• Biomedical Science Letters
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• v.11 no.3
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• pp.327-332
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• 2005

Sf21 cells become popular as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike human glucose transporters, very little is known about the characteristics of the endogenoussugar transporter(s) in Sf21 cells. Thus, some kinetic properties of the sugar transport system were investigated, involving the uptake of 2-deoxy-D-glucose (2dG1c). In order to obtain a true measure of the initial rate of uptake, the uptake of $[^3H]2dGlc$ from both low $(100{\mu}M)$ and high (10 mM) extracellular concentrations was measured over periods ranging from 30 sec to30 min. The data obtained indicated that the uptake was linear for at least 2 min at both concentrations, suggesting that measurements made over a 1min time course would reflect initial rates of the jexpse uptake. To determine $K_m\;and\;V_{max}$ of the endogenous glucose transporter(s) in Sf21 cells, the uptake of 2dG1c was measured over a range of substrate concentrations $(50{\mu}M\~10mM)$ 2dG1c uptake by the Sf21 cells appeared to involve both saturable and non-saturable (or very low affinity) components. A saturable transport system for 2dG1c was relatively high, the $K_m$ value for uptake being < 0.45 mM. The $V_{max}$ value obtained for 2dG1c transport in the Sf21 cells was about 9.7-folds higher than that reported for Chinese hamster ovary cells, which contain a GLUT1 homologue. Thus, it appeared that the transport activity of the Sf21 cells was very high. In addition, the Sf21 glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter

• Proceedings of the Korean Vacuum Society Conference
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• 2012.08a
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• pp.105-105
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• 2012

We have developed a photoemission-assisted plasma-enhanced chemical vapor deposition (PAPE-CVD) [1,2], in which photoelectrons emitting from the substrate surface irradiated with UV light ($h{\nu}$=7.2 eV) from a Xe excimer lamp are utilized as a trigger for generating DC discharge plasma as depicted in Fig. 1. As a result, photoemission-assisted plasma can appear just above the substrate surface with a limited interval between the substrate and the electrode (~10 mm), enabling us to suppress effectively the unintended deposition of soot on the chamber walls, to increase the deposition rate, and to decrease drastically the electric power consumption. In case of the deposition of DLC gate insulator films for the top-gate graphene channel FET, plasma discharge power is reduced down to as low as 0.01W, giving rise to decrease significantly the plasma-induced damage on the graphene channel [3]. In addition, DLC thickness can be precisely controlled in an atomic scale and dielectric constant is also changed from low ${\kappa}$ for the passivation layer to high ${\kappa}$ for the gate insulator. On the other hand, negative electron affinity (NEA) of a hydrogen-terminated diamond surface is attractive and of practical importance for PAPECVD, because the diamond surface under PAPE-CVD with H2-diluted (about 1%) CH4 gas is exposed to a lot of hydrogen radicals and therefore can perform as a high-efficiency electron emitter due to NEA. In fact, we observed a large change of discharge current between with and without hydrogen termination. It is noted that photoelectrons are emitted from the SiO2 (350 nm)/Si interface with 7.2-eV UV light, making it possible to grow few-layer graphene on the thick SiO2 surface with no transition layer of amorphous carbon by means of PAPE-CVD without any metal catalyst.

• BMB Reports
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• v.31 no.2
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• pp.149-155
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• 1998

The N199H variant of the EcoRI endonuclease has about twice the catalytic activity of the wild-type. A comparison of their biochemical characteristics, using synthetic oligonucleotides 5'-dAAAACTTAAGAAAAAAAAAAA-3' (KA) and 5'-dTTTTTGAATTCTTTTTTTTTT-3' (KT), helps to define the cleavage reaction pathway of these enzymes. Both EcoRI and EcoRI variant N199H were found to cleave singlestranded KA or KT about three times faster than the double-stranded forms, although the KT oligonucleotide was more susceptible. Using the ssDNA substrate in kinetic analyses, lower $K_m$ values were obtained for the N199H variant than for the wild-type at low (50 mM), as well as high (200 mM), sodium chloride concentrations. This difference between the endonucleases is attributed to a grealter accessibility for tbe substrate by the variant, and also a higher affinity for the DNA backbone. It also appears that the relative activities of the two enzymes, particularly at high ionic strength, are proportional to their populations in the monomeric enzyme form. That is, according to gel filtration data, half of the N199H molecules exist as monomers in 200 mM NaCl, whereas those of the wild-type are mainly dimeric. Consequently, the Asp199 residue of the EcoRI endonuclease may be implicated in the protein-protein interaction leading to dimerization, as well as in coupling to DNA substrates. In summary, it is proposed that active monomeric endonuclease molecules, derived from the dimeric enzyme, recognize and form a complex with a single stranded form of the DNA substrate, which then undergoes nucleophilic substitution and cleavage.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.230-235
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• 2003

A 1.25 kb DNA fragment including the lscR gene, which encodes a levansucrase of Rahnella aquatilis ATCC 15552, was subcloned into a high-expression vector, pET-29b, and the recombinant enzyme was overexpressed in Escherichia coli. Most of the levansucrase activity was detected in the cytoplasmic fraction after induction with isopropyl ${\beta}-D-thiogalactoside$. The recombinant enzyme with a tag of six histidine residues at the C-terminus was purified 146-fold by affinity and gel-filtration chromatographies. The molecular mass of the purified LscR was approx. 49 kDa as determined by SDS-PAGE. The optimum pH and temperature of this enzyme for levan formation was pH 6.0 and $30^{\circ}C$, respectively. The optimum substrate concentration for levan formation was 300 mM sucrose. Levan formation was increased by the increase of the enzyme concentrations. Maxium yield of levan formation at optimum substrate concentration, pH, and temperature after 24 h of reaction was approximately 80%.

• The Korean Journal of Mycology
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• v.11 no.3
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• pp.109-114
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• 1983

${\beta}-Galactosidase$ activity of Aspergillus phoenicis was studied using ONPG and lactose as substrate. It increased monotonically during the exponential growth phase and dropped rapidly at the beginning of the stationary one. It exhibited high tolerable temperature and acidic optimal pH which provides certain advantages from the industrial view point. Enzyme of ${\beta}-galactosidase$ had more subsrate affinity for ONPG than for lactose and its apparent maximum activity was also higher with the former as substrate. Activity of this enzyme depended upon the conditions of immobilization. Optimum crosslinking reaction was occurred at pH 7.2 and 0. 35 vol. % of glutaraldehyde concentration.

• Korean Journal of Food Science and Technology
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• v.32 no.5
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• pp.1079-1086
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• 2000

Pectinesterase inhibitor(PEI) of ripened kiwi fruit(Actinidia chinensis) was separated with affinity chromatography using CNBr-activated Sepharose 4B being covalently bound by orange pectinesterse(PE). The affinity resin strongly and selectively bound PEI, which could be eluted in high yield as a single peak by pH 9.5 without loss of inhibitory activity. The separated PEI had maintained almost inhibitory activity at $-25^{\circ}C$ and $5^{\circ}C$ during 30 days but lost it at room temperature in 4 weeks. The PEI possessed a molecular weight of 16.6 KDa, as estimated by 12.5% SDS-PAGE. PEI had optimum pH of 7.5, optimum temperature of below $10^{\circ}C$ and stability up to $70^{\circ}C$. Also, optimum inhibitory activity for PEI was obtained in 0.2 M NaCl of substrate solutions. The kind of inhibition on tomato pectinesterase was found to be noncompetitive, using citrus pectin as substrate. Fresh orange juice added with crude PEI extracts maintained almost the same cloud stability as pasteurized juice. In case of apple juice, the addition of crude PEI extracts to apple juice had decrease of L-ascorbic acid with nearly no effect on cloud loss.

• Journal of Environmental Impact Assessment
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• v.21 no.5
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• pp.707-713
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• 2012

There are a lot of parameters affecting microbial acclimation/cultivation characteristics such as dynamic conditions, F/M ratio and substrate affinity. From the process control point of view, the effect of high salinity on the removal efficiencies of BOD and SS have been documented by few researchers. In this research, lab-scale CAS(Conventional Activated Sludge) process and modified $A_2O$(Anaerobic/Anoxic/Oxic) process were operated and monitored to evaluate the characteristics of microbial acclimation and cultivation under high salinity wastewater during the period of three weeks. As a result of acute microbial activity test(6hr) at various $Cl^-$ concentration, the appropriate $Cl^-$ concentration for microbial growth and acclimation ranged under 3,100 mg/l. As a result of acclimation/cultivation test, the trend of COD removal efficiency reduced gradually as time elapsed. It is considered that $NH_4$-N removal phenomenon of the conventional pollutants removal mechanisms gave little effect to the microbial acclimation/cultivation under high salinity wastewater.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.88-92
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• 2005

Aptamer is the single-stranded oligonucleotide which binds to various target molecules such as proteins, peptides, lipids and small organic molecules with high affinity and specificity. DNA aptamers specific for the $17{\beta}-estradiol$ were selected by SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random DNA library. These DNA aptamers have a high affinity to $17{\beta}-estradiol$ as an endocrine disrupting chemical. Nanowell and $200{\mu}m$ gold electrode were used as substrate for DNA aptamer immobilization and electrochemical analysis. Especially, nanowell gold electrode was fabricated by e-beam lithography. The size of single nanowell is 130nm and 40,000 nanowells were deposited on one gold electrode. The immobilization method was based on the interaction between the biotinylated aptamer and streptavidin deposited on gold electrode previously. Immobilization procedure was optimized by surface plasma resonance (SPR) and electrochemical analysis. After the immobilization of DNA aptamer on streptavidin modified gold electrode, $17{\beta}-estradiol$ solution was treated on aptamer immobilized gold electrode. The current of gold electrode was decreased by the binding of $17{\beta}-estradiol$ to DNA aptamer immobilized on gold electrode. However, in negative control experiments of 1-aminoanthraquinone and 2-methoxynaphthalene, the current was rarely decreased. And more sensitive data was obtained from nanowell gold electrode comparing with $200{\mu}m$ gold electrode.

• Analytical Science and Technology
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• v.12 no.6
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• pp.565-570
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• 1999

The alcohol dehydrogenase (ADH) gene from Bacillus stearothermopilus was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pGEX-KG, and expressed it as a fusion protein with glutathione S-transferase (GST) in E. coli. The recombinant ADH was produced by induction with 1 mM isopropyl-${\beta}$-D-thiogalactopyranoside at $37^{\circ}C$ and purified by glutathione affinity chromatography. The recombinant ADH exhibited high substrate specificity for ethanol. The activity of the recombinant ADH proceeded optimally at pH 9.0 and $70^{\circ}C$. The recombinant ADH was highly stable against high temperature. This thermostable alcohol dehydrogenase can be used for the enzymatic determination of alcohol and for the industrial production of alcohol.

• YAKHAK HOEJI
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• v.47 no.6
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.