• Journal of Environmental Science International
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• v.19 no.10
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• pp.1307-1314
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• 2010

This study was conducted to isolate and characterize a novel feather-degrading bacterium producing keratinase activity. A strain K9 was isolated from soil at poultry farm and identified as Xanthomonas sp. K9 by phenotypic characters and 16S rRNA gene analysis. The cultural conditions for the keratinase production were 0.3% fructose, 0.1% gelatin, 0.04% $K_2HPO_4$, 0.06% $KH_2PO_4$, 0.05% NaCl and 0.01% $FeSO_4$ with an initial pH 8.0 at $30^{\circ}C$ and 200 rpm. In an optimized medium containing 0.1% chicken feather, production yield of keratinase was approximately 8-fold higher than the yield in basal medium. The strain K9 effectively degraded chicken feather meal (67%) and duck feather (54%), whereas human nail and human hair showed relatively low degradation rates (13-22%). Total free amino acid concentration in the cell-free supernatant was about 25.799 mg/l. Feather hydrolysate produced by the strain K9 stimulated growth of red pepper, indicating Xanthomonas sp. K9 could be not only used to increase the nutritional value of chicken feather but also a potential candidate for the development of natural fertilizer applicable to crop plant soil.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.183-187
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• 2008

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

• Journal of Microbiology
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• v.42 no.2
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• pp.117-125
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• 2004

Propionibacterium acnes (P. acnes) plays an important role in the disease pathogenesis of acne vulgaris, a disorder of pilosebaceous follicles, seen primarily in the adolescent age group. In the present study, the presence of antibodies against P. acnes (MTCC1951) were detected in acne patient (n=50) and disease free controls (n=25) using dot-ELISA and Western blot assay. The ability of P. acnes to induce pro-inflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), obtained from acne patients and healthy subjects, were also analysed. The patients (n=26) who were culture positive for skin swab culture, were found to have a more advanced disease and higher antibody titres (1:4000 to >1:16000) compared to the P. acnes negative patients (n=24) and normal controls (n=25). An analysis of patients' sera by western blot assay recognized a number of antigenic components of P. acnes, rang-ing from 29 to 205 kDa. The major reactive component was an approximately 96 kDa polypeptide, which was recognised in 92％ (24 of 26) of the patients sera. Further, the P. acnes culture supernatant, crude cell lysate and heat killed P. acnes whole cells, obtained from 72-h incubation culture, were observed to be able to induce significant amounts of IL-8 and tumor necrosis factor alpha (TNF-${\alpha}$) by the PBMCs in both the healthy subjects and patients, as analysed by cytokine-ELISA. The levels of cytokines were significantly higher in the patients than the healthy subjects. A major 96 kDa polypep-tide reactant was eluted from the gel and was found to cause dose dependent stimulation of the pro-ductions of IL-8 and TNF-${\alpha}$. Thus, the above results suggest that both humoral and pro-inflammatory responses play major roles in the pathogenesis of acne.

• Microbiology and Biotechnology Letters
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• v.40 no.1
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• pp.17-22
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• 2012

A bacterial strain was isolated from homemade Cheongkookjang as a producer of the ${\beta}$-galactosidase, capable of hydrolyzing lactose to liberate galactose and glucose residues. The isolate YB-1105 has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. ${\beta}$-Galactosidase activity was detected in both the culture supernatant and the cell extract of B. licheniformis YB-1105. The enzymes of both fractions demonstrated maximum activity for hydrolysis of para-nitrophenyl-${\beta}$-D-galactopyranoside (pNP-${\beta}Gal$) under identical reaction conditions of pH 6.5 and $50^{\circ}C$. However, ${\beta}$-galactosidase activity from the culture filtrate was affected more than that from the cell free extract at acidic pHs and high temperatures. The hydrolyzing activity of both ${\beta}$-galactosidases for pNP-${\beta}Gal$ was dramatically decreased by the addition of low concentrations of galactose, but was only marginally decreased by high concentrations of glucose or mannose.

• Korean Journal of Microbiology
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• v.50 no.1
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• pp.73-83
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• 2014

The objective of the this study was to investigate the binding capacity and removal ability of lactic acid bacterial strains obtained from Korean soybean paste for mutagenic heterocyclic amines (HCAs) formed during cooking of protein-rich food at high temperature. Among 19 strains identified by carbohydrate fermentation and 16S rRNA sequencing, the live cell or cell-free culture supernatant of Lactobacillus acidophilus D11, Enterococcus faecium D12, Pediococcus acidilactici D19, L. acidophilus D38, Lactobacillus sakei D44, Enterococcus faecalis D66, and Lactobacillus plantarum D70 inhibited the mutagenesis caused by either 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole (Trp-P-1) or 3-amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) in Salmonella typhimurium TA98 and TA100. The bacterial cells of the isolated strains showed greater binding activity than the pure cell wall, exopolysaccharide, and pepetidoglycan. The carbohydrate moieties of the cell wall or protein molecules on the cell surface have a significant role in binding Trp-P-1 and Trp-P-2, since protease, heating, sodium metaperiodate, or acidic pH treatments significantly (P<0.05) reduced the binding efficacy of the tested bacteria. Addition of metal ions or sodium dodecyl sulfate decreased the binding ability of E. faecium D12, L. acidophilus D38, and E. faecalis D66. Therefore, the binding mechanisms of these strains may consist of ion-exchange and hydrophobic bonds. Especially, the high mutagen binding by L. acidophilus D38 and L. plantarum D70 may reduce the accumulation or absorption of Trp-P-1 and Trp-P-2 in the small intestine via increased excretion of a mutagen-bacteria complex.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.288-299
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• 2015

Lactobacillus brevis BK11 obtained from Baikkimchi was selected to study the effects of culture medium, initial pH, atmosphere composition, incubation temperature and time, and prebiotics on growth and production of antimicrobial substances. Growth and antimicrobial substances production of L. brevis BK11 were significantly higher in MRS broth than in BHI or M17 broth. The production of cell mass, lactic acid, and bacteriocin by BK11 strain was at maximum in MRS broth adjusted to pH 6.0. Aerobic and microaerobic conditions were favored cell growth and antimicrobial substances production than anaerobic condition. Biomass and lactic acid production and antimicrobial substances activity of BK 11 were significantly better at 30 and $37^{\circ}C$ than at $25^{\circ}C$. Growth of the strain BK11 entered the stationary growth stage at 24 h after inoculation, and decreased after 36 h. Antimicrobial activities of cell-free culture supernatant and bacteriocin solution were highest when cultured in MRS broth with an initial pH 6.0 for 24-30 h at $37^{\circ}C$. In addition, the highest cell number and lactic acid and bacteriocin production were recorded in the presence of 1 and 2% (w/v) fructooligosaccharide (FOS), however, inulin and raffinose did not affect biological and physicochemical characteristics and antimicrobial activities of L. brevis BK11 cultures. According to these results, production of antimicrobial substances by L. brevis KB11 was closely associated with cell density. Under optimal conditions for antimicrobial substances production, L. brevis BK11 effectively inhibited the growth of Helicobacter pylori ATCC 43504.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• The Korean Journal of Physiology
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• v.17 no.2
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• pp.125-133
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• 1983

Red cell glycolytic intermediates, metabolites and metabolic ratios were studied. Glycolytic intermediates were measured in neutralized perchloric acid extracts of red cell suspensions after 3 hr incubation at $37^{\circ}C$ in the presence and absence of saponin. Adenosine triphosphate(ATP), adenosine diphosphate(ADP), pyruvate and lactate were measured by enzymatic procedures involving stoichiometric oxidation or reduction of a pyridine nucleotide. Glucose was determined using glucose oxidase after zinc hydroxide extraction. The redox state was calculated from the lactate dehydrogenase equilibrium. Adenosine triphosphatase activity(ATPase) was measured by determining the amount of phosphate released from ATP by washed erythrocyte membranes(ghost) during 20 min. incubation. Both total hydrolysis and the amount of hydrolysis that occured in the presence of ouabain were measured. The second measurement yields Mg-ATPase and represents nonspecific ATPase activity of the membranes. The difference between total and Mg-ATPase activity can be attributed to Na-K-ATPase. For the measurement of sodium fluxes, human erythrocytes were preincubated in $^{22}Na$ for 3 hr at $37^{\circ}C$, washed and suspended in a tracer-free medium. The amount of $^{22}Na$ transported out of cells at any time was determined by analysis of supernatant samples taken at various time after addition of the labeled cells to isotope-free medium. The cells and medium were separated and the radioactivity appearing in the medium was measured. From the total radioactivity in the suspension and the radioactivity appearing in the medium at known time, the rate constant for sodium release was computed. The results are summarized as follows: 1) ATP and ATP/ADP were found to increase at every concentration of saponin tested whereas ADP declined at every cone. of saponin. The increase in pyruvate and lactate were observed at every cone, of saponin and thus $NAD^+/NADH$ computed from pyruvate/lactate also increased. Glucose utilization was stimulated by saponin. 2) $Na^+-K^+-ATPase$ activities showed a biphasic response to saponin, first increasing in lower concentration and then decreasing in higher concentration of saponin. 3) The efflux of sodium was significantly increased by saponin in the range of 5 to 10 mg%. The stimulatory effect of saponin on the rate constants for active(ouabain-sensitive) sodium efflux was inhibited by addition of ouabain.

• Clinical and Experimental Pediatrics
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• v.49 no.4
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• pp.439-445
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• 2006

Purpose : ${\alpha}$-Galactosylceramide (${\alpha}$-GalCer)-stimulated human $V{\alpha}24$ natural killer T (NKT) cells exert antitumor activity against some leukemia in a CD1d dependent and TCR-mediated manner, but could not kill CD1d - negative neuroblastoma (NB) cells. There are few reports about the direct antitumor effect of highly secreted cytokines by these cells on activation. In this study, using a cell-free supernatant (SPN) collected from plate bound hCD1d/${\alpha}$ GalCer tetramers-stimulated NKT cells, we examined whether they could be helpful in the immunotherapeutic treatment of NB. Methods : Cells were cultured in IMDM. The cytokines produced by NKT cells were measured with Cytometric Bead Array (CBA) analysis. Cell viability was evaluated by calcein-AM fluorescence with digital image microscopy scanning (DIMSCAN). The percentage of specific apoptosis was calculated by flow cytometric detection of apoptosis using annexin V and 7-AAD. Results : The activated NKT cells secreted high levels of IL-2, INF-${\gamma}$, TNF-${\alpha}$. The SPN was significantly cytotoxic against four out of eight tested NB cell lines, through mainly apoptosis as evidenced by annexin-V staining and inhibition with the pretreatment of pancaspase blocker. This apoptosis was significantly inhibited when anti-TNF-${\alpha}$ and anti-IFN-${\gamma}$ neutralizing mAbs were used separately and it was completely abolished when the two mAbs were combined. Conclusion : IFN-${\gamma}$ and TNF-${\alpha}$ produced by NKT cells could exert synergistically direct antitumor activity through apoptosis on some NB cell lines.

• Mycobiology
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• v.40 no.1
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• pp.59-65
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• 2012

Antagonistic microorganisms against $Rhizoctonia$ $solani$ were isolated and their antifungal activities were investigated. Two hundred sixteen bacterial isolates were isolated from various soil samples and 19 isolates were found to antagonize the selected plant pathogenic fungi with varying degrees. Among them, isolate C9 was selected as an antagonistic microorganism with potential for use in further studies. Treatment with the selected isolate C9 resulted in significantly reduced incidence of stem-segment colonization by $R.$ $solani$ AG2-2(IV) in Zoysia grass and enhanced growth of grass. Through its biochemical, physiological, and 16S rDNA characteristics, the selected bacterium was identified as $Bacillus$ $subtilis$ subsp. $subtilis$. Mannitol (1%) and soytone (1%) were found to be the best carbon and nitrogen sources, respectively, for use in antibiotic production. An antibiotic compound, designated as DG4, was separated and purified from ethyl acetate extract of the culture broth of isolate C9. On the basis of spectral data, including proton nuclear magneric resonance ($^1H$ NMR), carbon nuclear magneric resonance ($^{13}C$ NMR), and mass analyses, its chemical structure was established as a stereoisomer of acetylbutanediol. Application of the ethyl acetate extract of isolate C9 to several plant pathogens resulted in dose-dependent inhibition. Treatment with the purified compound (an isomer of acetylbuanediol) resulted in significantly inhibited growth of tested pathogens. The cell free culture supernatant of isolate C9 showed a chitinase effect on chitin medium. Results from the present study demonstrated the significant potential of the purified compound from isolate C9 for use as a biocontrol agent as well as a plant growth promoter with the ability to trigger induced systemic resistance of plants.