• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.258-263
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• 1998

${\beta}$-Xylosidase was purified from the recombinant Escherichia coli carrying the Bacillus sp. KK-1 ${\beta}$-xylosidase gene (xylB). The molecular mass of the purified enzyme was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the apparent molecular mass of the ${\beta}$-xylosidase was 140 kDa, indicating that the native ${\beta}$-xylosidase has an oligomeric structure composed of two identical subunits. The isoelectric point was determined to be pH 5.5. The enzyme was highly active on p-nitrophenyl-$\beta$-D-xylopyranoside but it barely hydrolyzed xylan substrates, and did not exhibit activity towards carboxymethylcellulose and p-nitrophenyl-${\beta}$-D- glucopyranoside. The enzyme had a pH optimum for its activity at pH 6.5 and a temperature optimum at $40^{\circ}C$. The enzyme activity was completely inhibited by the presence of $Hg^{++}$, and also markedly inhibited by D-xylose and D-glucose.

• Fisheries and Aquatic Sciences
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• 제14권4호
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• pp.355-361
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• 2011

Effects of aluminum (Al) on plasma vitellogenin (VTG), alkaline-labile phosphorus (ALPP), calcium (Ca), glutamate pyruvate transaminase (GPT), the hepatosomatic index (HSI), and hepatic Al concentration were examined in estradiol-$17{\beta}$ ($E_2$)-administered immature rockfish Sebastes schlegeli. Fish were injected intraperitoneally with $E_2$ (5 mg/kg body weight [BW]) and/or Al (0, 0.1, 1, 5, and 10 mg/kg BW) and plasma and liver samples were extracted 7 days later. After sodium dodecyl sulfate polyacrylamide gel electrophoresis, the relative amount of VTG was determined by integrated optical density. VTG accounted for 23.6% of the total proteins in the control group, but this value decreased with increasing Al administration. Al reduced the concentrations of ALPP and Ca in a concentration-dependent manner and significant reduction occurred at Al concentrations greater than 5 mg/kg. The concentration of GPT increased in a concentration-dependent manner in all Al-administered rockfish. The concentrations of Al in the liver also increased, and HSI was decreased, in a concentration-dependent manner. These results suggest that Al inhibits $E_2$-induced VTG production by being toxic to hepatocytes in marine fish.

• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.133-140
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• 2012

A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.

• Fisheries and Aquatic Sciences
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• 제16권2호
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• pp.71-78
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• 2013

Subcritical water hydrolysis was carried out in a batch reactor to produce valuable materials, such as low-molecular weight (MW) peptides and essential amino acids with antioxidant properties, from heat-dried squid viscera. Hydrolysis of squid viscera was performed at 160 to $280^{\circ}C$ for 3 min. The yield was increased by increasing the temperature and pressure, while the protein content of squid viscera hydrolysate decreased with increasing temperature. Low-MW peptides were detected in all hydrolysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The highest yields of free and structural amino acids in heat-dried squid viscera hydrolysate were at $160^{\circ}C$ and were $411.95{\pm}1.15$ and $346.62{\pm}1.25$ mg/100 g, respectively. All essential amino acids were detected in viscera hydrolysates; leucine was the most abundant. Antioxidant activities of hydrolysates were highest at $220^{\circ}C$. Greater than $98{\pm}0.26%$ of the ABTS antioxidant activity was retained in hydrolysates after long-term heat treatment.

• 대한수의학회지
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• 제36권3호
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• pp.615-619
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• 1996

Sera of pigs with clinically normal and infectious conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) to demonstrate the specific changes in protein profile. In the sera from pigs with infection, haptoglobin with a 40KDa protein was found to be increased as compared to that of sera from normal pigs. As a rapid detection method for monitoring infections at large-scale farms, one of acute phase protein, haptoglobin, was selected to compare the concentrations between infectious and non-infectious conditions. Haptoglobin concentrations were low in pigs with clinically normal conditions but significantly increased in pigs with Aujesky's disease, hog cholera and parvo-virus infection. The studies provide that haptoglobin can be used as an indicator to monitor infections early at farm level.

• 한국어업기술학회:학술대회논문집
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• 한국어업기술학회 2000년도 춘계수산관련학회 공동학술대회발표요지집
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• pp.145-146
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• 2000

The presence of cross-reacting muramidase in Lactobacillus delbrueckii ssp. bulgaricus ULl2 was shown by using monoclonal antibodies raised against an muramidase-2 of Enterococcus hirae ATCC 9790. The separation of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot confirmed the presence of one cross-reacting band in Enterococcus hirae with an estimated molecular mass of 80 kDa, L. bulgaricus cultured cells harvested after 4 and 12 h were submitted to different autolysin releasing procedures and the liberated products were allowed to cross-react with muramidase-2 antibodies in order to estimate the efficiency of each treatment. Although the cultured cells harvested after 4 h yielded only a slight immune-reaction in Western immunoblots against these enzyme monoclonal antibodies, a strong signal was observed for the cell walls obtained from the same experimental conditions and treated with Triton X-100 surfactant. The same phenomenon was also observed by light fluorescence microscopy. Immune-labelling followed by optical and electron microscopy have shown that the muramidase-2 of L. bulgaricus ULl2 was essentially localized in the innermost part of the cell wall.(omitted)

• Fisheries and Aquatic Sciences
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• 제21권2호
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• pp.4.1-4.5
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• 2018

Insulin-like growth factors (IGFs), along with IGF-binding protein and IGF receptor, are well-known regulators in the growth and survival of vertebrates. In this study, we investigated the involvement of IGFs and protein variation during embryonic development of the olive flounder (Paralichthys olivaceus). Morphological stages were divided into six main developments as blastula, gastrula, cephalization, cranial regionalization, tail lift, and hatch. During embryonic development, protein variation was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization quadrupole time-of-flight mass spectrometry/mass spectrometry. In addition, the mechanism of signaling of IGF-I receptor was examined using immuno-blot analysis. We found marked changes in protein expression at four stages of embryonic development and identified proteins as belonging to the vitellogenin 2 family. As development progresses, expression of IGF-II, phosphotyrosine, and phospho-Akt increased, while expression of growth factor receptor-bound protein 2 (GRB2) and one of guanine-nucleotide-binding proteins (Ras) decreased. These results provide basic information on the IGF system in the embryonic development of the olive flounder.

• Animal cells and systems
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• 제16권6호
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Fisheries and Aquatic Sciences
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• 제15권1호
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• pp.9-14
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• 2012

The objectives of this research were to improve the film-forming properties of Channel Catfish Ictalurus punctatus skin gelatin (CSG) by cross-linking with transglutaminase (TG), determine and optimize the TG reaction time, and characterize the mechanical and barrier properties of CSG edible film. Cross-linking of CSG was performed by TG for 0, 10, 20, 30, and 40 min at $50^{\circ}C$, and the reaction was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The color and mechanical and barrier properties of edible films fabricated with CSG cross-linked with TG were characterized. Gelatin yields from the extraction ranged from 18.2% to 23.3%. SDS-PAGE exhibited dark bands at 120 and 250 kDa, indicating successful TG-mediated cross-linking. The color of CSG film was not affected by TG cross-linking. The tensile strength of CSG films cross-linked with TG decreased from 42.59 to 21.73 MPa and the percent elongation increased from 42.92% to 76.96% as reaction times increased from 0 to 40 min. There was no significant difference in water vapor permeability of CSG films.

• 대한약침학회지
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• 제14권3호
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• pp.71-79
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• 2011

Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially A${\alpha}$-chain and slowly B${\beta}$-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.