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Stress-Governed Expression and Purification of Human Type II Hexokinase in Escherichia coli

  • Jeong, Eun-Ju;Park, Kyoung-Sook;Yi, So-Yeon;Kang, Hyo-Jin;Chung, Sang-J.;Lee, Chang-Soo;Chung, Jin-Woong;Seol, Dai-Wu;Chung, Bong-Hyun;Kim, Moon-Il
    • Journal of Microbiology and Biotechnology
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    • v.17 no.4
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    • pp.638-643
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    • 2007
  • The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL2l (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E. coli proteins, and 81% of HXK $II_{6{\times}His}$ existed in inclusion bodies. To improve the production of soluble recombinant HXK II protein, in the functionally active form, we used low temperature, and the osmotic stress expression method. When expressed at $18^{\circ}C$, about 83% of HXK $II_{6{\times}His}$ existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at $37^{\circ}C$. The soluble form of HXK $II_{6{\times}His}$ was also highly produced in the presence of 1M sorbitol under the standard condition $(37^{\circ}C)$, which indicated that temperature downshift and low water potentials were required to improve the yield of active recombinant HXK II protein. The expressed protein was purified by metal chelate affinity chromatography performed in an IDA Excellose column charged with $Ni^{2+}$ ions, resulting in about 40mg recombinant HXK II protein obtained with purity over 89% from 51 of E. coli culture. The identity of HXK $II_{6{\times}His}$ was confirmed by Western blotting analysis. Taken together, using the stress-governed expression described in this study, human active HXK II can be purified in sufficient amounts for biochemical and biomedical studies.

Siderophore Production by Rhizosphere Biological Control Bacteria Brevibacillus brevis GZDF3 of Pinellia ternata and Its Antifungal Effects on Candida albicans

  • Sheng, Miaomiao;Jia, Huake;Zhang, Gongyou;Zeng, Lina;Zhang, Tingting;Long, Yaohang;Lan, Jing;Hu, Zuquan;Zeng, Zhu;Wang, Bing;Liu, Hongmei
    • Journal of Microbiology and Biotechnology
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    • v.30 no.5
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    • pp.689-699
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    • 2020
  • Brevibacillus brevis GZDF3 is a gram-positive, plant growth-promoting rhizosphere bacterium (PGPR) isolated from the rhizosphere soil of Pinellia ternata (an important herb in traditional Chinese medicine). The GZDF3 strain produces certain active compounds, such as siderophores, which are the final metabolite products of non-ribosomal peptide synthetase (NRPS) and independent non-ribosomal peptide synthetase (NIS) activity. With the present study, we attempted to investigate the siderophore production characteristics and conditions of Bacillus sp. GZDF3. The antibacterial activity of the siderophores on pathogenic fungi was also investigated. Optimal conditions for the synthesis of siderophores were determined by single factor method, using sucrose 15 g/l, asparagine 2 g/l, 32℃, and 48 h. The optimized sucrose asparagine medium significantly increased the production of siderophores, from 27.09% to 54.99%. Moreover, the effects of different kinds of metal ions on siderophore production were explored here. We found that Fe3+ and Cu2+ significantly inhibited the synthesis of siderophores. The preliminary separation and purification of siderophores by immobilized-metal affinity chromatography (IMAC) provides strong antibacterial activity against Candida albicans. The synergistic effect of siderophores and amphotericin B was also demonstrated. Our results have shown that the GZDF3 strain could produce a large amount of siderophores with strong antagonistic activity, which is helpful in the development of new biological control agents.

Fabrication and Characteristics of a Varactor Diode for UHF TV Tuner Operated within Low Tuning Voltage (저전압 UHF TV 튜너용 바렉터 다이오드의 제작 및 특성)

  • Kim, Hyun-Sik;Moon, Young-Soon;Son, Won-Ho;Choi, Sie-Young
    • Journal of Sensor Science and Technology
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    • v.23 no.3
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    • pp.185-191
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    • 2014
  • The width of depletion region in a varactor diode can be modulated by varying a reverse bias voltage. Thus, the preferred characteristics of depletion capacitance can obtained by the change in the width of depletion region so that it can select only the desirable frequencies. In this paper, the TV tuner varactor diode fabricated by hyper-abrupt profile control technique is presented. This diode can be operated within 3.3 V of driving voltage with capability of UHF band tuning. To form the hyperabrupt profile, firstly, p+ high concentration shallow junction with $0.2{\mu}m$ of junction depth and $1E+20ions/cm^3$ of surface concentration was formed using $BF_2$ implantation source. Simulation results optimized important factors such as epitaxial thickness and dose quality, diffusion time of n+ layer. To form steep hyper-abrupt profile, Formed n+ profile implanted the $PH_3$ source at Si(100) n-type epitaxial layer that has resistivity of $1.4{\Omega}cm$ and thickness of $2.4{\mu}m$ using p+ high concentration Shallow junction. Aluminum containing to 1% of Si was used as a electrode metal. Area of electrode was $30,200{\mu}m^2$. The C-V and Q-V electric characteristics were investigated by using impedance Analyzer (HP4291B). By controlling of concentration profile by n+ dosage at p+ high concentration shallow junction, the device with maximum $L_F$ at -1.5 V and 21.5~3.47 pF at 0.3~3.3 V was fabricated. We got the appropriate device in driving voltage 3.3 V having hyper-abrupt junction that profile order (m factor) is about -3/2. The deviation of capacitance by hyper-abrupt junction with C0.3 V of initial capacitance is due to the deviation of thermal process, ion implantation and diffusion. The deviation of initial capacitance at 0.3 V can be reduced by control of thermal process tolerance using RTP on wafer.

Selective Separation of $^{59/63}Ni$ from Radioactive Wastes (방사성 폐기물 내 $^{59/63}Ni$의 선택적 분리)

  • Lee Chang-Heon;Jung Kie-Chul;Choi Kwang-Soon;Jee Kwang-Yong;Kim Won-Ho
    • Proceedings of the Korean Radioactive Waste Society Conference
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    • 2005.06a
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    • pp.121-128
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    • 2005
  • A study on the selective separation of $^{99}Tc,\;^{94}Nb,\;^{55}Fe,\;^{90}Sr$ and $^{59}Ni(^{63}Ni)$ from various radioactive wastes discharged from the nuclear power plants in Korea is being performed for use in their quantifications which are indispensible for the evaluation of the radionuclide inventory. Separation behaviour of Ce, Ca, Mg, Al, Cr, Ti, Mn and Cu recovered along with Ni during the separation of Re (as a surrogate of $^{99}Tc$), Nb, Fe and Sr by anion exchange and Sr-Spec extraction chromatography was investigated by cation exchange and Ni-Spec extraction chromatography using synthetic radioactive waste dissolved solutions containing matrix elements such as Re, Nb, Fe, Sr, Ni, B, Na, K, Ce, Co, Ca, Mg, Al, Zn, Cr, Pb, Cd, Mo, Mn, Cu, Zr, Ti and U. To purify the Ni fraction recovered and prepare a radionuclide source available for gas proportional counting, an application of the Ni precipitation procedure with dimethylglyoxime in the medium of ammonium citrate and tartaric acid solutions as a masking agent for co-existing metal ions was described in detail.

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Seasonal Variations in Soil Solution Chemistry under Larix leptolepis, Pinus Koraiensis, and Quercus mongolica stands (일본잎갈나무, 잣나무, 신갈나무 임분 토양수 성질의 계절적 변이)

  • Ryu, Soung-Ryoul;Son, Yo-Whan;Joo, Yeong-Teuk;Jin, Hyun-O;Oh, Jong-Min;Jung, Duk-Young
    • Korean Journal of Environmental Agriculture
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    • v.19 no.1
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    • pp.75-79
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    • 2000
  • To investigate seasonal variations of soil solution chemistry, samples were collected from zero tension lysimeters in O, A, and B horizons of 26-year-old Larix leptolepis. Pinus koraiensis and Quercus mongolica stands in Kwangju, Kyunggi Province from September, 1996 through June. 1999. Potassium, $Mg^{2+}$, $Cl^-$, and $No_3\;^-$ concentrations increased in October and November concurrent with inputs of fresh litterfall and twigs. Sodium, $Ca^{2+}$, $K^+$, and $Mg^{2+}$ concentrations increased in March and April concurrent with the yellow sand effect. Potassium concentration showed the highest variation among ions, and $Cl^-$ concentration showed positive correlations with $K^+$ and $Mg^{2+}$ concentrations. Soil solution pH decreased while $Al^{3+}$ and $K^+$ concentrations increased during the study period. If our data reflects long term trends. then $Al^{3+}$ concentration in the O horizon will reach the toxic level (0.180 meq/l) within 10-20 years depending on species.

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Lower the Detection Limits of Accelerator Mass Spectrometry

  • John A., Eliades;Song, Jong-Han;Kim, Jun-Gon;Kim, Jae-Yeol;O, Jong-Ju;Kim, Jong-Chan
    • Proceedings of the Korean Vacuum Society Conference
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    • 2013.02a
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    • pp.243-244
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    • 2013
  • Over the past 15 years, several groups have incorporated radio-frequency quadrupole (RFQ) based instruments before the accelerator in accelerator mass spectrometry (AMS) systems for ion-gas interactions at low kinetic energy (<40 eV). Most AMS systems arebased on a tandem accelerator, which requires negative ions at injection. Typically, AMS sensitivity abundance ratios for radioactive-to-stable isotope are limited to Xr/Xs >10^-15, and the range of isotopes that can be analyzed is limited because of theneed to produce rather large negative ion beams and the presence of atomic isobaric interferences after stripping. The potential of using low-kinetic energy ion-gas interactions for isobar suppression before the accelerator has been demonstrated for several negative ion isobar systems with a prototype RFQ system incorporated into the AMS system at IsoTrace Laboratory, Canada (Ontario, Toronto). Requisite for any such RFQ system applied to very rare isotope analysis is large transmission of the analyte ion. This requires proper phase-space matching between the RFQ acceptance and the ion beam phase space (e.g. 35 keV, ${\varphi}3mm$, +-35 mrad), and the ability to control the average ion energy during interactions with the gas. A segmented RFQ instrument is currently being designed at Korea Institute for Science and Technology (한국과학기술연구원, KIST). It will consist of: a) an initial static voltage electrode deceleration region, to lower the ion energy from 35 keV down to <40 eV at injection into the first RFQ segment; b) the segmented quadrupole ion-gas interaction region; c) a static voltage electrode re-acceleration region for ion injection into a tandem accelerator. Design considerations and modeling will be discussed. This system should greatly lower the detection limits of the 6 MV AMS system currently being commissioned at KIST. As an example, current detection sensitivity of 41Ca/Ca is limited to the order of 10^-15 while the 41Ca/Ca abundance in modern samples is typically 41Ca/Ca~10^-14 - 10^-15. The major atomic isobaric interference in AMS is 41K. Proof-of-principal work at IsoTrace Lab. has demonstrated that a properly designed system can achieve a relative suppression of KF3-/41CaF3- >4 orders of magnitude while maintaining very high transmission of the 41CaF3- ion. This would lower the 41Ca detection limits of the KIST AMS system to at least 41Ca/Ca~10^-19. As Ca is found in bones and shells, this would potentially allow direct dating of valuable anthropological archives and archives relevant to our understanding of the most pronounced climate change events over the past million years that cannot be directly dated with the presently accessible isotopes.

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Crystal Growth of rutiles doped with Impurity Ions by Floating Zone Method (부유대용융법에 의한 불순이온 주입된 $TiO_2$단결정 성장 연구)

  • 이성영;유영문;김병호
    • Proceedings of the Korea Crystallographic Association Conference
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    • 1999.04a
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    • pp.1-1
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    • 1999
  • 부유대용융법에 의하여 불순이온의 종류와 각 이온의 주입 농도를 달리하는 Rutile 단결정을 성장하였다. 성장된 결정으로부터 제조한 박편시료를 이용하여 결정결함과 광투과도에 미치는 각 불순이온의 영향을 조사하였다. 결정성장용 주원료로 99.99%의 TiO2를 사용하고, 불순이온 주입을 위한 원료로서 99.99%의 Al2O3, H3BO3, Ga2O3, Sc2O3, V2O5, Fe2O3, ZrO2, Er2O3, Cr2O3를 각각 사용하였다. 불순이온의 종류에 따르는 영향을 조사하기 위하여 TiO2 99.8 atomic%-불순이온 0.2atomic%의 조성이 되도록 각 이온별로 원료를 정밀하게 평량하고 균일 혼합하였다. 불순 이온의 첨가량에 따르는 영향을 조사하기 위하여 Al2O3는 각각 pure, 0.2, 0.4, 0.6 atomic%를, Cr2O3는 pure, 0.003, 0.05, 0.2 atomic%를 각각 치환하여 원료를 조합하였다. 균일 혼합된 원료를 직경 8mm의 고무 튜브에 넣고 CIP(Cold Isostatic Press0에서 2000kg/$\textrm{cm}^2$의 압력으로 성형한 후 150$0^{\circ}C$에서 1시간 소결함으로서 결정성장용 다결정 원료를 합성하였다. 합성된 다결정을 double ellipsoidal mirror 내에 설치하고,halogen lamp로 가열하여 원료의 한쪽 끝을 용융한 다음, 20rpm의 회전속도, 3-5mm/hr의 성장속도로 하는 유속 1$\ell$/min의 O2 분위기속에서 부유대용융법에 의하여 결정을 성장하였다. 성장된 결정을 성장축에 수직한 방향으로 각각 절단, 연삭, 연마한 박편을 이용하여 편광하에서 low-angle grain boundaries 및 기타의 결정결함을 관찰하였으며, 0.3$\mu\textrm{m}$~0.8$\mu\textrm{m}$ 범위 및 0.6$\mu\textrm{m}$~3.4$\mu\textrm{m}$ 범위에서의 투과 및 흡수 스펙트럼을 측정하였다. 결정 성장 결과 B3+, Er3+, Cr3+ 이온은 Ti4+ 이온과 이온의 크기 차이가 심하여 결정의 정상적인 성장을 방해하는 물성을 나타냈고, V5+, Cr3+ 이온은 흑색의 결정, Fe3+ 이온은 적갈색의 결정으로 성장되었다. Al3+, Zr4+, Al3+의 순서로 투과도가 높아지는 것이 관찰되었다. 불순이온의 농도에 따른 영향으로서 Al3+ 이온의 경우 주입농도가 높아질수록 low angle boundary와 oxygen deficiency가 감소하였고, 투과율은 조금 감소하거나 큰 차이가 없는 것으로 나타났다. 반면에 Cr3+ 이온을 주입한 경우 0.003 atomic%에서 최적의 물성을 보였으며, 주입농도가 높아질수록 결정성장이 어려워지고 광의 투과도가 급격히 저하되었다.

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Expression of the lux Genes in Escherichia coli for the Basis of Development of Biosensor (바이오센서 개발을 위한 발광 유전자의 대장균에서의 발현 시스템 분석)

  • Cho, Mi-Mi;Kim, Young-Doo;Kang, Kyung-Sook;Kim, Sook-Kyung;Yang, In-Chul;Park, Sang-Ryoul;Lee, Chan-Yong
    • Korean Journal of Microbiology
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    • v.45 no.4
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    • pp.419-424
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    • 2009
  • To provide the basis of biosensor based on the lux genes from bioluminescent bacteria of Photobacterium leiognathi and Vibrio harveyi, we test the expression of lux genes in several strains of Escherichia coli. The expression of the recombinant plasmid of PlXba.pT7-3, containing all lux genes requiring for light emission without adding substrate, in E. coli 43R was so strong to see the blue-green light in single colony as well as in the alginate immobilized cell. In addition, the light intensity was decreased by adding heavy metal ion such as cadmium and zinc ions. These result raise the possibility that a biosensor can be developed using the lux genes system.

A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens

  • Lee, Su-Jin;Lee, Youngdeuk;Park, Gun-Hoo;Umasuthan, Navaneethaiyer;Heo, Soo-Jin;Zoysa, Mahanama De;Jung, Won-Kyo;Lee, Dae-Won;Kim, Hanjun;Kang, Do-Hyung;Oh, Chulhong
    • Journal of Microbiology and Biotechnology
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    • v.26 no.6
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    • pp.1115-1123
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    • 2016
  • L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the L-asparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40℃, pH 9. Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-L-ASPG86 was 687.1 units/mg under optimum conditions (37℃, pH 9, and 5 mM MnSO4).

Removal of Cobalt Ion by adsorbing Colloidal Flotation (흡착 교질 포말부선법에 의한 Cobalt Ion의 제거)

  • 정인하;이정원
    • Resources Recycling
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    • v.7 no.3
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    • pp.3-10
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    • 1998
  • Simulated waste liquid containing 50 ppm cobalt ion was treated by adsorbing colloidal flotation using Fe(III) or Al(IlI) as flocclant and a sodium lamyl sulfate as a collector. Parameters such as pH, surfactant concentration, Fe(III) or Al(III) concentration, gas flow rate, etc., W앙e considered. The flotation with Fe(III) showed 99.8% removal efficiency of cohalt on the conditions of initial cobalt ion concentration 50 ppm, pH 9.5, gas flow rate 70 ml/min, and flotation time 30 min. When the waste solution, was treated with 35% $H_2O_2$ prior to adsorbing colloidal flotation, the optimal pH for removing cobalt shifted m to weak alkaline range and flotation could be applied in wider range of pH as compared to non-use of $H_2O_2$. Additional use of 20 ppm Al(III) after precipitation of 50 ppm Co(II) with 50 ppm Fe(III) made the optimal pH range for preferable flotation w wider. Foreign ions such as, $NO_3^-$, $SO_4^{2-}$, $Na^+$, $Ca^{2+}$ were adopted and their effects were observed. Of which sulfate ion was f found to be detrimental to removal of cob퍼t ion by flotation. Coprecipitation of Co ion with Fe(III) and Al(III) resulted in b better removal efficiency of cobalt IOn 피 the presence of sulfate ion.

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