Earticle

3-hydroxypropionic acid (3-HP), an industrially important platform chemical is used as a precursor for many commercially important chemicals. Recently, the production of 3-HP from glycerol was demonstrated in recombinant K. pneumoniae without the addition of expensive coenzyme B12 [1]. 3-HP production in K. pneumoniae is always associated with the production of 1,3-propanediol and lactic acid due to the generation of large amounts of NADH [2]. To enhance 3-HP yield on glycerol by regenerating NAD+, nitrate was supplemented in the culture medium. But this resulted in significant decrease in 3-HP production and majority of carbon was utilized for cell biomass generation, due to the presence of undesirable anaerobic respiration pathway. To overcome this limitation, a novel recombinant strain, Klebsiella pneumoniae DdhaTDglpK (puuC) was developed. This strain significantly increased 3-HP production at a maximum of 22.2 g/L in the presence of nitrate under anaerobic condition with a yield on glycerol at 0.35 mol/mol.

In our previous study, indigo was produced by recombinant E. coli harboring a flavin-containing monooxygenase gene. The recombinant E. coli DH5α pBlue1.7 produced indigo (920 mg/L) and indirubin (≤5 mg/L) from 2g/L tryptophan. In this study, indigo production was performed in 5, 30, 100, and 3000L reactors containing tryptophan medium under the conditions as follows: working temperature 30℃, pH 7.0 and air supply 3vvm. The batch fermentation (3,000-L fermenter) produced 911±22 mg/L of indigo (yield 45.5%). For continuous fermentation (5-L fermenter), volumetric productivity was found to be 230 mg/L/h for up to 110 h (final accumulated bio-indigo was 23 g) with a constant dilution rate (D) of 0.084/h. In addition, it was found that indirubin production greatly increased when cysteine (3mM) was added to the tryptophan medium although it inhibited the growth of the recombinant E. coli DH5α pBlue1.7. Also, it was found that cystein modifies the synthetic pathway for hydroxyindole, which might function in increasing indiruin production. Optimum culture conditions for indirubin production in tryptophan medium was obtained from the response surface methodology analysis: 2 g/L tryptophan, 5 g/L yeast extract, 10 g/L NaCl, 3 mM cysteine, pH 8.0 and at 35℃. Under this condition, the recombinant E. coli cells were capable of producing 223.6 mg/L of indirubin from 2g/L of tryptophan.

The advent of biobutanol production leads to the search for cheap and efficient raw materials which do not necessitate the utilization of food crops and other lignocellulosic biomass. Macroalgae are promising options because they can be propagated easily along the seaside of the Korean peninsula thus eliminating the need for land and nutrient resources. In this study, four species of brown algae (Undaria pinnatifida, Laminaria japonica, Ecklonia stolonifera, Hizikia fusiforme, and Sargassum fulvellum) and two species of red algae (Porphyra tenera and Gelidium amansii) were investigated for the production of butanol by Clostridium saccharoperbutylacetonicum N1-4. To hydrolyze the polymeric materials of the algal biomass, dilute acid hydrolysis was carried out using 0.15 M H2SO4 followed by thermal pretreatment at 121°C for 1 hr. Using 100 g/L of hydrolyzed brown alga, the highest butanol production (5.51 g/L) was observed for L. japonica. Other brown algae and red algae did not exceed the butanol production by L. japonica, although, red algae were thought to contain easily digestible polymeric materials. Furthermore, a post-treatment of the acid-hydrolyzed biomass was carried out. Using the activated carbon and overliming methods, the final butanol concentration had increased by 24.14% and 12.16%, respectively. This showed that inhibiting substances might be present in the hydrolytic solution of the macroalgae.

Seaweed is a third generation biomass of bioethanol production. There are three types of seaweeds such as brown, green and red algae. Especially, Gelidium amansii, red seaweed, has advantage of high carbohydrate contents converted to fermentable sugar such as glucose and galactose. To obtain high ethanol concentrations, agar and cellulose have to be converted to galactose and glucose. Therefore, pretreatment is essential process for high reducing sugar production. In this study, thermal acid hydrolysis was performed with autoclave and sulfuric acid each treatment. In order to optimize pretreatment using Gelidium amansii, experiment was carried out with variables such as seaweed slurry contents, acid concentrations and treatment time. Optimal pretreatment condition was determined as follows: 8% (w/v) seaweed slurry with 113 mM sulfuric acid by autoclave at 121℃ for 30 min. Saccharification yields of 39.6% of fermentable sugar were obtained from Gelidium amansii.

Bio-ethanol is regarded as an alternative fuel to replace fossil fuels. Sugar cane, corn, cassava and seaweed have been studied as biomass for bio-ethanol production. The seaweed is regarded a third generation biomass among them. Seaweed has advantages in fast growth of biomass and high productivity in large area. In this study, seaweed, Saccharina japonica (Sea tangle, Dasima) was used as biomass to produce bioethanol. The pretreatment was evaluated by thermal acid hydrolysis with H2SO4 and HCl. Optimal pretreatment condition was determined with 37.5mM H2SO4and10%(w/v) seaweedslurrybyautocalveat121℃ for 60 min. To Increase the yield of saccharfication, 0.45 g dcw/L of isolated marine bacteria Bacillus sp. were used often the pretreatment and 43 g/L of reducing sugar was obtained. Ethanol fermentation was performed using simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) method with Pachysolen tannophilus KCTC 7937. The ethanol concentration of 6.3 g/L by SSF process and 6.0 g/L by SHF process were obtained respectively. Ethanol yields of 19.0 % and 18.1% of theoretical yield from total carbohydrate in Saccharina japonica were obtained by SSF and SHF, respectively.

Fossil fuel reserves are decreasing fast rate whreas the demans for alternative fuels such as biodiesel and bioethanol. As a raw material for biodiesel is increasing and accumulated lipids from microalgae is already technically feasible. Microalgae such as marine microaglae and cyanobacteria produce long-chain polyunsaturated fatty acids (LC-PUFAs) by PUFA synthase. Biodiesel production based on microalgae as a feedstock is associated with a high demand of nutrients, such as nitrogen and phosphorus. The N/P ratio of microalgae and cyanobacteria is critical to produce PUFA in nutrient limited cells. In this research, molecular N and P ratio effects on cell growth of microalgae Schizochytrium sp.1, benthic microalgae and Schizochytrium sp. 2. N/P ratio were set to 0/1, 8/1, 12/1, 24/1, 32/1 with other nutrients. Maximum dry cell weight of Schizochytrium sp. 1 was 1.274 g/L (dry cell weight) at N/P ratio of 8/1 and maximum dry cell weight of Schizochytrium sp. 2 was 0.442g/L (dry cell weight) N/P ratio of 12/1 at 15 days of cell cultures, respectively. pH was not changed, however phosphate was rapidly decreased with cell growth. Further study is needed to affect other factor to cell growth and analyze accumulated lipid in microalgae.

Enantioselective hydrolysis of racemic epoxides using epoxide hydrolase has been considered as the most advanced technique for the production of chiral epoxides. The epoxide hydrolase of Mugil cephalus (McEH) exhibits high activity, broad substrate spectra and low diol product inhibition. However, low stability, short life time and high price of epoxide hydrolase enzyme are main problems in enzymatic production of chiral epoxides. In order to prevent these problems, McEH enzyme was immobilized on various nanostructured materials. Immobilization of McEH enzyme onto nanostructured materials has several advantages such as high enzyme loading and low mass transfer inhibition due to large surface-to-volume ratio. Immobilization of McEH enzyme was carried out by the formation of cross-linking enzyme aggregate via covalent linkage between the amine groups in enzyme using glutaraldehyde. We analyzed and evaluated the stability of the immobilized McEH enzymes. The immobilized enzymes were efficiently reused more than seven times in repeated batch reactions for the production of chiral epoxides.

Byssal cuticles are compelling paradigms as wear and damage resistant coatings for compliant materials. The byssus of Mytilus species are covered by the protective coating protein Mytilus edulis foot protein-1 (Mefp-1). Protective coatings for tissues and materias are appealing to medical implant materials. The hardness(~ 2 GPa) and stiffness(~ 0.1 GPa) of the coating measured by nanoindentation are comparable with those of robust engineered epoxy, but the ultimate strain of the Mytilus cuticles are 20 times higher than that of expoxy.The main challenge in these bio-mimetic approaches is to sufficiently simplify the structure to enable easy-to-use synthesis routs. We produced, purified recombinant mussel foot protein-1 (Rmfp-1) for mimicking the byssal coating protein mefp-1. Rmfp-1 was produced from E.coli. Decapeptide was produced by cutting the Rmfp-1. The properties of Rmfp-1 and decapeptide were directly measured at the nano-scale by using the Surface Force Apparatus (SFA)

In recently studies, many strains isolated from Antarctica. Scientist have found that bacteria which survive in polar environment. This environment is extremely cold and nutrient-poor region. Nutrient and temperature are known as important factor in growing bacteria. Surprisingly, however, the qualitative and quantitative relationships between these factors are poorly understood. Recent investigations show that the temperature determines whether genetically adapted at low temperature or high temperature. Therefore we measure temperature dependence of their growth rates at both low and high temperature. A few strains of bacteria, isolated from various sites such as soil, sea and sediment in Antarctica, were grown at diverse temperature ranging from 5 to 37℃. A relationship was examined between growth rate and temperature. This relationship differed slightly according to each species. We investigated an analysis of data on temperature and growth rates for bacteria. From these data, we suggest a possible hypothesis for relationship in data on bacterial growth rates.

Marine brown algae are rich in phlorotannins which have various biological functions such as antioxidant, anti-aging and skin whitening. In this study, phlorotannins were isolated from the marine brown alga and their skin whitening effect on melanoma cells was evaluated by measuring tyrosinase inhibition. The result showed that phlorotannins were inhibited tyrosinase with an IC50 of 200 μM and it was more effective than arbutin without any significant cytotoxicity in melanoma cells. Furthermore, phlorotannins contents in marine brown alga were analyzed by reverse-phased high performance liquid chromatography (HPLC) technique. The HPLC method for determination of phlorotannins from the extracts of marine brown alga was validated for linearity, precision, accuracy, limit of detection (LOD) and limits of quantitation (LOQ). The contents of phlorotannins were calculated by linear calibration curves of standard compounds. These results suggest that phlorotannins have great potential to be further developed as a pharmaceutical or cosmetic agent for use in dermatological disorders associated with melanin.

Today a number of nanoparticles are manufactured for clinical and medicinal purposes through conjugating with drugs or bio-molecules such as DNA, RNA, and peptides. There are three distinct ways to deliver nanoparticles into animal and human bodies: injection, feeding, and transdermal administration. In this study, we fabricated biodegradable polymeric microneedle arrays containing nanoparticles for the efficient delivery of functional nanoparticle complexes through the skin. Nanoparticle-conjugated microneedle arrays were manufactured through three steps. First, PDMS (polydimethylsiloxane) molds for needles were casted out using silicon wafer. Second, microneedles were fabricated by curing melted agarose in the mold together with nanoparticles. Third, nanoparticles were separated and arrayed by their sizes using electrophoresis. These nanoparticle-conjugated microneedles have a great potential for an alternative method to deliver cosmetic or pharmaceutical materials into human skin locally without professional procedures and pain.

The Z-domains of protein A was expressed as a fusion protein at the outer membrane of E.coli by using the autodisplay technology. Because of the specific affinity towards the Fc region of immunoglobulins (IgG’s), the Z-domains have been used for the orientation control of antibodies in order to improve the sensitivity of immunoassays[1-2]. In this work the E.coli with autodispalyed Z-domains was immobilized as a monolayer to the SPR biosensor by the charge interaction. The surface modification was carried out by senquential layering of parylene-H film with formyl groups and poly- Llysine, and then the E.coli cells were immobilized by the charge interaction[3]. The effectiveness of this layer for the immobilization of E.coli was estimated by counting the number of E.coli cells in comparison with bare gold surface and poly-L-lysine coated gold surface. For the test of feasibility of the immobilized E.coli cells to SPR biosensor, the stability of immobilized E.coli cells was estimated by treatment of concentrated salt solution to the immobilized E.coli cells which were bound through the charge interaction. From this test, the E.coli cells immobilized to the parylene-H film with poly-L-lysine coating were determined to be stable under the salt concentration of human serum. Then, the applicability of the immobilized E.coli cells with autodisplayed Z-domains was demonstrated by detection of C-reactive protein (CRP). The effect of orientation control by autodisplayed Z-domains was estimated by comparing the sensivities by immobilization through the physical adsorption and charge interaction to poly-L-lysine coated layer.

Alginate is a major polysaccharide in brown algae and composed of two uronic acids, mannuronic acid and guluronic acid. Because of few information of uronic acid-metabolizing enzymes, utilization of the uronic acids as carbon source has been limited. As these backgrounds, we aimed at the development of enzymatic systems to produce alginate monomers and microorganisms able to utilize alginate monomers in this study. Two alginate lyases of Alg7D and Alg17C from a marine bacterium, Saccharophagus degradans 2-40, were overexpressed in Escherichia coli BL21(DE3) strain. SDS-PAGE analysis showed that Alg7D and Alg17C were expressed solubly in E. coli and their contents reached almost 80% of total protein. To hydrolyze alginate, crude protein solutions containing each enzyme were prepared and treated with sodium alginate sequentially. By thin layer chromatography, two-hours reaction with Alg7D at 50℃ resulted in hydrolysis of alginate with low DPs (about 2~4) and more addition of Alg17C gave full degradation of alginate polymer to its monomer (DP1). Alg17C was only able to hydrolyze sodium alginate to its monomer but its efficiency was lower than that of dual treatment of Alg7D and Alg17C.

Listeria monocytogenes is a facultative anaerobe, gram-positive bacterium that is isolated from the soil, vegetables, and wild or domestic animals. Listeria occurs predominantly in the elderly, immunocompromised patients, pregnant women and newborns. Infection by L. monocytogenes causes the disease listeriosis in humans that may meningitis, meningoencephalitis, brain abscess, pyogenic arthritis, osteomylitis, and liver abscesses. When this bacteria is present within a host organism quorum sensing cause the up regulation of several virulence genes. The prfA is the main activator of Listeria virulence gene expression in blood. Therefore, we have developed a new approach for detection of listeriosis using DNA aptamers. We selected target protein (prfA) to regulator of virulence gene expression and performed 10 cycle of SELEX (systematic evolution of ligands by exponential enrichment). Using real-time PCR and surface plasmon resonance (SPR), we tested the binding effects of aptamers. After aptamers obtained 9 cycle of selection demonstrated the high affinity and specific binding aptamers with prfA. In conclusion, aptasensor based on specific prfA binding aptamers can be effectively applied for the rapid and sensitive detection of L. monocytogenes from the contaminated environments such as corrupt food products, polluted drinking water, and stable vegetables.

Carotenoids are red-orange pigments abundant in many vegetables and fruits including carrot and tomato. Due to the antioxidant and anticancer activity of carotenoids, they are manufactured for the nutritional and health beneficial supplements. Recently, vegetables with larger amount of carotenoids content have been established by genetic modification or interbreeding. However, the instability and low water solubility of carotenoids are obstacles in the use of them as supplements in a variety of biological systems. In this study, we investigated the antioxidant activity and stability of three different carotenoids chemically synthesized by adding side benzene ring groups to the carotenoid backbone. In comparison with β-carotene, the representative carotenoids, three novel carotenoids displayed stronger antioxidant properties in DPPH and ABST assessments with higher storage stabilities.

This study presents the effects of micro-geometries on the swimming behavior of Pseudomonas aeruginosa. First, we have measured parameters of single-cell motility including cell speed, run duration time, and tumble angle under two dimensional space. Recently the modern microfabrication tools allow us to create complex environments at the micrometer-scale and study of small micro-geometries. In the last decade microfabrication and microfluidics have been found to be very useful in the study of the motility of microorganisms. The results are used to calculate motility coefficients in the width of microchannels ranging from 10 to 100 μm. Since the single-cell motility parameters measured depend on the interaction of flagella with the microchannel wall, the duration time of the running cell in restricted geometries is distinctively different. Therefore, the motility of bacteria is decreased by restricted geometries. This study suggests that microfluidic approach is useful tool for the analysis of bacterial motility under the restricted space and rapid analytical tool.

Undaria pinnatipida including physiologically active substances such as alginate and iodine has been known for health functional food in Korea. Various physiologically active substances isolated from Undaria pinnatipida were investigated to reduce obesity causing adult diseases. In this study, U. pinnatipida harvested in Korean coast was used for the substances by the extractions using hot water, ethanol and methanol. All samples were lyophilized after extraction. Accumulation of triglyceride in mature adipocyte was determined using ORO staining. Cell toxicity of the preadipocyte was determined by MTT assay. Various concentrations of extracts with 0, 50, 100, 150, 200 and 250 ㎍/ml dissolved in DMSO were added to differentiation medium of 3T3-L1. Hot water extract (250 ㎍/ml) did not show cell toxicity at the viability of 82% in 3T3-L1. However, extracts by ethanol (150 ㎍/ml) and methanol (150 ㎍/ml) showed cell toxicity with the cell viability less than 28%. Triglyceride accumulation of the adipocyte treated the extract by hot water was effective to the anti-obesity of 40% comparing to the control cell.

Depletion of fossil fuels and the increasing price of petroleum-based fuels have strengthened the interest of alternative. Renewable bioenergy has been introduced as an important contribution to future sustainable energy system. Among them, bioethanol is a promising fuel as it could replace the transportation fuels. Current bioethanol production has been performed using agricultural feedstock and lignocellulosic biomass but it has the considerable problem to its sustainability and difficulties of lignin decomposition, respectively. The seaweed is third generation biomass for the bioethanol production with advantages of high carbohydrate content and lignin-free compositions. The seaweed, Saccharina japonica (sea tangle) was studied as substrates for the bioethanol production in this study. S. japonica has high contents of carbohydrates such as alginate, laminaran and fucoidan. Conversion of its polysaccharide to monosaccharide, the saccharification must be performed prior to fermentation. Thermal hydrogen peroxide and marine bacteria treatment were applied for the saccharification. And separate hydrolysis and fermentation (SHF) is a potential operation mode for effective ethanol production from hydrogen peroxidel pretreated S. japonica.

Microsphere-based flow cytometric assay is commonly used immune assay for the analysis of mixed analytes in solution. It is based on antibodies conjugated fluorescence micro beads and uses the flow cytometer for detection. In this study, we developed a new flow cytometric assay by using fluorescence and autodisplaying proteins co-expressing E.coli. The autodisplay technology is one of the surface display methods for proteins or peptides at the E.coli outer membrane. In the autodisplay technology, proteins are expressed as recombinant proteins in a poly protein precursor for the autotransporter secretion pathway and automatically aligned on the outer membrane surface[1]. These aligned proteins can be used a affinity layer for capturing the analyte[2]. The autotranspoter vector and fluorescence protein expressing vector were transfected into the UT5600(DE3)　E.coli. For the fluorescence proteins, eGFP and tdTomato were used and Z-domains were autodisplayed on the surface of E.coli[3]. APC conjugated antibodies were treated to measure the activity of autodisplayed Z-domains and the activity Z-domain and expressed fluorescence proteins was measured by using a fluorescnece photometer and a flow cytometer. the result of fluorescence photometer shows that intracellular expressed eGFP and tdTomato have a excitation/emission wavelength of 488/507 and 554/581 nm. The flow cytometer result shows a clear increasement of fluorescence intensity after APC conjugated antibodies treatment.

Caffeine, aspirin, and tetracycline have been used in human life as diuretic, analgesic, and antibiotic. These pharmaceuticals have been detected in surface water resulting in the aquatic ecosystems confusion and human health change. In this study, it was analyzed specific stress responses caused by pharmaceuticals such as caffeine (890mg/l to 2225mg/l), aspirin (620mg/l to 1550mg/l) and tetracycline (180mg/l to 450mg/l) using four different stress responsive genes in Escherichia coli, i.e. fabA, grpE, katG and recA. The expression levels of these genes were quantified by semi-quantitative reverse transcription-PCR. It was found that four genes have shown different responsive patterns when E. coli cultures were under stressful conditions caused by caffeine, aspirin, tetracycline, respectively. Therefore, in this study, the stress responsive effects caused by these pharmaceuticals and the extent of each stress response can be analyzed using the expression levels and patterns of different stress responsive genes

The compound isolated from Marine Red alga Laurencia undulate; Floridoside was characterized for its ability in suppressing LPS mediated activation of BV-2 cells. Floridoside significantly inhibited the productions of NO and PGE2 by inhibiting iNOS and COX-2 at their protein and gene levels. The responsible molecular signaling for these inhibitory actions was found to be through suppression of the phosphorylation of MAPK molecules. These results indicate that Floridoside inhibits LPS-induced inflammatory response via blocking of MAPK signaling pathway in BV-2 cells, thus demonstrated its in vitro anti-inflammatory potential. These results suggest that Floridoside has the potential to be further developed as therapeutic against neuro-inflammation.

Ultraviolet-B (UVB) irradiation causes DNA damage and induces increase of matrix metalloproteinases (MMPs) in keratinocyte cells. Libanoridin is a natural product isolated from Corydalis heterocarpa which has recently been found to be effective antioxidant and anti-inflmmation. In this study, we assessed the effect of libanoridin aggainst UVB-induced damage using human keratinocyte (HaCaT) cells. In the results observed that libanoridin inhibited cell cytotoxicity and DNA damage due to 40 mJ/cm2 UVB exposure. Moreover, type IV collaganases MMP-2,-9 expression were decreased and MMP tissue inhibitor-1 (TIMP-1) decrease was inhibited followed by libanoridin treatment. In additionally we indicated that UVB-induced activation of phosphorylation of three MAPKase JNK, ERK, p38 and AP-1 transcription factor were decreased with libanoridin treatment. In conclusion, this study demonstrates that libanoridin has abilities to UVB induced cellular damage via MAPK and AP-1 signalling pathways, therefore, libanoridin may be used as an effetive natural compound on skin damage due to UVB exposure.

A silk from the Mediterranean Pinna(Pinna sp.) has been utilized as a "Western Silk" like legendary cloth before Silk Road was established. But, marine local pinna(Artina pectinata) silk fiber has hardly investigated as a commercial fiber source after Silk Road was established, nor marine local pinna silk fiber has explored as a source for bioengineering. Bio-mimetics of marine local pinna silk fiber has a potential to inspire new insights to design novel fiber materials. We found a 3,4- Dihydroxyphenylalanine (Dopa) containing proteins from marine local pinna foot by Acid-Urea Poly Acrylamide Gel Electrophoresis(AUPAGE) with 4-Nitro Blue Tetrazolium chloride(NBT) staining and Arnow assay. We evaluated tensile strength of Pinna's silk, pulled the silk as 1mm/min by 10N Load cell. And that checked strain, stress, toughness of silk, afterward compared the mechanical properties of the Pinna's silk with several existing natural fibers. Marine local pinna silk fiber may offer wide application for fiber industry including medical sealant, ECM material for tissue/medical engineering, bio-plastic, and cosmetic additve.

Coordination between Fe3+ and catechol ligand has been believed to play an important role in raising the hardness and the extensibility of mussel cuticle and contribute to self-healing mechanism of mussel’s tissue. We have reported self-healing protein hydrogel cross-linked by Fe3+-catechol ligand coordination, inspired by maturing mechanism of mussel cuticle. The hydrogel were prepared as the following manner. Recombinant mussel foot protein 1 (mfp1) was dissolved in PBS buffer to 20wt%. Then, FeCl3 was added into the solution to 30mole% against catechol concentration. By adding 1M NaOH, the pH of it was increased by 10. The mechanical properties of the hydrogel had been compared with those of covalently cross-linked hydrogel prepared by I04 - oxidation. Gelation time, dynamic modulus, and relative degree of self-healability of the hydrogels were measured by an Advanced Rheometric Expansion System (ARES).

Immunoassay has been widely used for the detection of target analytes in complex mixtures such as blood by using highly specific antigenantibody interactions. In this work, antibodies against a target analyte was immobilized to magnetic beads and the change of surface charge of a magnetic bead was measured after the binding of a target protein by using a zeta potential analyzer. For the feasibility test of the zeta potential analysis for immunoassays, horseradish peroxidase (HRP) was used as a model target analyte. The application to medical diagnosis was demonstrated by detection of C-reactive protein (CRP) which is known to be a biomarker for inflammatory diseases.

Marine biomass including macroalgae has been evaluated as the third generation biomass for biorefinery. However, biological conversion of brown seaweed has been limited by lack of feasible microorganisms which can transform structured alginate from kelp into biochemicals. As a proprietary technology for conversion of seaweed to ethanol, the consolidated bioprocessing (CBP) has been being developed to avoid the needs for expensive enzymes or chemical pretreatment of biomass. For this purpose, from Laminaria japonica with thallus shedding, alginatedegrading microorganisms were isolated, which can utilize alginate as a sole carbon source for their growth and ethanol production. Based on the biochemical characteristics, we categorized candidate isolates into 3 groups: i) isolates with high saccharolytic activity against alginate; ii) isolates producing succinic acid; and iii) isolates producing ethanol directly from alginate. Each isolate is identified by 16S rRNA sequence analysis and evaluated the compatibility as a strain for CBP of L. japonica. Also, batch and continuous processes are operated for targeted production of ethanol or succinic acid with high selectivity and high yield as well.

The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different genes in a same reaction mixture. Since the incubation periods of individual template plasmids are freely controllable, relative expression levels of multiple proteins can be tuned to desired levels. We expect that the presented results will find wide application to the flexible design and execution of synthetic pathways in cell-free chassis.

In the previous work, we reported semiconductor nanowires synthesized by VLS mechanism could be applied for matrix-free laser desorption/ionization ? time of flight analysis called nanowire-assisted laser desorption/ionization mass spectroscopy (NALDI-MS). By using this method, the quantitative analysis of small molecule could be achieved without adding organic matrix molecules. In order to apply TiO2 nanowires to NALDI-TOF MS, the nanowires were synthesized by mild hydrothermal process directly on the surface of Ti plate. For this study, the sample target plate was patterned to have 81 TiO2 nanowire zones on the surface. The morphology of the synthesized TiO2 nanowires were observed to be a diameter of 30 nm by SEM, and the crystal structure was determined to be a mixture of anatase and rutile phases by XRD. The feasibility of applying the synthesized TiO2 nanowires to NALDI-TOF MS was tested by the quantitative analysis of bradykinin fragments 1-7 and P14R synthetic peptide. Mass spectrometric analyses of the compounds mainly showed [M+H]+ and [M+K]+ ion peaks by using TiO2 nanowires, and the sums of the two peaks was considered to be signals for the quantitative analysis. The summed signals from bradykinin fragments 1-7 were exponentially increased with a correlation coefficient of 0.997 in a concentration range of 5 ? 100 nmol/mL. Similarly, P14R synthetic peptide showed exponentially increased signals in the same concentration range. The results show that the synthetic TiO2 nanowires could be applied to laser desorption/ionization MS, and the quantitative analysis of small compounds by using top-down synthesized TiO2 nanowires were feasible.

MALDI-TOF mass spectroscopy has been used for the analysis of receptor-ligand interactions. The receptor-ligand interaction has been analyzed by the following steps: immobilization of receptor proteins, binding of ligand molecules, and analysis with MALDI-TOF mass spectroscopy (MS). For this study, the parylene-H, which is a modified polymer of para-xylene to have formyl groups on the surface, was used as a receptor binding surface. In order to apply parylene-H film for the immobilization of receptor proteins, the parylene-H was thermally deposited on the target plate to be less than 50 nm thick by microprocessor controlled parylene coater. The thickness and the roughness of parylene-H film were determined by the AFM, and the rms deviation of the surface roughness was estimated to be ± 2 nm within the area of 5 x 5 μm2. In order to analyze the receptor-ligand interaction, streptavidin and biotinylated CCP were used as a receptor protein and ligand molecule, respectively. As the first step, streptavidin was immobilized to the parylene-H film by dipping the parylene-H coated target plate to the streptavidin solution, and then analyzed by MALDI-TOF MS. From the results, no significant mass peak was found at the mass spectrum, because streptavidin was covalently immobilized to parylene-H film by forming imine bond. As the second step, the ligand molecule, biotinylated CCP in this study, was treated to the immobilized receptor proteins on the target plate. The target plate with covalently immobilized streptavidin resulted in the far higher mass signal of biotinylated CCP than physically adsorbed streptavidin target plate. These results show that the parylene-H film is feasible for the analysis of receptor-ligand interaction with MALDI-TOF MS.

Recently, Z-domain was expressed as a fusion protein at the outer membrane of E.coli by using autodisplay technology. Because of the binding activity towards the Fc region of IgG, theZ-domain has been used for the orientation control of the antibodies in order to improve the sensitivity of immunoassays. By immobilizing detection antibodies, the E.coli cell with autodisplayed Z-domains could be applied for “direct immunoassay” and this type of immunoassays could achieve hyper sensitive results. For the direct immunoassay, however, the E.coli should be centrifuged repeatedly for washing steps and reagent chaging steps. In this work, magnetic beads were used as a solid support of the E.coli cells with autodisplayed Z-domains in order to perform the direct immunoassay without centrifugation steps. By using super-paramagnetic beads, the centrifugation step was simply replaced to the separation step by using an external magnet. For the immobilization of E.coli cells to the magnetic beads, the surface of magnetic beads was modified with poly-L-lysine to bind the negatively charged E.coli cells. In this work, the changes in surface charge and diameter of the magnetic beads were analyzed during the modification steps to confirm the construction of E.coli -magnetic bead complex. The formation of E.coli-magnetic bead complex was also analyzed by using SEM images. For the feasibility test of magnetic bead based immunoassay, horseradish peroxidase (HRP) was used as a model analyte and a biomarker for inflammatory diseases called C-reactive protein (CRP) was used for demonstration of an application to medical diagnosis.