• Journal of Microbiology and Biotechnology
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• v.5 no.4
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• pp.181-187
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• 1995

The cells of Vibrio vulnificus can be induced to the viable but nonculturable (VBNC) state by natural environmental parameters. The V. vulnificus cells in the VBNC state can not be recovered by ordinary laboratory techniques. This nonculturability could often hamper development of effective processing strategies to minimize the number of V. vulnificus in seafoods. Even with V. vulnificus cells in a culturable state, the length of time required to identify the bacteria in contaminated food by phenotyphic characterization may prevent appropriate in-time responses by public health agencies to infections of the bacteria. In the present study, we used polymerase chain reaction (PCR) to develop a rapid and direct detection method for V. vulnificus in small octopus (Octopus variabilis) which is consumed as a raw food in Korea. The region targeted was a 704-base pair (bp) portion of the hemolysin gene, vvhA, of V. vulnificus. The primers designed for PCR amplification were specific for all V. vulnificus sp. tested. Several methods were examined to extract total DNA directly from V. vulnificus seeded into the octopus homogenate and the guanidine isothiocyanate (CITC) method appeared to be most effective. From the octopus homogenate seeded by V. vulnificus at an initial level of $10^2$ CFU/ml of the homogenate and then incubated for 12 h, the targeted sequence was successfully amplified by PCR and the 704-bp DNA fragment was observed by gel electrophoresis. The total completion of this assay requires less than one day.

• Microbiology and Biotechnology Letters
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• v.24 no.5
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• pp.567-573
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• 1996

Chitinase (EC 3.2.1.14) from culture fluid of Bacillus licheniformis KFB-C14 was purified 66-folds to homogenity in overall yield of 21% by ammonium sulfate fractionation, DEAE-Toyopearl, Butyl-Toyopearl and TSK-Gel HW-55F column chromatography. The enzyme protein had a molecular weight of about 86,000 and was composed of one subunit. The enzyme was significantly stable not only at high temperature but also on treatment with organic solvents and protein denaturants such as SDS, urea and guanidine-HC1. The optimum temperature and pH for reaction was 60$\circ $C and 6.0, respectively. The enzyme activity was inhibited by only Mn$^{2+}$ ion, but not inhibited by EDTA, N- ethylmaleimide and pCMB. The enzyme had high activity with colloidal chitin (V$_{max}$: 421) and commercial chitin (V$_{max}$: 480), but not with typical substrates of exo type chitinase. The thermostable chitinase had an useful reactivity for producing functional chitooligosaccharide, showing the production of (GlcNAc)$_{1}, (GlcNAc)$_{3}$, and (GlcNAc)$_{2}$ as major product.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.49-57
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• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

• Journal of the Korean Society of Food Science and Nutrition
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• v.23 no.2
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• pp.308-314
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• 1994

Four Taro isolectins (I, II, III, IV) were purified by ammonium sulfate, chromatography on CM-celluose and isoelectric focusing. I and IV lectins proved homogeneous by disk polyacrylamid gel electrophoresis and densitometric patterns. But in the presence of urea, IV lectin further dissociated into two different subunits. These lectinis had different hemagglutinating activities and inhibition in their activities after mixed with pepsin particuclary, but not with carbohydrates, heating pH, urea, guanidine, trypsin, pronase and $Ca^{2+}$.

• Genomics & Informatics
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• v.4 no.3
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• pp.133-136
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• 2006

The adsorption of proteins on the surface of glass slides is essential for construction of protein chips. Previously, we prepared a nickel-coated plate by the spin-coating method for immobilization of His-tagged proteins. In order to know whether the structural factor is responsible for the immobilization of His-tagged proteins to the nickel-coated glass slide, we executed a series of experiments. First we purified a His-tagged protein after expressing the vector in E. coli BL21 (DE3). Then we obtained the unfolding curve for the His-tagged protein by using guanidine hydrochloride. Fractions unfolded were monitored by internal fluorescence spectroscopy. The ${\Delta}G_{H20}$ for unfolding was $2.27kcal/mol{/pm}0.52$. Then we tested if unfolded His-tagged proteins can be adsorbed to the nickel-coated plate, comparing with $Ni^{2+}-NTA$ (nitrilotriacetic acid) beads. Whereas unfolded His-tagged proteins were adsorbed to $Ni^{2+}-NTA$ beads, they did not bind to the nickel-coated plate. In conclusion, a structural factor is likely to be an important factor for constructing the protein chips, when His-tagged proteins will immobilize to the nickel-coated slides.

• BMB Reports
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• v.28 no.6
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• pp.477-483
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• 1995

The recombinant human interferon alpha 2a ($rhIFN-{\alpha}2a$), expressed in Saccharomyces cerevtsiae, was purified from insoluble aggregates. The inclusion body of $rhIFN-{\alpha}$ was solubilized by guanidine salt in the presence of disulfide reducing agent. The refolding of denatured $rhIFN-{\alpha}2a$ was achieved by simple dilution. The authentic interferon alpha, which has two correctly matched disulfide bonds, was seperated from incompletely oxidized $IFN-{\alpha}$ and dimeric $IFN-{\alpha}$ by use of a CM-Sepharose column, followed by size exclusion columns at two different pH conditions. The purified protein has been subjected to detailed physicochemical characterization including sequence determination. Unlike other $rhIFN-{\alpha}2a$ from E. coli reported, the $rhIFN-{\alpha}2a$ from S. cerevisiae has no methionine residue at its N-terminus originating from the start codon, ATG. The pI of the protein was determined to be 6.05 with a single band in the pI gel, which demonstrated that the purified $rhIFN-{\alpha}$ was homogeneous. The structural study using circular dichroism showed that the protein retains its three dimensional structure in the wide range of pH conditions between pH 3 and 9, and only minor strucural deformation was observed at pH 1.0.

• Archives of Pharmacal Research
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• v.21 no.4
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• pp.429-435
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• 1998

Lectins and its A- and B-chains from Korean mistletoe (Viscum album var. coloratum) were isolated by affinity chromatography on the Sepharose 4B modified by lactose-BSA conjugate synthesized by reductive amination of ligand (lactose) to .epsilon.-amino groups of lysine residues of spacer (BSA) after reduction by $NaCNBH_3$. The lactose-BSA conjugate was coupled to Sepharose 4B activated by cyanogen bromide. The molecular weight determined by SDS-PAGE were a 31 kD of A-chain and a 35kD of B-chain. Amino acid analysis and N-terminal sequencing were performed. The effects of pH, temperature and guanidine chloride on the conformation of the lectin were investigated by measuring its intrinsic fluorescence and compared with its hemagglutinating activities. Blue shift was detected on the acidic pH and there was a close relationship between activities and conformation of the lectin. Under denaturing conditions, the tryptophan emission profile of lectin showed typical denaturaiional red shift which also correspond to the conformations and activity of lectin.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.6
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• pp.410-418
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• 2001

The effects of several variables on the refolding of hen egg white lysozyme have been studied, Lysozyme was denatured in both urea, and guanidine hydrochloride(GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1 M Tris-HCI, pH 8.2 mM EDTA 3 mM reduced glutathione and 0.3 mM oxidised glutathions. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50$\^{C}$. The apparent activation energy for lysozyme re-folding wasf ound to be 56kJ/mol, Refolding by dilution results in low concentrations of both de-naturant and reducing agent species. It was found that the residual concentrations obtained dur-ing dilution(100-fold dilution:[GuHCI]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1-10mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT higher refolding yields were obtained when starting from higher initial lysozyme concentra-tions. This trend was reversed when residual denaturant components were removed from the re-folding buffer.

• Food Science and Biotechnology
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• v.14 no.3
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• pp.387-391
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• 2005

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in pork meat. Pork meat was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate-phenol-chloroform and subjected to PCR amplification. Sixteen primer sets for L. monocytogenes hlyA gene were tested for sensitive detection and the DG69/DG74 primer set was selected. The detection limit achieved with this primer set was as low as 860 colony-forming units (cfu) per 0.1 g of pork meat. When the samples were cultured at $30^{\circ}C$ for 16 hr in Brain Heart Infusion (BHI) medium, even a single bacterium could be detected with this primer set by PCR. For cPCR, the hlyA gene, which features a 148 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA which has the same primer binding sites was co-amplified with L. monocytogenes total DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to cfu from the Most Probable Number (MPN) method. The whole procedure took only 5 hr.

• Elastomers and Composites
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• v.54 no.2
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• pp.97-104
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• 2019

Emulsion styrene-butadiene rubber silica wet masterbatch (ESBR silica WMB) technology was studied to develop highly filled and highly dispersed silica compounds, involving the preparation of a composite by co-coagulating the modified silica and the rubber latex in a liquid phase. Previous studies have shown that when manufacturing ESBR silica WMB/Butadiene silica dry masterbatch (BR silica DMB) blend compounds, preparing BR silica dry masterbatch and mixing it with ESBR silica WMB gave excellent results. However, WMB still has the problem of lower crosslink density due to residual surfactants. Therefore, in this study, tetrabenzylthiuram disulfide (TBzTD) was added instead of diphenyl guanidine (DPG) in the ESBR silica WMB/BR silica DMB blend compounds and sulfur/CBS contents were increased to evaluate their cure characteristics, crosslink densities, mechanical properties, and dynamic viscoelastic properties. TBzTD was found to be more effective in increasing the crosslink density and to produce superior properties compared to DPG. In addition, with increasing sulfur/CBS contents, mechanical properties and rolling resistance were enhanced due to high crosslink density, but the abrasion resistance was not significantly changed because of the toughness.