• Korean Journal of Microbiology
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• v.47 no.4
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• pp.328-334
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• 2011

Leucine zipper motif consists of multiple periodic leucine residues, which forms amphipathic alpha helix. The hydrophobic nature of leucine zipper motif can dimerize proteins which contain this motif. Leucine zipper motif addition at C-terminus of single-chain Fv (ScFv) antibody induces its dimerization. Since the dimeric ScFv antibody contains two antigen binding sites (bivalency) like Y-shaped complete antibody, it could increase avidity. As a result, it could show higher antigen binding activity than monomeric ScFv antibodies. Based on this concept, monomeric chicken 8C3 ScFv antibody previously developed from chicken hybridoma was dimerized by the addition of leucine zipper motif at C-terminus of ScFv antibody. The dimeric 8C3 ScFv antibody specifically reacted with Eimerian sporozoite which causes Avian Coccidiosis. As expected, dimeric 8C3 ScFv antibody showed 3-folds higher antigen binding activity than monomer due to increased avidity. In addition, protien yields of dimer expression were 2-folds higher than monomer.

• KSBB Journal
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• v.23 no.3
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• pp.205-212
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• 2008

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic interaction, yielding about 5.9 mg/L in flask culture. Six functional fusion partners were introduced by linking to N-terminal part of hEGF gene for the high-level expression of soluble and active hEGF in cytoplasm and peri plasm region. Three fusion partners for cytoplasmic expression such as acidic tail of synuclein (ATS), thioredoxin (Trx) and lipase, and three fusion partners for periplasmic expression such as periplasmic cystein oxidoreductases (DsbA and DsbC) and maltose binding protein (MBP) were investigated. hEGF fused with ATS and DsbA showed over 90% of solubility in cytoplasm and periplasm, respectively. Especially DsbA was found to be an efficient fusion partner for soluble and high-level expression of hEGF, yielding about 18.1 mg/L and three-fold higher level compared to that of insoluble non-fusion hEGF in cytoplasm. Thus, heterologous proteins containing complex disulfide bond and many hydrophobic amino acids can effectively be produced as an active form in E. coli by introducing a suitable peptide or protein.

• Journal of the Korea Organic Resources Recycling Association
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• v.31 no.1
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• pp.35-45
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• 2023

In this study, the sludge formation in the wastewater drain from the advanced packaging process mechanisms are revealed as well as the key factors, materials, and sludge prevention methods using surfactant. Compared with that of conventional packaging process, advanced packaging process employ similar process to the semiconductor fabrication process, and thus many processes may generate wastewater. In specific, a large amount of wastewater may generate during the carrier wafer bonding, photo, development, and carrier wafer debonding processes. In order to identify the key factors for the formation of sludge during the advanced packaging process, six types of chemicals including bonding glue, HMDS, photoresist (PR), PR developer, debonding cleaner, and water are utilized and mixing evaluation is assessed. As a result, it is confirmed that the black solid sludge is formed, which is originated by the sludge seed formation by hydrolysis/dehydration reaction of HMDS and sludge growth via hydrophobic-hydrophobic binding with sludge seed and PR. For the sludge prevention investigation, three surfactants of CTAB, PEG, and shampoo are mixed with the key materials of sludge, and it is confirmed that the sludge formations are successfully suppressed. The underlying mechanism behind the sludge formation is that the carbon tails of the surfactant bind to PR with hydrophobic-hydrophobic interaction and inhibit the reaction with HMDS-based slurry seeds to prevent the sludge formation. In this regard, it is expected that various problems like clogging in drains and pipes during the advanced packaging process may effectively solve by the injection of surfactants into the drains.

• Journal of fish pathology
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• v.9 no.1
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• pp.65-77
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• 1996

Tachyplesin I is an antimicrobial peptide isolated from horseshoe crab. To investigate the mechanism of action of tachyplesin I for phospholipid bilayers, tachyplesin I and five analogs have been synthesized by the solution method. The synthesized five analogs are [$Phe^2$]-tachyplesin I, [$Phe^{8,13}$]-tachyplesin I, [$Cys(Acm)^{3,7,12,16}$]-tachyplesin I with no disulfide bonds, 7(Acm) and 10 (Acm) which denote the fragments [$Cys(Acm)^{3,7,12,16}$]-tachyplesin I. Circular dichroism spectra showed that tachyplesin I took an antiparallel $\beta$-structure in buffer solution and a less ordered structure in acidic liposomes. The carboxyfluorescein leakage experiment indicated that tachyplesin I interacted strongly with neutral and acidic phospholipid bilayers. In fluorescence experiment, the hydrophobic part of the peptide was shown to be embedded in lipid bilayers. All the peptides except for 7(Acm) and 10(Acm) were almost equally active in lipopolysaccharide binding. Therefore, the present study suggested that phospholipid bilayers induced a conformational change of tachyplesin I from the stable $\beta$-structure to a less ordered one.

• Bulletin of the Korean Chemical Society
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• v.33 no.8
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• pp.2508-2512
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• 2012

Cold shock proteins (CSPs) are a family of proteins induced at low temperatures. CSPs bind to single-stranded nucleic acids through the ribonucleoprotein 1 and 2 (RNP 1 and 2) binding motifs. CSPs play an essential role in cold adaptation by regulating transcription and translation via molecular chaperones. The solution nuclear magnetic resonance (NMR) or X-ray crystal structures of several CSPs from various microorganisms have been determined, but structural characteristics of psychrophilic CSPs have not been studied. Therefore, we optimized the purification process to obtain highly pure Lm-Csp and determined the three-dimensional structure model of Lm-Csp by comparative homology modeling using MODELLER on the basis of the solution NMR structure of Bs-CspB. Lm-Csp consists of a ${\beta}$-barrel structure, which includes antiparallel ${\beta}$ strands (G4-N10, F15-I18, V26-H29, A46-D50, and P58-Q64). The template protein, Bs-CspB, shares a similar ${\beta}$ sheet structure and an identical chain fold to Lm-Csp. However, the sheets in Lm-Csp were much shorter than those of Bs-CspB. The Lm-Csp side chains, E2 and R20 form a salt bridge, thus, stabilizing the Lm-Csp structure. To evaluate the contribution of this ionic interaction as well as that of the hydrophobic patch on protein stability, we investigated the secondary structures of wild type and mutant protein (W8, F15, and R20) of Lm-Csp using circular dichroism (CD) spectroscopy. The results showed that solvent-exposed aromatic side chains as well as residues participating in ionic interactions are very important for structural stability. Further studies on the three-dimensional structure and dynamics of Lm-Csp using NMR spectroscopy are required.

• Bulletin of the Korean Chemical Society
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• v.30 no.9
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• pp.2083-2086
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• 2009

Human vascular endothelial growth factor receptor 2 (hVEGFR2) is an important signaling protein involved in angiogenesis and attractive drug target in cancer therapy. It has been reported that flavonols, a class of flavonoids, have anti-angiogenic activity in various cancer cell lines. We performed receptor-oriented pharmacophore based in silico screening for identification of hVEGFR2 inhibitors from flavonol database. By comparing with three X-ray complex structures of hVEGFR2 and its inhibitors, we evaluated the specific interactions between inhibitors and receptors and determined a single pharmacophore map. This map consisted of four features, a hydrogen bonding acceptor (HBA) on Cys917, two hydrogen bonding donors on Glu917 (HBD1) and Glu883 (HBD2), and one hydrophobic interaction (Lipo) with Val846, Ala864, Val897, Val914 and Phe1045 of hVEGFR2. Using this map, we searched a flavonol database including 9 typical flavonols and proposed that five flavonols, kaempferol, quercetin, fisetin, morin, and rhamnetin can be potent inhibitors of hVEGFR2. 3-OH of C-ring and 4’-OH of B-ring of flavonols are the essential features for hVEGFR2 inhibition. This study will be helpful for understanding the mechanism of inhibition of hVEGFR2 by natural products.

• KSBB Journal
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• v.11 no.2
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• pp.218-224
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• 1996

The adsorption of cellulase on celluloses with different crystallinities was carried out In nonionic surfactant(Tween 20) solution. Highly crystallized celluloses were prepared by enzymatic prehydrolyzation. From the experiments, the Langmuir isotherm parameters, maximum adsorption amount (Amax) and adsorption equilibrium constant(Kad) for the adsorption, were obtained in the presence and absence of nonionic surfactant. It was found that the Kad values were decreased by adding Tween 20. This indicates that the adsorption affinity is reduced by nonionic surfactant, and Amax decreased with increasing crystallinity under conditions accompanying in both the presence and absence of surfactant. The thermodynamic parameters such as $\Delta$Ha, $\Delta$Ga, and $\Delta$Sa for the adsorption were calculated by using the experimental data. From these results, it was found that the adsorption processes are exothermic reactions in both the presence and absence of surfactant. The heats of adsorption in surfactant solution(-4.68∼-3.62KJmol-1) are smaller than that of the adsorption in the absence of surfactant(-15.60∼-12.10KJmol-1). These results indicated that the tightness of adsorption was reduced by the addition of surfactant. The $\Delta$Sa values were estimated to be positive. This may suggest that the water and solute are released from cellulose on adsorption. The $\Delta$Sa values in surfactant solution are larger than that of the adsorption in the absence of surfactant. This may suggest that the binding of surfactant on hydrophobic region of cellulase cause dispersion of water and solute molecule orienting around the enzyme molecule. The surfactant played an important role in the desorption of enzyme from cellulose functional groups, and enhance the saccharification of the cellulose.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.656-662
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• 1998

To make a distinction between the influence of the dielectric constant and of methanol concentration on trypsin-catalyzed hydrolysis and methanolysis at $0^{\circ}C$, a model reaction of $N^u$-benzyloxycarbonyl-L-lysine p-nitrophenyl ester with water-methanol mixtures was chosen and a kinetic study done. The $k_{cat}$ values increased with methanol concentration, in a linear manner whereas $K_{M}$ values increased in a log-linear fashion. However, the $k_{cat},$_{M}$ ratio increased at lower methanol concentrations than 30% and then began to decrease at higher concentrations. The decrease in $k_{catK_M}$observed at higher than 30% methanol concentrations is attributed to the hydrophobic partitioning effect on substrate binding. On the other hand, the increase in $k_{catK_M}$ in the 0~30% methanol concentration range seems to be due to the effect of nucleophilic cosolvent on $k_{cat}$ and of the dielectric constant on $k_m$. This explanation was verified by measuring the effect of varying the dielectric constant of the medium on kinetic constants with isopropyl alcohol chemically unrelated to the enzyme reaction as the methanol concentration is maintained at a constant level. Therefore, we conclude that the effect of increasing the methanol concentration in the model reaction on the kinetic parameters $k_{cat \;and\;{K_M}}$ is caused by changes in both the nucleophilicity and the dielectric constant of the medium. Based on product analysis, the increase in $k_4, k_3$by decreasing the temperature can be accounted for by the suppression of hydrolytic reactions. This observation indicates that the nucleophile is favored by low temperatures. There was no loss of trypsin activity over a 10 h period in 60% methanol concentration at $pH^＊\; 5.5,\; 0^{\circ}C$.EX>.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1701-1707
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• 2016

Lipoxygenase (LOX) is an industrial enzyme with wide applications in food and pharmaceutical industries. The available structure information indicates that eukaryotic LOXs consist of N terminus β-barrel and C terminus catalytic domains. However, the latest crystal structure of Pseudomonas aeruginosa LOX shows it is significantly different from those of eukaryotic LOXs, including the N-terminal helix domain. In this paper, the functions of this N-terminal helix domain in the soluble expression and catalysis of P. aeruginosa LOX were analyzed. Genetic truncation of this helix domain resulted in an insoluble P. aeruginosa LOX mutant. The active C-terminal domain was obtained by dispase digestion of the P. aeruginosa LOX derivative containing the genetically introduced dispase recognition sites. This functional C-terminal domain showed raised substrate affinity but reduced catalytic activity and thermostability. Crystal structure analyses demonstrate that the broken polar contacts connecting the two domains and the exposed hydrophobic substrate binding pocket may contribute to the insoluble expression of the C terminus domain and the changes in the enzyme properties. Our data suggest that the N terminus domain of P. aeruginosa LOX is required for its soluble expression in E. coli, which is different from that of the eukaryotic LOXs. Besides this, this N-terminal domain is not necessary for catalysis but shows positive effects on the enzyme properties. The results presented here provide new and valuable information on the functions of the N terminus helix domain of P. aeruginosa LOX and further improvement of its enzyme properties by molecular modification.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.196-203
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• 2002

A novel process for urokinase purification was studied using p-aminobenzamidine as the ligand and sepharose 4B as the matrix. The adsorption, washing, and elution conditions were optimized by an unusual method. An adsorption buffer containing 2.5 M NaCl and $1\%$ Tween 80 facilitated the adsorption of urokinase on the affinity media and prevented contaminants from binding to the p-aminobenzamidine affinity gel. It was found that $5\%$ Tween 80 removed most of the contaminants from the affinity column. A 0.2 M glycine elution buffer containing 0.5 M NaCl (pH 3.0) was found to have a strong elution ability with a high recovery and purity of urokinase. A crude urokinase material of231 IU/mg protein from human urine was purified to 124,300 IU/mg protein with a purification factor of 538 and yield of $86.7\%$. As a result, a high purity urokinase was obtained with only a single affinity chromatography step. The purification process was successfully scaled-up to a 2-1 chromatography column. The resulting urokinase eluate could be directly lyophilized, thereby complying with Chinese pharmacopoeia (1995 version) standards.