Earticle

Reporter proteins, such as GFP, beta-galactosidase and luciferase, have been used in many studies to monitor biological activities. For researchers working with predatory strains, top agar plates are needed to determine the population numbers, typically requiring 4 days. In this study, therefore, reporter proteins were used to monitor the kinetics of predatory bacterial strains and evaluated as a tool to estimate their numbers in a much shorter time. It was found that the fluorescent proteins, including CFP and RFP, were not degraded by the predatory bacteria but were released into the supernatant after lysis of the prey. As such the “ackground”ú fluorescence increased faster with a higher multiplicity of infection (MOI) ratio but required about 6 hours for determination. In contrast, the bioluminescence, which is integrally related with the energy level of the cell, decreased shortly after mixing the predator and prey, indicative of a reduced metabolic activity from the attacked prey. This decrease was faster for higher MOIs, allowing us to reliably estimate the predator numbers within 1 hour.

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.

In the last decade, advances in cancer research have enhanced our understanding of cancer biology and genetics. Anticancer effects may be exhibited through selective cytotoxicity, antiproliferative action, and apoptosis. Natural products or phytochemicals, substances found in edible fruits and vegetables, exhibit a potential for modulating human metabolism in a manner favorable for the prevention of chronic and degenerative diseases. Many different sub-tropical fruits are currently being evaluated for their effects against cancer as well as the possible mechanism of this action using various human cancer cell lines in our groups. The chloroform fraction (CF) of dangyuja leaf extract strongly decreased the survival rate of HeLa cells, whereas the CF of guava leaf extract exhibited the maximum cytotoxicity on SNU-16 cells. Both CF treatments induced the down-regulation of anti-apoptotic Bcl-2 expression, resulting in the proteolytic activation of caspases and the degradation of poly (ADP–ibose) polymerase (PARP) protein in these cell lines. Arrested cell growth and induction of apoptosis were confirmed by flow cytometry and DNA fragmentation analysis, respectively. The major components, which might be responsible for induction of apoptosis, of two different CFs identified by high performance liquid chromatography coupled with photo diode array and electrospray ionization mass spectrometry (HPLC-PDA-ESI-MS) will be discussed.

We developed the homogeneous fluorescence assay for multiplex detection based on the target induced conformational change of DNA aptamers. DNA aptamers were immobilized on quantum dots (QDs), and QDs conjugated ssDNA was adsorbed on the surface of gold nanoparticles (AuNPs) by electrostatic interaction between positively charged bases of ssDNA and the surface of AuNPs. Subsequently the fluorescence of QDs was effectively quenched by AuNPs due to fluorescence resonance energy transfer (FRET) of QDs to AuNPs. In the presence of targets, the QDs conjugated aptamers was detached from AuNPs by target induced conformational change of aptamers consequently the fluorescence of QDs was recovered proportional to the target concentration. In this study, three different QD/aptamer conjugates were used for multiplex detection of mercury ion, adenosine and potassium ion. Some parameters (i.e. molar ratio of QDs to AuNPs, spacer size of ssDNA, and surface condition of QDs) were optimized to increase the quenching effect of QDs by the strong interaction of ssDNA aptamers conjugated on QDs to AuNPs. In control experiment, all of three targets were simultaneously detected with high selectivity.

One of the major issues in the field of obesity is why some humans become obese and others resist development of obesity when exposed to high-calorie diets. Despite the same genetic background, namely obesity-prone (OP) and -resistant (OR) rats, differing responses have been demonstrated in a high fat diet (HFD)-induced rodent model. The aim of the present study was to discover novel obesity-related biomarkers for susceptibility and/or resistance to obesity by proteomic analysis of OP and OR rat plasma. After feeding of HFD, OP rats gained approximately 25% more body weight than OR rats and were used for proteomic analysis using 2-DE combined with MALDI-TOF-MS. We categorized identified proteins into three groups by analysis of both average spot density in each group and individual spot density of six rats as a function of body weight. Consequently, category (1) included inter-alpha-inhibitor H4 heavy chain (ITIH4) and fetuin B (Ft B) precursor, which can be used as novel plasma biomarkers for risk of obesity. Nine proteins of category (2) and (3) can also be plausible plasma markers in the study of obesity. This proteomic study is an important advancement over the previous steps needed for identification of OP and OR rats.

The surface forces apparatus (SFA) has been used for many years to measure the physical forces between surfaces, such as van der Waals, electrostatic forces, adhesion forces, ligand-receptor interactions, friction and lubrication force. Currently, the SFA technique is quite demanding for interdisciplinary area of physics, chemistry, biology, materials science, and chemical engineering. However, only a handful of labs worldwide are equipped with functional SFAs and no SFA is available in South Korea due to long training time for skillful operation and tricky data process. The SFA measures the magnitude and distance of the intermolecular forces between two atomically smooth surfaces by approaching, retracting or shearing from one another. Forces are measured with a resolution of 10nN and the separations are determined with 0.1 nm resolution by multiple beam interference fringes of equal chromatic order (FECO). A base surface for the SFA studies is usually mica but adsorbing or depositing a thin film of some other materials, i.e., lipid layers, metal oxides, polymer films including proteins and carbohydrate have been also used in the SFA. Here, an introduction to the SFA, the comparative analysis with AFM and its application to marine adhesives will be discussed.

Unicellular green algae, Haematococcus lacustris is able to produce astaxanthin up to 80% of dry cell weight, and it is induced under various stress conditions, such as high irradiance, high salinity, nutrient starvation, and oxidative stress by reactive oxygen species (ROS) as a defense mechanism of the cells. In this study, we used proteomic and transcriptomic analyses to obtain gene expression profiles in H. lacustris under astaxanthin-inductive culture conditions, combining high irradiance and nutrient starvation. In the experiments, we analyzed several genes that were differentially expressed more than twofold change. As the results, significant decrease in the expression of chlorophyll biosynthesis and light harvesting complex (LHC) related genes were shown in astaxanthin-accumulated H. lacustris (red cyst cells), that can decrease photosynthesis. On the other hand, respirationrelated genes, lipid metabolism-related genes and stress response-related genes were activated in the red cyst cells. These results provide comprehensive information for understanding cell responses during astaxanthin accumulation by stress conditions in H. lacustris.

Recombinant Saccharomyes cerevisiae harboring xylose reductase gradually grew, and consumed amount of xylose and excreted xylitol under xylose/glucose condition, however it grew slowly and consumed a little xylose in xylose, because hexose transporters transport xylose with low affinity than glucose. In addition, recombinant S. cereviaise strains co-expressed xyl1, xyl2, and xyl3 consumed smidgen of xylose. In this study, the genetically modified S. cerevisiae strains JH1, JHC1, and JHC2 were used for experiments. We found that protein profiles from JH1, JHC1, and JHC2 were shown different expression patterns for three different culture conditions by using 2-DE, The changed spots between JH1 and JHM were significantly increased to 174 under the YPDX condition. In addition, JH1 grown in YPX condition had shown quite different patterns, comparing to when grown in YPDX. Therefore, recombinant S. cerevisiae harboring xylose utilization related genes probably show the metabolic alterations caused by foreign genes expression under the xylose supplemented conditions.

Redox balance is important factor on distribution of final products in anaerobic fermentation (1). For microbial production of useful fatty acids, more reduced substrates like glycerol (2) have been used as carbon source or enzymatic modification (3) applied for more reducing power. We used a mediator to change electron flows to more redox state and also applied cathode as extra electron donor for more reducing power. Here we show that how external electron supply affected final products and it enhanced fatty acid production in anaerobic fermentation. Sucrose-utilizing C. tyrobutyricum BAS7 was used as a model organism and it produced butyrate, acetate, hydrogen, and carbon dioxide as final products under anaerobic condition. Methyl viologen (MV) instead of proton was reduced by hydrogenase and gas formation was reduced in MV-amended condition. An electron flow model (4) showed electrons flow from ferredoxin pool (Fdox/Fdred) to NADH pool (NAD+/NADH2) and it increased butyrate production from 5.7 (±0.6) to 8.5 (±0.4) g/L to consume NADH. Another external electron donor was cathode. Bioelectrical reactor (BER) was applied and C. tyrobutyricum BAS7 was grown in cathode compartment with MV 0.5 mM. Cathode did not enhance butyrate production more than MV did. Interestingly, unknown compound was produced in BER and analyzed. We studied proteomics to know how external electron donor changed microbial metabolism and activated under BER condition. This result shows external electron donors changed microbial metabolism and could lead valuable products of biofuel from electricity.

A major value of anaerobic digestion is the production of fuel-grade biogas formed as the end product during degradation of organic material. Methanogens participate in the terminal step of anaerobic digestion, methanogenesis, and play a key role in generation of methane. Since methanogenesis is the rate-limiting step in most anaerobic processes, lots of works on the process optimization study focused on the methanogenesis step have been made. Those have been, however, confined to modification of process and optimization of operating conditions. In order to overcome the innate biological limit, therefore, molecular biological approaches should be concurrent with the process optimization study. M. thermophila is one of the most metabolically diverse methanogens in that it utilizes the methyl groups of acetate, methanol, or methylamines for growth and methanogenesis [1]. Genetic manipulation of an aceticlastic methanoarchaeon Methanosarcina thermophila was performed. A metabolic gene which encodes carbonic anhydrase(ca) from M. thermophila was overexpressed in M. thermophila TM-1 host strain. The recombinant plasmid used to transfer ca into the host cell, pMC1, was maintained in approximately four copies per cell. This result corresponded to the previously reported results for methanogenic natural plasmids. The recombinant strain showed about 2.4-fold higher expression level of ca than the wild-type strain. Corresponding to this result, the carbonic anhydrase activity was also about 2.1-fold higher in the recombinant strain. These results indicated that the pMC1 was well-functioning in the recombinant M. thermophila host cells and, as a result, the elevation of carbonic anhydrase activity was induced by over expression of ca.

The increasing environmental issues, generated green-house gases from the fossil fuels and pertaining to limited energy reserves, have been becoming ‘ot-potato’concerns in every country. With significantly desired alternatives as an energy product, biodiesel (fatty acid alkyl esters, FAAEs) has a considerable attraction because of non-toxic, biodegradable properties and without lower exhaust emissions. Biodiesel is produced by followings: esterification of fatty acids or transesterification of oils and fats with short chain alcohols. When fatty acid methyl ester (FAME) and unrefined glycerol as a by-product are generated, these reaction products have been analyzed by gas chromatography (GC) method. For the glycerol quantification, GC method has demerits such as laborious works of sample preparation, long time and cost of sample analysis. Thus, there is a need to analyze glycerol more easily. Herein we report that the analysis of glycerol by using malachite green conducted by UV-Vis Spectrophotometer at the wavelength 617 nm whose peak was maximum intensity of malachite green, resulting in the peak shift and intensity change of dye as a function of glycerol amount.

Wheat germ is a by-product of the wheat milling industry. It constitutes about 2-3% of the wheat grain and contains about 11% oil. The by-products recovered from food processing can be turned into valuable products or at least converted into useful products. Manufacturing lipid products from natural resources normally yields significant amounts of low-value fats and oils next to the focused high valued processes. With better refining technologies these resources can be turned into value added products. Fats are possible to be converted into glycerine and fatty acid esters by alcoholysis. Monoglycerides, which are widely used as emulsifiers in the food industry and yield higher market prices than oils. The chemically catalyzed reaction of lipids with alcohols is simple to carry out, but can generate many side products like soaps and free fatty acids which can be tedious to remove from the reaction mixture. As an alternative the lipase catalyzed reaction is more selective and ideally leads only to monoglycerides and fatty acid esters.For that, wheat germ oil was extracted by 250bar and 40℃ condition by supercritical carbon dioxide. The enzymatic ethanolysis is carried out in an elenmyer flask containing a mixture of wheat germ oil and ethanol (99.90%) using 3-5 wt% mobilized lipase and the reaction mixture were incubated at 30-50℃ with 120 rpm shaking.

The optimal conditions for production of carboxymethylcellulase (CMCase) of B. amyloliquifaciens DL-3 by E. coli JM109/DL-3 were established using the response surface method. The optimal carbon and nitrogen sources for production of CMCase were found to be rice bran and tryptone, respectively. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor ("probe>F"less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal conditions of rice bran, tryptone, and initial pH for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L,5.0 g/L, and 7.1. The optimal temperature for cell growth and production of CMCase were found to be 37℃. The maximal production CMCase by a recombinant E. coli JM109/DL-3 was 487 U/mL.

The nanoparticles (NPs) have been used in a variety of fields due to their authentic physicochemical properties. Biological or environmental toxicity of NPs, however, has not been fully understood. In this study, the toxicity of water stable fullerene colloids, nC60 and fullerol, C60-OH NPs was assessed in Caenorhabditis elegans 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 in THF followed by repeated dialysis with water. C. elegans is a free-living soil nematode feeding on bacteria such as Escherichia coli. We have found that the oral-administrated NPs were accumulated in the animals for several days after feeding the mixture of bacterial food and nC60 or C60-OH NPs to the L4 larvas of C. elegans. The fullerol NPs reduced the viability of animals while no decrease in survival was found in the animals fed with nC60. It was also found that both nC60 and C60-OH diminished 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, apoptosis regulators such as CED-3, CED-4, and EGL-1.

Probiotics are live microorganisms known as a health promoting bacteria for the host animals. In general, Lactic acid bacteria and Bifidobacteria belong to probiotic microbes, and yeasts and bacilli are also thought be helpful for their hosts when they are administered in adequate amounts. In this study, we investigated the beneficial effect of probiotic bacteria on the nematode C. elegans focusing on enhancement of lifespan and reproduction of host animals. Several Lactic acid bacteria and Bifidobacteria isolated from Korean traditional fermented foods were fed into C. elegans on the NGM plate, and the survival rate and reproduction ratio were subsequently assessed by scoring animals under the stereo microscope. In order to identify whether small molecules or metabolites produced by bacteria have those beneficial healthy functions, the extracts of bacterial culture broth were administered into C. elegans together with their common foods, E. coli OP50. When C. elegans were fed with some of selected probiotic bacteria, body size of animals were enlarged in their adult hoods. Moreover, the digestion of bacterial foods and locomotion of old animals was also improved by the supplementation with probiotics. These results imply that there are many probiotic resources in Korean traditional fermented foods to increase animals’growth and digestion with anti-aging effects.

A novel simultaneous microwave-assisted extraction (MAE) and adsorbent treatment process was developed for recovering the anticancer agent paclitaxel from plant cell cultures and removing the plant-derived tar and waxy compounds. Furthermore, the process efficiency was maximized by optimizing the major process variables (extraction time, amount of absorbent, biomass-to-methanol ratio, extraction temperature). The optimum conditions of the simultaneous process for efficiency of the recovery of paclitaxel and the removal of tar and waxy compounds were 6 min for the extraction time, 4.5 (w/v) for the ratio of adsorbent to crude extract, 1:1 (w/v) for the biomass-to-methanol ratio, and 40℃ for the extraction temperature. Performing the simultaneous process under the optimum conditions could recover most (>99%) of the paclitaxel with the purity of ~22% could be recovered from hexane precipitation which was the following process. This result was similar to the efficiency (purity: ~20%, yield: ~95%) of the conventional process in which the paclitaxel recovery process is separated from the adsorbent treatment process. Therefore, it is expected that the conventional process could be simplified by simultaneously performing the recovery of paclitaxel and the removal of plant-derived tar and waxy compounds from plant cell cultures as one process, thus dramatically reducing the cost of the paclitaxel separation/purification process

Gliadin, component of gluten, is known as a important allergen which cause allergic reactions to patients who have wheat allergy. This study was conducted to evaluate the effect of physical treatments on the antigenicity of gliadin in wheat flour and dough. The wheat flour was treated with supercritical carbon dioxide (SC-CO2) and in combination with autoclave and microwave. The conformational changes were observed by SDS-PAGE and the binding ability of gliadin to anti-gliadin IgG was examined by ci-ELISA and immunoblotting. The binding ability of wheat flour treated with SC-CO2 was slightly decreased compared with untreated flour. When treated with SC-CO2 and autoclave, it was shown that the binding ability to anti-gliadin IgG was decreased and the intensity of gliadin bands were weakened in SDS-PAGE. In case of the microwave treatment after SC-CO2 treatment, the binding ability was slightly reduced. When treated with SC-CO2, autoclave and microwave, the binding ability of gliadin was also decreased.

Transaminases (TAs) have been studied extensively for its importance in the industrial production of chiral amino acids which are used as synthetic blocks in peptidomimetics. This enzyme shows a rapid reaction rate, broad substrate specificity, high enantioselectivity, and it doesn’ require the external cofactor regeneration. It is classified into a-TA and w-TA according to the relative position of the amino group transferred with respect to the carboxyl group of the substrate. w-TA helps in transferring amino group of an amino donor onto a carbonyl moiety of amino acceptor, whereby at least one of the two substrates is not a a-amino acid or a a-keto acid. Nowadays, w-TA is gaining increasing interest owing to its great utility for the production of chiral amines via kinetic resolution and asymmetric synthesis. In this study, we demonstrate the efficient production of enantiomerically pure unnatural amino acid such as b-amino acids and fluorinated a-amino acid through omega-transaminase catalyzed reaction.

Amino-organoclays have been synthesized as organic-inorganic hybrid materials with a variety of centered metal cations such as Mg2+, Ca2+, Fe2+, Cu2+, Sn2+, Mn2+, Co2+, Al3+, Fe3+ etc. at sandwiched aminopropyl organo-functionalities, showing superior water-dispersibility via exfoliated dispersions of organoclay sheets by protonated amino groups in aqueous solution. Among those, we focused on synthesis of Cu-aminoclay to further investigate antibacterial activities, comparing with Cu salts. For the synthesis of Cu-aminoclay, we followed the method of the reported literatures. In order to evaluate the antibacterial activity of Cu-aminoclay, the inhibition growth assay of bacteria with Cu-aminoclay was confirmed by measuring absorbance at 620 nm, after mixing with suspensions with 2.5×10⁵colony forming units (CFU)/ml bacteria and Cu-aminoclay buffer solution. With micro-well plate and agar plate assays, the antibacterial activity of Gram positive species, Gram negative species, and fungal strains as well as antibiotic resistants (E. coli CCARM-12296, E. coli. CCARM-12386, S. aureus CCARM-3089, S. aureus CCARM- 3108) were examined.

Retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration, are the leading causes of blindness and related with disruption of retinal structure and function. The objective of this study is to confirm that glycol chitosan nanoparticles (CNPs) with amine and glycol groups can reach retina and even deeper sub-retinal part than other cationic nanoparticles. To evaluate this possibility, we synthesized self-assembled nanoparticles by conjugation of hydrophilic glycol chitosan and hydrophobic 5β-cholanic acid. As control cationic nanoparticle, PEI-5β-cholanic acid nanoparticles were synthesized by similar amide conjugation. Both nanoparticles were administered to long-Evans rats intravitreally, and we analyzed their movements by fluorescence imaging of ocular tissues. CNPs were able to penetrate vitreous and localized in retina after 1 hour, while PEI nanoparticles stuck in vitreous. Furthermore, some of CNPs could reach sub-retinal part related with age related macular degeneration after 3 days. These results demonstrated that CNPs are promising carriers for ocular delivery to overcome various retinal and sub-retinal diseases.

In this study, we degraded several toxic chemicals, which can be released from the pretreatment of lignocellulose for the biofuel production, using microorganisms isolated from the soil. First, the strains were characterized with growth on pure hydrolysate compounds including p-coumaric acid and vanilic acid within media. All strains grew most quickly on 4-hydroxy benzoic acid having the simplest chemical stucture. We also performed the fed-batch experiment and checked the maximum degradation. Finally, we confirmed the ability of these isolates to mineralize those chemicals using E. coli biosensor. In our previous study, we constructed a bioluminescent fusion strain and demonstrated that the strain have the potential as a biosensor to analyze phenolics present in hydrolysate sample. As shown, bioluminescence responses decreased according to time, suggesting that the concentration of chemicals in media also decreased.

In this research, we designed the helical bioreactor for improving nitrification process and investigated the microbial community structure of nitrifying bacteria. The three 6L of laboratory scale bioreactor including the one control bioreactor were operated at batch mode and each batch trial was repeated four times. From the results, nitrification was successfully carried out at all experiment trials, then we could ascertain saving of aeration amount at the helical reactor up to 25 percent. The reaction time was gradually shortened along with repetition of batch trials. From its molecular analysis, it was quantified that five cluster of ammonia oxidizing bacteria were Nitrosomonas europaea, N. mobilis, N. nitrosa, N.cryotalerans and Nitrosospira cluster in the system through the real-time quantitative PCR. The major species was changed over the process operation from N.cryotolerans to N. europaea cluster. Especially, copy number of N.europaea increased one hundred times m than that at the initial condition. In case of nitrite oxidizing bacteria, the major species was shifted from Nitrosospira spp. to the Nitrobacter spp. These changes of the microbial community structure seem to have closely related to the reduction of reaction time and ecological characteristics.

The purpose of this study was to investigate the relationships between hydrolysis of particulate organic materials (POM) and microorganisms in acidogenic reactors treating food-waste recycling wastewater (FRW). Hydrolysis efficiency in terms of volatile suspended solid (VSS) reduction was observed in continuous completely stirred tank reactors (CSTRs) with changing hydraulic retention time HRT (1 - 3 day), pH (4 - 6), and temperature (25 - 45oC) and was interpreted by 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. Denaturing gel gradient electrophoresis (DGGE) using bacterial 16S rRNA genes were carried out to investigate acidogenic bacterial community structure. The ten identified acidogenic bacterial microorganisms (ABMs) were selected as target for developing speciesspecific primers and probe set for use of real time quantitative polymerase chain reaction (QPCR). The results of QPCR showed that Lactobacillus amylovorus, L. acetotolerans, and Bifidobacterium thermophilum had a large influence in the hydrolysis of POM.

Chitosan is excellent biocompatible and biocompatible and is a plentiful material with a natural linear biopolyaminosaccharide and well established technological base. Chitosan is obtained by alkaline deacetylation of chitin, which is the principal component of protective cuticles of crustaceans such as crabs, shrimps, prawns, lobster etc. Due to its unique polymeric cationic character and its gel properties, chitosan has been examined extensively in the food industry, biomedical and pharmaceutical applications. Chitosan is usually used as a component in hydrogels for medical applications. In this study, we investigated the effect of pH of sodium polyphosphate (TPP) solution during the formation of chitosan hollow beads. The electrostatic attraction between the cationic amino groups of chitosan and the anionic groups of the TPP is the main interaction leading to the formation of chtosan hollow beads. The pH of TPP solution was an important factor acting the formation of clear hollow bubbles or loose mesh-like structure. The stable chitosan hollow beads formed through the complexation of chitosan with TPP may be a promising material for drug delivery.

In this study, a steady supply of ATP for cell-free protein synthesis was achieved by using starch as an energy source. Glycogen phosphorylase (GlgP) activity in the E. coli cell extract caused continuous release of glucose, which was then metabolized to regenerated ATP molecules. Compared to the previously reported methods employing glucose as the primary energy source, the use of starch alleviated the problem of a pH drop during the cell-free protein synthesis reaction, thereby allowing a prolonged reaction period. As a result, after 12 hours of incubation, approximately 1.7±0.1 mg/mL of the target protein accumulated in the reaction mixture. In addition, most likely due to the relatively homeostatic maintenance of the reaction conditions and controlled rate of translation, substantially larger amounts of proteins were obtained in a soluble and functional form.

Quality control of glycan profiles is a critical issue in the biological and pharmaceutical industry. Recently, the glycan profile method which combined with Girard"s reagent T (GT) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was exploited for qualitative and quantitative analysis of glycans. In this study, we present a rapid and high-throughput N-glycan release, purification, esterification and GT derivatization method using 96-well plate based procedures. The N-glycans were released from model glycoprotein using a polyvinylidene fluoride(PVDF) membrane filter 96-well plate, then subsequently loaded onto porous graphitic carbon (PGC) containing 96-well plate to remove the salts. The purified N-glycans were neutralazated by methyl esterification using 96-well plate, then GT derivatizated for generate a permanent cationic charge to the reducing end of neutral glycan. finally, the N-glycans were characterized by qualitative and quantitative analysis using MALDI-TOF MS. It is believed that the established 96-well plate platform described here may contribute to the rapid screening of potential N-glycan biomarkers.

All eukaryotic cells contain functional cell organelles such as lysosomes (LY) and peroxisomes (PE). In our study, it has tried to characterize the suitable conditions to separate subcellular distribution of lysosomal and peroxisomal proteins in eukaryotic cells and evaluate different in vitro function of each cell organelle. To certify to organelles' special function, several chemicals were used to stimulate in vitro function to LY or PE isolated from Saccharomyces cerevisiae. We found that in comparison with control, the number of and fluorescent intensity of LY were increased 3.4, 5.5 or 2.0 folds in HeLa cells exposed by 1unit/ml glucose oxidase, salicylic acid or 6-hydroxydopaime (6-OHDA), respectively. It maybe because these chemicals contain the hydroxy functional group in the structures. Additionally, in vitro antimicrobial and anticancerous activity of LY was enhanced over 2 folds when exposed by 6-OHDA. However, PE were 0.8 or 2.0 folds inactivated by exposure of phenylhydrazine (PHD) or sodium azide, therefore melanin degradation effect of peroxisomal extract was reduced less than 2 folds by PHD, based on the reduction of melanin pigmentary intensity. Therefore, our results provide insight for the application of LY and PE extract as useful asgents for antimicrobial, anticacerous agents and treatment of hyperpigmentation disorders.

Acremonium chrysogenum is cultured worldwide to produce approximately 2,500 tons of cephalosporin derivates annually (Schmitt et al., 2004) and production of cephalosporin C has doubled several times over the last few decades (Christensen et al., 2000). However, the cost of substrates used in antibiotic production, such as glucose or various plant oils, has increased. Recently, optimization of nutritional factors such as novel carbon sources was reported based on statistical analysis of the production of ß-lactam antibiotics by filamentous fungi (Saudagar and Singhal, 2007), and the development of efficient methods of producing cephalosporin C from low-priced materials using various carbon sources and optimization of parameters has also gained attention (Brakhage et al., 2004). Crude glycerol is created as a byproduct of biodiesel production (Galan et al., 2009); accordingly, the amount of glycerol produced as a result of increased biodiesel production worldwide has increased dramatically during the last 10 years, and the industrial value of glycerol has decreased from approximately 650-700 €ton in 1996 to 300-350 €ton in 2004 and to 150-200 €ton in 2005 (Peters, 2007). Due to this reduction in price, glycerol is now an attractive feedstock for the production of useful chemicals.In this study, the effects of glycerol on cephalosporin C production by Acremonium chrysogenum M35 were evaluated. The addition of glycerol increased cephalosporin production by up to 12-fold. Glycerol caused the upregulation of the transcription of the isopenicillin synthase (pcbC) and transporter (cefT) genes in early exponential phase, and affected the cell morphology since hyphal fragments differentiated into arthrospores. These results indicate that glycerol effectively enhances cephalosporin C production via stimulation of cell differentiation.

From the limitation of 20 natural amino acids, the development of a method that allows us to encode extra amino acids genetically might facilitate the posttranslational modification of protein for various applications. Moreover, the ability to incorporate amino acids with desired properties at unique sites in proteins will provide powerful new tools for exploring protein structure and function in vitro and in vivo. Over 30 unnatural amino acids (UAA) —including those containing spectroscopic probes, metal chelators, photoaffinity labels and other chemical moieties —have been selectively incorporated into proteins with high fidelity and efficiency in response to unique codons1,2. Here we show new application of UAA for efficient surface-based protein analysis. For effective single-molecule FRET (smFRET) analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. We demonstrated that site-specific labeling of protein via incorporation of UAA enables a more effective smFRET analysis of protein3. Immobilization of proteins in a functionally active form and proper orientation is also crucial for effective surface-based analysis of protein-protein interaction. Here we present a general method for controlled and oriented immobilization of protein by site-specific incorporation of UAA and click chemistry.

The extraordinary biocompatibility of silk protein is the strongest factor of being used as a novel texture for centuries. With this benefit, usage of silk reaches to the material industry and medical area. Property of silk protein is organism-dependent. To be specific, silk from spider is stronger and tougher than silkworm. The researches for silk protein have been mainly focused on spider and silkworm. In the present work, we investigated new silk-bearing marine organisms to figure out flesh property and possibility of novel silk protein to apply industries. We designed and constructed new silk-like protein (designated to ‘neroin’ from sea anemone. With simple heat shock and acid purification process, we obtained recombinant aneroin successfully. We spun about 50-100 mm of aneroin with no defects by wet spinning. In addition, electrospinning of aneroin was also conducted.