Earticle

The titanium oxide (TiO2), photocatalyst, has been widely used in the field of environment for degradation of many air and water pollutants. Additionally, TiO2 photocatalysis has been intensively carried out to inactivate microorganisms. However, traditional studies of TiO2 photocatalyst generally involved low antimicrobial activity and no selectivity for target microorganisms due to random attack of microorganisms. We established antimicrobial activity method of target microorganisms using antibody-conjugated TiO2 (TiO2-AbE). To prepare for TiO2-AbE we were combined bacteria-specific antibody using via EDC/NHS with PAA coated TiO2 particles. We evaluated selective and efficient antimicrobial effect of TiO2-AbE with target E. coli, and compared with the non-specific other bacteria. The results clearly showed that the effect of TiO2-AbE on antimicrobial activity to E. coli was much greater than raw TiO2 particles while other bacteria were no significant differences between raw TiO2 and TiO2-AbE. The TiO2-AbE allows selective E. coli killing with low concentrations and short UV irradiation time compared to raw TiO2 particles. This method might be used to remove selective target bacteria for very short time and prevent infection of various bacteria.

1.063 g/mL and 0.901 g./mL of saponins were obtained from 50 g (dry weight) of tea seed with/without sonication, respectively, with newextraction methods for greentea seed saponin. Sonicated samples was enhanced by 15.23% than the untreated sample. The results of cytotoxicity of tea seed saponin by MTT assay with different concentration, so, it could be used for various purposes. The inhibitory effect of greentea seed saponin on breast cancer cells, cervical cancer, liver cancer and stomach cancer cells was observed more than 80% inhibition at 5 ppm, especially, stomach cancer cells showed completely supressed. Five kinds of strains by using antimicrobial test results, 5 kinds of strains that were significantly inhibited at 50 ppm of tea seed saponin extract. While ob mouse treated with a high fat diet and treated with tea seed saponin, and weight loss was investigated. As a result, compared to the control group appeared to be reduced by more than 30% weight, so it show that tea seed saponin is to be a good substance for anti-obesity. From the above results, Tea-saponin E1 from green tea seed extract was showed novel angiogenesis inhibitory effect and antitumor activity.

Liapse was immobilized and stabilized in a form of nanoscale enzyme reactors (NERs) within meso-structured onion-like silica (Meso-Onion-S), which has highly-curved meso-pores of 10 nm diameter in a multi-shell structure. NERs were prepared via a two-step process of enzyme adsorption and subsequent enzyme crosslinking, which effectively prevents the leaching of crosslinked enzyme aggregates from highly-curved mesopores of Meso-Onion-S. As a result, NERs in Meso-Onion-S significantly improved the enzyme stability as well as the enzyme loading. For example, NERs of lipase (NER-LP) was stable under rigorous shaking for 40 days, while the control sample of adsorbed LP (ADS-LP) with no enzyme crosslinking showed a rapid inactivation due to rigorous enzyme leaching under shaking. Highly stable NER-LP was successfully employed to produce biodiesels, fatty acid methyl esters, from the LP-catalyzed transesterification of soybean oil with methanol in isooctane. As a result, the specific activity of NER-LP was 23 and 10 times higher than those of free LP and ADS-LP, respectively, revealing the importance of LP stabilization in the form of NER-LP in the presence of organic solvents.

Subtilisin Carlsberg (SC) from Bacillus licheniformis was immobilized and stabilized in magnetically-separable mesocellular silica (Mag-MSU-F), and used for the model synthetic reaction in an organic solvent. The SC stabilization was based on a ship-in-a-bottle approach, which consists of enzyme adsorption and crosslinking steps. In mesoporous silica with a bottle-neck pore structure, the crosslinked enzymes in larger mesocellular pores did not leach out through smaller window mesopores. As a result, crosslinked enzymes retained more than 66% of the initial activity even after ten times of reuse and for more than 15 days under rigorous shaking condition. On the other hand, free and adsorbed SC showed less than 1% and 2.8% of initial activities, respectively, under the same condition. Crosslinked SC were employed for the transesterification reaction of N-Ac-Phenylalanine ethyl ester with n-propanol in isooctane. Especially, the use of magnetically-separable mesoporous silica allowed for easy separation and recycled uses of immobilized enzymes, which is important for the practical applications of enzymes in fields.

A cDNA encoding manganese peroxidase isozyme H4 (MnPH4), isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of the alcohol oxidase I promoter. The recombinant MnPH4 is efficiently secreted into the medium upon hemin supplementation, at a maximum concentration of 500 U/L, and the purified rMnPH4 was used to decolorize the triphenylmethane dye, Malachite green (MG). Response surface methodology (RSM) was employed to optimize different operational parameters on the decolorization of MG. RSM showed that the optimized variables viz. enzyme (0.662 U), MnSO4 (448 mM) and hydrogen peroxide (159mM) decolorized 100 ppm of MG completely in 3 hours. UV-VIS spectra, HPLC, GC-MS and LC-ESI MS analysis showed the degradation of MG. Finally, treatment of MG with rMnPH4 resulted in the complete removal of toxicity which was checked under in vitro conditions.

Lipase (LP) is one of well-known enzymes for a wide range of applications such as detergents, foods, oils and cosmetics and pharmaceutical industries. In the present work, LPs were immobilized and stabilized into polyaniline nanofibers (PANFs) via a three-step process of enzyme adsorption, precipitation and crosslinking (EAPC). As a result, EAPC retained high enzyme loading and showed highly-enhanced enzyme stability. In other words, EAPC showed 5.1 and 5.9 times higher activity than enzyme adsorption (EA) and enzyme adsorption/crosslinking (EAC), respectively. After 84 days under 200 rpm shaking at room temperature, the residual activities of EA, EAC and EAPC were 11%, 24% and 74% of initial activities, respectively. The esterification of ibuprofen with n-propoanl in isooctane was performed by using EA, EAC and EAPC, and the different amounts of dioctyl sodium sulfosuccinate (aerosol-OT, AOT) were added to better disperse the immobilized enzymes. The addition of 100 mM AOT resulted in 57-fold increase of EAPC activity when compared to the case with no AOT addition. Highly-stable and active EAPC showed a great potential of successes in various enzyme applications.

Phthalocyanine derivatives have perhaps received more attention than any other class of dyes as sensitizers for photodynamic therapy and most favoured are the aluminium and zinc derivatives. Photoinduced electron transfer in donor-acceptor molecules has been extensively studied, in particular for covalently linked dyads in solution as model systems of natural photosynthesis to harvest efficiently solar energy. In this study, the improvements of electron reactions from immobilization of R. sphaeroides on two phthalocyanines were evaluated. There are two solvent for phthalocyanines, water and SDS. In order to determine electron reaction of cell bound phthalocyanines, we are confirmed cyclic voltammetry measurement. R. sphaeroides bound ZnPc in water solvent and CuPc in SDS solvent were shown active electron reactions. It was correlated with the amount of cell bound phthalocyanines. In addition, it was found that the indirect immobilization of R. sphaeroides on phthalocyanines was confirmed when it was supplemented by sodium bicarbonate. Phthalocyanines as electron donor on R. sphaeroids can apply for improving photosynthetic efficiency.

Xylosidases, a hemicellulase enzyme group that hydrolyze the short xylooligomer to final product of xylose, is an importance enzyme in biomass degradation. In this study, we reported the characterization of a recombinant putative gene that encodes for beta-xylosidase from biomass degradation white-rot fungus Phanerochaete chrysosporium. The beta-xylosidase gene was obtained from cDNA library of P. chrysosporium. Sequence analysis indicated that beta-xylosidase cDNA gene consists 1797 nucleotides, encodes for 598 amino acids. The beta-xylosidase gene was then searched blast against available nucleotide database on NCBI. The results showed that beta-xylosidase matched to beta-xylosidase of Clostridium acetobutylicum, Geobacillus stearothermophilus and Bacillus halodurans at 28-30% similarity. Enzymatic assay on p-nitrophenyl-beta-xylopyranoside resulted optimum pH and temperature of 5 and 45OC, respectively. Mg2+, Mn2+ and Fe3+ increased enzyme activity where as Zn2+, Ca2+ and Fe2+ inhibited xylosidase activity. Thin layer chromatography analysis of end product of enzyme on xylooligomer indicated the enzyme act as exo-type. The enzyme did not hydrolyze xylan substrates such as birchwood xylan, beechwood xylan and arabinoxylan but it hydrolyzed p-nitrophenyl-alph-L-arabinofuranoside, resulting that beta-xylosidase have two biocatalytic domains.

Direct saccharification of dried fruits of honey tree, which have been used for functional foods, was investigated to enhance recovery yield and reduce production cost using cellulases produced by Bacillus amyloliquefaciens DL-3, Bacillus velezensis A-68, and Cellulophag lytica LBH-14. At lower temperatures less than 50℃, saccharification of dried fruits of honey tree with enzyme was higher than that without cellulase. Yield of saccharification increased with increased amounts of cellulase and dried fruits of honey tree. The maximal yield of saccharification occurred at 60 g/L dried fruits of honey tree with 10 g/L cellulase produced by B. velezensis A-6. Its yield of saccharification was 38.2% and concentration of reducing sugars was 22.9 g/L.

In the past decade, many DNA mismatch detection methods have been proposed for analyzing SNPs (single nucleotide polymorphisms), which are one-base DNA sequence variations that are responsible for various inherited diseases and drug resistance. Among them, DNA ligase-based assay has been considered as one of the most potent methods in terms of its various applicability. However, DNA ligase-based methods for mismatch detection showed limitation due to its inevitable occurrence of false-positive result (observation of ligation product in G:T mismatch, that is serious disadvantage in cytosine/thymine(C/T)-type SNP analysis). In order to overcome the problem, we employed chemically-modified nucleobases, several universal or non-bonding nucleobases (such a 5-Nitroindole, Iso dG, Iso dC), at the 3’-end of a downstream ligation fragment and optimized successful DNA ligation reaction conditions for distinguishing G:C and G:T matches. In particular, the use of iso-dG and 5-nitroindole showed highly-distinctive detection results of C/T type SNP in DNA ligase-based analysis. The developed DNA-ligation methods using modified-end DNA oligomer as ligation fragment will be usefully employed for development of highly-accurate system of SNP analysis.

The approach of nanoscale enzyme reactors (NERs), consisting of enzyme adsorption into mesoporous materials with a bottle-neck structure and follow-up enzyme crosslinking, have demonstrated successful stabilization of enzyme activity in a ship-in-a-bottle mechanism. In details, crosslinked enzymes in larger mesocellular pores cannot leach out through smaller bottle-neck mesopores.1 When compared to simple adsorption with no enzyme crosslinking, the NER approach has proven to be effective in improving the stability and sensitivity of electrochemical biosensors. 2 For example, glucose oxidase (GOx) and organophosphorus hydrolase (OPH) were immobilized and stabilized in mesoporous carbons (MSU-F-C) with mesocellular pores (26 nm) connected by window mesopores (17 nm). NERs of GOx and OPH in MSU-F-C were employed to develop highly stable and sensitive enzyme-based biosensors, which can find its potential applications in medical and environmental biosensors.

Natural products extracts of Coptis japonica, Schizandra chinensis, Phellodendron amurense, Asparagus cochinchinensis were effectively controlled with the growth of harmful bacteria such as Streptococcus pyogenes, Streptococcus zooepidemicus, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium difficile, Clostridium perfringens. As a result, C. difficile and C. perfringensand growth was inhibited by Coptis japonica and Schizandra chinensis, S. pyogenes and S. zooepidemicus was inhibited by Asparagus cochinchinensis, Coptis japonica and Schizandra chinensis. Also, C. perfringens and S. aureus growth was inhibited by three mixture extract of Coptis japonica, Asparagus cochinchinensis, Phellodendron amurense, and S. epidermidis was inhibited by addition of three mixture extracts of Schizandra chinensis, Asparagus cochinchinensis, Phellodendron amurense, Asparagus cochinchinensis. 6 kinds of bacteria growth was inhibited by addition of four mixed combinaton of Coptis japonica, Phellodendron amurense, Schizandra chinensis, Asparagus cochinchinensis, and its ratio was 1:1:1:1, in particular, the ratio of 3:3:2:2 was showed the most effective antibacterial effects.

Plant-derived peptones (PPs) have been utilized to improve cell growth and volumetric productivity in plant cell cultures. PPs contain amino acids, carbohydrates and minerals. Carbon (C) and nitrogen (N) sources have been known as major factors in plant cell cultures. Supplementation of chemically defined medium with PP was found to enhance cell growth and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) significantly in transgenic rice cell suspension cultures. Therefore, it was necessary to investigate the effects of C and N sources for the improvement of recombinant protein production. The concentrations of C and N sources used in this study were as follows: 0 (control), 1, 3, 5% (w/v) of sucrose and 10, 20, 30 g/L of PP. In case of adding 3% sucrose with 10 g/L of PP, maximum dry cell weight was 1.9-fold higher than that of control and the maximum level of hCTLA4Ig reached up to 64.8 mg/L. Whereas hCTLA4Ig was not produced in control cultures. In conclusion, it was found that the determination of optimal concentration ratio between C and N sources are important to improve cell growth and hCTLA4Ig production in transgenic rice cell cultures.

Mesenchymal stem cells (MSCs) are important tools for cell therapy. They have capabilities of self-renewal and differentiation into various types of cells for therapeutic purposes. MSCs are commonly cultured in two-dimensional (2D) system. However, MSCs can lose their differentiation capacity with time in 2D culture. In addition, available surface area for the propagation of cells is limited. Therefore, various three-dimensional (3D) culture systems have been suggested. In this study, we have developed a 3D culture system for the expansion of periosteum-derived progenitor cells (PDPCs) as spheres. The PDPCs are one of diverse MSCs and they have high proliferation capacity. The spheroid formation of PDPCs was induced by using a rotation platform. The spheres maintained both their viability and proliferation ability. Furthermore, the properties of stem cells have been improved in 3D cultures. In conclusion, a simple and economical 3D culture system could be developed. The 3D culture system can increase the potential of PDPCs for clinical use.

We have demonstrated one-step method for the fabrication of biomimetic poly(ε-caprolactone) (PCL) fibrous matrix with gradient in bone-like calcium phosphate (TCP) content. PCL solution in chloroform containing β-tricalcium phosphate nanopowders was centrifuged to induce gradient content in TCP. The PCL/TCP dispersion was electrospun into the collector, resulting in the composite matrix with a high content of TCP at the bottom and zero content at the top in the composite matrix. The morphology and the content of TCP were examined by SEM and EDX, respectively. Cell culture on PCL and PCL/TCP fibrous matrices confirmed that osteoblast (MC3T3-E1) favored the PCL/TCP fibrous matrix in terms of proliferation and osteogenic differentiation. The PCL/TCP fibrous matrix can potentially be employed for the biomimetic application to the bone and bone-to-tendon regeneration.

We investigated certain components of the medium for cell growth and antibody production by grouping feeding conditions. The depletion of one or more essential nutrient resulted in cell death or reduced mAb production in batch culture. Therefore, the key for high performance of cell growth and mAb production is development of effective nutrient feeding strategies. This study evaluates the effect of grouped feeding conditions composed media on the growth rate and monoclonal antibody production. Feeding groups include Amino acids, Vitamins, Trace elements, Lipids, Other components and Growth factors. In the process, nutrient concentrations were maintained to avoid depletion of substances and glucose and/or glutamine supply to maintain proper concentration. The maximum viable cell density attained in the fed-batch culture was a 100% improvement over the control culture. The mAb titer is approximately 150% higher than in the batch culture. We have experimented a dihydrofolate reductase deficient Chinese Hamster Ovary DG44 Cells (rCHO-DG44) line in self made Chemicallydefined media. These feeding strategies will utilize available in other cells and media.

Cellulophaga lytica LBH-14, w hich had been isolated from seawater in the previous study, was known to produce two types of cellulases, carboxymethylcellulase (CMCase) and cellobiase. The optimal carbon and nitrogen sources for cell growth of C. lytica LBH-14 were found to be rice hulls and yeast extract, whereas those for production of CMCase were rice bran and yeast extract. Based on productivity and economic aspect, rice bran and ammonium nitrate were chosen to carbon and nitrogen sources for optimization of major conditions for enhanced production of cellulases. The optimal conditions of rice bran, ammonium nitrate, and initial pH of medium for cell growth were 125 g/L, 5.0 g/L, and 6.5, whereas those for production of CMCase and cellobiase were 100 g/L, 6.5 g/L, and 6.5. The optimal conditions for production of CMCase extracted using orthogonal array method (OAM) were the same as those of cellobiase. However, the optimal conditions of rice bran, ammonium nitrate, and initial pH of medium for production of CMCase were predicted as 75.0 g/L, 7.5 g/L, and 6.5 using response surface method (RSM), whereas those for production of cellobiase were 88.1 g/L, 4.9 g/L, and 4.7. The maximal cell growth and production of CMCase and cellobiase by C. lytica LBH-14 were predicted as 8.91 g/L, 350.1 U/mL, and 121.1 U/mL, respectively.

Xylan is a major component of hemicellulose which is considered to be the second most abundant natural polymers in nature and it is regarded as one of the most important renewable resources. In this study, we engineered Corynenacterium glutamicum for the direct utilization of xylan as a carbon source. Overall scheme of this research was two-step process comprising (i) enzymatic hydrolysis of hemicellulose and (ii) xylose utilization as a carbon source by introducing heterologous xylose metabolic pathway. For the hydrolysis of xylan, two heterologous enzymes (endoxylanase and xylosidase) were expressed in C. glutamicum. Due to the lack of xylose metabolic pathway, wild type of C. glutamicum cannot utilize the xylose as a carbon source. Next, to give the ability of xylose metabolism, heterologous cytoplasmic expression of the xylose isomerase (xylA) and xylulokinase (xylB) genes were introduced in C. glutamicum. Finally, two steps (xylan hydrolysis and xylose metabolism) were successfully combined as a whole cell system in which xylan can be directly utilized for growth of C. glutamicum.

In this study, we developed a lime addition-capacitive deionization (CDI) hybrid process that can efficiently remove acetic acid and sulfuric acid from the model mixture of glucose, xylose, acetic acid, and sulfuric acid, which are the major components from the biomass hydrolyzate by acid hydrolysis. The key parameters of lime addition process (type of lime, amount of lime, stirrer speed, reaction time) and CDI process (voltage, flow rate, feed concentration) were also optimized. In the lime addition process, the optimal lime type, acids(sulfuric acid and acetic acid)/lim e molar ratio, stirrer speed, and reaction tim e for removal of sulfuric acid were CaCO3, 1:1, 200 rpm, and 6 min, respectively. For the CDI process, the optimal voltage and flow rate were 1.2 V and 20 mL/min. The efficiency of acid removal increased as the initial acetic acid concentration decreased. The developed hybrid process was able to remove 98.08% of sulfuric acid and 76.97% of acetic acid from the mixture of glucose, xylose, acetic acid, and sulfuric acid. It was able to recover most of the sugar (>99%) at high purity (97.53%).

Transgenic rice (Oryza sativa L.) cell suspension cultures have been utilized to produce recombinant proteins. General preservation method for plant cells has been known to be the repeated subcultures, but one drawback is the genetic instability of transformed cell lines. For that reason, a cryopreservation method was developed for long-term storage of the transformed rice cell lines. The optimal method was developed by using various cryoprotectant mixtures and thawing steps. Through these optimized procedures, the cells cryopreserved for 5 years were regenerated to callus successfully and then suspended into the liquid medium. The growth and production characteristics after regeneration were similar with those of non-cryopreserved cells. Regeneration of the cryopreserved cells was time-consuming and the cells were damaged by osmotic stress. Therefore, to reduce the osmotic stress to the rice cells after thawing procedure, the effects of glutathione as a potent antioxidant were investigated. For direct regeneration of cells without the cultivation period on solid medium, the cells were regenerated in basal AA medium containing 0.4 M sucrose and 0.5 mM glutathione. Viability of the cells was 1.5-fold higher than that of control.

Dayflower(Commelina communis) is distributed Korea, Japan, China and North America and commonly grows in wetland of roadside, grass and stream. Commelina communis is consists of root, stem, flower, leaf and fruit. Young buds of Commelina communis is used for food, entire plant of Commelina communis is used for medicinal purposes. The content of antioxidative and biological activities (DPPH (α,α’-diphenyl-β-picrylhydrazyl) free radical scavenging activity, contents of total phenolic compounds and flavonoid, Fe/Cu reducing power, peroxidation of rat liver homogenate, contents of protein, comparison of resolving power in CMC, starch plate) were tested by in vitro experimental models using water(CW), ethanol(CE) and methanol(CM) extracts of Commelina communis. CW showed highest extracted yield(3.6%). The highest free radical scavenging activity was found in the CE by 61.72%. The highest contents of phenolic compounds in CM by 62.32mg/100g and contents of flavonoid in CE by 108.76mg/100g. The Fe/Cu reducing powers were strong in CE. Antioxidant activities on lipid peroxdaion using rat liver homogenate as measured by TBARS method showed strong in CE. The highest contents of protein as measured Bradford method in CE(25.1mg/ml). Resolving power in CMC, starch plate showed all extracts from Commelina communis.

A gene encoding carboxymethylcellualse (CMCase) from a marine bacterium Bacillus velezensis A-68 LBH-14 had been cloned into E. coli JM109. The optimal carbon and nitrogen sources for cell growth of a recombinant E. coli JM109/A-68 were found to be maltose and tryptone, whereas those for production of CMCase were rice bran and yeast extract. Based on productivity and economic aspect, rice bran and ammonium chloride were chosen to carbon and nitrogen sources for optimization of major conditions for enhanced production of CMCase using response surface method (RSM). The optimal conditions for rice bran, ammonium chloride, and initial pH of medium for cell growth were found to be 150.0 g/L, 2.5 g/L, and 8.0, whereas those for production of CMCase were 125 g/L, 5.0 g/L, and 7.0. The maximal cell growth and production of CMCase by E. coli JM109/A-68 were predicted as 8.20 g/L and 1079.7 U/mL.

Water-in-oil-in-water (W/O/W) microbeads were fabricated using a simple fluidic device, where the inner water phase served as drug reservoir. Using a fluidic device with three flow channels, we could obtain uniform W/O/W microbeads with a single or multiple droplets, where the inner water, middle oil, and outer water phases were an gelatin, poly(ε-caprolactone) (PCL), and poly(vinyl alcohol) solutions, respectively. The number and size of the core droplets can be tuned by changing the flow rate of each phase in the fluidic device. To demonstrate a controlled release, hydrophilic and hydrophobic dyes were encapsulated as model drugs in the core and PCL layer, respectively. Besides drug carriers, these microstructured microbeads can be further employed for cell encapsulation and tissue engineering.

In this study, we evaluated the efficiency of different adsorbents for the removal of plant-derived impurities during the pre-purification of paclitaxel from plant cell cultures. Using the synthetic adsorbents sylopute and active clay and their major components SiO2 and MgO, we performed adsorbent treatment and analyzed the paclitaxel precipitates recovered from hexane precipitation. When SiO2 was used, the highest purity (~58.1%) and yield (~91.5%) of paclitaxel were obtained. We also determined differences in the effectiveness of the adsorbent treatment according to changes in the surface area, pore volume and pore diameter of SiO2. Adsorbent treatment was more effective when pore diameter was larger (silica I [2.19 nm] <silica II [4.92 nm] <silica III [9.07 nm]). The highest purity (~74.3%) and yield (~92.9%) of paclitaxel were obtained when silica III was used in the adsorbent treatment. Pore diameter had a greater effect on the removal of plantderived impurities during the pre-purification of paclitaxel compared with surface area and pore volume. This result could be confirmed by HPLC analysis of the absorbent after treatment and TGA of the organic substances that were bonded to the adsorbent.

The control of glutamine concentration is very important for improve cell growth and recombinant protein as reducing ammonium concentration. Ammonium is toxic and inhibitory byproduct of mammalian cell metabolism at the end of typical recombinant protein production. The ammonium affects negatively both cell growth and recombinant protein productivity. The most of ammonium comes from glutamine metabolism and decomposition in media. So we have to control the concentration of glutamine for decrease of ammonium production. We have investigated about the chemically defined culture media formulation for Chinese Hamster Ovary DG44 (CHO-DG44) cell line for achieving a more efficient use of nutrients and avoiding the accumulation of byproducts in the media. Glutamine is one of the most commonly employed nitrogen source for mammalian cell culture. This amino acid not only supports cell growth and monoclonal antibody production but also can be used as alternative energy source and precursor of nucleotide. But glutamine products two ammonium ions during metabolism to alpha-ketoglutarate for Tricarboxylic acid cycle (TCA cycle). When the ammonium level raise over than limitation of toxicity, mammalian cell can’t grow more. Because of this reasons, the media can’t contain high level glutamine, though glutamine is very important in mammalian cell culture. In this study we suggest simple solution to reduce ammonium product as control glutamine level in culture media. It is the novel method to product more recombinant protein for CHO DG44 cell in chemically defined media. And the strategy can improve productivity and will bring numerous commercial benefit.

Monodisperse gelatin beads with a uniform pore size were fabricated from gelatin using a simple fluidic device fabricated with a Tygon tube, a syringe needle, and a glass capillary. Toluene and an aqueous gelatin solution are served as the discontinuous and continuous phases in the fluidic device, respectively. Oil-in-water emulsion droplets with a uniform diameter were pinched off from the tip of the capillary tube and subsequently frozen in liquid nitrogen. The frozen O/W emulsion droplets evolved into porous beads with a uniform pore size after removal of toluene and then freeze-drying. Due to the uniform size of toluene droplets in water phase, the porous beads exhibited a uniform pore size. The number and size of pores in the porous beads can be easily controlled by changing the flow rate of each phase.

Porous tricalcium phosphate beads(beta TCP) with a controllable pore size were fabricated using an emulsion templating method. An aqueous dispersion of TCP powders and gelatin as achieved using ball mill with Zirconia balls. Primary oil-in-water (O/W) emulsions with a different size in oil droplets were prepared by changing the homogenization speed, where toluene and the aqueous TCP phases were served as oil and water, respectively. The primary O/W emulsion was introduced into a fluidic device using cooled toluene as the continuous phase, evolving into oil-in-water-in-oil (O/W/O) emulsion droplets. The O/W/O emulsion droplets were freeze-dried and then calcined at various temperature, finally obtaining TCP beads with micro and macro pore sizes. These micro and macro porous beads have great potential for applications in bone tissue engineering.

We were investigated for components depleted during a batch-culture of recombinant Chinese hamster ovary DG44 cells in chemically defined media. About after 5 days of cell cultured, cell growth and viability are usually reduced. It is predicted that specific components which help cell growth and antibody production are depleted in media. Therefore we investigated that rCHO-DG44 cell growth screening for the depletion components in Chemically-defined media. In the case of amino acid composition of the medium is analyzed by HPLC, but depletion or accumulation of other components are difficult to measure by analyzer. In this study, we collected supernatants of rCHO-DG44 cell culture in Chemically-defined media at day 4 and day 7 of culture. Then the each supernatant mixed with equal volume fresh media without specific components we wanted to monitor. rCHO-DG44 cell is cultured in mixed media. As a result, amino acids, vitamin and small molecules are depletion. By this method, we know depletion components for cell growth and antibody production. It is expected that it is useful for batch-culture enhance and fed-batch culture strategy. And the same method as above, other media and cell lines will be useful.

The contents of bioactive and antioxidative were tested by in vitro experimental models using water, ethanol and methanol extracts from Cuscuta japonica chois. Tests to check for bioactive materials are phenolic compounds and flavonoids test and to check for biological activities are DPPH free radical scavenging activity, reducing power Cu/Fe and tyrosinase inhibition activity. The highest extraction yield is in water extract (18.07%). The highest contents of phenolic compounds and flavonoids in the methanol extract were 170.20mg/100g and 81.38mg/100g. The highest free radical scavenging activity showed in methanol extract by 89.59% at 0.5% additional level and in ethanol extract by 78.60% at 0.5% additional level. The activities of free radical scavenging was stronger in ethanol and methanol extracts than water extract from Cuscuta japonica. And reducing power was found in the ethanol and methanol extracts. Tyrosinase inhibition activity were stronger in the methanol extracts than others extracts of Cuscuta japonica chois. These results may provide the basic data to understand the biological activities of bio-active materials derived from Cuscuta japonica chois. for development of functional foods.

The proportion of unsaturated fatty acids such as oleic acid (18:1) is an important factor for increasing oxidative stability of algal oil during storage. Oleic acid and palmitoleic acid (16:1) are desaturated to more unsaturated fatty acids in chloroplast by FAD6 (plastid-pathway) and cytosol by FAD2 (ER-pathway) in higher plants. In this study, we isolated FAD6 from Chlamydomonas reinhardtii, named CrFAD6 and generated CrFAD6-downregulated transgenic lines using RNAi to increase accumulation level of oleic acid in C. reinhardtii. In CrFAD6- RNAi lines, oleic acid and palmitoleic acid content were increased ~2.1- and ~5.6-fold, respectively, compared to the wild-type control. Interestingly, both linoleic acid (18:2) and linolenic acid (18:3) contents in CrFAD6-RNAi lines were reduced ~32% in comparison with the control; in fad6 mutant of Arabidopsis, palmitoleic acid, oleic acid and linoleic acid content were induced, whereas linolenic acid content was reduced, indicating that desaturation of oleic acid to linoleic acid is mainly regulated by ER pathway in higher plants. In this study, remarkable phenomenon compared to higher plants is that downregulation of CrFAD6 resulted in the reduction of linoleic acid content (18:2) in C. reinhardtii. These results also suggest that the plastidic pathway may play a major role in desaturation of oleic acid to linoleic acid/linolenic acid in green alga C. reinhardtii.