• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.25-31
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• 2006

Changes of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in the glyphosate-tolerant Roundup Ready soybean were examined using purified CP4 EPSPS produced in cloned Escherichia coli as a control. CP4 EPSPS in genetically modified soybean was detected by twodimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with databases. CP4 EPSPS in soybean products was resolved on 2-DE by first isoelectric focusing (IEF) based on its characteristic pI of 5.1, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on its molecular mass of 47.5 kDa. We quantified various percentages of soybean CP4 EPSPS. The quantitative analysis was performed using a 2D software program on artificial gels with spots varying in Gaussian volumes. These results suggested that 2-DE image analysis could be used for quantitative detection of GM soybean, unlike Western blotting.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.429-435
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• 1999

An adenosine 5'-monophosphate deaminase (AMP aminohydrolase, EC 3.5.4.6) was purified to homogeneity from the cell-free extract of Saccharomyces cerevisiae DKCTC7248. The molecular mass of subunit was estimated to be 80 kDa on SDS-PAGE, and that of the holoenzyme was shown to be 240 kDa by gel filtration. The isoelectric point of the enzyme (AMP deaminase D) was determined to be 6.2. The AMP deaminase D was specific towards AMP with an apparent $K_m$ value of 4.1 mM and a Hill coefficient, $n_H$, of 2.2. Both ATP and ADP were positive allosteric effectors of the AMP deaminase D: The apparent $K_{m}$ was decreased to 1.6 mM and 3.3 mM in the presence of 0.1 mM ATP and ADP, respectively, lowering $n_{H}$ to 1.0. Univalent cations like $K^+, Na^+ and Li^ +$ activated the enzyme but some divalent cations such as $Cu^{ 2+} and Cd^{2+}$ showed strong inhibitory effects. This enzyme displayed optimum activity at $30^{\circ}C$ and pH 7.0. In addition, it was stable up to $45^{\circ}C$ and over a wide pH range(pH 5.5-9.0). Amino acid sequences of its N-terminal region were analyzed to be ADYKMQMFADDA.

• The Korean Journal of Zoology
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• v.32 no.4
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• pp.429-440
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• 1989

Spirometru erinocei의 유충인 sparganum에서 추출한 조항원 단백질을 항원으로 하여 sparganosis, cysticercosis, hydatidosis환자의 IgG 항체와 정상인의 IgG 항체를 혈청반응시켜 ELISA와 EITB에 의해 교차반응을 일으키는 비 특이항원 성분과 종특이항원 성분을 추구하였다. 1. sparganum에서 0.01 M PBS (PH 7.4)로 추출한 조항원 단백질을 SDS-PAGE로 전개하여 290 Kd에서 23 Kd범위의 분자량을 가진 25개의 단백질이 분획되었다. 2. sparganum의 조항원을 항원으로하여 ELISA로 sparganosis, cysticercosis, hydatidosis 환자의 190 항체와의 혈청 반응치는 sparganosis환자에게서는 0.44 $\pm$ 0.07에서 1.90 $\pm$ 0.03으로 negative control normal sera의 0.D (0.15 $\pm$ 0.03)를 기준으로 하였을 때 모두 양성이며 민감도(sensitivity)가 100 %이었으며 cysticercosis, hydatidosis환자혈청에서 양성반응이 나타났으며 교차반응도 있었다. 3. EITB에서는 spargancsis환자의 IgG항체에 의해 16개의 항원성분이 인지되었으며 이 중 6개의 항원성분이 정상인의 혈청에서도 인지되어 교차반응을 일으키는 항원성분이었으며 cysticercosis환자혈청에서 인지된 4개의 항원성분 중 2개의 항원성분이 sparganosis 환자혈청에서 인지된 것과 같았으며 hydatidosis환자의 190항체에 의해 인지된 19개의 항원성분 중 12개의 항원성분이 sparganosis환자혈청에서 인지된 항원 성분과 같았다. 4. 290 Kd, 200 Kd, 28 Kd의 항원성분은 sparganosis환자의 196항체에서만 인지되었고 228 Kd, 152 Kd, 66 Kd항원성분은 hydatidosis환자의 19G항체에서만 인지되었으며 66 Kd항원성분은 sparganosis, cysticercosis, hydatidosis, 정상인의 혈청에서 모두 인지되었다. We studied the serological reaction between the specific and nonspecific antigenic components from metacestode (plerocercoid) of spiromeko erimacei and IgG antibodies in sparganosis, cysticercosis. hydatidosis patients and normal human sera by ELISA and EITB. We prepared the crude extracts of sparganlim from snake, Matrix tigrina laterolis and used as antigenic components. By SDS-PAGE, we detected a total 25 peptide bands (fractions) with 290 Kd to 23 Kd molecular weight, and 8 bands of these detected bands developed strongly by silver stain. In serological test, ELISA, we recognized the cross-reaction of antigenic components reacting with IgG antibodies in heterogenous sera, cysticercosis and hydatidosis patients sera. The crude antigenic components of sparganum showed the high sensitivity in sparganosis, hydatidosis patients sera, but showed lower sensitivity in cysticercosis patients sera than the sparasanosis, hydatidosis patients sera. Sixteen antigenic components of these 25 separated bands were recognized by antibodies In sparsanosis patients sera,8 antigenic components in normal human sera, 4 antigenic components in cysticercosis patients sera and 19 antigenic compoenents in hydatidosis patients sera. The crude antigenic compnenets with 290 Kd, 200 Kd, 125 Kd and 28 Kd molecular weight was only recognized in sparganosis patients sera, but 64 Kd antigenic component was nonspecific antigenic components which were also cross-reacted with sparganosis, hydatidosis, cysticercosis patients sera and normal human sera.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1547-1556
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• 2009

A novel thermostable $\alpha$-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in E. coli and characterized. The TtgluA gene contained 2,253 bp, which encodes 750 amino acids. The native TtGluA was a trimer with monomer molecular mass of 89 kDa shown by SDS-PAGE. The purified recombinant enzyme showed hydrolytic activity on maltooligosaccharides, p-nitrophenyl-$\alpha$-D-glucopyranide, and dextrin with an exotype cleavage manner. TtGluA showed preference for short-chain maltooligosaccharides and the highest specific activity for maltose of 3.26 units/mg. Maximal activity was observed at $60^{\circ}C$ and pH 5.5. The half-life was 2 h at $60^{\circ}C$. The enzyme showed good tolerance to urea and SDS but was inhibited by Tris. When maltose with the concentration over 50 mM was used as substrate, TtGluA was also capable of catalyzing transglycosylation to produce $\alpha$-1,4-linked maltotriose and $\alpha$-1,6-linked isomaltooligosaccharides. More importantly, TtGluA showed exclusive regiospecificity with high yield to produce $\alpha$-1,6-linked isomaltooligosaccharides when the reaction time extended to more than 10 h.

• Journal of Animal Science and Technology
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• v.45 no.3
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• pp.491-498
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• 2003

The purpose of this study is to observe purification and properties of osteopontin(OPN) from bovine milk. The purification of osteopontin from bovine milk was performed by using ion-exchange and hydrophobic chromatography. SDS-PAGE analysis revealed that the protein migrated at Mw. 60,000. NH2-terminal sequence analysis of the first seven amio acids revealed the protein to be identical to that previously reported for bovine OPN. 35-wk-old chickens, including 3 Single Comb White Leghorn (SCWL), were used to produce egg yolk antibody(IgY) against OPNas a antigen. However, the anti-OPN antibody activities determined by ELISA. Immunological assy of OPN in milk was performed using radial immunodiffusion test based on the standard curve of pure OPN. The radial precipitation lines of four different milk samples indicated that the concentrations of OPN in the milk samples were within the range of 31.7 to 39.7${\mu}g$/ml. On inhibition with OPN on precipitation of calcium phosphate, OPN was slightly higher than casein phosphopeptide(CPP) and poly-glutamic acid.

• Korean Journal of Food Science and Technology
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• v.21 no.5
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• pp.606-612
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• 1989

A New lectin from baby clam, Tapes japonica, was isolated and purified through the following procedures; acetone powder, 0.15M NaCl extraction, ammonium sulfate fractionation, N-acetyl-D-galactosamine-agarose affinity column, and ion exchange Mono Q of FPLC. This lectin nonspecifically agglutinated human erythrocytes but didn't agglutinate mouse and rabbit erythrocytes. And the lectin neither stimulated human lymphocytes nor agglutinated Sarcoma 180 cells. On polyacrylamide gel electrophoresis, the lectin migrated as a major single band indicating homogeneous. A molecular weight was estimated to be about 131,000 daltons by Biogel P-300 and 125,000 daltons by SDS-PAGE without ${\beta}-mercaptoethanol$. This lectin is supposed to be a tetramer composed of heterogeneous subunits, about 30,000 and 33,000 daltons. Baby clam lectin was inhibited by EDTA and recovered agglutinating activity by $Ca^{++}\;and\;Mn^{++}$. This lectin is revealed as glycoprotein that contained about 4.2% neutral sugar.

• Journal of Microbiology and Biotechnology
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• v.34 no.2
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• pp.436-456
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• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

• KSBB Journal
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• v.19 no.5
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• pp.352-357
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• 2004

Human ferritin H- and L-chain genes (hfH and hfL) were cloned into the yeast shuttle vector YEp352 containing the GAL1 (galactokinase) and GAL10 (epimerase) divergent promoters and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. SDS-PAGE displayed expression of the introduced hfH and hfL in both recombinant strains of Y1H10L and Y1L10H. The ferritin subunits, that represented ca. $22\%$ and $15\%$ of the soluble proteins in Y1H10L and Y1L10H, were spontaneously assembled into active ferritin heteropolymers. The H subunit content of the purified recombinant human ferritin heteropolymers was proven to reflect the relative expression yield of the subunits. When the cells of 2d culture were incubated with 14.3 mM Fe(2), the cellular iron concentration of Y1H10L and Y1L10H was 1.7 and 2.0 times, respectively, that of the control strain. It is assumed that increase in the iron uptake of the recombinant yeasts is closely related to ferritin expression and H subunit content.

• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.179-187
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• 1989

The experiment was carried out to study the profile of uterine specific protein during early pregnancy in sows and to test it's immunosuppressive activity. Uterine protein samples were obtained by flushing the uterine horn on Day 3, 6, 9, 12 and 15 of the estrous cycle and the pregnancy respectively and the protein concentration of each sample was determined. The change of uterine protein was examined by sodium dodecyl sulfate(SDS)-PAGE. The immunosuppressive activity of uterine secretory protein was investigated according to the lymphocyte blastogenesis response to mitogen. The results of this experiment are summarized as follows ; 1. The uterine protein during estrous cycle and early pregnancy was relatively constant up to Day 9, but increased on Day 12. Maxium total protein values were found on Day 15. The concentration of serum proteins were about 82-95 mg during estrous cycle, but decreased to about 70-82 mg during early pregnancy. 2. The proteins components similar electrophoretic patterns(PAGE) that were no differences (band ; a, b, c, d, e, f, g, I) on Days 3, 6 and 9 of the estrous cycle and pregnancy. But there were more 2 bands specifically on Day 12 of the pregnancy and on Day 15 of estrous cycle and showed more 4 bands on Day 15 of early pregnancy. They seemed to be acidoprotein and their average molecular weight were 38,000, 22,300 and 12,600. 3. When uterine protein were added 500$\mu\textrm{g}$/ml, there was no immunosuppresive activity on Day 3 of estrous cycle and lymphocyte blastogenesis was slightly suppressed on Day 3 of pregnancy. The immunosuppressive activity on Day 9 of estrous cycle and pregnancy appeared in 500$\mu\textrm{g}$/ml and 150$\mu\textrm{g}$/ml, respectively the uterine protein on Day 12 and 15 showed immunosuppresive activity, which at the level of 150$\mu\textrm{g}$/ml during non-pregnancy and at the level of 100 to 125$\mu\textrm{g}$/ml during early pregnancy, respectively.

• Journal of Microbiology and Biotechnology
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• v.21 no.10
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• pp.1057-1063
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• 2011

Herein, a novel ginsenosidase, named ginsenosidase type IV, hydrolyzing 6-O-multi-glycosides of protopanaxatriol-type ginsenosides (PPT), such as Re, R1, Rf, and Rg2, was isolated from the Aspergillus sp. 39g strain, purified, and characterized. Ginsenosidase type IV was able to hydrolyze the 6-O-${\alpha}$-L-($1{\rightarrow}2$)-rhamnoside of Re and the 6-O-${\beta}$-D-($1{\rightarrow}2$)-xyloside of R1 into ginsenoside Rg1. Subsequently, it could hydrolyze the 6-O-${\beta}$-D-glucoside of Rg1 into F1. Similarly, it was able to hydrolyze the 6-O-$_{\alpha}$-L-($1{\rightarrow}2$)-rhamnoside of Rg2 and the 6-O-${\beta}$-D-($1{\rightarrow}2$)-glucoside of Rf into Rh1, and then further hydrolyze Rh1 into its aglycone. However, ginsenosidase type IV could not hydrolyze the 3-O- or 20-O-glycosides of protopanaxadiol-type ginsenosides (PPD), such as Rb1, Rb2, Rb3, Rc, and Rd. These exhibited properties are significantly different from those of glycosidases described in Enzyme Nomenclature by the NC-IUBMB. The optimal temperature and pH for ginsenosidase type IV were $40^{\circ}C$ and 6.0, respectively. The activity of ginsenosidase type IV was slightly improved by the $Mg^{2+}$ ion, and inhibited by $Cu^{2+}$ and $Fe^{2+}$ ions. The molecular mass of the enzyme, based on SDS-PAGE, was noted as being approximately 56 kDa.