Earticle

Water-soluble proteins, Phycobiliproteins are used as a highly-fluorescent substance in fluorescence-activated cell sorting and flow cytometry analyses of cell populations, immunochemistry, detection of reactive oxygen species, and amino acid sequencing. Synechocystis sp. PCC 6701 accumulates phycobiliproteins after the transformation to the aplanospore stage under special spectrum. Phycocyanin is a blue protein that absorbs orange and red light and emits blue light. Phycoerythrin absorbs green light and emits red light. Allophycocyanin absorbs and emits red light (650 and 660 nm max, respectively). Flashing light is also drawing an increasing interest as a potential alternative lighting scheme to enhance the efficiency of photosynthesis and productivity in algal biomass production. So, we will run experiment by using continuous light and flashing light. We will cultivate cells with continuous light by fluorescent lamp, LEDs and flashing light by LEDs. We will run comparative analysis for concentration of phycobiliproteins production by each condition.

Actinorhodin (ACT) and Doxorubicin (DOX) belong to a structural family of type II polyketide compounds generated by a high G+C Gram-positive soil bacterium, Streptomyces species. Here we report the identification of a couple of previously-unknown down-regulator genes via comparative transcriptome profiles using S. coelicolor cDNA microarrays. Using interspecies DNA microarray analysis, we found several putative transcriptional down-regulator genes involved in antibiotic biosynthesis. To show the biological significance of the transcriptomics-driven potential down-regulator genes in Streptomyces species, these genes were functionally expressed both in ACT-producing S. coelicolor and DOX-producing S. peucetius. Further analysis revealed that the expression of these putative transcriptional down-regulators inhibited both actinorhodin and undecylprodigiosin production in S. coelicolor. Overexpression of these genes also inhibited the biosynthesis of DOX in S. peucetius. Real-time RT-PCR confirmed that the differences of antibiotic productivity in S. coelicolor were due to the differential expression levels of antibiotic pathway-specific regulatory genes including actII-orf4 and redD/Z. Therefore, we concluded that the interspecies DNA microarray analysis reported here successfully identified a couple of novel global down-regulators of antibiotic biosynthesis in Streptomyces species.

Microalgae are photosynthetic organisms to produce industrially important metabolites such as astaxanthin, β-carotene, unsaturated fatty acid and phycobiliprotein etc. In order to enhance their productivity, researchers have developed many kinds of bioreactors and various cultivation methods. But recently these efforts came to limitation. Because of these reasons, systems biology started to attract researchers who want to increase productivity using novel tools or technology. Through the use of systems biology, researchers can model molecular and cellular phenomena of microalgae using integrated and interacting network of genes, proteins and biochemical reactions. In order to model levels of in silico behaviors, large volume of genome-scale data is required. The first step is to identify open reading frames and determine their biochemical reactions. After that, network gaps are determined by biochemical or physiological evidence. The accuracy of metabolic network can be analyzed by mathematical methods such as flux-balance analysis. Consequently comprehension of microalgae using systems biology is expected to increase their productivity as well as develop photosynthetic mechanism.

Many algal species can form blooms, commonly referred to as "red tides". Most of these blooms are harmless, but a few species of phytoplankton are toxic to marine ecosystems, which form "harmful algal bloom" (or HAB). Heterosigma akashiwo is known as one of HAB-generating species.1)2) In order to find the mechanisms of H. akashiwo to generate red tide, proteomic comparison between the two different states (before and after bloom formation) was performed to identify up-regulated pathways in the HAB state. From the proteomics experiments, it was found that a series of spots with low pIs were appeared as bloom formed. We are in the process of identifying these spots through MALDI-TOF.3)4)

Seasonal change in body size and grazing rate on phytoplankton of the copepod Acartia hongi female were investigated in Incheon coastal waters with bimonthly sampling from April 2004 to February 2005. Seasonal distribution of A. hongi female abundance ranged from 53inds./m3 to 6,798inds./m3 and variation of gut pigment contents of A. hongi female ranged from 0.014ng․pigment/inds. to 0.766ng․pigment/inds. The grazing rate of A. hongi female ranged from 1.627n g․pigment/inds./day to 78.112ng․pigment/inds./day. The Variation of grazing pressure on phytoplankton by A. hongi female ranged from 0.17㎍Chl-a/ℓ/day to 65.74㎍Chl-a/ℓ/day and was significant with the abundance of A. hongi female. The relationship between Cephalothorax length(L, ㎛) of A. hongi female and water temperature(T, ℃) was approximately liner, where L = 923.94 - 14.54T (r2=0.96, n=60, p<0.01) and the constant(K) of gut evacuation rate of A. hongi female was positively related to water temperature(T) and the relationship was described by the equation K=0.001T + 0.0027(r2=0.82, n=18, p<0.01). Grazing rate(G, ng․pigment/inds./day) was significant with cephalothorax length, where G = 0.0023e0.0071L (r2=0.75, n=108, p<0.01).

The bacterial genus Streptomyces is well known for its ability to produce a wide variety of secondary metabolites, including medically important products such as antibiotics, anti-tumor agents, immunosuppressants, and enzyme inhibitors. Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Using a Streptomyces polyketide methylmalonyl-CoA acyltransferase gene as a probe, three overlapping cosmids were isolated from the genomic library of TMC-producing Streptomyces sp. CK4412. Sequence information of an approximately 70 kb contiguous DNA region revealed two multi-modular type I polyketide synthases (PKSs) and twelve additional gene including a regulatory gene products presumably involved in TMC biosynthesis. The deduced roles for most of the TMC PKS catalytic domains were consistent with the expected functions necessary for TMC chain elongation and processing. In addition, disruption of a putative TMC acyl-CoA transferase gene, located upstream of the PKS gene locus, completely abolished TMC biosynthesis. Taken together, these data provide strong supporting evidence that the cloned gene cluster identified in this study is responsible for TMC biosynthesis in Streptomyces sp. CK4412, and sets the stage for detailed genetic and biochemical studies on the biosynthesis of this important metabolite.

Clostridium difficile (C.difficile) is not only the most common infectious cause of nosocomial diarrhea, accounting for 20~25% of all antibiotic associated diarrhea, but also identified in 95% of pseudomembranous colitis(PMC) cases. In particular, there has been a recent increase in the number of children and infants suffering from C.difficile-related infection during antibiotics treatment. The overuse of antibiotics may alter the normal intestinal flora and increase the risk of developing C.difficile diarrhea. Although antibiotics (vancomycin, etc.) have various problem such as high medical costs, antibiotic resistance, and relapses or re-infections during treatment periods, new alternative agents for treatment of C.difficile associated diarrhea or infection diseases was not yet determined. Therefore, in this study, we confirm the inhibitory activity through the AG ethanol extract. In order to verify the effect, we will use test methods such as paper disc diffusion method, MIC(Minimum Inhibitory concentration) method and liquid medium dillution method. The ethanol soluble fractionate that is obtained by the traditional herbal medicines have high inhibitory activity for C.difficile about 93.9±1.7. Also, AG have the high inhibitory activity against growth of S. aureus. S. aureus with Salmonella spp., vibrio parahaemolyticus is food poisoning bacillus. We will test the antimicrobial effect about the AG of C.difficile and food poisoning bacillus. Also, we will test lactobacillus that is affected by extract which shows high inhibitory activity for the C.difficile.

The morphology and infraciliature of Favella ehrenbergii were observed using the protargol staining. F. ehrenbergii was collected from Incheon coastal water, Korea and cultured in the laboratory. Although F. ehrenbergii is widely distributed in marine water, its infraciliautre is still unknown. In this laboratory culturing study, we described the change of lorica-shape with the life cycle and morphogenesis of F. ehrenbergii.

In Korea and many other developed countries, obesity is one of the most threatening health problems. However, there are only few good anti-obesity products in terms of effectiveness and safety. This study is to develop many edible, nontoxic agricultural food grade, biomaterials for development of side-effect free anti-obesity functional food or pharmaceutical products. For the development of the anti-obesity natural drug, the inhibitor of adipocyte differentiation is screened from Korean traditional medicinal plants. We will examine the inhibitory effect of raw material extracts on adipogenesis by oil red O staining, adipocyte differentiation related transcription factor expression, and lipid accumulation experiment. Finally, we will isolate and identify the active-compounds containing an anti-adipogenesis activity from selected plant extracts using open column chromatography and HPLC/MS.

The polyene antifungal antibiotics, mostly produced by Gram-positive soil actinomycetes, are a family of type I polyketide macrolide ring compounds with 20-40 carbon backbone contain 3-8 conjugated double bonds. Using polyene-specific genomic screening strategy, we previously isolated three novel polyene-producing actinomycetes strains from soil, implying the potential application of these strains’ spores as microbial pesticides. Among three isolated strains, an approximately 112.9 kb contiguous nystatin-like CPP (Cryptic Pseudonocardia Polyene) biosynthetic gene cluster in P. autotrohpica and a 44.8kb FR008-like polyene biosynthetic gene cluster in Streptomyces MMBL003 have been isolated and genetically characterized. Using a fractional factorial design, both KH2PO4 and yeast extract were identified to be the key factors influencing CPP productivity in P. autotrophica. In addition, we confirmed that the sonication was a very efficient method for actinomycetes spore generation with a sonicator power-dependent manner. These sonication-driven actinomycetes spores retained significant portion of their cell viabilities as well as antifungal activities after freeze-drying procedure, implying the potential application of these strains’ spores as microbial pesticides.

1,2-Propanediol has been used in unsaturated polyester resins, liquid laundry detergents, cosmetics, and pharmaceutical manufacturing as a solvent for drugs unstable in water. Several organisms are reported to ferment common sugars to 1,2-PD. However, the titers are fairy low and the organism is not characterized well enough to enable the improvement of the 1,2-PD production by metabolic engineering1). S. cerevisiae has a well established genetics system and has been completely sequenced2). In this work, a strain of Saccharomyces cerevisiae was engineered for the production of 1,2-PD. For the production of 1,2-PD, methylglyoxal synthase (mgs) and glycerol dehydrogenase (gldA) from Escherichia coli were cloned onto high copy number vector pYES2/CT and pYES3/CT plasmid respectively. After introducing the mgs and gldA genes, production of 1,2-PD was analyzed by HPLC.

The use of lipid vesicles in delivery system for skin treatment has attracted increasing attention in recent years. Also, recently many efforts were focused to develop natural materials for improvement of skin pigmentation abnormalities. The aim of this study was to investigate the antioxidant activity effect of Gleditsia Sinensis Lam and Cnidium officinale to encapsulate using Multilamellar vesicle. The ethanol extracts of Cnidium officinale (23.5%) and Gleditsia sinensis lam (39.17%) showed the two spices in the 1.1-diphenyl-2-picrylhydrazyl radical scavenging capacities. They also absorbed wavelength in the UV-B and UV-C region. Multilamellar vesicle were prepared using phosphatidylcholine, cholesterol and were observed by transmission electron microscopy and scanning electron microscopy for the size distribution and shape: the size of Multilamellar vesicle were rang from 111±18.9nm and they were ball shape. The presents study focuses on the characterization of Multilamellar vesicle and present the antioxidant activity of the natural materials extracts.

Immuno-stimulating activities of thiolated chitosan microspheres loading Bordetella bronchiseptica were evaluated by measuring the mucosal immune response following nasal administration in mice. Thiolated chitosan microspheres were prepared according to the ionotropic gelation of low molecular chitosan with tripolyphosphate anions1). The thiol group was induced from 2-iminothiolane hydrochloride. The particle size distributions of thiolated chitosan microspheres were analyzed using an electrophotometic light scattering spectrophotometer, Photal, ELS-8000, Osaka). The contents of thiol group in chitosan microsphere were measured by Ellman’s assay method. The level of antigen-specific antivodies (IgA and IgG) in serum, saliva, and nasal wash samples was determined using enzyme-linked immunoabsorbent assay (ELISA).

The detection and kinetics of mucosal pathogenic bacteria binding on polysaccharide ligands were studied using a surface plasmon resonance biosensor. The kinetic model applied curve-fitting to the experimental surface plasmon resonance sensorgrams to evaluate the binding interactions. The binding interactions of the mucosal pathogenic bacteria with polysaccharide binding pairs (Pseudomonas aeruginosa/alginate, Streptococcus pneumoniae/pneumococcal polysaccharide, Staphylococcus aureus/pectin) were also compared with their kinetic parameters. The adhesion affinity of Pseudomonas aeruginosa with alginate was higher than that for the other binding pairs. The binding affinities of the pathogenic bacteria with their own polysaccharide were higher than that of Staphylococcus aureus with pectin.

1,2-Propanediol (1,2-PD) is a major commodity chemical that is currently derives from propylene, a non-renewable resource1). The yeast species Saccharomyces cerevisiae engineered to produce 1,2-propanediol using the δ/UB sequential gene integration method was used2). In this strain there are 3 copies of methylglyoxal synthase gene (mgs) and glycerol dehydrogenase gene (gldA) from Escherichia coli which were sequentially integrated into the chromosomes2). The purpose of this study was to investigate the important parameters for the production of 1,2-PD by S. cerevisiae M3G3. The production of 1,2-PD was studied at different growth phases and the effect of the inducer was investigated. 1,2-PD production was analyzed by HPLC.

1,2-Propanediol (1,2-PD, propylene glycol) is a chemical which has three carbon-diol and it contains a stereo structure at center of carbon. It is mostly used in unsaturated polyester resins, cosmetics, liquid detergents, detergents and solvent for unstable to water in pharmaceutical industry. Saccharomyces cerevisiae is a well known and a useful strain for industrial fermentation. Since S. cerevisae does not have genes necessary for the production of 1,2-propanediol, it is essential to import the genes from 1,2-propanediol producing cells. The objective of this research is to construct a plasmid for S. cerevisae to produce 1,2-propanediol by genetic manipulation. For this work, the key enzymes in the metabolic pathway of S. cerevisiae were selected for the production of 1,2-propanediol methylglyoxal synthase and glycerol dehydrogenase. Methylglyoxal synthase convert dihydroxyacetonephosphate (DHAP) to methylglyoxal. Glycerol dehydrogenase is involved in the conversion of glycerol to dihydroxyacetone. The genes which encode methylglyoxal synthase (mgs) and glycerol dehydrogenase (gldA) were obtained from Escherichia coli. The genes were cloned in a pESC-URA vector and transformed to S. cerevisiae.

Antimicrobial effect of Salicornia herbacea against various pathogens relate to atopic dermatitis. They were separately extracted in 70%ethanol from dried samples at room temperature, and freeze⁃dried. Ethanol extract of salicornia herbacea showed antimicrobial activity against Staphylococcus aureus (ATCC49520) and Staphylococcus epidermis (ATCC12228) and Candida albicans (ATCC10231). The antimicrobial activity of the ethanol extracts were tested by thr disc diffuson technique. The antimicrobial activity of the Salicornia herbacea were determined by minimum inhibitory concentration. These results suggest that Salicornia herbacea extract can use usefully in atopic dermatitis.

Lymphatic drug delivery is an important research field because the lymphatic system plays an important role as a transferal route of various diseases. The major purposes of this study is to prepare dextran acetate (DxA) nanoparticles for cyclosporin A (CSA) delivery and to evaluate delivery properties of the nanoparticles into lymphatic sites with radioscintigraphy. Dextran was chemically acetylated and CsA-loaded DxA nanoparticles were prepared by a dialysis method. Tc-99m was radiolabeled to the nanoparticles for tracing of delivery routes. Morphology of the CsA-loaded DxA nanoparticles observed by SEM and TEM was spherical in shape and the particle mean size was 200 nm. Radiolabeling efficiency of the nanoparticles determined by thin layer chromatography was 95%. In vivo animal study, it was rapid injection site clearly and low distal lymph node accumulation showed that the CsA-loaded DxA nanoparticles could be delivered to the lymphatic system. The biodistribution of 99mTc-loaded CsA-Dextran Acetate Nanoparticles in mice was investigated by subcutaneous injection into the foot pad. The biodistributions of 99mTc-HSA and 99mTc-loaded CsA-Dextran Acetate Nanoparticles were also investigated for comparison. Dynamic whole-body images were obtained for 30 min after subcutaneous injection into the rats foot pads. Therefore in vivo study proved that CsA-loaded DxA nanoparticles showed enhanced accumulation of drug efficacy in the lymph node.

the purpose of this study was to assess the effects of a herb remedy . on the cell proliferation, inducible nitric oxide synthase (inos) mRNA gene expression and nitric oxide (NO) production in RAW 264.7 macrophage cells. For the screening of anti-inflammation, a ethanolic extract of traditional herbal medicine was examined its inhibitory effect, and we confirmed that 1 herb. NO concentrations were determined by measuring the amount of nitrite in the LPS treated-macrophage cell culture supernatant using Griess reagent. In cytotoxicity test, with the a herbal remedy concentration of up to 500 mg/mL, no significant effect on RAW 264.7 macrophage cells morphology and viability was observed compared to the control during the culture period. Also, more than the concentration of 125ppm, a herbal remedy was significantly decreased the production of NO. As a result, we knew inhibitory effects of a herbal remedy extracts on Nitric Oxide synthesis in RAW 264.7 macrophage cells.

The whitening effect of the extracts of Pharbitis nil was studied with evaluation of anti-oxidation ability. Extraction and purification method of whitening agent from Pharbitis nil were also described in this study. The extracts of Pharbitis nil were prepared with extraction of Pharbitis nil by various solvents. The Pharbitis nil was concentrated with water, ethanol, and methanol. The extracts from dry samples were filtered and concentrated, consequently. The whitening effect of extracts was examined using 1,1-diphenyl-2-picryl hydrazyl ( DPPH ) by radical scavenging activity . The extracts were encapsulated with lecithin. The extracts capsule were defined the particle size by electrophotometic light scattering spectrophotometer.

Malic and succinic acids are an important C4 dicarboxylic acid and they are widely used in food, cosmetics, products and textile industry. There are many reports about the effort to produce the malic and succinic acids using the biological method instead of petrochemical-based process. In recent years, reactive extraction processes have been received as an increasing attention for recovery of carboxylic acids from aqueous solution. Reactive extraction is the separation process using reaction between extractants and materials extracted. The extractant in the organic phase reacts with material of aqueous phase and reaction complex formed was solubilized into the organic phase. In this study, the extraction equilibrium of malic and succinic acids with TOA/1-octanol was investigated. And extraction characteristics of solutions containing malic and succinic acids with effcet of pH were studied. The compositions of malic and succinic acids with a gram ratio of malic acid to succinic acid of 13.6:1 based on the fermentation results by Taing, O. and Taing, K.1)

The binding interaction of pathogenic bacteria, such as Helicobacter pylori, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, were studied with alginic acid ligand. The binding interactions between the pathogenic bacteria and alginic acid ligand were measured using surface plasmon spectrum by monitoring a variation of the RU values. The association and dissociation rate constants of the pathogenic bacteria with alginic acid ligand were determined from a kinetic model applied curve-fitting method.1) The affinities between pathogenic bacteria and alginic acid ligand were also considered with their kinetic parameters. The high adhesion affinity appeared in the binding of Helicobacter pylori with alginic acid ligand. Propionibacterium acnes also exhibited significant adhesive properties with alginic acid ligand.

This study investigated that antibiotic effect of aroma oils on Staphylococcus aureus, which exists in the skin suffering from atopic dermatitis. To retardate the volatility and diminish the toxicity of the aroma oil, encapsulation technique using alginate and carboxymethylscleroglucan (CMSG) beads was employed. Inhibition effect of the aroma oil on the growth of S. aureus depended on the concentration of the oils. In the aroma oil-loaded beads, the antibiotic effect on the bacteria increased with the treated weight of the beads. The Chamomileloaded beads showed the most effective antibiotic property, while the inhibition effect on the growth of the bacteria was not observed in the Eucalyptus-loaded beads. S. aureus did not proliferate completely in the amount above 0.8 g of the Chamomile-loaded beads and 1.0 g of the Tea tree-loaded beads. In addition, the difference between alginate and CMSG beads in the antibiotic effect on S. aureus did not observed in all the tested groups. The results of this study indicated that the aroma oils and the aroma oils-loaded beads may inhibit the growth of S. aureus in the skin suffering from atopic dermatitis. Therefore, alginate and CMSG-algiante bead containing the aroma oils may be used for preventing the proliferation of S. aureus in atopic dermatitis and as the therapeutic systems in a biomedical and health care fields.

The method of micellar extraction was developed for pre-purifying paclitaxel from Taxus chinensis, giving a high purity and yield. The approach in this work was to transfer paclitaxel in the crude extract to an aqueous surfactant solution as a micelle, allowing organic solvents to be used for removal of lipids and non-polar impurities. CPC was chosen as the surfactant for this process on the basis of the high solubility of paclitaxel in aqueous solutions of this surfactant and the low solubility of this surfactant in organic solvents. The effects of surfactant type and concentration, initial crude paclitaxel concentration, pH, extraction number, and ionic strength on the separation of paclitaxel were investigated. The use of micellar extraction in the pre-purification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This pre-purification process serves to minimize solvent usage, and the size and complexity of the high-performance liquid chromatography (HPLC) operation for paclitaxel purification.

Mandelic acid has a asymmetric carbon atom and thus has two chiral isomers: D-, L-. Only D-mandelic acid is pharmaceutically active such as antibacterial agent, antiaging agent and diuretics. Simulated moving bed (SMB) chromatography is a suitable process for continuous separation of chiral compounds. However, SMB operation is a formidable task since it is a periodic cyclic process. To find optimal separation condition in SMB, we performed simulations in m2-m3 plane base on the triangle theory and calculated operating parameters (flow rates of four zones, switching time and feed concentration and so on) using Aspen chromatography. we compared the simulation with experimental results for determining SMB operating parameters and optimal condition.

A halophyte Salicornia herbacea (known as glasswort) was collected from Daebudo, Ansan in Korea. Active compound from Salicornia herbacea was isolated using HPLC technique. The structure of this compound was determined by extensive 2D-NMR analyses. The free radical scavenging activities of the compound on 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (.OH) and carbon-centered radical were investigated using electron spin resonance (ESR) spectroscopy. Increased concentration of the compound exhibited a dose dependent scavenging activities on generation of DPPH, hydroxyl and carbon-centered radicals. It not only scavenged 30, 69, 78 and 85% on generation of DPPH radical at 3.125, 6.25, 12.5, and 25 μg/ml, respectively, but also scavenged 66, 77, 86 and 92% on hydroxyl radical at 3.125, 6.25, 12.5, and 25 μg/ml, respectively. In addition, it showed more than 85% scavenging effect on carbon-centered radical at the various concentrations. Therefore, these results suggested that this active compound from Salicornia herbacea could be developed as a candidate for potential natural antioxidant.

Shade-dried whole plants of Glehnia littoralis that is growing on the sandy beaches of eastern Korea were extracted twice overnight with CH2Cl2 and MeOH at a room temperature, respectively. The combined crude extracts were concentrated in vacuo at 40℃ to leave a dark brown gum (175.0g) and then partitioned between CH2Cl2 and H2O. The organic layer was further partitioned with n-hexane/85% aq. MeOH and then the aqueous layer, n-BuOH/H2O, respectively, to afford the n-hexane (22.0 g), 85% aq. MeOH (9.0g), n-BuOH (17.0g) and H2O (125.0g) fractions. A continuing investigation of 85% aq. MeOH fraction has resulted in the isolation of two furocoumarins, known as bergapten (1) and isopimpinellin (2). Structure of these compounds has been determined by extensive 2-D NMR experiments such as 1H COSY, TOCSY, HMQC, and HMBC and by comparison with the reported data in the literature.

Homoharringtonine was purified from Cephalotaxus koreana by a combination of extraction, synthetic adsorbent treatment, low-pressure chromatography, and high performance liquid chromatography (HPLC). A crude extract was obtained by methanol extraction of biomass, followed by liquid-liquid extraction using chloroform. The waxy compounds were efficiently removed by adsorbent (active clay P-1) treatment. The extract was purified to greater than 52% with a 86.4% step yield by silica gel low-pressure chromatography. High-performance liquid chromatography steps, which were composed of an HPLC step with silica column and an HPLC step with ODS column, were applied to give 98% purity with high yield. Amorphous homoharringtonine, with a fine particle size, was simply made by dissolving homoharringtonine in methylene chloride/methanol (98/2, v/v), followed by spray drying. Residual solvents, methylene chloride and methanol, could be reduced to 250 ppm and 1,160 ppm by spray drying and successive drying in a vacuum oven.

FVIII is a protein cofactor essential for blood coagulation. The deficiency of factor VIII activity in humans is associated with a congenital bleeding disorder, called hemophilia A.1) In general, most of chemical reaction such as surface modification of a protein is performed at a high pH. In contrast Factor VIII should be performed at pH 6.5~7.5 to prevent the lees of activity.2) In order to enhance, Factor VIII’s activity, usually thiolation performed at pH 7.5. The purpose of Factor VIII thiolation enhanced PEGylation yield. Before Thiolated Factor VIII PEGylation, human IgG selected as a model-protein for thiol PEGylation. Because IgG(150kDa) has similar molecular weight compared with rFactor VIII(170kDa). Thiolation of human IgG performed with N-succinimidyl S-acetylthioacetate (SATA), and then amino residue of human IgG was labeledby su lfhydryl. After that SATA-labelede human IgG performed with hydroxylamine, and then human IgG has more sulfide residue. Free N-succinimidyl S-acetylthioacetate (SATA) was separated using Sephadex G-25 column. Thiolated IgG PEGylation was performed with PEG-SH (MW 30kDa). And analysis of thiol PEGylation, used by HPLC, GPC and MALDI-TOF.

Green tea (Camellia sinensis) has recently receiving increasing attention due to its health-benefiting effects primarily caused by catechins including epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin g1allate (ECG), and epicatechin (EC). However, caffeine contained in the green tea is found to cause adverse physiological effects such as sleep deprivation, miscarriage, and nervousness. To remove caffeine from green tea leaves, we have employed supercritical carbon dioxide (SC-CO2), which is known to be an ideal solvent, coupled with a cosolvent such as ethanol or water. By varying the extraction conditions including temperature, pressure, cosolvent, particle size, and CO2 flow rate, not only changes in the amount of caffeine, but also the quantities of the principal bioactive components of green tea, including catechins, such as EGCG, EGC, ECG, and EC, were investigated. The extraction yield of caffeine increased with an increase in temperature at a constant pressure, and also at different pressures at a fixed temperature. As the content of the ethanol or water used as a cosolvent increases, the extraction yield of caffeine significantly increased. Comparing with the results of ethanol and water, caffeine was much more effectively removed when ethanol was used, but the loss of EGCG by extraction was similar to water and ethanol. When the CO2 mass flow rate increased, the total extraction yield of caffeine also increased, but the extraction efficiency of CO2 (amount of caffeine extracted per amount of CO2 used) decreased. The reduction of green tea leaf particle size by grinding also resulted in the enhanced extraction of caffeine. The caffeine content in the decaffeinated green tea was reduced to 2.6% of the initial content after extraction with SC-CO2 modified with 4.6 g of 95% (v/v) ethanol as a cosolvent per 100 g of CO2 at 300 bar, 70°C, mean particle size of 236.5 µm for the ground green tea, and CO2 flow rate of 11.5 g/min for 120 min. However, after the extraction, a substantial loss of EGCG, as much as 40% of original content, proved unavoidable.