• Korean Journal of Microbiology
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• v.40 no.2
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• pp.81-86
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• 2004

Among the annotated ORFs of Streptomyces coelicolor, SCO7697 was supposed to encode for phytase (myo-inositol hexakisphosphate phosphohydrolase). The DNA fragment containing SCO7697 was cloned by the PCR from the chromosomal DNA of S.coelicolor A3(2)M. The cloned fragment was introduced into E. coli expres-sion vector, pET28a(+), to yield two recombinant plasmids, pET28-SP and pET28-LP, which were designed to encode different length of proteins. When the pET28-SP and pET28-LP were introduced into E. coli BL21, the transformants successfully overexpressed recombinant proteins, but the molecular weights of the expressed pro-teins were appeared bigger than those of expected in SDS-polyacrylamide gel electrophoresis. The shift of cul-tural temperature from 37 to $30^{\circ}C$ made most of expressed protein be solubilized. The expressed protein, however, did not show any phytase activity. When the DNA fragment with its own promoter placed on the E. coli-Streptomyces vector, pWHM3, and introduced into S. lividans, the phytase activity was not detected either. These results suggest that even though the SCO7697 was annotated as a probable phytase with high probability (E value is $6e^{-89}$), the real product doest not have phytase activity.

• Journal of fish pathology
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• v.5 no.1
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• pp.29-35
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• 1992

This study was carried out in order to identify the biochemical and serological characteristics of Edwardsiella tarda isolated from cultured flounder, Paralichthys olivaceus in the east and south coast of Korea. During the year of 1990 and 1991, the number of isolated E. tarda were 131 strains. To identify the biochemical characteristics of them kinds of tests were conducted. The results represented that all the strains had the same biochemical characteristics, and their biochemical characteristics were no differences among strains. A serological analysis was carried out based on agglutination test with antiserum belonging to E. tarda serotype a (E-22), b (SU-138), c (SU-100) classfied in japan. The selected 10 isolates showed agglutinin titer of 5120-2560, 40-80 and below 40 against E. tarda serotype a, b and c, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) profiles of cell proteins of selected 10 isolates were showed no differences in kinds and volumes of proteins among strains.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.94-100
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• 2011

A gene encoding a putative phospholipase D was isolated from Bacillus licheniformis and cloned into pGEM-T easy vector. The gene was expressed in E. coli BL21 (DE3) using a pET-21(a) vector containing His6 tag. Affinity purification of the recombinant phospholipase D with nickel-nitrilotriacetic acid (Ni-NTA) resin resulted major one-band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified enzyme showed a molecular weight of 44 kDa. The optimum activity of enzyme was around pH 7.0 and the enzyme was also the most stable around this condition. The optimum temperature was about $40-45^{\circ}C$ and the enzyme still showed considerable activities at wide range of temperature. Among various detergents, Triton X-100 significantly increased the enzyme activity, resulting in 181% activity of control at 0.6 mM of the detergent. Calcium ion did not significantly affect the enzyme activity, suggesting that the enzyme might be classified into $Ca^{2+}$-independent PLD.

• The Korean Journal of Mycology
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• v.25 no.4 s.83
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• pp.330-339
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• 1997

Botrytis cinerea T91-1 has shown to produce at least four different polygalacturonases in a liquid medium containing citrus pectin as a carbon source. One of the enzymes, its molecular weight was estimated as 37 kDa by denatured polyacrylamide gel electrophoresis, was purified by a series of procedures including acetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. By viscometric analysis, the enzyme was revealed as an endo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as $Ca^{2+}$, $Co^{2+}$, and $Cu^{2+}$. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was $55^{\circ}C$ and the enzyme showed optimal pH values between 4.0 and 4.5. The enzyme was stable up to 12 hours in the range of pH 4 to 7 and at the temperature below $30^{\circ}C$. Amino acid sequence from N-terminal up to 6 amino acids determined by Edman degradation showed little homology with polygalacturonases from fungi and plants.

• The Journal of Korean Academy of Prosthodontics
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• v.37 no.5
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• pp.698-713
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• 1999

Adherence of Candida albicans(C. albicans) to the surface of a denture is believed to be an initial and essential step in the formation of denture-induced stematitis. Previous studies have provided enormous infomation on the relationship between composition of palatine gland/parotid saliva and upper denture stomatitis. Relatively little information is available on the correlation between lower denture stomatitis and sublingual-submandibular ( SLSM ) saliva. The plaque samples were collected from the two sites($100mm^2$) on the inner surface of lower partial denture corresponding to the stematitis and healthy region of the lower partial dentures of 12 denture stomatitis patients and 6 nor-mal persons who wore lower partial dentures. The samples were plated to isolate C. albicans on a selective Saboraud's dextrose agar plate and the isolates were identified by germ tube test and gram staining. The subjects were divided into group I (stomatitis with C. albican), group II (lesion without C. albicans), group III (no lesion but C. albicans), and group IV (normal and healthy denture wearer). Individual SLSM saliva($20{\mu}g$ of protein) was analyzed by SDS-PAGE (SDS -poly-acrylamide gel electrophoresis) with Coomassie brilliant blue and PAS(Periodic Acid Schinff) stain-ing. The salivary proteins separated in the polyacryamide gels were subjected to immunoblot anaysis using anti-lactoferrin, anti-sIgA, and anti-secretory component of sIgA. In this study using custom made acrylic denture resin beads(5mm in diameter) coated with stimulated individual SLSM saliva, the binding ability of individual C. albicans strains to the beads was observed. Levels of C, albicans adhered to the acrylic resin beads were determined by measuring the optical density of the bound C. albicans to the beads at 580nm. The results showed that a higher number of C. albicans was observed in the lesion site than healthy site. The saliva of group I contained more high molecular weight glycoprotein(mucin, MGI) as compared to group II, III and IV. And lactoferrin and sIgA affected to the binding ability of C. albicans to acylic resin beads. Binding ability of individual C. albicans to the acrylic resin coated with respective individual saliva was found to be greater in group I than the other 3 groups. And when bound cells of C. albicans isolated from individual subject #2 to the saliva coated beads were used binding ability of subject #2 saliva coated beads was founed to be greater than the other sutjects. These results suggested that denture induced stomatitis is related to individual patient's salivary protein composition, especially MG-1. Future studies will be directed toward saliva exam-ination of patients who have general disease and analysis of pellicles formed on prosthesis with respect to oral disease.

• Korean Chemical Engineering Research
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• v.55 no.3
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• pp.369-378
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• 2017

Vascular endotherial growth factor (VEGF) is a potent mitogen that stimulates vascular permeability and angiogenesis and has a potential in therapeutic applications. An industrial production method that provides high yield as well as purity is needed. Researches for various factors of mild solubilization with combination of ubiquitin fusion protein to increase solubility were carried out as well as by changing pH and denaturant concentration. Usage of pET28-a bacteral expression vector in BL21 (DE3) host cell was capable of producing approximately 14 g/L VEGF fusion protein in 20L fermentor. A purification process consisting of four chromatography steps including refolding and digestion with UBP1 resulted in mild solublization under the conditions of 2M urea and pH 10.0 due to ubiquitin fusion tag protein that increases in solubility of target protein VEGF. High yield of refolding and dimerization could be obtained between two step Ni-affinity chromatography. Multimeric and misfolded proteins and endotoxin were removed by DEAE anion exchange chromatography. Final monomers were removed from dimers by gel filtration chromatography. Characterization analysis of purified dimeric VEGF was performed using SDS-PAGE and RP-HPLC with a purity of 97%.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.104-111
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• 2002

The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

• Journal of Veterinary Clinics
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• v.21 no.1
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• pp.20-22
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• 2004

Bacterial infection of canine atopic dermatitis is largely caused by Staphylococcus intermedius and may be a superficial or deep pyoderma. The Purpose of this study was to identify the major proteins of S. intermedius cell surface components in humoral immune response of atopic dermatitis dog. Sera samples were obtained from dogs with atopic dermatitis and superficial pyoderma referred to the Veterinary Medical Teaching Hospital, Konkuk University. An isolate of S. intermedius from a clinical case of canine atopic dermatitis was cultured in brain heart infusion broth overnight at $37^{\circ}C$ in aerobic conditions on an orbital shaker. Following culture, Staphylococci were harvested by centrifugation, washed in PBS, and resuspended in PBS containing lysostaphin. The soluble components were separated by centrifugation and were collected. The soluble extract of S. intermedius was separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically transferred onto nitrocellulose membrane. Western blotting for the specificity of serum IgG antistaphylococcal antibody was performed with anti-dog-IgG and sera obtained from an atopic dermatitis case and a normal dog. The molecular masses of four major proteins of S. intermedius recognized by serum obtained from an atopic dermatitis case were 18, 31, 75, and 110 kDa as determined by Western blot analysis. The present study indicates that most dogs of S. intermedius infection with atopic dermatitis could have a significant humoral immune response to bacterial proteins of the causative organism.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.574-582
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• 1992

Exo-xylanase encoded by the xylA gene of Bacillus stearothermoPhillus was produced from Escherichia coli ]M109 carrying a recombinant plasmid pMGL Synthesis of the enzyme was observed to be cell-associated, and about 94% of the enzyme synthesized was located in the cytoplasmic region. The maximum production was attained when the E. coli strain was grown at $37^{\circ}C$ for 8 hours on the medium containing 0.5% fructose, 1.0% tryptone, 1.0% sodium chloride, and 0.5% yeast extract. The exo-xylanase was purified to homogeneity using a combination of salting out with ammonium sulfate, DEAE-Sepharose CL-6B ion exchange chromatography, Sephadex G-IOO gel filtration, and Sephadex G-150 gel filtration. The' purified enzyme was most active at pH 6.0 and $45^{\circ}C$. $Ca^{2+}$ and $Co^{2+}$ activated the exo-xylanase activity by about 20% while $Ag^{2+}$, $Fe^{2+}$, $Mg^{2+}$ and $Zn^{2+}$ inhibited the enzyme activity by up to 60%. The $K_m$, value on p-nitrophenyl-$\beta$-D-xylanopyranoside was 2.75 mM. The enzyme had a pI value of 4.7. The estimated molecular weight of the native protein was 200,000 daL SDS-polyacrylamide gel electrophoresis analysis suggested that the native enzyme was a trimer composed of three identical 66,000 da!. polypeptides. The purified enzyme efficiently converted all the xylo-oligosaccharides tested to xylose. It was also confirmed that the enzyme split xylans in an exo-manner even though the degree of hydrolysis was fairly low. The xylanolytic enzyme was, therefore, classified to be one of the few bacterial exo-xylanases lacking transferase activity.

• The Korean Journal of Mycology
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• v.17 no.2
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• pp.66-75
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• 1989

Heterogeneity in antigenic composition of Aspergillus fumigatus isolates from clinical specimens and in antibody response of patients infected with this fungus was investigated by immunoblotting. A considerable quantitative and qualitative difference was found in composition of the culture filtrate antigens derived from a reference strain (ATCC 13073) and 8 clinical isolates of A. fumigatus on SDS-PAGE and immunoblots. The crude CF antigen of a strain AFG7 was selected to identify the serologically reactive and specific components by immunoblotting. Out of more than 36 components separated by electrophoresis, transblotted to nitrocellulose sheet, and reacted with sera that showed a positive reaction to A. fumigatus or other fungal antigens on immunodiffusion tests, merely four or so were found useful to serodiagnosis of aspergillosis. An antigen of 82KD was found most reactive and specific component so as to be contained in the standard preparation. Several other components, for example 11KD, 26KD, 30KD and 31KD, also possessed relatively high reactivity and specificity and seemed to be worth while purifying and characterizing. Antibody binding activity (reactivity) of the antigenic components was clearly shown on immunoblots because some were faintly stained with Coomassie blue but darkly stained on immunoblots, while some others behaved contrary to them. A number of components seemed to carry not only species specific but cross reactive antigenic determinants. Immunoblotting proved very useful to identify serologically reactive and specific components that should be present in the antigen to be employed to the serodiagnosis of aspergillosis.