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Effects of N-/C-Terminal Extra Tags on the Optimal Reaction Conditions, Activity, and Quaternary Structure of Bacillus thuringiensis Glucose 1-Dehydrogenase

  • Hyun, Jeongwoo;Abigail, Maria;Choo, Jin Woo;Ryu, Jin;Kim, Hyung Kwoun
    • Journal of Microbiology and Biotechnology
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    • v.26 no.10
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    • pp.1708-1716
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    • 2016
  • Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned from Bacillus thuringiensis subsp. kurstaki, and wild-type (GDH-BTWT) and His-tagged (GDH-BTN-His, GDH-BTC-His) enzymes were produced in Escherichia coli BL21 (DE3). All enzymes were produced in the soluble forms from E. coli. GDH-BTWT and GDH-BTN-His showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BTC-His showed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BTWT was stable up to 65℃, whereas GDH-BTN-His and GDH-BTC-His were stable up to 45℃. Gel permeation chromatography showed that GDH-BTWT is a dimer, whereas GDH-BTN-His and GDH-BTC-His are monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BTWT single subunit was constructed based on the crystal structure of Bacillus megaterium GDH (PDB ID 3AY6), showing that GDH-BTWT has a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BTWT and GDH-BTN-His regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.

The Effect of Crystallization by Heat Treatment on Electromagnetic Interference Shielding Efficiency of Carbon Fibers (열처리 온도에 의한 구조 결정성이 탄소섬유의 전자파 차폐 성능에 미치는 영향)

  • Kim, Jong Gu;Chung, Choul Ho;Lee, Young-Seak
    • Applied Chemistry for Engineering
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    • v.22 no.2
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    • pp.138-143
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    • 2011
  • To investigate the electromagnetic interference shielding efficiency (EMI SE) property based on heat treatment effects of carbon fibers in various temperatures, the polyacrilonitrle-based carbon fibers were prepared by electrospinning method and treated at 1073, 1323, 1873 and 2573 K. The surface morphology of carbon fibers was investigated by using FE-SEM and the carbon crystallization was studied by Raman spectroscopy based on effects of reaction temperatures. The electrical conductivity was obtained by measuring the surface resistance with four probe method on carbon crystallization. The permittivity, permeability and EMI SE were investigated by using S-parameter in the range of 800~4500 MHz. In case of carbon fibers treated at 2573 K, the improved carbon crystallization was confirmed by Raman spectrum and the enhanced electrical conductivity showing 54.7 S/cm was also observed. The permittivity was dramatically improved by factor of 4 based on effect of high reaction temperature. Eventually, the highly improved EMI SE value was obtained showing around 41.7 dB.

Production of Monoclonal Antibody against Escherichia coli O157:H7 and Development of Enzyme Linked Immunosorbent Assay (Escherichia coli O157:H7의 단크론성 항체 생산과 효소면역분석법의 개발)

  • Ryu, Hee-Jeong;Kim, Jeong-Sook;Kim, Kyeong-Yeol;Nam, Bo-Ram;Nam, Min-Ji;Shim, Won-Bo;Kim, Nam-Soo;Cho, Yong-Jin;Chung, Duck-Hwa
    • Korean Journal of Food Science and Technology
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    • v.42 no.3
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    • pp.329-334
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    • 2010
  • Escherichia coli O157:H7 causes hemolytic uremic syndrome and hemorrhagic colitis in humans. The objectives of this study were to produce monoclonal antibody(MAb) against E. coli O157:H7 and to develop an enzyme linked immunosorbent assay(ELISA) for the rapid detection of E. coli O157:H7 in agri-stockbreeding. The characterization of MAb produced from hybridoma cell (HKEC 4G8-5) was validated by ELISA and Western blot. The produced MAb was specific to E. coli O157:H7 and showed weak cross-reaction to Staphylococcus aureus. The detection limit of ELISA based on 4G8-5 MAb was $10^5\;cell/mL$. Although the ELISA could not detect E. coli O157:H7 in the meat and sprout samples inoculated with $1{\times}10^1\;cell$/10 g without enrichment, the same samples after enrichment for 6 hr were confirmed to be positive by ELISA. These results indicated that the ELISA combined with short enrichment (6 hr) is useful tool for rapid screening of E. coli O157:H7 in various samples.

Honeycomb-type Single Chamber SOFC Running on Methane-Air Mixture (Methane-Air 혼합 Gas에서 구동하는 하니컴 형태의 SC-SOFC)

  • Park Byung-Tak;Yoon Sung Pil;Kim Hyun Jae;Nam Suk Woo;Han Jonghee;Lim Tae-Hoon;Hong Seong-Ahn;Lee Dokyol
    • 한국신재생에너지학회:학술대회논문집
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    • 2005.06a
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    • pp.306-309
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    • 2005
  • One of the most critical issues in sol id oxide fuel cell (SOFC)running on hydrocarbon fuels is the risk of carbon formation from the fuel gas. The simple method to reduce the risk of carbon formation from the reactions is to add steam to the fuel stream, leading to the carbon gasification react ion. However, the addition of steam to fuel is not appropriate for the auxiliary power unit (APU) and potable power generation (PPG) systems due to an increase of complexity and bulkiness. In this regard, many researchers have focused on so-called 'direct methane' operation of SOFC, which works with dry methane without coking. However, coking can be suppressed only by the operation with a high current density, which may be a drawback especially for the APU and PPG systems. The single chamber fuel cell (SC-SOFC) is a novel simplification of the conventional SOFC into which a premixed fuel/air mixture is introduced. It relies on the selectivity of the anode and cathode catalysts to generate a chemical potential gradient across the cell. Moreover it allows compact and seal-free stack design. In this study, we fabricated honeycomb type mixed-gas fuel cell (MGFC) which has advantages of stacking to the axial direction and increasing volume power density. Honeycomb-structured SOFC with four channels was prepared by dry pressing method. Two alternative channels were coated with electrolyte and cathode slurry in order to make cathodic reaction sites. We will discuss that the anode supported honeycomb type cell running on mixed gas condition.

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Effect of L-Glutamic Acid and Paraben Solution on the Endothelial Cell Proliferation in the Glutaraldehyde- Fixed Bovine Pericardium (글루타르알데하이드 고정 소심 낭막에서의 내피세포 증식에 대한 글루탕산 및 파라벤용액의 효과)

  • Kim, Beom-Sik;Lee, Mun-Hwan;Yu, Se-Yeong;Kim, Won-Gon
    • Journal of Chest Surgery
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    • v.29 no.1
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    • pp.7-13
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    • 1996
  • The conventional glutaraldehyde (GA) fixation method of tissue valves is considered to be responsible for accelerated valve degeneration. The release of toxic GA from the valve tissue is believed to limit endothelial cell (EC) ingrowth. Removal of toxic GA by reaction with L-glutamic acid and storage in a Paraben solution may offer good EC growth. To investigate the conditions for endothelialization of tissue valves, the growth properties of ECs on the conventionally and alternatively treated pericardial tissue were compared. Conventional preparation included zero-pressure fixation for 72 hours in phosphated-buffered saline (PBS) solution containing 0.5% GA at 4$^{\circ}C$ and storage into PBS containing 0.2% GA(group I). Alternatively treated pericardial tissues were divided into three postfixation treatment groups : (1) storage in PBS solution containing Paraben(group II), (2) treatment with PBS containing 8$^{\circ}C$ L-glutamic acid(PH 7.35) and storage in PBS solution containing Paraben (g oup III), (3) treatment with L-glutamic acid dissolved in distilled water (PH 3.5) (group IV). Pericardial tissue were transferred into the 24-well plate after storage for 4 weeks. ECs were harvested enzymatically from the bovine pulmonary artery and grown to confluence on culture flask surfaces. Detached ECs by trypsin were incubated into the each well of the 24-well plate including test pericardial tissues. Cells were detached by trypsin, 1, 2, 3, 5, 7 days after incubation and counted on the hemacytometer. Cell viability test was performed by frypan-blue exclusion method. Acute cell death in the group I were found even after prolonged washing. The group II showed prolonged cell survival compared with the group I. Both group III and group IV showed better cell growth than group II. There was no statistically significant difference between group III and group IV method in terms of EC growth. This results suggest that treatment by L-glutamic ac id and storage in a Paraben solution be a promising approach for improvement of durability of GA-treated tissue valves.

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A Study on the Synthesis of Potassium Hexatitanate Whisker by the Slow Cooling Calcination Process (서냉 소성법에 의한 육티탄산칼륨 Whisker의 합성에 관한 연구)

  • Lee, Chul-Tae;Choi, Ung-Su;Kim, Young-Myoung
    • Applied Chemistry for Engineering
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    • v.5 no.1
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    • pp.160-175
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    • 1994
  • Fibrous potassium hexatitanate whisker with the size of $ID=0.5{\sim}1{\mu}m$ and length=$100{\sim}1000{\mu}m$ (aspect ratio=100~1000) was produced through the reaction between titanium dioxide and potassium carbonate using the slow-cooling calcination followed by water leaching treatment. The optimum condition for the production of fibrous potassium titanate was calcination temperature of $1100^{\circ}C$ for 5hrs, $TiO_2$ mole ratlo to $K_2CO_3$ of 4.5 and slow-cooling rate of $0.5^{\circ}C/min$ to $860^{\circ}C$. Fibrous crystal are grown by the association between the solid potassium titanate and liquid phase during the slow-cooling process. The Proper water leaching condition for removing of K component was leaching time of 10hrs in boiling water. Pressurizing of the mixture of $K_2CO_3$ and $TiO_2$ to be calcinated became effective on the growth of fibrous crystal.

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Control of Graphene's Electrical Properties by Chemical Doping Methods

  • Lee, Seung-Hwan;Choi, Min-Sup;La, Chang-Ho;Yoo, Won-Jong
    • Proceedings of the Korean Vacuum Society Conference
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    • 2011.08a
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    • pp.119-119
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    • 2011
  • This study examined the synthesis of large area graphene and the change of its characteristics depending on the ratio of CH4/H2 by using the thermal CVD methods and performed the experiments to control the electron-hole conduction and Dirac-point of graphene by using chemical doping methods. Firstly, with regard to the characteristics of the large area graphene depending on the ratio of CH4/H2, hydrophobic characteristics of the graphene changed to hydrophilic characteristics as the ratio of CH4/H2 reduces. The angle of contact also increased to 78$^{\circ}$ from 58$^{\circ}$. According to the results of Raman spectroscopy showing the degree of defect, the ratio of I(D)/I(G) increases to 0.42% from 0.25% and the surface resistance also increased to 950 ${\Omega}$ from 750 ${\Omega}$/sq. As for the graphene synthesis at the high temperature of 1,000$^{\circ}$ by using CH4/H2 in a Cu-Foil, the possibility of graphene formation was determined as a function of the ratio of H2 included in the fixed quantity of CH4 as per specifications of every equipment. It was observed that the excessive amount of H2 prevented graphene from forming, as extra H-atoms and molecules activated the reaction to C-bond of graphene. Secondly, in the experiment for the electron-hole conduction and the Dirac-point of graphene using the chemical doping method, the shift of Dirac-point and the change in the electron-hole conduction were observed for both the N-type (PEI) and the P-type (Diazonium) dopings. The ID-VG results show that, for the N-type (PEI) doped graphene, Dirac-point shifted to the left (-voltage direction) by 90V at an hour and by 130 V at 2 hours respectively, compared to the pristine graphene. Carrier mobility was also reduced by 1,600 cm2/Vs (1 hour) and 1,100 cm2/Vs (2 hours), compared to the maximum hole mobility of the pristine graphene.

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Evaluation of 99mTc-MAG3-2-nitroimidazole for hypoxic tumor imaging

  • Lee, Yun-Sang;Kim, Young Joo;Jeong, Jae Min
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.5 no.1
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    • pp.18-25
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    • 2019
  • 2-Nitroimidazole derivatives have been reported to accumulate in hypoxic tissue. We prepared a novel $^{99m}Tc-MAG_3$-2-nitroimidazole and evaluated the feasibility for hypoxia imaging agent. $Bz-MAG_3$-2-nitroimidazole was synthesized by direct coupling of $Bz-MAG_3$ and 2-nitroimidazole using dicyclohexylcarbodiimide. $Bz-MAG_3$-2-nitroimidazole was labeled with $^{99m}Tc$ in the presence of tartaric acid and $SnCl_2-2H_2O$ at $100^{\circ}C$ for 30 min. And the reaction mixture was purified by $C_{18}$ Sep-pak cartridge. The labeling efficiency and the radiochemical purity were checked by ITLC-SG/acetonitrile. The tumor was grown in balb/c mice for 8~13 days after the subcutaneous injection of tumor cells, CT-26 (murine colon adenocarcinoma cell). Biodistribution study and tumor autoradiography were performed in the xenografted mice after i.v injection of 74 kBq/0.1 mL and 19 MBq/0.1 mL of $^{99m}Tc-MAG_3$-2-nitroimidazole, respectively. In vivo images of $^{99m}Tc-MAG_3$-2-nitroimidazole in tumor bearing mice were obtained 1.5 hr post injection. The labeling efficiency was $45{\pm}20%$ and the radiochemical purity after purification was over 95%. Paper electrophoresis confirmed negative charge of $^{99m}Tc-MAG_3$-2-nitroimidazole. $^{99m}Tc-MAG_3$-2-nitroimidazole was very stable at room temperature and its protein binding was 53%. The $^{99m}Tc-MAG_3$-2-nitroimidazole exhibited high uptake in the liver, stomach and intestine. In biodistribution study using tumor bearing mice, the uptakes (% ID/g) of the tumor were $0.5{\pm}0.1$, $0.4{\pm}0.0$, $0.2{\pm}0.1$ and $0.1{\pm}0.1$ at 5, 15, 30 min and 4 hrs. Tumor/muscle ratio were $1.4{\pm}0.1$, $2.2{\pm}0.83$, $3.0{\pm}0.9$, and 3.7 (n=2) for 5, 15, 30 min and 4 hrs. The uptake in hypoxic area was found higher than in non-hypoxic area of tumor tissue by autoradiography. In vivo images showed the relatively faint uptake to the hypoxic tumor region. $^{99m}Tc-MAG_3$-2-nitroimidazole was successfully synthesized and found feasible for imaging hypoxia.

The Individual Discrimination Location Tracking Technology for Multimodal Interaction at the Exhibition (전시 공간에서 다중 인터랙션을 위한 개인식별 위치 측위 기술 연구)

  • Jung, Hyun-Chul;Kim, Nam-Jin;Choi, Lee-Kwon
    • Journal of Intelligence and Information Systems
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    • v.18 no.2
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    • pp.19-28
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    • 2012
  • After the internet era, we are moving to the ubiquitous society. Nowadays the people are interested in the multimodal interaction technology, which enables audience to naturally interact with the computing environment at the exhibitions such as gallery, museum, and park. Also, there are other attempts to provide additional service based on the location information of the audience, or to improve and deploy interaction between subjects and audience by analyzing the using pattern of the people. In order to provide multimodal interaction service to the audience at the exhibition, it is important to distinguish the individuals and trace their location and route. For the location tracking on the outside, GPS is widely used nowadays. GPS is able to get the real time location of the subjects moving fast, so this is one of the important technologies in the field requiring location tracking service. However, as GPS uses the location tracking method using satellites, the service cannot be used on the inside, because it cannot catch the satellite signal. For this reason, the studies about inside location tracking are going on using very short range communication service such as ZigBee, UWB, RFID, as well as using mobile communication network and wireless lan service. However these technologies have shortcomings in that the audience needs to use additional sensor device and it becomes difficult and expensive as the density of the target area gets higher. In addition, the usual exhibition environment has many obstacles for the network, which makes the performance of the system to fall. Above all these things, the biggest problem is that the interaction method using the devices based on the old technologies cannot provide natural service to the users. Plus the system uses sensor recognition method, so multiple users should equip the devices. Therefore, there is the limitation in the number of the users that can use the system simultaneously. In order to make up for these shortcomings, in this study we suggest a technology that gets the exact location information of the users through the location mapping technology using Wi-Fi and 3d camera of the smartphones. We applied the signal amplitude of access point using wireless lan, to develop inside location tracking system with lower price. AP is cheaper than other devices used in other tracking techniques, and by installing the software to the user's mobile device it can be directly used as the tracking system device. We used the Microsoft Kinect sensor for the 3D Camera. Kinect is equippedwith the function discriminating the depth and human information inside the shooting area. Therefore it is appropriate to extract user's body, vector, and acceleration information with low price. We confirm the location of the audience using the cell ID obtained from the Wi-Fi signal. By using smartphones as the basic device for the location service, we solve the problems of additional tagging device and provide environment that multiple users can get the interaction service simultaneously. 3d cameras located at each cell areas get the exact location and status information of the users. The 3d cameras are connected to the Camera Client, calculate the mapping information aligned to each cells, get the exact information of the users, and get the status and pattern information of the audience. The location mapping technique of Camera Client decreases the error rate that occurs on the inside location service, increases accuracy of individual discrimination in the area through the individual discrimination based on body information, and establishes the foundation of the multimodal interaction technology at the exhibition. Calculated data and information enables the users to get the appropriate interaction service through the main server.