Earticle

많은 미래학자들이 미래사회는 정보화시대와 나노시대를 거쳐 ‘바이오시대’로 진입할 것이라고 예측하고 있으며, 경제협력개발기구(OECD)도 2006년에 “세계 경제는 2020년경 ‘바이오경제(BioEconomy)’ 시대로 진입할 것”이라는 전망을 내놓은 바 있다. 정부도 1994년 「제1차 생명공학육성기본계획」을 시작으로 전략적 차원에서 생명공학을 육성하기 위한 지원을 계속하고 있다. 특히 해양생명공학(Marine Biotechnology)은 새로운 블루오션으로 각광받고 있다. 지구상 생물종의 80%이상이 서식하고 있는 해양이 바이오시대의 새로운 자원의 보고(寶庫)로 주목받고 있기 때문이다. 이에 정부는 2004년 해양생명공학을 집중육성하기 위한 ‘마린바이오21 사업’을 추진하였고, 현재 국토해양부 ‘해양생명공학기술개발사업’으로 확대․지속되어 오고 있다. 또한 2008년 ‘해양생명공학육성기본계획(Blue-Bio 2016)’ 및 2009년 ‘해양바이오 연구개발 활성화 대책’을 수립하여 2016년까지 해양생명공학 세계 7대 강국 실현의 목표를 가지고 해양생명공학을 범부처 국정과제로 채택한 바 있다. 국토해양부 해양생명공학기술개발사업은 현재 해양천연물신약연구단, 해양극한분자유전체연구단, 해양바이오프로세스연구단, 해외해양생 물자원확보연구단, 해양바이오에너지연구단의 5개 연구단과 8개 기탁등록보존기관으로 구성되어 있으며, 올해 해양산업신소재연구단의 공모를 통해 6개 연구단 체제로 구성될 예정이다. 그리고 전문관리기관인 한국해양과학기술진흥원에 정책적 지원을 위한 해양바이오정책지원센터를 설립하여 체계적이고 전문적인 해양바이오 육성을 꾀하고 있다. 2004년부터의 연구성과를 살펴보면, 2009년 기준으로 SCI급 논문 551건, 국내특허 254건, 국외특허 109건 등 짧은 연구기간에도 불구하고 상당한 논문과 특허를 냈다. 기초연구 성과를 바탕으로 한 시제품 제작은 29건, 기술이전은 9건의 실적을 거두고 있다. 2004년에 시작된 3개 연구단이 2010년부터 실용화단계에 진입하여 향후에는 산업화 성과가 더 높아질 것으로 기대되고 있다. 해양생명공학은 아직 초기단계에 있어 우리나라가 충분히 기술선점이 가능한 분야로 여겨지고 있다. 국토해양부는 대형해양조사선 건조, 국립해양생물자원관 건립 등의 인프라와 연계하여 해양생명공학이 기후변화와 자원 위기를 극복할 신성장동력이 될 수 있도록 적극적 지원을 아끼지 않을 것이다.

Previously, we showed that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of β -Nacetylglucosaminidase (GlcNAcase) action, which removes terminal N-acetylglucosamine (GlcNAc) residues. In an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycanswithtwo terminal GlcNAc residues were synthesized. In the present work, we investigated synergistic effects of Nacetylglucosaminyltransferase II and/or β-1,4-galactosyltransferase co-expressions (by recombinant baculoviral infection) and GlcNAcase suppression (by knock-out screening) on N-glycan patterns. Using HPLC and MALDI-TOF MS analyses, we found that the N-glycosylation pattern of human erythropoietin secreted by engineered S2 cells expressing glycosyltransferase but not GlcNAcase was complete except for sialylation; N-glycan structures had two terminal galactose residues. Therefore, it will be possible to express complete functional human therapeutic glycoprotein in engineered Drosophila cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

Autoantibody is the principal mediators of autoimmune disease. IVIG is a milestone of the therapy of autoimmune disease. Recently, it has been reported that sialylation on Fc doamin of immunogloublin is critical for the effect of intravenous immunoglobulin (IVIG) and seems to be mediated by lectins which are expressed on splenic marginal zone macrophages. And they demonstrated that SIGN-R1 preferentially binds to 2,6-sialylated Fc compared with similarly sialylated, biantennary glycoproteins, thus suggesting that a specific binding site is created by the sialylation of IgG Fc. Moreover, it was showed that a human homologue of SIGN-R1, DC-SIGN, displays a similar binding specificity to SIGN-R1 but differs in its cellular distribution, potentially accounting for some of the species differences observed in IVIG protection. However, the function of DC-SIGN in IVIG effects is controversial now, given that DC-SIGN and alpha2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells. Therefore, further studies of SIGN-R1-mediated IVIG effect could identify the IVIG-mediated intracellular signaling and the unknown anti-inflammatory factors, leading to unravel the specific mechanism of autoimmune diseases. These studies could be applied to develop the new therapies or new drug candidates by using sialylated SIGN-R1 ligands which mimic the IVIG effect to cure several auto-immune disease.

The enzymatic hydrolysis of chitinous materials has been studied for almost a century. Since many early studies established that at least two major enzymes called chitinase that mainly yields N,N’‐diacetylchitobiose (GlcNAc)2, and 􀁉‐N‐acetylglucosaminidase that yields GlcNAc as the final product are required for the chitin catabolism for the most of microorganisms including marine bacteria such as Vibrio furnissii or V. cholera. However, the chitin catabolic cascade is quite complex. Early study showed that chitin catabolism by the marine bacteria involves at least three signal transduction systems and many genes, several of which were molecularly cloned, and the corresponding proteins were characterized. This study demonstrates the purification and characterization of three unique enzymes: 1) a novel ATP‐dependent glucosamine kinase of V. cholerae encoded by a gene designated gspK. The protein, GspK (31.6 kDa), was purified to apparent homogeneity from recombinant Escherichia coli. The kinase can be used to specifically determine micro quantities of GlcN in acid hydrolysates of glycoconjugates; 2) a novel phosphorylase of V. furnissii. The gene, chbP, was cloned into E. coli; the enzyme, ChbP (89‐kDa protein), was purified to apparent homogeneity, and characterized kinetically; and 3) the identification of bglA, a gene contiguous to gspK in a presumptive large chitin catabolic operon. BglA was molecularly cloned into E. coli, and the protein BglA was overexpressed and purified to apparent homogeneity (65 kDa). The Henrissat algorithm places BglA sequence in Family 9 of the glycosidases, suggesting it is an endoglucanase. However, the results demonstrated that BglA is an exoenzyme yielding Glc at each cleavage step. To resolve this apparent discrepancy, detailed kinetic studies were conducted with cellotetraose. Only exoglucanase activity was detected. The function of this enzyme in V. cholerae remains to be determined, especially because our strain of this organism does not utilize cellobiose. In addition, diversity of chitinolytic enzymes in microorganisms is discussed.

More than 60% of protein therapeutics are glycoproteins attached with glycans which play crucial roles for folding, therapeutic efficacy, in vivo half-life and immunogenecity. Therefore, the analysis of their glycan structures is of great importance especially when developing glycoprotein therapeutics and remodeling glycosylation pathway for improved expression hosts [1]. We builded up the glycan analysis system based on high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization tim e-of-flight m ass spectrometry (MALDI-TOF MS) and DNA sequencer. It was nicely able to characterize and monitor the glycosylation patterns of protein therapeutics including three representative categories (cytokines, therapeutic antibody and enzyme). Glycan profile of HPLC provided quantitative information robustly and reproducibly for each peak of glycan while MALDI-TOF could be successfully employed for the identification of glycan structures. On the other hand, DNA sequencebased methods could generate quantitative information for glycan profiling in a high-throghput manner using 96-well formats [2], which allows rapid and efficient screening for glycoegnineerng and glycan remodeling.

Therapeutic proteins can prevent or treat wide ranges of diseases from cancer and viral or bacterial infections. Plants have advantages which include the lack of animal pathogenic contaminants, low cost of production, and ease of agricultural scale‐up compared to other currently available systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to incapability of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, the effective plant production system for recombinant therapeutics requires the appropriate plant expression machinery with optimal combination of transgene expression regulatory conditions such as control of transcriptional and post transcriptional events. We are interested in developing a plant system to express a large amount of therapeutic proteins in plant cells using so called cell-reprogramming and several glyco-engineering strategies in plants, particularly including glycoprotein subcellular targeting, inhibition of plant specific glycosyltranferases and addition of human specific glycosyltransferases. Among the variety of available heterologous expression systems, the baculovirus-based insect cell expression system also has been utilized frequently for the high-level production of therapeutic recombinant proteins and harbors glycosylation processing pathways, which constitute an advantage over other prokaryotic systems that lack glycosylation. Currently, we have successfully developed both plant and insect expression systems for production of monoclonal antibodies for immunotherapy.

N‐glycosylated epidermal growth factor receptor (EGFR) at membrane microdomains plays an essential role in the growth control of epidermal cells. Ligand‐dependent activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3 containing sialyllactose, but to a much lesser degree by other glycosphingolipids. However, the mechanism of the inhibitory effect of GM3 on EGFR tyrosine kinase has been ambiguous. The mechanism is now defined by binding of N‐linked glycan having multiple GlcNAc termini to GM3 through carbohydrateto‐carbohydrate interaction. These findings indicate that N‐linked glycan with GlcNAc termini linked to EGFR is the target to interact with GM3, causing inhibition of EGF‐induced EGFR tyrosine kinase. In addition, the functional role of N‐linked glycans or O‐linked glycans involved in CCI process was explored. We showed that N‐linked glycans having 56 GlcNAc termini were found to interact with itself, as a conjugate with phospholipid or with ceramide mimetic. We also showed a novel self‐interaction of high mannose‐type Nlinked glycans which did not interact with complex type N‐linked glycan with various number of GlcNAc termini. Hamster embryonic fibroblasts NIL 2K cells expressing high mannose‐type glycans at the surface of cell membrane adhered strongly to high mannose‐type Nlinked glycans, but not to complex type N‐linked glycan with multiple GlcNAc termini. High mannose‐type glycans may mediate adhesion of NIL‐2K cells through the self‐recognitive capability of such structure.

Here, we have developed a highly elegant and simple bioconjugation strategy in combination with genetic code engineering. We show for the first time the residue specific incorporation of hormonal precursor 3, 4‐dihydroxy‐L‐phenylalanine (L‐DOPA) can alter the functional properties of protein to conjugate into the polymers. This facile method will facilitate the synthesis of homogeneously modified protein. It yields quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for wide spread use to fabricate protein conjugates and development of protein microarray and synthetic biology applications.

Using a simple fluidic device fabricated with a PVC tube, a syringe needle, and a glass capillary tube, we produced uniform microspheres from poly(ε‐caprolactone) (PCL), ethyle‐2‐cyanoacrylate (ECA), and gelatin. Precise control over sphere size could be achieved by varying the concentration of the discontinuous phase, the flow rates for each phase, and/or dimensions of the fluidic device. We developed inverse opal scaffolds from chitosan and poly(D,L‐lactide‐co‐glyclide) (PLGA) by using PCL and gelatin lattices as templates, respectively. The scaffolds exhibited uniform pore size and well‐interconnected pore structure in three‐dimensional (3D) fashion. We believe that the inverse opal scaffold could provide a promising platform for both in vitro and in vivo experiments related to 3D tissue engineering. We subcutaneously implanted four kinds of inverse opal scaffolds with different pore sizes into mice to evaluate the effect of pore size on degree of neovascularization. Histology analysis confirmed that the density and area ratio of blood vessels were directly governed by the morphology of the scaffolds. Beside the inverse opal scaffolds, uniform PLGA microbeads with a hollow interior and porous wall were prepared using a fluidic device with three‐way channels. The microstructured microbeads could be potentially useful for the encapsulation of cells as well as active agents. We also successfully prepared uniform, porous PLGA beads with controllable pore sizes by employing unstable W/O emulsion as the discontinuous phase, which can be useful for therapeutic cell delivery and tissue engineering. We believe that the advanced materials, including uniform microspheres, inverse opal scaffolds, uniform beads with a core and porous wall, and uniform porous beads, can be applied to most of strategies in biomedical engineering, eventually resolving significant problems that we currently encounter.

We have successfully developed a homogeneous colorimetric assay using DNA aptamer and unmodified gold nanoparticles (AuNPs) for sensitive and specific detection of oxytetracycline, one of most commonly used antibacterial agents.1 A highly specific DNA aptamer that bind to oxytetracycline with high affinity was employed to discriminate other structurally similar antibacterial agents in tetracycline's group, such as tetracycline and doxycycline. Oxytetracycline binding DNA aptamers protected the aggregation of AuNPs at high salt concentration by the adsorption on the surface of AuNPs in the absence of oxytetracycline.2 The aggregation of AuNPs was induced by desorption of oxytetracycline binding aptamers on the AuNPs as a result of the interaction between aptamers and oxytetracycline, leading to the color change from red to purple. The color change of mixture was observed as low as 1 uM oxytetracycline with naked eyes, but other antibacterial agents didn't induced the color change. The detection limit of oxytetracyline was enhanced up to 25 nM by UV spectrometer analysis, which was 20-fold low than the limit USA-EPA regulated, with two orders of magnitudes in its linear dynamic range by successful optimization on the salt and AuNPs, and the molar ratio of aptamers to AuNPs. This colorimetric assay is advantageous over the other conventional methods in terms of its simple signal generation and detection with naked eyes, which can be realized in on-site detection of antibacterial oxytetracycline.

In the last few years, considerable attention has been devoted to the immobilization of biomolecules, especially enzymes on electrode surface in relation to the development of biosensors and biotechnological processes. In this work, we immobilized HRP onto the bifunctional silane 3‐aminopropyltrimethoxy silane (APTMOS) through glutaraldehyde crosslinker. Separately, the organic mediator Neutral red was covalently crosslinked to 3‐aminopropyltrimethoxy silane using glutaraldehyde. The primary amino groups on the mediators, enzyme and the sol‐gel precursor were of advantage in the motive for covalent immobilization. Methyl trimethoxy silane was used as an additional sol‐gel monomer to maintain the hydrophilic/hydrophobic nature of the composite. The composite electrode was prepared using the above two silane mixtures along with graphite powder. The electrochemical behavior of the modified electrode was probed in using cyclic voltammetry. The biosensing application of the modified electrode was verified using hydrogen peroxide as the substrate. The biosensor showed a good linear response towards hydrogen peroxide in the range of 2.14 x 10‐7 M to 1.93 x 10‐4 M with a detection limit of 1.28 x 10‐7 M. Kinetic parameters such as electron transfer rate constant, ks was evaluated to be 0.30 ± 0.03 s‐1 and the diffusion coefficient, α was found to be 0.27 by applying the Laviron’s method [1]. The chronoamperometric studies showed that the biosensor could sense effectively in dynamic mode. The performance of the biosensor was optimized with respect the scan rate, pH and temperature.

Once released into soil, organic pollutants undergo sorption and sequestration which results in reduced availability and toxicity of the pollutants [1‐2]. A novel reporter bacterial strain was constructed to quantitatively determine and visualize such behavior of the chemicals in the presence of various model solids. The reporter bacterium harbors a cell-killing gef gene and is to die and lose its fluorescence when the strain starts to biodegrade phenanthrene [3]. Four types of model solids (i.e., nonporous/porous hydrophilic beads and nonporous/porous hydrophobic beads) were chosen to represent two important soil parameters organic carbon content and porosity mostly affecting sorption of the chemical. Phenanthrene freshly spiked into the model solids showed the different biodegradability depending on the properties of the model solids. Also, the reporter bacterium showed quantitative and linear relationships between the amounts of phenanthrene biodegraded and the extents of cell death. The reporter bacterial strain developed in the study may find a useful means to determine both bioavailable fraction and toxicity of phenanthrene in soil.

Korean rice wine is low alcoholic beverage with about 6%(w/w) ethanol. It is called “makolli” or “Milky wine”. Someone call it “drunken rice”, “makcohol” or “markelixir”, recently. Takju means cloudy alcohol that has a pearly alcohol, like milk. The characters of Makoli are low degree alcohol, carbonated drink, and color of pearl. Makoli is made of rice, nuruk (fungi for saccharification), yeast and water.Makoli is replacing as popularity of Korean traditional alcoholic beverage instead of rubus coreanus fruit wine and “Bekseju” a new type of alcohol in the late 1990s. A fashion is changing in short time span. Arising from the rice wine-Makoli bar in late 2005, Makoli has been leading to increased product diversification and revenue Act, supported and promoted the policy proposal which came out last year. What is alcoholic beverage post Makoli? Many people prospect distilled spirits. There are world 3big famous alcoholic beverages that known as Scotch whiskey, Cognac and Maotai. Soju will be global famous alcoholic beverages in the near future. Although the boom of Korean rice wine comes from Korean wave which is drama, popular music, movie etc. The passion of Korean has expressed there will. The issue for agricultural-food industry has began in promoting the consumption of rice which was changed to increase interest for Korean traditional alcoholic beverages and was developed by the globalization of Korean food. The government has been making not only development policy but also a romotion law for Korean traditional alcoholic beverages,in order to consume surplus rice. Therefore, GARES is going to research that developing new product with Gyeonggido agricultural product - purple sweet potato makoli, opuntia humifusa makoli; teaching for producing method - nongminju; progression of Korean traditional alcohol beverage fair. What we need to do is to discusstechnical challenges and future prospects of the Korean traditional alcoholic beverages such as manufacturing methods (microbial fermentation), lternative sweeteners, conservation methods, hygienicconditions and professional development.

Development of a high-throughput in vitro system that can closely mimic human response to a drug is crucial for reducing the cost and the time of developing new drugs. Current cell-based assay system in a multi-well format is a static system which consists of a single cell type, and lack the important aspect of biotransformation and multi‐organ interactions. In addition, 2‐D monolayer cell culture does not provide a physiologically realistic environment and alter the behavior of cells, making the conventional cell-based assay systems inadequate for prediction of human response to a drug. A multi‐chamber, microfluidic device utilizing 3‐D constructs of cellembedded hydrogel was developed and used to test the effect of anti-cancer drugs and their metabolites. A microscale cell culture analog (μCCA) is a physical realization of a physiologically-based pharmacokinetic (PBPK) model, where chambers representing key organs are fabricated on a silicon chip and interconnected by microchannels for medium recirculation which emulates blood flow. We tested Tegafur, an oral prodrug of 5-FU which is a chemotherapeutic agent for colon cancer, using the μCCA device with the HepG2/C3A cells and HCT-116 cells embedded in Matrigel constructs cultured in the liver and the tumor compartment, respectively. Tegafur is non‐toxic to cells and is converted to 5‐FU in the liver by cytochrome P450 enzymes, which then exerts a toxic effect on cells. The results show that the μCCA device was able to capture the metabolism in the liver compartment and consequent damages to the tumor cells, which was not observed in a traditional, static 96‐well plate assay. This 3‐D μCCA system was able to reproduce the action of a drug that could only be observed in animal or human model before. It can mimic the dynamic multiple organ interactions and enables the study of pharmacokinetic/pharmacodynamic properties of a drug in vitro in a more physiologically realistic microenvironment.

Biodiesel has received considerable attention in recent years, as it is a biodegradable, renewable and non‐toxic fuel. Microalgae have been suggested as very good candidates for fuel production because of their advantages of higher photosynthetic efficiency, higher biomass production and faster growth compared to other energy crops. Moreover, microalgae are eukaryotic photosynthetic microorganisms that can be used to produce high value compounds as carbohydrates, hydrocarbons and natural oils. Microalgae appear to be the only source of biodiesel that has the potential to completely displace fossil diesel. Microalgae with high oil productivities are desired for producing biodiesel. The key processes involved in biodiesel production using microalgae are cultivation, harvest, and cell disruption for lipid extraction. Although all these steps are essential, the cell disruption is particularly important, as the contents of the extracted lipids are determined according to the disruption method. Therefore, the appropriate cell disruption method is important to increasing the lipid extraction efficiency. Botryococcus braunii is a green microalga that produces hydrocarbons whose content can reach 75% of the dry biomass, has already been proposed as a future renewable source of fuel. Cell disruption methods including autoclave, osmotic shock, acid and base lyses were compared with microwaves, sonification to determine the most efficient method. Among the tested methods, the microwave oven method was identified as the most simple, easy, and effective for lipid extraction from microalgae.

The exocrine pancreas creates strong alkaline fluid. However, how the human pancreatic duct cells secrete copious amount of bicarbonate (HCO3-) has long been a puzzle for more than a century. Present study firstly reports a novel mechanism whereby intracellular Cl- concentration ([Cl-]i)-sensitive kinases play a critical role in pancreatic HCO3- secretion. Recently, two related kinase families, with-no-lysine (WNK) kinases and sterile 20 (STE20)-like kinases, have emerged as osmotic sensors that modulate diverse ion transporters. In general, WNK kinases, including WNK1, are activated by osmotic stress such as a decrease in [Cl-]i, and subsequently phosphorylate and activate downstream STE20-like kinases, especially OSR1 and SPAK. In human pancreatic tissues, CFTR-positive duct cells co-expressed WNK1, OSR1, and SPAK kinases. Interestingly, CFTR activation greatly reduced [Cl-]i, and this in turn induced the activation of WNK1-OSR1/SPAK kinase cascade. Notably, the WNK1-mediated OSR1 and SPAK activation by low [Cl-]i strongly increased CFTR HCO3- permeability making CFTR primarily a HCO3- channel, which is essential for the secretion of pancreatic juice containing 140 mM HCO3-. In contrast, OSR1 and SPAK activation inhibited the CFTR-dependent Cl-/HCO3- exchange activity, which may reabsorb HCO3- from the high HCO3--containing pancreatic juice. These findingssuggest that the dynamic regulation of CFTR and anion exchange activity, via the [Cl-]i-sensitive WNK1-OSR1/SPAK pathway, is the molecular switch that generates a HCO3--rich fluid in the human pancreatic duct.

Pretreatment of the Undaria pinnatifida prior to saccharification must be performed to increase the yield of reducing sugar. This study was carried out to optimize pretreatment conditions for bioethanol production. Pretreatment by thermal acid hydrolysis was performed at different sulfuric acid concentration (0.2-1% (w/v) H2SO4), treatment time (15-60 min) and solid contents of slurry (10-20% (w/v)) using Undaria pinnatifida. The effect of three variables was analyzed by using a Response-Surface Methodology(RSM). Optimal pretreatment conditions were determined as 0.7% sulfuric acid and 18% slurry at 12 1℃ for 60 min. A maximum reducing sugar of 19.2 g/L and viscosity of 33.19 cP were obtained. The pretreated seaweed was further saccharified by the treatment implementing isolated marine bacteria and commercial enzymes after neutralization by 5N NaOH. The enzyme treatment was performed by adding 1% (v/v) commercial enzymes, and then incubated at 55℃ with 120 rpm in water-bath for 4 days. Ten percent (v/v, 0.468 g dcw/L) isolated marine bacteria was added and the mixture was incubated at 30℃ with 200 rpm for 8 days. An increase of reducing sugar and decrease of viscosity were observed.

The supercritical fluid extraction (SFE) of wheat germ oil was studied. Wheat germ oil is used in products such as foods, biological insect control agents, pharmaceuticals and cosmetic formulations, and has been shown to reduce plasma and liver cholesterol in animals and to delay aging. Wheat germ contains important bioactive compounds such as antioxidants and sterols. Antioxidants of wheat germ include tocopherols, tocotrienols (together abbreviated as tocols and summarized under the term vitamin E), phenolics and carotenoids. Supercritical fluid extraction is an alternative to conventional separation process technique which takes advantage of the enhanced solvent power of supercritical fluids. Supercritical carbon dioxide (SCO2) extraction was applied for bio-industry (foods, medicines, cosmetics, ect) which enable to high value-added business creation. Independent variables were operating temperature (20~60℃), pressure (10~30MPa) and flow rate (26.81g/min). Oil extraction yield increased with increasing extraction pressure. The extract was analyzed by GC technique. The main fatty acids of wheat germ oil extracted SCO2 were linoleic acid and palmitic acid. β-caroten, an important phytochemical for natural antioxidant in the wheat wheat germ oil extracted by SCO2 was analyzed by UV and GC/FID. Further study is going to investigate the antioxidation of comparing the oils extracted by SCO2 and soxhlet extraction with organic solvent by measuring acid value.

Particle design is presently a major development of supercritical fluids applications, mainly in the paint, cosmetic, pharmaceutical, and specialty chemical industries. Supercritical fluids have been successfully used to obtain composites or encapsulates, which comprise an active compound loaded into a matrix of a carrier material, in order to improving product preservation as well as controlling the dissolution rate of the active compound. The particles from gas-saturated solution (PGSS) process has been used to micronize suspensions of different substances in polymer melts, in order to obtain composite materials. The particle formation of functional pigment with biodegradable polymer, polyethelene glycol (PEG) was performed by supercritical carbon dioxide (SCO2) in a thermostated stirred vessel. PGSS were carried out in different temperatures and pressures to measure the optimum condition for the formation of functional pigment particle (pressure from 20 MPa to 25 MPa, temperature from 40 to 50°C). The average diameter of functional particles was about 0.8-1.4 μm however the average diameter of unprocessed particles was 430 μm. At higher temperature and pressure PEG contained higher amount of functional pigments. The recovery of functional material as particle with PEG was very high. The highest recovery was found at high temperature and pressure.

The absence of lignin and the low content of cellulose in algae make them a simpler material for bio-conversion than land plants[1]. Brown algae Laminaria Japonica, was used in anaerobic fermentation for methane production. The changes in microbial populations, associated with volatile fatty acids profiles and methane gas productions, were investigated in anaerobic batch reactor during 42 days. The concentration of acetic and propionic acid accounted for 90.4% of overall VFAs production and total amounts of accumulated methane gas was 1.34 L/L. The microbial population dynamics was monitored using 16S rRNA gene targeted real-time PCR. At time 0, Methanobacteriales(MBT), Methanomicrobiales(MMB) and Methanosarcinales(MSL) groups in order of the 16S rRNA gene abundance, were detected and quantified. However, the MSL 16S rRNA gene concentration increased rapidly at day 16, which coincided with the decrease in acetic acid. Methane started to from at 10 days and was continuously produced. This indicated the possible growth of aceticlastic methanogens actively utilizing the acetate produced in this period. After the day 16, the population of MSL was decreased. Only order MBT, hydrogenotrophic methanogens, kept increasing and accounted for 73% of total methanogenic population. Thus, MBT was the primary methanogenic group responsible for methane production from the latter half of overall period.

In this study, it was demonstrated that E. coli responds to compounds present within hydrolysates through the higher expression of the marA regulon and aaeXAB operon by RT-qPCR with pure hydrolysate compounds, including coumaric acid and vanillin. Using gene fusions of two marA-regulated genes, i.e., inaA and zwf, with the luxCDABE genes in plasmid pDEW201, it was demonstrated that both of these genes are induced by the compounds but that strain SP4 (inaA::lux) is more responsive. Furthermore, it was found that this strain responds preferentially for phenolic acids not naturally produced by E. coli. These results clearly show the potential of this strain as a biosensor to monitor the characteristics of the wood hydrolysate and to evaluate the possible inhibition of fermentative bacterial strains by the phenolics in the hydrolysate. Finally, tests with an actual hydrolysate, using RT-qPCR, found that the marA regulon was still strongly induced while the expression of the aaeXAB operon was not as significant when compared with the pure chemicals. Based upon the characteristics of both systems, it is assumed that non-phenolics produced during the hydrolysis, including furfural, are involved in the toxicity and that these lead to an induction of the marA regulon but not the aaeXAB operon.

Photosynthetic microorganisms produce relatively large amounts of physiological activation materials and stimulate the physiological activity of other organisms. In this study, two mammalian cell were cultured under the different culture medium, Dulbecco’'s Modified Eagle medium (DMEM) including fetal bovine serum (FBS) and DMEM including physiological activation materials produced from photosynthetic microorganism. Cell growth was estimated by cell counting in a hemocytometer. The level of cellular stress was measured using LysoTracker dyes, known as a specific fluorescent dye to stain only lysosome. In comparison with FBS, we did not show significant differences for growth of mammalian cell under the feed condition with physiological activation materials. The growth of the cells can be similarly controlled by varying the photosynthetic microorganism concentration. In addition, we found that any cellular stresses were not induced by adding the physiological activation materials in mammalian cells. Our results suggest that new materials produced from photosynthetic microorganism can enhance the physiological activity of mammalian cells as a nontoxic bioavailable materials.

Recombinant bovine trypsin was produced in transgenic rice cell suspension culture using the rice α-amylase system. The effects of alternative carbon sources on production of recombinant trypsin were investigated in transgenic rice cell suspension cultures. While this expression system is inducible by sugar depletion, we have found that the productivity of recombinant trypsin increased 5.4 to 6.3 fold, compared with the control medium without carbon source and NaCl. The accumulated recombinant trypsin in the medium containing fumaric acid and NaCl reached 79.5 – 95.3 mg/L on day 7 after sugar starvation. By adding the alternative carbon source and NaCl in the induction medium, the percentage of the active recombinant trypsin in the total secreted proteins increased from 20 to 40.3%. Supplementation with these alternative carbon sources has potential for high production of recombinant enzymes in rice cell suspension culture. Acknowledgments This work was supported by a grant from the Next Generation New Technology Development program of the Ministry of Commerce, Industry and Energy, and by the National Research Foundation of Korea Grant funded by the Korean Government (2009-0070005)

This research illustrates the development of microbial production of fatty acid, which can be transformed into the bio-diesel for alternative energy. We developed a new green technology that can produce a fatty acid with high productivity from both strains, Bacillus subtilis (gram-positive) and Escherichia coli (gram-negative). To produce a fatty acid in bacteria, it is essential to generate a three-carbon precursor, malonyl-CoA, which is derived from acetyl-CoA by the action of acyl-CoA carboxylase (accA, accB, accC). The malonyl-CoA is transferred to the acyl carrier protein by malonyl-CoA:ACP acyltransferase (FabD). This process is the most essential step in the biosynthesis of long–-chain fatty acids. Therefore, overexpression of accA, accB and accC genes, encoding acetyl-CoA carboxylase subunits enzymes, is expected to increase the productivity of fatty acids. The single, double and triple organized artificial operons of accA, accB, accC and fabD were designed and constructed in a vector. By introducing four distinct genes obtained from Bacillus subtilis into E. coli, we have engineered E. coli strain for high productivity of fatty acids.

Food-waste recycling wastewater (FRW) contains high particulate organic materials (POM) cause a serious drawback for the use of high rate anaerobic reactors [1]. To enhance the anaerobic digestion process, the effects of changing hydraulic retention time (HRT, 1-3 days), pH (4-6) and temperature (T, 25-45℃) on the efficiencies of bacterial hydrolysis of POM treating FRW was investigated by using response surface methodology (RSM). The maximum hydrolysis efficiency was estimated as 47% reduction of VSS when (HRT), pH and (T) were at 3 days, 6.0 and 45℃, respectively. To determine the bacterial community structures, denaturing gel gradient electrophoresis (DGGE) analysis targeting the bacterial 16S rRNA genes from reactor samples of 16 experimental trials carried out. Lactobacillus amylovorus appeared to be dominant species to hydrolysis of POM in the FRW. BMP test was conducted to compare the effect of the hydrolysis optimization with the raw FRW. The results indicated that methane yields of raw FWR wastewater and effluent of the hydrolysis reactor were 0.635 and 0.988 L CH4/g VSreduction, respectively.

This study describes the evaluation and optimization of a crystallizing process capable of efficiently purifying vancomycin in high purity and yield. In particular, we observed how the main process parameters influenced the formation of crystals, determined their morphology, and monitored purity and yield. Acetone was shown to be more effective than alcohol solvents for the crystallization of vancomycin. The optimal distilled water/acetone ratio, storage temperature, storage time, pH, conductivity, initial vancomycin concentration and stirrer velocity were shown to be 1:3.5 (v/v), 10ºC, 24 h, pH 2.5, 20 ms/cm, 0.1 g/mL, and 640 rpm, respectively. Temperature had a decisive influence on crystal formation; crystals were successfully produced at 10ºC, while at other temperatures, conglomeration, disintegration and cohesion occurred. Crystal growth developed over time and was complete at about 24 h. Vancomycin purity remained at about 97.0% irrespective of storage time while the yield increased over time, reaching a maximum of 95.0% at around 24 h, after which there was no substantial change. Crystallization occurred over a certain range of pH (2.5-3.0), but purity and yield were highest at pH 2.5. When the pH was outside this range, a conglomeration (gelation) phenomenon prevented the efficient production of crystals. Vancomycin crystals were produced irrespective of the stirrer velocity, which had no influence on purity; however, the highest yield of vancomycin was obtained at 640 rpm.

The optimal fed-batch process for high cell density culture of E.coli K-12/pHCE-InaN-GAPDH-Ghost27 as a ghost vaccine was developed. The nutrient feeding strategy with Riesenberg's defined medium during fed-batch process was evaluated. The fermentation was conducted in four phases as follow: initial phase (phase 1), fed-batch phase (phase 2), temperature increase phase (phase 3) and high temperature holding phase (phase 4). The maximum ghost bacterial vaccine (GBV) was obtained about 38 g dcw/L (OD600=100). The efficacy of GBV was evaluated by the challenge test with the infection of live S. iniae to vaccinated olive flounders. E. coli K-12 host strain group, E. coli K-12/pHCE vector control group and fomalin-killed cell (FKC) treated vaccine group showed 100%, 100% and 65% of cumulative mortalities, respectively. The GBV treated groups showed 50% of cumulative mortalities with increased survival ratio. Hence, the immunoprotective efficacy of GBV against S. iniae was better than that of FKC vaccine. Therefore, the GBV could be used as an effective and vaccine in aquaculture for the prevention of streptococcal disease.

The application of nanoparticles (NPs) in various fields has been growing rapidly due to its exceptional physicochemical properties. The potential biological or environmental toxicity of NPs, however, has been frequently reported. In this study, the toxicity of nC60 and nC60-OH NPs in Caenorhabditis elegans was assessed through the measurement of lifespan and brood size. The water-stable colloids form of nC60 NPs are prepared through the long-term exposure of fullerene, C60 in toluene. C. elegans is a free-living soil nematode feeding on bacteria such as Escherichia coli. We confirmed that the oral-administrated NPs were accumulated in the animals for several days after feeding the mixture of bacterial food and nC60 or nC60-OH NPs to the L4 larvas of C. elegans. The nC60-OH NPs reduced the viability of animals while no decrease in survival was found in the animals fed with nC60. We have also found that both nC60 and nC60-OH reduced the reproduction of C. elegans. In order to illuminate the genetic mechanism of toxicity induced by those NPs, the assessment of viability and fertility was carried out using the strains mutated in oxidative stress or programmed cell death regulators.

Saccharification of seaweed, Laminaria japonica (sea tangle) is an essential process for the bioethanol fermentation. The saccharification was carried out by thermal acid hydrolysis and the treatment of isolated marine bacteria, Bacillus sp. JS-1. The 10% seaweed slurry was hydrolyzed with 0.2% (v/v) H2SO4 at 121℃ for 60 min. The hydrolysate was neutralized by 5N NaOH and further degradation was carried out by the 10% (v/v, 1 g dcw/L) Bacillus sp. JS-1. The reducing sugar concentration and viscosity under the optimal saccharification process were 45.6±5.0 g/L and 24.9 cp, respectively. Total yield of the reducing sugar from Laminaria japonica was 69.1%. The isolated marine bacteria was identified as Bacillus sp. by 16s ribosomal RNA sequencing and named Bacillus sp. JS-1. SSF was carried out for the bioethanol production. Thermal acid hydrolysate of 10% Laminaria japonica slurry was fermented at 30℃, 200 rpm for 136 hr by the addition of the 4% (v/v, 0.39 g dcw/L) Bacillus sp. JS-1 and 4% (v/v, 0.45 g dcw/L) yeast. The highest ethanol concentration of 7.7 g/L with 33% of theoretical yield was obtained from 10% Laminaria japonica slurry fermented with Pichia angoporae.

Butyric acid is a valuable intermediate to be converted to butanol and has been biologically produced in pure culture systems. However, pure culture is vulnerable to contamination and requires high cost for construction and operation; thus, mixed culture systems are required for mass production. The objective of this study is to determine optimum conditions of a bioreactor to produce butyric acid from molasses In mixed culture. Batch tests are performed to investigate optimum temperature, substrate load, and cell concentration; and a continuous stirred-tank reactor (CSTR) is operated to determine optimum hydraulic and cell residence times. The results of the batch test show that at pH 6 and 35℃, the fermentation enables the highest production of 4.4 g/l with a yield of 0.088 g/g. Experiments are on-going to obtain other optimum conditions, which will be presented on the conference. This study will provide optimum design and operating factors of the mixed culture bioreactor to mass produce butyric acid stably and economically.