ischemic model was reported. However, their low differentiation efficiency limits further application of ASCs in clinical therapy. We sought to develop an artificial ECM for hASCs differentiation and to demonstrate that hASCs cultured on FGF2- immobilized substrate were readily differentiated into vascular cells. hASCs formed three-dimensional cell masses (3DCM) on FGF-immobilized substrate and produced angiogenic factors, such as VEGF. Tubular microvessels were observed in the ischemic hind limb of rat as well as nude mice that received the 3DCMs transplant, and were recognized by antibodies against human-SMA, KDR, CD31, and CD34, but not by antibodies against murine antigens. These results suggest that the vasculatures were originated from the human cells. The ability of implanted cells to be directly incorporated into newly formed vasculature has been a matter of debate. Transplanted ASCs or ASC-derived endothelial lineage cells resulted in host angiogenesis via the secretion of pro-angiogenic factors, and were partially involved in the neovascularization in mice hind-limb ischemia. However, it was not reported whether the cells formed vascular networks in vivo. In our study, an efficient method to form mature vasculature from human ASCs was described. In our knowledge, this study is the first to demonstrate direct vasculature formation from adult stem cells.

The methods currently used for treating alopecia have some limitations. The number of hair transplantation restricts because total transplantable hair number is no increase. To overcome these problems, researchers have attempted the in vitro culturing of hair follicle cells and implanting these cells in the treatment area. In the present study, culture-expanded mesenchymal stem cells (MSCs) that do not possess aggregative activity were used to produce self-aggregated cell-aggregated spheroidal dermal papilla like tissues (DPLTs) with the aid of a special culture condition in vitro, and hair bulb structure inductive capacity pertinent to the aggregative activity was then evaluated. Then hair inducing activity of self-aggregated DPLTs employing MSCs was tested in athymic mice.The DPLTs have the same hair bulb structure inductive ability as natural DPLTs in vitro. Transplanted DPLTs can induce new hair follicle in athymic mice. As a result, UC-MSCs and BM-MSCs may be an applicable and novel cell sources for the generation of human hair cell therapy.

The central strategy in tissue engineering involves a biomaterial scaffold as a delivery carrier of cells and a depot to deliver bioactive molecules. The ability of scaffolds to control cellular response to direct particular repair and regeneration processes is essential to obtain functional tissue engineering constructs. Therefore, many efforts have been made to understand local interactions of cells with their extracellular matrix (ECM) microenvironment and exploit these interactions for designing an ideal scaffold mimicking the chemical, physiological, and structural features of native ECM. ECM is composed of a number of biomacromolecules including proteins, glycosaminoglycans, and proteoglycans, which are assembled together to form complex 3-dimensional network. Cells are bound to these ECM components, which in many cases, is implicated in critical cell fate process such as adhesion, proliferation, differentiation, and survival. In this presentation, our approaches to develop cell-interactive scaffolds for modulation of cell function by providing several exemplary works of engineering bone, muscle, ischemic, and neural tissue.

Tissue engineering often requires a well-defined scaffold that is highly porous. The multi-head deposition system (MHDS), a form of solid freeform fabrication, has raised great interest as a method for fabricating scaffolds, since it yields a highly porous inter-connective structure without the use of cytotoxic solvents, and permits the diffusion of nutrients and oxygen. However, fabrication of scaffold using MHDS necessitates thermal procedure that might induce degradation of poly(lactic-co-glycolic acid) (PLGA) followed by accelerating acidification of environment surrounding implantation site. Acidic condition of the extracellular matrix deteriorates an engineered tissue. Moreover, this method is not suitable for introducing proteins, as it includes a heating process. In present study, we sought to determine whether three-dimensional scaffolds fabricated from PLGA with acetyl end group using MHDS method could be appropriately used as a scaffold to regenerate cartilage tissue in vivo. In addition, the surface of a scaffold fabricated using MHDS was coated with a mixture of fibrin and hyaluronic acid (HA) that was used as a vehicle for delivery of both proteins and stem cells. During scaffold fabrication by MHDS method, the acetyl end group modification of PLGA (PLGA-Ac) induced lower decrease of molecular weight than untreated PLGA. After in vivo implantation of the scaffolds with chondrocytes, PLGA-Ac scaffolds showed higher extracellular matrix and GAG content of engineered-cartilage without inflammatory response compared to untreated PLGA scaffolds. Meanwhile, fibrin/HA coating of the scaffold significantly enhanced initial cell attachment. The transplantation of adipose-derived stem cells(ASCs) inoculated on protein-loaded, fibrin/HA-coated scaffolds resulted in more improved tissue formation than did the transplantation of ASCs seeded on uncoated scaffolds or on fibrin/HA-coated scaffolds without proteins, but containing BMP-2 in the cell suspension medium. These results demonstrates that end group modification and physical coating method of scaffolds undergoing thermal process may be useful to enhance in vivo tissue regeneration

Abstract Stem cell implantation can be used to induce neovascularization and has been tested as a therapy for ischemia treatment. However, stem cell implantation as a therapy for ischemia treatment may have limitations for clinical applications. Since the methods of stem cell harvest are invasive, it may not be feasible to harvest autologous stem cells from aged patients or patients with cardiovascular risk. Furthermore, poor cell survival after engraftment in ischemic tissue may lower the therapeutic efficacy of stem cells. The goal of this study is to demonstrate that medium collected from human adipose-derived stromal cells (hADSCs) cultured as spheroids can exhibit improved therapeutic efficacy for ischemia treatment. hADSCs implanted to ischemic tissues support tissue revascularization in large part through secreted angiogenic factors. Due to a mild hypoxic environment formed in hADSC spheroid, spheroid culture was effective to precondition the hADSCs to upregulate hypoxia-inducible factor-1α gene expression following significant enhancement in both angiogenic and anti-apoptotic factor secretion to the culture medium compared to monolayer cultures. Conditioned medium derived from hADSC monolayer culture (M-CM) or spheroid culture (S-CM), fresh medium (FM), or hADSCs were injected intramuscularly into the muscle in the medial thigh after mouse hindlimb ischemia modeling. S-CM administration significantly enhanced neovasclurization, protected muscles from incipient ischemic apoptosis, and improved limb survival as compared to M-CM or FM administration or hADSC implantation. These data suggest that injection of conditioned medium obtained from hADSC spheroid culture may be more effective therapeutic option for treatment of ischemic diseases than hADSC implantation. References 1. Bhang SH, Cho SW, La WG, Lee TJ, Yang HS, Sun AY, Baek SH, Rhie JW, Kim BS. Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells. Biomaterials (in press)

Biomaterials can provide stem cells with the microenvironments which can regulate stem cell fates. The stem cell behavior including proliferation and differentiation can be modulated by engineering of biomaterials for stem cell culture. Here we propose that engineered biomaterials can provide the role of stem cell niches to enhance proliferation and specific differentiation of stem cells. Several strategies of biomaterial engineering for stem cell manipulation will be introduced.

This paper presents a microfluidic immunohistochemistry (IHC) platform for accurate histopathological diagnoses using numerous specific biomarkers simultaneously. Breast cancer is a heterogeneous disease with many biological subtypes, and it is difficult to accurately monitor the treatment response of the disease as well as to predict the clinical outcome of individual neoplasms. We recently succeeded in developing a microfluidic interface that enables multiplexed IHC measurements on breast tissue samples. The device consists of a poly(dimethylsiloxane) (PDMS) microfludic layer with four parallel channels, which is simply pressed onto the tissue slide. Consequently, four biomarkers, such as estrogen receptor, human epidermal growth factor receptor 2, progesterone receptor and Ki-67, were examined simultaneously on human breast cancer tissues including needle biopsy. This new IHC platform has improved performance concerning assay time, consumption of tissue, antibodies and staining compounds, sensitivity, specificity and costeffectiveness. The similar microfluidic platform has also been applied for quantitative proteomic profiling in breast cancer samples. Proteomic profiling via immunocytochemistry (ICC) was examined for four breast cancer cell lines. The device enabled 20 ICC assays on a biological specimen at the same time and could be used to quantitatively compare the expression level of each biomarker.

Nanosensors functionalized with biomolecules hold promise for many potential applications in biotechnology and nanomedicine. Biolumenescent nanosensors have been drawing much attention for in vitro and in vivo monitoring due to their extremely low background and high sensitivity. Our developed nanosensor is based on the energy transfer between the donor bioluminescent protein (luciferase) and its counterpart (nanoparticle), which was applied for the detection of matrix metalloproteinase (MMP) activity. MMP was chosen as the model protease because of its important role in promoting tumor progression and invasion. In this talk, emphasis will be made upon the broad applications of BL nanosensors to in vitro diagnosis and in vivo imaging with functional conjugation methods between biomolecules and nanoparticles. This approach will help expand the utility of bioluminescent detection in biology and nanotechnology research.

There are very big trend changes in the pharmaceutical business. In spite of huge information from "-omics" researches, there are rather reduced new drugs in development. And we can no more expect blockbuster drugs like a cholesterol drug "Lipito". Instead, people often raise issues of personalized medicines, stratified medicines, and pharmacogenetics. And another big issue is also raising, integration of therapy and diagnostics, namely theragnostics or companion diagnostics. In this presentation, background of these changes, important roll of diagnostics, trends in diagnostics market including several companies, value chains of diagnostics area, and to do area of diagnostics will be discussed.

In this study, the five different aptamers (to Oxytetracyclin, Tetracyclin, Ibuprofen, Diclofenac, and b-estradiol) having different affinity ratios were used, and the order in the sensitivity of target detection was found to be well correlated with its affinity ratio. An affinity ratio, the affinity of the aptamer to targets divided by the affinity to umAuNPs (KdAuNP/KdTarget), was found to be an important factor determining the sensitivity in the competitive interactions among aptamers, targets, and unmodified gold nanoparticles (umAuNPs) [1]. This working principle was primarily confirmed by using a tetracycline binding aptamer showing different affinities to its three derivatives, tetracycline, oxytetracycline, and doxycycline [2]. The color change of the mixture containing umAuNPs, targets, and the tetracyclin binding aptamers (TBAs) was found to be proportional to the affinity ratio of TBA to each of three target compounds. In addition, by implementing this principle established, the sensitivity of ibuprofen detection could be enhanced simply by increasing the ratio of affinity, i.e., reducing the ibuprofen binding aptamer’ affinity to umAuNPs by using bis (p-sulfonatophenyl) phenylphosphine as an AuNP-capping ligand, instead of using the citrate. A clear color change was resulted even at a 20-fold less amount of ibuprofen. Finally, this working principle has been also strongly supported and confirmed by adsorption-equilibrium based mathematical model simulation.

We developed various assay systems using protein arrays for protein profiling and determination of enzyme activities in a high-throughput manner. Protein arrays is a key technology in proteomics and serodiagnosis, since the technology allows a high-throughput and large-scale analysis of protein interactions in a small sample format. We fabricated protein arrays by immobilizing proteins or antibodies to well-type amine arrays. We developed assay systems for rapid determination of matrixmetalloproteinase (MMP)-3 activity using an array-based spectral surface plasmon resonance biosensor and fluorescence-based gelatin arrays, and these systems showed a potential for screening of MMP-3 inhibitors. We also developed an on-chip activity assay of blood coagulation factor XIII (FXIII) using fluorescence-based fibrinogen arrays and it showed a strong potential to be applied toward FXIII-related personalized medicines. In addition, fluorescence-based antibody arrays were successfully applied to identification of serological marker proteins for liver disease. Thus, protein array system, based on SPR biosensors and fluorescence detection, is useful for inhibitor screening, serodiagnosis, and personalized medicine.

We report a quick and reproducible surface-enhanced Raman scattering (SERS)-based opotofluidic sensor for multiplex biomarker detection. A novel microfluidic sensor with functional internal structures has been designed and fabricated. This sensor is composed of three compartments consisting of the gradient channel that serially dilutes the target marker, the injection and mixing area of antibody-conjugated hollow gold nanospheres and magnetic beads and the trapping area of sandwich immunocomplexes using multiple solenoids. Quantitative analysis of a specific target marker is performed by analysing its characteristic SERS signals. This SERSbased gradient optofluidic sensor can replace the set of microwells used in manual serial dilutions that have been traditionally used in ELISAtype assays. This novel SERS-based optofluidic immunoassay system is expected to be a powerful clinical tool for the fast and sensitive medical diagnosis of a disease.

Spider silk fiber spun from spider is extremely strong and as strong as Kevlar. However, this great material property could not be realized so far by using the made recombinant spider silk proteins. In this lecture, I will report designing of synthetic gene modules, systems metabolic engineering of E. coli network to increase the required metabolic fluxes, and cellular engineering to cope with increased tRNA pool. This allowed production of ultra high molecular weight spider silk protein in E. coli, which gave Kevlar strength silk fiber when spun. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. Further support by the World Class University Program (R32-2008-000- 10142-0) through the National Research Foundation of Korea funded by the MEST is appreciated.]

Synthetic wood composite films, fibers, and beads containing cellulose, hemicelluloses, and lignin, the three major components of natural wood, were prepared in a room temperature ionic liquid solvent, 1-ethyl-3-methylimidazolium acetate, [Emim][Ac]. Various synthetic wood composites were obtained by dissolution of individual wood components together with additives, including polyethylene glycol (PEG), chitosan, multi-wall carbon nanotubes, lipase, and poly(3-octylthiophene) (P3OT) in [Emim][Ac]. The addition of water affords a gel that was dried in either a low humidity environment or under vacuum. Synthetic wood films showed smoother surface textures, higher water resistance, and higher tensile strengths than cellulose films formed by the same methods. Synthetic wood electrospun fibers containing P3OT showed micrometer sized and branched fiber structures. Lipase entrapped in synthetic wood composite beads retained high enzyme activity. Tailor-made synthetic wood composites were also prepared having a variety of desirable properties, including antimicrobial activities, controlled hydrophobicity/philicity, high relative dielectric constant, and a high degree of cohesiveness.

The regeneration of nicotinamide cofactors is critically needed for the promotion of economically feasible redox enzymatic processes; however, it remains a key unsolved problem over the last decades. In biocatalytic systems, enzymes catalyze and build intricate products with an amazing specificity under environmentally benign conditions. With recent advances in genomics and genetic engineering, the use of biocatalysis for industrial synthetic chemistry is growing rapidly. Most of the biocatalysis in current use, however, are limited to cofactorindependent enzymes, such as hydrolases, which perform relatively simple chemistry. In comparison, cofactor-dependent enzymes are capable of performing much more complex chemistry and catalyzing a large number of synthetically useful reactions. In this talk, I will discuss about how to regenerate cofactors photochemically by using visible light to couple with redox enzymatic reactions. The success of this approach will open a promizing route to fix solar energy in the form of fine chemicals towards the development of biomimetic artificial photosynthesis. Artificial photosynthesis will create a technological foundation for the efficient synthesis and production of fine chemicals utilizing solar energy.

Marine mussels attach to substrates using adhesive proteins. It has been suggested that complex coacervation (liquid-liquid phase separation via concentration) might be involved in the highly condensed and non-water dispersed adhesion process of mussel adhesive proteins (MAPs). However, as purified natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation model. We demonstrate complex coacervation in a system including recombinant MAPs. Our recombinant hybrid MAPs can be produced in large quantities, and are readily purified. We observed successful complex coacervation using cationic MAP and an anionic partner. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strength of MAPs in both dry and wet environments. Especially, underwater adhesion was possible using the coacervated MAPs. Collectively, our results indicate that a complex coacervation system based on MAPs shows superior adhesive properties, combined with additional valuable features including liquid/liquid phase separation and appropriate viscoelasticity. Our coacervated mussel adhesive could be useful in the development of underwater adhesion for use in biomedical applications.

Due to the inter-particle-coupling effect on the surface plasmon resonance of gold nanoparticles (AuNPs), controlled aggregation of AuNPs has been a subject of increasing interest especially for colorimetric detections of biologically or medically important substances. In addition, AuNPs exhibit high efficacy for fluorescence quenching over a wide range of wavelengths than organic molecules because of the strong absorbance extinction and unique dimension. Therefore, AuNPs have been also adapted in fluorescence-change-based monitoring systems. Versatile interactions of nucleic acids with various molecules such as proteins, enzymes, metal ions and chemical compounds and advents of selection technologies for synthetic oligonucleotides called aptamers, DNAzymes, and RNAzymes have spanned the possibility of nucleic acids as biological components with high specificity and affinity in biosensors. In addition, the significant advances for the detection of nucleic acids up to single copy of the target nucleic acids have allowed the nucleic acids as signal amplifying mediators for detections of the non nucleic acid targets. In recent, we developed variety of biosensors combining the functional nucleic acids with AuNPs for detections of medically and environmentally concerned substances including protease, antimicrobial agents, heavy metal ions, and genotoxic drugs. In this talk, we will present the high performances of the biosensors which showed high sensitivity, specificity and also operational feasibility. For examples, DNA mediated amplification of fluorescence signal on AuNPs resulted in 100-fold enhancement of sensitivity for the detection of protease and lead ion and controlled interaction between AuNPs allowed high throughput detection of genotoxic substances including even ultraviolet light in an instrument-free way. We will discuss more details of the working principles and methods to integrate the functional nucleic acids in the nanoparticles based biosensors along with the experimental results.

For sustainable development of current industries, many research groups have made an effort to change the currently available crude oil to the renewable biomass as resources. To overcome the general problems of corn- and sugar-based biomass, non-food biomass such as cellulosic and marine biomass has been concerned as alternative biomass for production of biofuels and biochemicals. Contrary to cellulosic biomass (tree, straw, agricultural residue et al.), marine biomass (red, green, brown algae) does not contain lignin, of which presence raises many problems in bioprocess for biomaterial production. Among marine biomass, red algae are mostly composed of cellulose and galactan. Galactan is a representative polymer consisting of D-galactose and anhydro-galactose. For efficient conversion of red algae to biomaterials, both glucose from cellulose and D-galactose from galactan should be fermented. In this study, a food-grade lactic acid bacterium, Lactobacillus casei was chosen to be metabolically engineered to produce a value-added biochemical of lactic acid and a promising biofuel of bioethanol simultaneously from both glucose and galactose. For simultaneous production of lactic acid and bioethanol, two foreign genes coding for pyruvate decarboxylase and alcohol dehydrogenase were expressed and many genes encoding endogenous lactate dehydrogenase were disrupted in Lactobacillus strains. Batch fermentation and transcriptional analysis were undertaken to investigate the simultaneous production of lactic acid and bioethanol. In this research, it is provided that red algae and lactic acid bacteria have a potential to be used for production of biofuels and biochemcials.

Fluorescent protein variants with different spectral characteristics are highly desirable in broad range of biotechnology applications. Among them the green fluorescent protein (avGFP) from Aequorea victoria is unique fluorescent proteins and from the onset wild-type GFP, variety of mutant variants has been created and in many contexts they became a powerful reporter in the cellular and molecular biology. Several mutations were introduced at the chromophore and surrounding regions that resulted in various GFP mutants with different spectral properties which were successfully used in multi-color labeling, resonance energy transfer, and intracellular tracking studies. To obtain these variants, directed evolution and rational mutagenesis has been the method of choice to alter the spectral properties. Through these methods, only the canonical amino acids were used for replacing the chromophore residues of the protein. In particular, the presence of aromatic amino acid at Tyrosine 66 position is frequently used to create GFP variants. On the other hand, incorporation of non canonical amino acids into chromophore residues of GFP will overcome these limitations. The NCAA provides new functional groups to the protein that manipulates or enhances the spectral properties of the protein. Here, we reassigned of Tyrosine codon with different surrogates through selective pressure incorporation method. It offered different faces to the wild GFP with interesting features such as protein biosensor, protein conjugation, highly red shifted variant etc.