Earticle

Oil degradation agent was developed with organic sludge and modified peat moss(MPM) to recover oil contaminated soil. Waste sludge discharged from wastewater treatment plant of chemical plant in Ulsan National Industrial Park was used as organic sludge, and MPM was purchased. Organic sludge was adequate to use as growth medium for microorganism, the surface of MPM had porous structure which could enhance the cultivation condition of oil degradation microorganisms. Water contents and TPH variation with time were observed to investigate the degradation capacity of developed degradation agent. Water contents were rapidly decreased with higher content of MPM. Degradation capacity of TPH was increased with degradation time, however, the difference of MPM content did not affect to degradation capacity with time in the range of 5 to 25 percent of MPM. Therefore, it was recommended that the content of MPM for developing degradation agent was controlled to below 10% as mixing with organic sludge. For testing the recovering agent with pilot plant and field test, landfarming method was tried. Oil contaminated soil recovering agent was thought to contain more microorganisms than raw waste sludge and was no problem to come onto the market because there did no have any items of specified wastes. According to the results of TPH variation with time in the field test, it was observed the initial degradation velocity of oil with produced recovering agent was rapidly up to 50% after 4 days, remarkably. Because the microorganisms in the organic sludge discharged from chemical plant already acclimated with oil, therefore, it could be estimated initial degradation velocity of recovering agent might be rapid. It was concluded the oil contaminated soil recovering agent produced in this study have high marketability because of its two aspects on recycling of wastes and initial rapid degradation capacity.

Biodiesel is a type of biofuel obtained from bioresources and able to use in diesel vehicles as an alternative/additive to petro diesel. In recent biodiesel research, there are three main issues which include high quality biodiesel, low cost feedstock and a highly efficient biodiesel production process. The sustainable production and use of biodiesel are attracting much attention in the renewable energy field. In this paper, we review some of the literatures related to environmental and economic evaluation for biodiesel production and analysis the issues including life cycle assessment (LCA), global warming potential (GWP), energy consumption, biodiesel production cost, production technologies and feedstock.

Mussel adhesive proteins (MAPs), found in byssal adhesive plaques, have come to be recognized as very attractive biomaterials thanks to their fascinating thanks to their fascinating properties including strong and flexible adhesion in any substrates in wet environments. Especially, MAP fp-5 from the highest 3,4-dihydroxyphenyl-L-alanine (DOPA) content is mainly involved in binding, serving as a adhesive between a surface and the adhesion plaque of mussels. However, it has been very difficult to obtain purified natural MAP fp-5 in quantity. In this study, we firstly over-expressed Mytilus galloprovincialis-originated fp-5 in Escherichia coli and efficiently purified the protein. Mechanical properties of fp-5 was investigated, and multi-functional coatings were formed through simple dip-coating of objects in a fp-5 aqueous solution. From this study, we expect MAP fp-5 can be used as a significant bioadhesive and an efficient multi-functional surface coating material.

Single nucleotide polymorphisms (SNPs) are one-base DNA sequence variations that are responsible for various inherited diseases. DNA ligase-based mismatch detection methods have been proposed as useful strategies for SNP analyses. However, there is a critical problem for cytosine/thymine(C/T) SNP analyses: guanine:thymine (G:T) mismatch is not distinguished from guanine:cytosine (G:C). In this study, we employed chemically modified nucleobases at the end of a ligation fragment and analyzed whether it shows differnet ligation efficiency between G:C and G:T. Successful ligation for G:C and no ligation for G:T were observed when oxanine was employed adjacent to guanine in the ligation junction. This ligation method using an oxanine-containing fragment has strong potentials for the accurate analysis of C/T SNPs.

Green fluorescent protein (GFP) from jellyfish is a versatile reporter protein for monitoring gene expression and protein localization in variety of systems. Applications using GFP have expanded greatly due to the availability of mutants with altered spectral prosperities, Such as YFP, BGFP and CFP etc. Generally all these GFP variants were generated through replacement of naturally occurring amino acids by site directed mutation or directed evolution methodology. The main disadvantage of this methodology is laborious, time consuming and importantly limited with 20naturally occurring side chains. In other hand unnatural amino acid mutagenesis has been used to selectively substitute target amino acid with our interesting side chains to our protein of interest. Current studies have explored the incorporation of unnatural amino acids into green fluorescent protein and modify the characteristic features of the protein. We have selected different tyrosine analogues and successfully incorporated into GFP and altered the emission and excitation spectral properties. We have selected the tyrosine analogues due to the important role in the chromophore and structure formation. Current study not only demonstrated the importance of unnatural amino acid mutagenesis and also demonstrated new approach to generate tailor-made proteins with interesting and useful spectral properties.

Recent studies uncover that RNA does much more than a passive carrier of genetic information. For example, RNA shows an increasing range of cellular functionality including selective binding affinity toward target ligands and regulation of gene expression through a direct sensing of metabolites. The versatile functions of RNA in living cells have enabled the natural RNA devices to be engineered into synthetic riboswitches, permitting programmable genetic responses directly from chemical cues without the aid of protein receptors. Here we present synthetic riboregulatory systems to control prokaryotic and eukaryotic gene expressions by small molecules. The riboregulatory circuits are designed by incorporation of aptamer and ribozyme sequences in 5’-UTR or 5’-proximal coding region of a reporter gene. Sequence design of the riboswitch is easily achieved to fine-tune mRNA secondary structures and the consequent switching behaviors including target specificity and sensitivity. Owing to the good sequence designability, the riboswitch seems promising for biomedical applications to high-throughput screening systems, biosensor and in vivo bioimaging.

Recent attention has been focused on nanomaterials due to their broad potential for biological and medical applications. Nanoparticles fabricated from metals or polymers have been used to enhance the quality of biomedical images by increasing contrast of specific targets. Moreover, nanomaterials have been combined with medicines in an effort to prolong their in vivo half-lives. Most research into medical applications of nanoparticles has involved injecting them into an animal. We have found that polystyrene, metal, and nC60 nanoparticles can undergo in vivo dispersion in C. elegans through oral administration. We found that orally administered nano-size clusters pass through the pharynx and are taken up into the intestine. To explore the requirements for nanoparticle transport, we tested uptake of 50 and 100 nm particles in worms defective for genes involved in endocytosis and found that movement from the intestine to other sites was blocked in such mutants. Thus, inter-organ transport of nanoparticles from the intestine to the gonad and other tissues appears to involve endocytosis. High concentration of nanoparticles over a certain threshold point was shown to be extremely toxic in life-span of multi-cellular organisms resulting in defects in organs for digestion and reproduction. For the application of those particles as medicinal complex, we have developed a novel architecture using gold particles that provides delivery of RNAi in nematodes for gene silencing.

Prostaglandins are derived from arachidonic acid through the cyclooxygenase (COX) pathway. Two COX isoforms have been recognized. COX-1 is expressed constitutively in various tissues, including the stomach, whereas COX-2 is induced by cytokines, growth factors, tumor promoters and other agents. Prostaglandins have a short life time in vivo because they are metabolized rapidly by oxidation to 15-ketoprostaglandins catalyzed by a cytosolic enzyme named NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). This enzyme is present ubiquitously in mammalian tissues and is responsible for the biological inactivation of prostaglandins because 15-ketoprostaglandins possess significantly lower biological activities. Prostaglandins have been implicated in a wide variety of physiological and pathological processes. Prostaglandin E2 (PGE2) is a major mediator of inflammation and a key player in the control of various physiological functions. Recently, clinical studies demonstrated that PGE2 causes the growth of body hair and eyelashes in humans and animals. In humans, trials carried out on the scalp have shown that PGE2 could increase the hair density. Furthermore, 15-PGDH is also expressed in hair melanocytes. Inhibition of this enzyme as a target to reduce hair loss has been reported. PGE2 has also been identified as an important mediator of gastric ulcer healing, bone formation, and dermal wound healing. Therefore, inhibitors of 15-PGDH will be valuable for the therapeutic management of such disorders.

Due to the finite and limited supply of fossil fuel resources, availability of fossil fuels for use as an energy source will be non-existent in the future. In this regard, biofuels have recently attracted significant attention for reasons associated with energy security, diversity and sustainability. For long term applications, biomass must be utilized as the sources for fuels and chemicals. The most common concern related to this system of biofuels production is that as production capacity increases, so does competition within the agricultural industry; thus, land resources for food production would become limited as demand for biofuels increase. Biodiesel may eventually be applicable as a petro-diesel and chemical substitute. The feedstocks for biodiesel production are classified into vegetable oils, animal fats, waste oils and algae oil. Recently, non-edible oils such as Jatropa, animal fats, waste oils, yellow grease have been used in biodiesel production. Recently, biofuels and chemicals derived from marine biomass have been attracted considerable attention. In this study, I mention around the biodiesel production process from lab- to pilot-scale process.

Biodegradation of phenol in laboratory-contaminated soils by the gram positive soil bacterium Corynebacterium glutamicum have been investigated. This study showed that phenol degradation by C. glutamicum was greatly enhanced by the addition of 1 % yeast extract. From toxicity test using Daphnia magna, phenol removed soil by C. glutamicum was not revealed hazardous anymore. In addition, treatment of C. glutamicum to the phenol contaminated soils alleviated various soil amino acids compositions such as glycine, threonine, isoleucine, alanine, valine, leucine, tyrosine, and phenyl alanine. The phenomena induced increasing seed erminate rate and root elongation of Avena sativa (oat). We conclude that the phenol contaminated soil was effectively fertilized through increased soil amino acid compositions as well as the phenol in soil environment was biodegraded by C. glutamicum.

We report on a novel approach to predict the mode of genotoxic action of chemicals using a series of DNA damage specific bioluminescent bacteria. For this, a group of seven different DNA-damage sensing recombinant bioluminescent strains were employed. Each of these strains were tested against model DNA damaging agents, such as mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), nalidixic acid (Nal) and 4-nitroquinoline N-oxide (4-NQO). These biosensors were grouped based on their responses to a specific mode of genotoxic action, such as (a) DNA damage cascade response (biosensor with nrdA-, dinI- and sbmC-lux), (b) SOS response or DNA repair (strains carrying recA-, recN- and sulA-lux), and (c) DNA damage potentially by alkylation (biosensor with alkA-lux). The differential response patterns and its strength of these strains to various model genotoxicants allowed classifying the chemical’s potential genotoxic mode. Therefore, it is possible to elucidate and classify the mode of genotoxic impacts of an unknown sample and that together they may be utilized in the pre-screening steps of new drugs, newly synthesized chemicals, food and environmental contaminants.

Pathogen detection is an important issue due to the threats posed by severe communicable diseases. Here, to achieve multiple detection of 11 selected pathogens, we constructed a 16S rDNA chip. Although 7 target pathogens were specifically detected using PCR labeling method, some target species or subtypes were difficult to discriminate due to nonspecific binding. Therefore, a pattern-mapping statistical model was established using an artificial neural network algorithm. The pattern-mapping tool combined with the DNA chip resulted in successful detection of all target pathogens. In addition, to reduce the intrinsic bias and extended time and effort induced by PCR or labeling reaction, we suggested direct detection method using 16S rRNA as target. The bacterial total RNAs from cell lysate were hybridized to constructed 16S rDNA-based chip and detected using 7 fluorescent-labeled detector probes. The direct detection combined with the DNA chip resulted in more specific detection of all target pathogens than that of PCR labeling method. Collectively, our novel pattern-mapping tool and 16S rRNA direct detection combined with a 16S rDNA-based chip are a simple and effective method for detecting multiple pathogens.

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality inmost industrialized nations throughout the world. Several clinical biomarkers arecommonly used for the early detection of heart disease including the MB isoenzyme ofcreatine kinase (CK-MB), and myoglobin. But troponin I is a specific and sensitivebiomarker for acute myocardial infarction. In this study, we used polyvalent phagedisplay to isolate unique linear peptide motifs which recognize both the human and rathomologs of troponin I. Enzyme-linked immunosorbent assays (ELISAs) were used toevaluate the binding interactions. The two phagedisplayed peptides exhibited some cross-reactivity, but they were both more specific for the troponin I homolog they were selected against. Kinetic indirect phage ELISAs revealed that both troponin I binding peptides were found to have nanomolar affinities for the troponin proteins while attached to the phage particles. These new peptides may have potential utility in the development of new clinical assays for cardiac injury as well as in monitoring of cardiac cells grown in culture.

Parkinson’s disease is caused by a deficiency of the neurotransmitter dopamine. Since L-DOPA (L-3,4-dihydroxyphenylalanine) is a precursor of dopamine and can pass across the blood-brain barrier, it has been used as a treatment for Parkinson’s disease. Hundreds tons of L-DOPA are produced per year, and most of the current supply is produced by a chemical method of asymmetric synthesis. However, the chemical process for L-DOPA synthesis requires an expensive metal catalyst and shows low conversion rates and low enantioselectivity. In this study, we developed a novel technology for the production of L-DOPA, an electroenzymatic synthesis with a tyrosinase-immobilized cathode under the reduction potential of DOPAquinone, which is -530 mV. Compared to other approaches for L-DOPA synthesis reported previously, this electroenzymatic system showed the highest conversion rate and a highly enhanced productivity of up to 95.9% and 47.27 mgL-1h-1, respectively.

Therapeutic proteins can prevent or treat wide ranges of diseases from cancer and viral or bacterial infections. Production of the therapeutic proteins has been well established mainly in mammalian and bacterial cells including transgenic organisms by recombinant DNA techniques. Recent plant biotechnology and advanced molecular immunology have established plant molecular biofarming system as an alternative way to produce recombinant pharmaceutical proteins such as immunotherapeutic monoclonal antibodies (mAb). The plants as an expression system have several 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. Thus, the paradigm of plant is being shifted as a food source to so-called plant bioreactor for production of therapeutic proteins. Currently, we have successfully developed a plant expression system for production of anti-cancer and anti-virus monoclonal antibodies. 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. We are exploring the possibility of targeting proteins to the ER and possibly storing them to the protein storage vacuoles with glycosylation modification in plant cell. Production of inexpensive and safe plant-derived therapeutic proteins should be promising alternatives.

The diagnosis of infectious diseases is nowadays mostly performed by molecular methods that require multiplex detection and quantification of a wide range of pathogenic microorganisms. However, simultaneous multiplex quantitative detection of pathogens suffers from compromises between the level of multiplexing and accuracy of quantification. Here, we demonstrate the novel multiplex pathogen detection method using capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) coupled with multiplex ligation-dependent probe amplification (MLPA). MLPA-CE-SSCP is composed of four major steps which are probe hybridization, probe ligation, multiplex amplification and detection. Using ten foodborne pathogens as a model set, all the steps were carefully optimized to precisely quantify by MLPA-CE-SSCP. We could obtain the results which illustrate a strong potential in clinical diagnosis, food safety, and biosafety.

This study was extract phospholipids (PL) from squid-viscera which is uselessness on food industry. PL were successfully extracted from squid-viscera by using supercritical carbon dioxide (SC-CO2). The freeze dried squid-viscera powder was extracted with SC-CO2 at 200 bar and 45°C for removing non polar molecule. The power subjected to ethanol and acetone extraction for the enrichment of PL. The extraction yield obtained by SC-CO2 powder (6g of extract per 100g sample) can be compared with the freeze dried powder (4g of extract per 100g sample). HPLC analysis coupled with an evaporative light scattering detector (ELSD) showed selectivity in the SC-CO2 extraction of the PL components. SC-CO2 extraction showed more extraction of Phosphatidic acid (PA) than solvent extraction (PA 6% high, PI 2% high), But SC-CO2 showed less Phosphatidyl ethanolamine (PE) and Phosphatidyl choline (PC) (PE 6% low, PC 11% low).

Saccharification of Laminaria japonica (sea tangle) is an essential process for the use as a substrate for bioethanol fermentation. The seaweeds obtained from local market in Busan (South Korea) were dried in sunlight or hot-air and then ground by hammer mill to the powder with 150~750 ㎛ (wet) sizes of particles. The saccharification process by the thermal acid hydrolysis and the isolated marine bacteria treatment showed the highest yield of saccharification from Laminaria japonica. The optimal saccharification condition with the highest reducing sugar content and lowest viscosity was determined as follows: 10% (w/v) seaweed slurry, 0.2% (v/v) H2SO4, thermal hydrolysis at 121℃ for 60 min, neutralization by 5N NaOH, and then incubation at 30℃, 200 rpm for 5 days after 10% (v/v) inoculation of the isolated marine bacteria to the seaweed slurry. The reducing sugar content and viscosity under the optimal condition were 40.5 g / liter and 24.9 cp, respectively. Isolated marine bacteria was identified as Bacillus sp. by 16s ribosomalRNA sequencing. The saccharification yield of Laminaria japonica was 82.1% with the optimal saccharification conditions.

WSSV is one of the most serious pathogens affecting the shrimp aquaculture industry worldwide. At present, at least 40 WSSV structural proteins ranging from 68 to 6077 amino acid residues in size have been identified. Viral envelop proteins of WSSV play important roles on the virus infection. This study was carried out for the identification of mRNA expression level at WSSV infection stages through comparison with various structural proteins. mRNAs of shrimps were isolated from shrimp organs at various infection stages after WSSV infection. Through the comparison of mRNA transcriptional levels of infected shrimp organs, it was confirmed that the vaccination of shrimp delayed viral gene transcription for 5 to 10 days. However, the general mRNA transcriptional pattern of vaccinated shrimps showed similar trend to that of unvaccinated shrimps. This results indicated that VP28 was not suitable for the analysis of infection stage due to early and high level transcription in every sample. But, VP26 was a useful marker of infection stage owing to infection stage related RT-PCR transcriptional pattern. RT-PCR of VP26 could provide information on the shrimp mortality and virus propagation.

In this study, the waste bacterial biomasses of Corynebacterium glutamicum and Escherichia coli were used as biosorbents for biosorption of Mo from the chemical wastewater generated in SK Chemical Co. Ltd. The raw C. glutamicum was treated with citric acid (CA) and polyethyleneimine (PEI) for enhancing the binding sites on the biomass surface. The batch biosorption experiments were carried out using the raw, CA-modified (CAB) and PEI-modified (PEIB) C. glutamicum, and raw E. coli. The biosorption did not happen when the biosorbents were brought into contact with the original wastewater. However, after adjusting the pH at 4 using 2N H2SO4, the uptakes of Mo were enhanced as 45.59, 9.41, 47.18 and 44.21 mg/g for raw C. glutamicum, CAB, PEIB, raw E. coli, respectively. In addition to the pH effect, other factors affecting biosorption performance like temperature and contact time were also studied.

Nattokinase is a potent fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. The enzymes were discovered from various sources, such as Japanese natto, Korean Chungkookjang soy sauce, and Chinese douchi. Based on its food origin and relatively strong fibrinolytic activity, nattokinase has advantages over other commercially used medicine in preventative and prolonged effects, convenient oral administration, and stability in the gastrointestinal tract. In this study, Bacillus subtilis was cultivated to produce nattokinase. Glucose and NH4Cl were used as carbon and nitrogen sources with peptone supplementation. In flask culture, peptone concentration was fixed at 50 g/L. The highest cell growth (optical density monitored at 600 nm) and nattokinase activity were obtained with 10 g/L of glucose supplementation. At this condition, nattokinase activity was 688 unit/ml. In fed-batch culture of B. subtilis in fermentor, the ratio of peptone and glucose in feeding solution was varied. The highest cell concentration and nattokinase activity were 75 g/L and 15,000 unit/ml, respectively. Nattokinase activity increased with cell growth, showing growthassociated production.

Here, we report the development of novel target-specific binding proteins based on the kringle domain (KD) (~80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the 7 loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anti-cancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-α (TNFα) and efficiently neutralizes TNFα-induced cytotoxicity in vitro and in vivo. The KD scaffold with protruding 7 loops from the central core was strongly sequence-tolerant to mutations in the loop regions, offering favorable characteristics, such as the extensive randomizable sequence space and potentially multiple binding sites on the single domain for target recognition. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.

Protein A is an antibody binding protein, which specifically targets the Fc portion of antibody. Mussel adhesive proteins (MAPs) are able to form strong bonds to diverse substrates such as glass, metals, and plastics. In the present work, we constructed a novel fusion protein, BC-MAP, as an immobilizing agent by genetically fusing MAP with two domains (B and C) of protein A for effective immobilization of antibody on surface. Sole BC domain without MAP was also constructed as a comparative control. In results, quartz crystal microbalance (QCM) analyses showed that BC-MAP has an excellent antibody binding ability compared to sole BC protein. Owing to unique property of MAP, BC-MAP-based antibody immobilization does not require any surface chemical modification procedures. Thus, the proposed BC-MAP fusion protein could be a valuable linker material for efficient immobilization of diverse antibodies onto diverse surfaces.

Bombyx mori paralytic peptide (BmPP) is a multifunctional insect cytokine. BmPP consists of 23 amino acid residues including a core region that shows a structural similarity to the C-terminal β-loop domain of human epidermal growth factor (hEGF). Even though BmPP lacks the N-terminal subdomain of hEGF which is indispensable for binding EGF receptor (EGFR), BmPP showed human cell growthpromoting activity like hEGF. On the basis of this finding, a novel EGF-like growth factor was constructed by combining the N-terminal disordered region of BmPP and the C-terminal β-loop domain of hEGF. The constructed chimeric peptide consisting of 27 amino acid residues was designated "PEChimera". PEChimera increased the phosphorylated level of mitogen-activated protein kinase (MAPK) and also possessed human cell growth-promoting activity with a potency equivalent to those of hEGF and BmPP.

In our previous study, indigo was produced by recombinant E. coli harboring a flavin-containing monooxygenase gene. This gene was responsible for producing, the blue pigment, indigo. The recombinant plasmid, designated as pBlue1.7, contained a 1686bp DNA fragment of Methylophaga aminisulfidivorans MPT. Scale-up of batch, repeated batch and continuous fermentation for indigo production was performed in 5, 30, 100, and 3000L reactors containing tryptophan medium (2g tryptophan, 10g NaCl, 5g yeast extract, and 50mg ampicillin per liter) under the conditions as follows: working temperature 30℃, pH 7.0 and air supply 3vvm. The batch fermentation in a 3,000-L fermenter produced 911±22 mg L-1 of indigo from 2 g L-1 tryptophan as substrate (yield 45.5%). For continuous fermentation in a 5-L fermenter, volumetric productivity was found to be 230 mg L-1 h-1 for up to 110 h (final accumulated bio-indigo was 23 g) with a constant dilution rate (D) of 0.084 h-1 (constant feeding rate of 2.8 mL min-1 with medium containing 3 g L-1 tryptophan). Recombinant E. coli cells have the ability to withstand the high concentration of accumulated indigo toxicity in a batch fermentation. In a continuous fermentation, the recombinant cells exhibited high plasmid stability up to 110 h, after which time they lost plasmid. These two types of bio-indigo production system could be used for industrial application which could overcome the environmental problems with synthetic indigo and meet the natural indigo demands in the dye and textile industry.

Elastin-like polypeptides (ELPs) are a family of polypeptides derived from a pentapeptide Val-Pro-Gly-Xaa-Gly repeat in the amino acid sequence of mammalian elastin. ELPs undergo a thermodynamically driven phase transition, characterized by intramolecular coacervation at the inverse transition temperature (Tt). Because of their specific thermal behavior, ELPs have been applied to the various fields including protein purification (ITC), biosensors for heavy metals removal, and drug carrier scaffolds. Despite the extensive application and valuable usefulness of these materials, it has some challenges to synthesize the long ELPs in vivo. First of all, repetitive units in ELPs gene are easily eliminated in the E.coli cells. In addition, owing to slipped strand mispairing, insufficiency of tRNA for translation, causative mutation in the translation process can occur frequently. Thus the productivity of the long ELPs are much lower than those of the short ELPs in the same condition. To break through these shortcomings, we used cell-free protein synthesis system. By selective addition of specific amino acids constituting ELPs, we improved the productivity of the long ELPs. It is thought that this technique can be more effective in producing longer repetitive polypeptides or in preventing mutation caused by the insufficiency of specific amino acids.

Rapid and quantitative identification of pathogen has been considered as important criteria for its clinical and hygienical requirements. Among various methods, capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis which can separate target DNA with sequence difference has been applied for multiplex quantitative detection of pathogen. However, polymer matrix generally used for CE-based analysis has limited resolution in CE-SSCP since it is optimized for molecular weight dependent separation. In this study, multiplex quantitative detection of 12 pathogens using CE-SSCP was performed with noble high resolution polymer matrix. The targets were amplified with single set of universal PCR primers from conserved region of 16S rRNA gene, and the length of PCR products were 238-265 bp. A series of experiments using the different amounts of gDNAs of the pathogens exhibited 0.31 –1.56 pg of limit of detection and ~ 102 of dynamic range. The results showed that 12-plex quantitative analysis was achieved with high resolution and sensitivity, and simple procedure. The results also displayed the potential of new polymer matrix for various genetic analyses.

The applications of enzymes industrial processes are often hampered by their instability at high temperature. The selection and optimization of unstable residues are critical in improving the thermostability of enzymes by protein engineering. In this study, computational modeling was used to optimize unstable regions of Bacillus subtilis xylanase (Bcx) instead of random mutagenesis. The thermal fluctuations of unstable region known as important to the thermal unfolding of Bcx were investigated by the Molecular dynamics simulations at 300K and 330K to identify unstable residues. The N52 site showed the highest thermal fluctuations. Subsequently, computational design was conducted to predict the optimal sequences of unstable residues. Five optimal single mutants were predicted by the computational design, the N52Y mutation showed the thermostabilization effect. The N52 residue is conserved in Bacillus species xylanases and the structure analysis revealed that the N52Y mutation introduced more hydrophobic clusters for the thermostability, and a more favorable aromatic stacking environment for substrate binding. The quadruple mutant (F48Y/T50V/N52Y/T147L) was constructed by the introduction of the N52Y mutation into the thermostable triple (F48Y/T50V/T147L) designed in our previous study. The quadruple mutant improved thermal properties with the synergistic effects due to the N52Y mutation. In this study, we confirm that flexible residues at high temperature can be promising targets to improve the thermostability of enzymes.

There have been many attempts to produce hydrogen gas in fermentative bacteria such as E .coli, the most common biosynthetic system, as a solution for the energy crisis. Previously, we demonstrated successful biohydrogen production in recombinant E. coli expressing [NiFe]-hydrogenase 1. Although [NiFe]-hydrogenase 1 showed relatively high oxygen-tolerance compared to other hydrogenases, especially [FeFe]-hydrogenases, the hydrogen production was still unsatisfactory in terms of both the efficiency and the amount. In this work, we introduced photoreceptor into the recombinant E. coli strain so that this fermentative bacterium can exploit light energy for producing hydrogen. As the photoreceptor system, we chose proteorhodopsin and concomitant retinal synthesizing enzymes which are required for the synthesis of the retinal prosthetic group. We found that biohydrogen production efficiency was increased with the photoreceptor introduced-recombinant E. coli. We also investigated the correlation between the light intensity and the amount of hydrogen production. In conclusion, introducing photoreceptor in E. coli is a prominent strategy for enhancing biohydrogen production in light condition.

"Metabolic Engineering" aims the purposeful redesign of the biological systems and requires the accurate information of the cellular metabolic networks and proper tools for the reconstruction of the biological. Numerous regulatory elements such as promoter libraries and RBS calculator can be applied for the modulation of gene expression. However, without carefully considering the structural information of 5’-unstranslated region (5’-UTR) sequence, it is insufficient to precisely design and modulate the gene expression level. To address this issue, in this study, we randomized 5’-UTRs maintaining ribosome binding affinity using superfolder GFP as a reporting system in Escherichia coli. A mathematical model was constructed by mapping between the secondary structures of 5’-UTRs and the expression level of superfolder GFP. Examples using the other genetic contexts will show the potentials of this model to predict the precise expression level based on the structural information and consequently will provide a valuable tool for "Metabolic Engineering".