Earticle

Optically active β-amino acids are the fundamental building blocks for the preparation of pharmacologically active peptides, proteins, and agrochemical molecules. Much effort has been dedicated in this field to synthesis the optically pure amino acids. In the recent years, it is an attractive and challenging goal for the organic chemists and biologists to produce optically pure β-amino acids. Here, we have identified a novel ω-TAs which is highly reactive towards various aromatic β-amino acids and successfully yield (R)-β-amino acids with high enantiomeric excess (>99%) through the kinetic resolution of racemic mixture of β -amino acids. We are the first to demonstrate the kinetic resolution of various aromatic β-amino acids using novel ω-TA.

The aetiological agent of epizootic ulcerative syndrome (EUS) is Aphanomyces invadans in both estuarine and freshwater fish (1). The actual economic losses due to EUS in aquaculture industry were to be in excess of US$9 billion per year. Currently, no effective proplylatic measures are available for EUS disease, although adding lime and salt to the ponds has been used (2).In the present study, Cirrhina mrigala intramuscularly injected with Aphanomyces invadans (isolate B99C) at 2.3 x 107 CFU ml-1 was investigate the hematological and biochemical parameters. In infected untreated group, the white blood cell count (WBC: 106mm-3) is significantly increased (P <0.05) from the control, while in no change in groups fed with probiotics, herbal, and azadirachtin supplementation diets on 30 days. Similar trend was noted in the hemoglobin (Hb: g/dL) and hematocrit (Hct: %) levels. Interestingly, infected fish fed with probiotics, herbal and azadirachtin supplementation diets groups did not differ (P> 0.05) from the control. Asimilar trend prevailed in the percentage of lymphocytes (LYM), monocytes (MON), eosinophils (EOS) and neutrophils (NEU). The total protein (TP: g dl-1), glucose (GLU: mg dl-1), calcium (CAL: mmol l-1) and cholesterol (CHO mmol l-1) levels were affected (P <0.05) in the infected group whereas these values after probiotics, herbal and azadirachtin supplementation diet treated groups were restored near control group. The present study was suggested that the infected fish after administration with probiotics, herbal, and azadirachtin supplementation diets for 30 days protect the hematological and biochemical parameters in C. mrigala against A. invadans.

2,3-Butanediol (2,3-BD) is a promising bulk chemical due to its extensive industrial applications. Through chemical reaction such as dehydration and esterification, 2,3-BD can be converted into various chemical compounds including 1,3-butadiene, diacetyl, methyl ethyl ketone (MEK; butan-2-one). These chemical compounds are used to make highly valued materials such as synthetic rubber, antifreeze agent, flavoring agent. Several microorganism species such as Klebsiella pneumonia, Klebsiella oxytoca and Enterobacter aerogenes are natural producers of 2,3-BD. Among these microorganisms, E. aerogenes is a facultative anaerobe, which is able to produce significant amounts of 2,3-BD in mixed fermentations, while generating a number of soluble and gaseous products. Furthermore, it has a high growth rate, high vitality in different types of environment, and is able to utilize diverse range of carbon sources. First, we performed medium optimization and pH control to obtain the optimal culture condition for high cell growth and high production of 2,3-BD product. In addition, we have successfully performed lactate dehydrogenase (ldh) gene knockout by using the Red lambda recombination method. ldh gene knock out strain showed improved yield of 2,3-BD when compare with wild type. We also overexpressed budA, budB, budC and bdh gene of E.aerogenes 2,3-BD pathway operon separately and the whole operon collectively to characterize the role of each gene and to enhance the production of 2,3-BD.

Biohydrogen production can be one of solutions for energy crisis. Previously, we successfully demonstrated the production of biohydrogen by constructing [NiFe]-hydrogenase-expressing recombinant E. coli. Although [NiFe]-hydrogenase showed relatively high oxygen-tolerance compare to other hydrogenases, still biohydrogen production efficiency was low. In this research, we introduced proteorhodopsin, light acceptor for prokaryote working as proton pump, into [NiFe]-hydrogenaseexpressing recombinant E. coli for utilizing light energy for producing biohydrogen. Proteorhodopsin need a chemical named retinal for working as proton pump by using light energy but E. coli does not have a pathway to synthesize it. In order to give E. coli an ability to synthesize retinal, we expressed five foreign genes expressing enzymes for retinal synthesis pathway. By introducing light accepting system into E. coli expressing [NiFe]-hydrogenase, we could get enhanced biohydrogen productivity.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers in the world. When HCC is diagnosed in the later stage, there is very poor with a 5-year survival rate than 10%-15%. Thus, accurate test for early diagnosis of HCC would significantly improve the clinical outcome of liver cancer. Currently, early diagnosis of HCC is used to measure method as the identification of alpha-fetoprotein (AFP) expression level in serum using AFP antibody. But, the identification and production of AFP antibodies are onerous, and AFP antibodies are also sensitive to temperature. Therefore, new substitutive molecule that can specifically replace AFP antibodies is needed. This study focused on development of aptamer-based AFP detection assay for HCC early diagnosis. AFP aptamers are generated through an in vitro selection process called systematic evolution of ligands by exponential enrichment (SELEX). After 10 rounds of selection, affinity test and interaction between AFP and AFP antibody were performed. These results demonstrate that AFP binding DNA aptamers have high affinity interaction (8.87x10-11) and inhibit interaction between AFP and AFP antibody. Hence, AFP aptamer is adaptable to novel substitution for detecting HCC by binding AFP.

Unicellular green microalgae, haematococcus pluvialis, has been focused as a microbial source of astaxanthin production and astaxanthin has been suggested as a food supplement for humans. Haematococcus pluvialis accumulates the highest level of astaxanthin under severe condition. In this study, we developed new typed photobioreactor and improved reactor design&parts. Also, we studied correlation among light, astaxanthin, biomass using controlled indoor photobioreactor system. For efficient cultivation, we reformed v-neck degree and sparger size so we gained progressed mixing effect data. Indoor photobioreactor system composed of fluorescent lamp, carbon dioxide suppliment, reactor and temperature control system. Carbon dioxide supplied 2% concentration with normal air. Temperature is maintained 20~25℃. Controlled light intensity illuminated according to cell density. We draw mathematical relation for light intensity, astaxanthin, biomass based on this data.

Nanofiber technology is the promising one that can create the extracellular matrix(ECM)-like porous structure. Synthetic polymers have been used for fabrication of electrospun nanofibers. However, poor bioactivity is the limitation for the use of tissue engineering application. Previously, we suggested that our recombinant mussel adhesive proteins (MAPs) which have a great cell adhesion ability and biocompatibility can be the potential biomaterials for tissue engineering. In the present work, through the typical electrospinning procedure, we fabricated novel composite nanofibers using polycaprolactone (PCL) and MAPs by varing the mixing ratio of them. Using the FT-IR, XPS, and contact angle analyses, MAPs were identified to be well-exposed on the surface of composite nanofibers to provide the friendly environment for cell-nanofiber interaction. Mechanically, MAP-contained nanofibers showed the stiff and brittle properties, on the contrary, tensile strength was increased by the addition of MAPs. In our in vitro cell culture experiments, cell adhesion and proliferation were also significantly enhanced on the PCL/MAP nanofibers compared to sole PCL nanofibers. Collectively, our newly fabricated MAP-based nanofiber can be used as tissue engineering scaffolds and applied for the specific tissue regeneration as a further work.

Lime addition is a simple, efficient method for isolating sugars from the mixture of glucose, xylose, acetic acid, and sulfuric acid, which are the typical products from the biomass hydrolysis. In this study, we optimized the important process parameters of lime addition method to isolate sugars. The optimal lime type, sulfuric acid/ calcium carbonate molar ratio, and operation time were calcium carbonate, 1/1.1, and 12 min, respectively. In the model solution of glucose, xylose, acetic acid, and sulfuric acid, the removal of sulfuric acid was 90% under the aforementioned optimal conditions.

This study is focused on analyzing the enhanced hydrogen production by extracted mixed organic compounds from Rhodobacter sphaeroides. In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production by the expression of HupSL hydrogenase isolated from R. sphaeroides in Escherichia coli. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli was increased 20.9-fold compared to control E. coli. In addition, we found that biohydrogen production from recombinant E. coli was more increased under the mixed organic compounds supplemented condition. The concentration of the ATP as cofactor for hydrogen production from in mixed organic compounds extracted from R. sphaeroides showed about 10-7M, while E. coli extract was about 10-10M. From these results, we conclude that mixed organic compounds from R. sphaeroides have multifunctional potential to increase the fermentation efficiency of microorganism.

In tissue engineering, immobilization of growth factors or peptides on scaffolds is advantageous for promotion of viability, proliferation, and differentiation of target cells. One such ligand is osteogenic growth peptide (OGP), a short and naturally occurring 14-mer growth factor peptide found naturally in human serum at μmol/L concentration. Here, we designed fusion immobilizing protein, fp-151-OGP, for efficient osteoblast adhesion, based on fusion of mussel adhesive protein (MAP) with OGP. We expressed fp-151-OGP in Escherichia coli system and easily purified by one-step extraction method. Osteoblast behaviors, including attachment and proliferation were investigated on the fp-151- OGP-coated surfaces using mouse pre-osteoblast cell lines (MC3T3-E1). We found that osteoblast on fp-151-OGP-coated surface displayed good attachment and proliferation properties, compared to other commercially available cell adhesion material, poly-L-lysine (PLL). Collectively, our results suggest that the new functional fusion MAP, fp-151-OGP, can be successfully used in bone tissue engineering fields for improving osteoblast growth.

Crystallization is a simple, energy-efficient and environmentally friendly process for purifying vancomycin from fermentation broth. However, the crystallization process has been inherently problematic due to the lengthy crystallization time that is required. An improved crystallization process could significantly reduce the crystallization time by increasing the surface area available for crystallization. Vancomycin crystallization time was shortened compared to the control by increasing the surface area per working volume (surface area/volume of reaction solution, S/V) of the reacting solution through the addition of a cation exchange resin, an anion exchange resin. Most of the vancomycin could be obtained after about 12 hr of crystallization using Amberlite IR-120 (plus), Amberlite 200, Amberlite IRC-50, Amberlite IRA-400, and Amberlite IRA-910. Since high purity vancomycin can be obtained in high yield and the crystallization time can be reduced, this improved method is expected to significantly enhance the final purification process.

It has been needed to develop rapid, sensitive, and accurate techniques for detection and diagnosis of pathogenic bacteria, especially in food safety and hygiene. Thus, there have been many attempts to apply the DNA microarray as one of pathogenic bacteria detection methods. To employ DNA microarray for food safety, target DNA must be prepared by extraction from food matrix, amplified by polymerase chain reaction (PCR), and labeled using fluorescent dye. It is not easy to directly extract the DNA from food and takes a long time for target DNA preparation. In the present work, we established the 16S rDNA-based oligonucleotide microarray detection system using RNAs from food bacteria. Also, we constructed the simple pathogenic bacteria RNAs extraction and treatment from various food matrixes. Using this technique, we confirmed the detection of pathogenic bacteria from diverse food matrixes such as grain, meat, milk, egg, and vegetable. Although background signal was raised, it was successful to detect a model pathogenic bacterium. Therefore, we expect that this technique for direct detection of pathogenic bacteria from many food matrixes is functional.

The capacitive deionization (CDI) method is first applied to remove zinc chloride from the mixture of zinc chloride and insulin, which are the typical metabolites from the microbial fermentation based on recombinant DNA technology. In this study, we optimized the important process parameters of capacitive deionization to remove of zinc chloride. The optimal electrical voltage, operation time, and flow rate were 1.4 V, 3 min, and 20 mL/min, respectively. Under the optimal conditions, the removal of zinc chloride was 88%, 79%, and 69% at the initial zinc chloride concentration of 180 mg/L, 360 mg/L, and 540 mg/L, respectively. The capacitive deionization (CDI) process as an effective method for the zinc chloride removal is expected to replace the existing size-exclusion chromatography, which is the typical process for removal of zinc chloride.

β-Carotene is a red-orange color pigment abundant in many plants and fruits. Non-polar β-carotene is commonly separated from the mixture of carotenoids using an organic compound such as hexane. Carotenoids are known as a strong antioxidant which scavenges reactive oxygen species (ROS) or free radicals. However, the instability of carotenoids is an obstacle in the use of them in a variety of biological systems. Here, we investigated the enhancement of health beneficial effect of β- carotene in the Caenorhabditis elegans animal model using synthesized derivatives of β-carotene with a great stability. When novel β-carotene derivatives were fed into animals together with foods, the life-span was pro-longed in their adulthoods. In addition to the pro-longed lifespan, the locomotion of C. elegans was more activated than that of control worms grown without novel β-carotene in spite of old age. Those anti-aging effects were due to the stable activation of antioxidant effect by novel form of β-carotene. Results imply that the stabilized form of β -carotene has a potential for health aid to suppress aging of animals and human beings.

Cancer is one of serious human diseases that cause angiogenesis, metastasis, and abnormal cell divisions including genetic change. Thus, lots of researchers have been investigated to inhibit cancer. Human 90K (h90K) protein has very crucial role for inhibition of cancer metastasis. h90K is a glycoprotein having potential 7 N-glycosylation sites (4 sites are clear but remaining 3 sites are not yet). In the present work, we performed expression of recombinant h90K glycoprotein in insect Drosophila S2 cell system. We constructed stably-transfected S2-h90K cells by antibiotic selection and confirmed h90K expression using Western blot and in vitro bioactivity analyses. We found that recombinant h90K has anti-cancer activity. We also identified N-glycan patterns of recombinant h90K glycoprotein using HPLC and MALDI-TOF MS. Based on the identified N-glycan structures, we next engineered N-glycosylation pathway to develop advanced N-glycan patterns using multiple expression platform strategy with recombinant baculoviruses. We found that infection with recombinant baculovirus having galactosyltransferase resulted in advancement of bi-antennary galactosyl form of N-glycans in recombinant h90K glycoprotein.

F2N78, a human somatic hybrid cell line, has been derived from a fusion of human embryonic kidney 293 (HEK293) cells and Namalwa cells. HEK293 has been widely used as a human host for the effective transient expression of recombinant proteins, and Namalwa is a Burkitt’ lymphoma cell line harboring integrated Epstein-Barr Virus (EBV) genome in its chromosome. F2N78 has been adapted to serum-free suspension condition, so that the transfection could be performed in suspension cultures. In this study, we investigated the transient transfection of F2N78 under suspension condition. Higher transfection efficacy and long-lasting cell viability could be obtained compared with other cell lines such as HEK293 and Chinese hamster ovary (CHO-K1) cells usually used for the expression of foreign genes. Furthermore, the growth characteristics and metabolism of F2N78 cell culture were analyzed using different basal media to find an optimal medium. It was found that a chemically defined media named MPC002 provided the best growth conditions. Optimization of MPC002 for the production of recombinant antibody in F2N78 cells is going to be performed for further study.

Waste glycerol, by-product from biodiesel production process, contains high amount of fatty acids (20 % of waste glycerol). Although fatty acids, particularly, unsaturated fatty acids have been widely used in the field of food and pharmaceutical preparation, fatty acids derived from waste glycerol cannot be used directly without treatment due to its dark color. Urea-addition crystallization and a high liquid chromatography were reported for purifying the fatty acids. However, the decolorization of fatty acid has not been reported. Here, we aimed to remove the colored materials in fatty acids obtained from waste glycerol. Effects of temperature, reaction time, the amount of adsorbent (kaoline) and initial fatty acid color were investigated. Temperature and reaction time almost have no effect on the decolorization. Central composite design was applied to optimize the content of kaoline and initial fatty acid color. By statistical analysis, we found the optimal condition, at which colored materials were completely removed and thus clean fatty acid was obtained with no loss of fatty acid.

This study attempted to evaluate an adsorbent that can efficiently remove the tar and waxy compounds from plant cell cultures. Using synthetic adsorbents (sylopute and active clay) and their major components (SiO2 and MgO), we performed adsorbent treatments and analyzed the recovered precipitates from a hexane precipitation. When SiO2 has been used, the highest purity (~ 58.1%) and yield (~ 91.5%) of paclitaxel were obtained. We found that SiO2 was most effective for the removal of plant-derived tar and waxy compounds. We also investigated the effectiveness of the adsorbent treatment applying varied levels of physical properties (surface area, pore volume and pore diameter) of SiO2 and found that adsorbent treatment is more effective when pore diameter is 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, which has the largest pore diameter, was used in the adsorbent treatment. Pore diameter has greater effect on the removal of plant-derived tar and waxy compounds compared with surface area and pore volume. The waxy compounds removal effect could also be confirmed by HPLC and TGA quantitative analysis of organic substances that were bonded to the adsorbent.

Lactic acid production by Lactobacillus helveticus using batch culture and continuous culture was studied. It is known that the pretreatment and enzyme hydrolysis process optimize the potential of water hyacinth. L. helveticus which is known for L(+)-lactic acid could grow and produce lactic acid from pretreated water hyacinth. Comparing with traditional batch culture, the continuous fermentation strategy has higher productivity and also improves the lactic acid production. Cells were grown at 370 C and pH 5.5. Lactic acid production and glucose concentration were measured by HPLC. Much higher productivity and yield were obtained from continuous culture than batch culture. These are comparable to the lactic acid produced in complex medium such as MRS medium. Feeding strategy can be an alternative to improve productivity in lactic acid fermentation. This study informed that water hyacinth medium could be alternative medium which can replace the complex and expensive medium for growing Lactobacillus strains in production of lactic acid.

Living organisms can be damaged or even destroyed by ultraviolet (UV). Some organisms can survive in the extreme environment of strong light or UV ray by photo-protective process. Marine plants have been known to have UV defense mechanisms such as photo reactivating and synthetic sunscreen chemicals. UV protection efficacies were studied in two ways, UV absorption and recovery after UV damage, with marine plants such as Undaria pinnatifida, Laminaria japonica and Porphyra tenera. For UV absorption studies, SPF analyzer was used to measure the UV absorbance of each sample. Samples were prepared in the cream formulation containing extract of each marine plant. For the recovery after UV damage, DPPH scavenging activity was measured. Then, extracteds from marine plants were tested on the human fibroblast damaged by UV to study possible cell revitalizing effects. 2-D PAGE was used to find the proteins related to UV damage and recovery of human fibroblast treated with marine plants extracts.

Galactosemia, one of the major newborn metabolic disorders, is caused by genetic enzyme deficiencies of galactose metabolism leading to the accumulation of galactose and galactose 1-phosphate in blood.1 Therefore, the diagnosis of galactosemia is generally achieved by measuring galactose or galactose-1-phosphate concentration using clinical dried blood spot specimens which adsorb a trace amount of blood. Up to now, several screening methods have been available such as biological inhibition assays developed by Guthrie and Paigen, high performance liquid chromatography and enzymatic colorimetric assay to determine galactose concentration.2,-4 However, these methods often require labor-intensive and time-consuming procedures or complicated and expensive instrumentation. To overcome these limitations, herein, we report a new galactose assay by utilizing two bioluminescent Escherichia coli immobilized within agarose gel arrayed on a well plate. galT knockout strain (galT-) was genetically engineered to inhibit the cell growth in the presence of galactose in a sample. A relative luminescence unit (RLU) value corresponding to the galT- cell growth induced by glucose was subtracted from the RLU value corresponding to the normal cell growth induced by both glucose and galactose. From the differences of RLU values, we successfully quantified galactose extracted from the clinical blood spots of newborn babies. Therefore, we suggest that our method is a powerful approach for convenient, economical and time-saving diagnosis of galactosemia in newborn screening.

Vibrio harveyi is a pathogenic bacterium, causing vibriosis, a common disease, faced in marine aquaculture systems. The aim of this study is to control the Vibrio in the water environment biologically, to facilitate the disease free propagation of larvae of the swimming crab and shrimp. 34 strains were isolated from many Jeotgals (salted fish) which have been generally recognized as safe (GRAS) strain of Korean fermented food and 18 strains from the gut of Portunus trituberculatus. To evaluate the inhibitory mechanism, we have used the CFU (Colony-forming unit) counting and CFSs (Cell-free supernatants) application, methodology. One isolated bacteria showed the production of anti Vibrio harveyi compound while others have not. Inhibition of Vibrio harveyi was confirmed during co-culture experimentation, by CFU (Colony-forming unit) counting method. We anticipate that it is possible that some bacterial strains will be effective for use as biological control agents in marine aquaculture systems.

In recent years, several carbon dioxide capture and storage technologies are being developed. One of the most promising biological carbon dioxide sequestration methods is to use carbonic anhydrase (CA). CA is a zinc-containing metalloenzyme catalyzing the reversible hydration of carbon dioxide (CO2 + H2O→HCO3 -). CA plays an important role for sequestrating carbon dioxide since uncatalyzed interconversion between carbon dioxide and bicarbonate is slow. In the present work, we expressed recombinant CA originated from Neisseria gonorrhoeae in Escherichia coli. We purified recombinant CA with 28 kDa molecular weight including periplasmic signal sequence. The purified recombinant CA showed high biological activity. In addition, calcium carbonate precipitation was successfully observed in carbon dioxide-saturated water solution by addition of calcium chloride. We found that kinetics for forming crystal morphology was much faster than the case without enzyme. Collectively, we could confirm that the possibility of carbon dioxide sequestration using CA and successful conversion of carbon dioxide into calcium carbonate.

Haematococcus pluvialis is a genus of green alga. It is known to be a microalgae to accumulate astaxanthin. They are unicellular flagellates. It is known that Haematococcus pluvialis has phototaxis in light. However, it is difficult to observation phototaxis in Haematococcus pluvialis in real time in bulk size. Microfluidics is able to observe movement of single cell which is easy to detect phototaxis in real time. The temperature was controlled at 20-25 and wavelength of light was 450-500nm which is to observe positive phototaxis, 650-700nm which is to observe negative phototaxis. In near future we will continue our work to detection of chemotaxis in real time using microfluidics.

In the bioindustry, it has been large category in that raw materials convert into value-added products by fermentation process. The efficient strain improvement is necessary to provide cost-competitiveness in the biotechnological production of commodity chemical. Although many techniques are available for generating improved strain in library, there is still limiting step, separating improved strain during combinatorial approach. To improve strain as high lysine producer, an artificial enrichment system has been constructed for efficient strain separation in combinatorial approach. The artificial enrichment system is composed of lysine riboswitch and dual selectable selection marker. The riboswitch responding lysine concentration regulated downstream gene expression. The operation of riboswitch in the enrichment tool was detected with fluorescence change by sgfp, reporter gene, expression level change. After that, the downstream gene has been replaced with TetA, dual selection marker. The expression level change caused of lysine concentration change was detected by specific growth rate change. Therefore, with appropriate selection pressure high lysine producer was enriched. This artificial enrichments system can be applied to further combinatorial approach.

Rapamycin, macrocyclic polyketide produced by Streptomyces hygroscopicus, is one of the potent immunosuppressants. In this study, classical mutation, genome shuffling and gene manipulation have been carried out to improve the rapamycin production. In addition, small scale culture and bioassay condition were developed for enhancing the screening efficiency of rapamycin hyper-producing mutants. A mutant Ra-II-111-01, which has 100% higher productivity than that of the parent strain Ra-I-88-3, was obtained by UV mutation under malonic acid and propionate as selective agent. When an additional copy of rapH gene, one of the positive regulatory gene of rapamycin biosynthesis, was inserted in Ra-I-88-3 strain, rapamycin production was increased by 17% on average. The best condition of protoplast formation for genome shuffling was determined by optimizing the culture media composition and lysozyme treatment condition. By optimizing the smallscale culture condition, it was observed that rapamycin productivity in 24 and 96 square deep well plate cultivation was 100% and 40% of that in flask scale, respectively.

To breed optimal yeast strain for bioethanol production, we planed a strategy for breeding yeast harboring various industrially useful characters, such as ethanol tolerance, thermotolerance and β-1,3-glucanase activity. In this study, a BY4742Δexg1/pAInu-exgA strain which has extracellular β -1,3-glucanase activity from Aspergillus oryzae and YKY020 strain which has phenotype of ethanol tolerance and thermothlerance, were fused by yeast protoplast fusion. By cell fusion, 12 fusion cells were selected and six cells (No. 3, 9, 10, 11, 12 and 13) among 12 cells grew well compared with YKY020 and BY4742Δexg1/pAInu-exgA strains in thermotolerance test at 40℃. Subsequently, in ethanol tolerance (7% ethanol concentration) and β-1,3-glucanase activity tests, the fusion cell No. 11 showed enhanced characters compared with YKY020 and BY4742Δexg1/pAInu-exgA strain. It was proved that new yeast strain is easily breeded by fusion among yeasts having different characters and same mating type. This fusion cell called BYK-F11 strain, and this strain will be further improved by several times protoplast fusion to use bioethanol production or industrial process.

Bacterial surface display finds its important biotechnological application in the fields of screening tools of evolved enzyme, bioremediation, whole cell bioconversion and tool for live vaccine production [1]. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on Bacillus subtilis spore was developed. Among 11 spore coat proteins examined, cotE and cotG were selected. Using this motif, beta-galactosidase, which is active only in tetrameric form, was functionally displayed on the surface of Bacillus subtilis spore. The surface localization of beta-galactosidase was verified by enzymatic assay of purified spore, protease accessibility test and FACS analysis of spore expressing beta-galactosidase. While Bacillus subtilis spore wall integrity, examined by lysozyme and heat treatments, was slightly affected by the incorporation of CotE-LacZ fusion protein, it was not affected by the incorporation of CotG-lacZ fusion. Other enzymes such as streptavidin, lipase (lipB) of Bacillus subtilis and GFPUV were also successfully displayed using cotE and cotG motifs, and its possible application in various biotechnological fields will be discussed [2, 3]Bacterial surface display finds its important biotechnological application in the fields of screening tools of evolved enzyme, bioremediation, whole cell bioconversion and tool for live vaccine production [1]. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on Bacillus subtilis spore was developed. Among 11 spore coat proteins examined, cotE and cotG were selected. Using this motif, beta-galactosidase, which is active only in tetrameric form, was functionally displayed on the surface of Bacillus subtilis spore. The surface localization of beta-galactosidase was verified by enzymatic assay of purified spore, protease accessibility test and FACS analysis of spore expressing beta-galactosidase. While Bacillus subtilis spore wall integrity, examined by lysozyme and heat treatments, was slightly affected by the incorporation of CotE-LacZ fusion protein, it was not affected by the incorporation of CotG-lacZ fusion. Other enzymes such as streptavidin, lipase (lipB) of Bacillus subtilis and GFPUV were also successfully displayed using cotE and cotG motifs, and its possible application in various biotechnological fields will be discussed [2, 3].

To produce a novel bacteriocin against fish pathogenic bacteria, Lactococcus lactis subsp. was isolated from flounders collected from aquaculture farms. The antimicrobial activity and some physico-chemical properties of the purified bacteriocin were studied. The bacteriocin was purified by 15% PEG 4000/20% Na2SO4 and ethanol saturation followed by twice C-18 reverse phase HPLC. The molecular weight of the bacteriocin measured by MALDI-TOF was 3.3 kDa. We found that the bacteriocin inhibited the growth of gram-positive bacteria including fish pathogenic bacteria, Streptococcus sp. and Lactococcus garviae, but not the growth of gram-negative bacteria. Especially, S. pleomorphus indicator strain (400 arbitrary units ml-1), which is a harmful pathogenic bacteria to aquaculture fish, was shown to be most sensitive . The bacteriocin was found to be stable at 100°C for 20 min and in pH ranged from 3.0-4.0, but it was unstable at pH 10.0. The results indicates that antimicrobial agent produced by L. lactis subsp. may be a good candidate instead of antibiotics to apply in aquaculture and seafood industries.

Quorum sensing (QS) is a cell density-dependent signaling system used by bacteria to coordinate gene expression within their population. In the QS mechanism of many gram-negative bacteria, acyl homoserine lactones (AHLs) are known to be the triggering molecules which form a complex with a transcriptional activator protein and promote the binding of the complex to DNA regulatory site activating transcription of many virulence genes. In this study, we describe the development and characterization of in vivo cell-based bioassay systems for detecting QS inhibitors based on three members of the LuxR family proteins, TraR, LasR and the recently identified QscR. Three different gram-negative bacteria, Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa, were used as reporter strains to over-produce one of the QS activator proteins and respond to AHLs and/or their inhibitors. The nine different in vivo assay systems (3 reporter strains × 3 QS proteins) were evaluated for their applicability and reliability by studying quantitative responses to various AHLs and furanones, the latter of which were observed as potent inhibitors against AHLs. The results indicate that, although they do not detect direct binding between QS proteins and AHLs and/or inhibiting molecules, the cell-based in vivo bioassay systems are a sensitive and reliable tool for screening of QS activators and inhibitors. This study also suggests that furanones are potentially important QS inhibitors for many LuxR-type activator proteins.