• 대한한의학회지
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• 제31권5호
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• pp.112-123
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• 2010

Objectives: Three yin-tonifying formulae (Ssanghwa-tang, Yukmijihwang-tang and Jaeumganghwa-tang) were applied to investigate their immunological activities on antigen (Ag)-specific or Ag-non-specific immune responses in the murine macrophage cell line (RAW 264.7) and in ovalbumin (OVA)-immunized mice. Methods: This study was carried out in nitricoxide (NO) synthesis in RAW 264.7 cells and cellular proliferation in mouse splenocytes in association with three herbal formulas. C57BL/6 mice were immunized intraperitoneally with OVA/aluminum ($100\;{\mu}g/200\;{\mu}g$/mouse) on days 1, 8, and 15. Three herbal formulas were administrated to mice orally for 3 weeks from day 1. On day 22, OVA-, lipopolysaccharide (LPS)-, and concanavalin A (Con A)-stimulated splenocyte proliferation and antibodies (OVA-specific antibodies of the IgG, IgG1, and total IgM classes) in plasma were measured. Results: All three yin-tonifying formulas significantly enhanced cellular proliferation by LPS and Con A in splenocytes from OVA-immunized mice (p<0.001). Also, these herbal formulas all significantly enhanced plasma OVA-specific IgG, IgG1, and total IgM levels compared with the OVA/Alum group. Conclusion: These results suggested that the three yin-tonifying formulae could be used as stimulators of immune response.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.467-473
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• 2010

To improve the expression efficiency of recombinant endo-$\beta$-1,4-glucanase in P. pastoris, the endo-$\beta$-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-$\beta$-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$, and the optimal pH was 5.0 when CMC was used as the substrate.

• 사상체질의학회지
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• 제22권4호
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• pp.77-84
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• 2010

1. Objectives Yanggyuksanhwa-tang for Soyangin was applied to investigate the immunological activities on antigen (Ag)-specific or Ag-non-specific immune responses on murine macrophage cell line (RAW 264.7) and ovalbumin/aluminium (OVA/Alum)-immunized mice. 2. Methods This study were carried out in nitric oxide (NO) synthesis on RAW 264.7 cells and cellular proliferation on mouse splenocytes. C57BL/6 mice were immunized intraperitonially with OVA/Alum (100 ${\mu}g$/200 ${\mu}g$) on day 1, 8, and 15. Yanggyuksanhwa-tang was administrated to mice orally for 3 weeks from day 1. On day 22, OVA-, lipopolysaccharide (LPS)-, and concanavalin A (Con A)-stimulated splenocyte proliferation and antibodies (OVA-specific antibodies of the IgG, IgG1, and total IgM classes) in plasma were measured. 3. Results Yanggyuksanhwa-tang significantly enhanced cellular proliferation by LPS and Con A on splenocytes from OVA/Alum-immunized mice (p<.001). Yanggyuksanhwa-tang also significantly enhanced plasma OVA-specific IgG (p<.001), IgG1 (p<.001), and total IgM (p<.01) levels compared with the OVA/Alum group. 4. Conclusions These results suggested that Yanggyuksanhwa-tang for Soyangin could be used as immunopotent.

• 사상체질의학회지
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• 제22권4호
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• pp.56-64
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• 2010

1. Objectives 4 herbal formulas (Yanggyeoksanhwa-tang, Yeoldahanso-tang, Cheongsimyeonja-tang and Taeeumjowi-tang) were applied to investigate the anti-inflammatory activities. In many studies, plant-derived anti-inflammatory efficacies have been investigate for their potential inhibitory effects on lipopolysaccharide (LPS)-stimulated macrophages. This study was performed to examine the anti-inflammatory activities of 4 herbal formulas on LPS-stimulated RAW 264.7 cells. 2. Methods The productions of nitric oxide (NO), prostaglandin (PG)$E_2$, interleukin(IL)-6 and tumor necrosis factor (TNF)-${\alpha}$ were examined in the presence of the 4 herbal formulas in RAW 264.7 cells. The cells were incubated with LPS 1 ${\mu}g/mL$ and 4 herbal formulas for 18 hrs. The anti-inflammatory activity of 4 herbal formulas were investigate by carrageenin-induced paw edema in rats. The paw volume was measured at 2 and 4 hrs following carrageenininduced paw edema in rats. 3. Results Yanggyeoksanhwa-tang and Cheongsimyeonja-tang showed inhibitory effect on $PGE_2$ production in LPS-stimulated RAW 264.7 cells and a reduction in carrageenin-induced paw edema on rats. Yanggyeoksanhwa-tang showed inhibitory effect on IL-6 in LPS-stimulated RAW 264.7 cells. 4 herbal formulas not affect on NO and TNF-${\alpha}$ inhibition in LPS-stimulated RAW 264.7 cells. 4. Conclusions These results suggested that Yanggyeoksanhwa-tang and Cheongsimyeonja-tang have anti-inflammatory activity.

• 대한한의학회지
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• 제31권3호
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• pp.47-54
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• 2010

Objective: To derive information on the efficacy of Sam-chul-kun-bi-tang (SKT), by evaluating its anti-inflammatory effect. SKT is a widely-used herbal formula in traditional Korean medicine. In man y studies, plant-derived anti-inflammatory efficacies have been investigated for their potential inhibitory effects on lipopolysaccharide (LPS)-stimulated macrophages. This study was performed to examine the anti-inflammatory effects of SKT extract on LPS-stimulated RAW 264.7 cells. Methods: The production of nitric oxide (NO), prostaglandin $(PG)E_2$, tumor necrosis factor (TNF)-$\alpha$ and interleukin (IL)-6 were examined in a macrophage cell line, RAW 264.7 cells, in the presence of SKT. RAW 264.7 cells were incubated with LPS 1 ${\mu}g/mL$ and SKT for 18 hrs. The anti-inflammatory activity of SKT was investigated by carrageenan-induced paw edema in rats. The paw volume was measured at 2 and 4 hrs following carrageenan-induced paw edema in rats. Results: SKT showed inhibitory effect on $PGE_2$, TNF-$\alpha$ and IL-6 in LPS-stimulated RAW 264.7 cells. But SKT was not inhibitory effect on NO by LPS-stimulated RAW 264.7 cells. Administration of SKT (1 g/kg) also showed a reduction in carrageenan-induced paw edema on rats. Conclusion: These results suggest that SKT has anti-inflammatory activities in both in vitro and in vivo models.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1551-1558
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• 2014

The esterase gene Est8 from the thermophilic bacterium Bacillus sp. K91 was cloned and expressed in Escherichia coli. The monomeric enzyme exhibited a theoretical molecular mass of 24.5 kDa and an optimal activity around $50^{\circ}C$ at pH 9.0. A model of Est8 was constructed using a hypothetical YxiM precursor structure (2O14_A) from Bacillus subtilis as template. The structure showed an ${\alpha}/{\beta}$-hydrolase fold and indicated the presence of a typical catalytic triad consisting of Ser-11, Asp-182, and His-185, which were investigated by site-directed replacements coupled with kinetic characterization. Asp-182 and His-185 residues were more critical than the Ser-11 residue in the catalytic activity of Est8. A comparison of the amino acid sequence showed that Est8 could be grouped into the GDSL family and further classified as an SGNH hydrolase. Est8 is a new member of the SGNH hydrolase subfamily and may employ a different catalytic mechanism.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1516-1524
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• 2014

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) can utilize a variety of external electron acceptors and also has stricter substrate specificity than any other glucose oxidoreductases, which makes it the ideal diagnostic enzyme in the field of glucose biosensors. A gene coding for a hypothetical protein, similar to glucose oxidase and derived from Aspergillus terreus NIH2624, was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 260,000 U/l in the culture supernatant after fed-batch cultivation for 84 h. After a three-step purification protocol that included isopropanol precipitation, affinity chromatography, and a second isopropanol precipitation, recombinant FAD-GDH was purified with a recovery of 65%. This is the first time that isopropanol precipitation has been used to concentrate a fermentation supernatant and exchange buffers after affinity chromatography purification. The purified FAD-GDH exhibited a broad and diffuse band between 83 and 150 kDa. The recombinant FAD-GDH was stable across a wide pH range (3.5 to 9.0) with maximum activity at pH 7.5 and $55^{\circ}C$. In addition, it displayed very high thermal stability, with a half-life of 82 min at $60^{\circ}C$. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.

• Structural Engineering and Mechanics
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• 제31권2호
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• pp.137-152
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• 2009

Mechanic behavior of Y-shape thin-walled box girder bridge structure is complex, so one can not exactly hold the mechanical behavior of the Y-shape thin-walled box girder bridge structure through general calculation theory and analytical method. To hold the mechanical behavior better, based on elementary beam theory, by increasing the degree of freedom analytical method, taking account of restrained torsiondistortion angledistortion warp and shearing lag effect at the same time, authors obtain a thin-walled box beam analytical element of 10 degrees of freedom of every node, derive stiffness matrix of the element, and code a finite element procedure. In addition, authors combine the obtained procedure with spatial grillage analytical method, meanwhile, they build a new analytical method that is the spatial thin-walled box girder element grillage analysis method. In order to validate the precision of the obtained analysis method, authors analyze a type Y-shape thin-walled box girder bridge structure according to the elementary beam theory analytical method, the shell theory analytical method and the spatial thin-walled box girder element grillage analysis method respectively. At last, authors test a type Y-shape thin-walled box girder bridge structure. Comparisons of the results of theory analysis with the experimental text show that the spatial thin-walled box girder element grillage analysis method is simple and exact. The research results are helpful for the knowledge of the mechanics property of these Y-shape thin-walled box girder bridge structures.

• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1221-1226
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• 2008

The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072$\pm$47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50$^{\circ}C$, respectively. The $K_m$ value was 0.17 mM, with a $V_{max}$ of 1,714 $\mu$mol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.

• Journal of Microbiology and Biotechnology
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• 제32권4호
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• pp.484-492
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• 2022

Lichenase is an enzyme mainly implicated in the degradation of polysaccharides in the cell walls of grains. Emerging evidence shows that a highly efficient expression of a thermostable recombinant lichenase holds considerable promise for application in the beer-brewing and animal feed industries. Herein, we cloned a lichenase gene (CelA203) from Bacillus subtilis B110 and expressed it in E. coli. This gene contains an ORF of 729 bp, encoding a protein with 242 amino acids and a calculated molecular mass of 27.3 kDa. According to the zymogram results, purified CelA203 existed in two forms, a monomer, and a tetramer, but only the tetramer had potent enzymatic activity. CelA203 remained stable over a broad pH and temperature range and retained 40% activity at 70℃ for 1 h. The K_m and V_max of CelA203 towards barley β-glucan and lichenan were 3.98 mg/ml, 1017.17 U/mg, and 2.78 mg/ml, 198.24 U/mg, respectively. Furthermore, trisaccharide and tetrasaccharide were the main products obtained from CelA203-mediated hydrolysis of deactivated oat bran. These findings demonstrate a promising role for CelA203 in the production of oligosaccharides in animal feed and brewing industries.