• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.411-416
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• 2002

Bacillus macerans cyclodextrin glucanotransferase (CGTase) was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane-anchored protein, Aga1p. The surface display of CGTase was confirmed by immunofluorescence microscopy and its enzymatic ability to form ${\alpha}$-cyclodextrin from starch. The maximum surface-display of CGTase was obtained by growing recombinant S. cerevisiae at $20^{\circ}C$ and pH 6.0. S. cerevisiae cells displaying CGTase on their surface consumed glucose and maltose, inhibitory byproducts of the CGTase reaction, to enhance the purity of produced cyclodextrins. Accordingly, the experimental results described herein suggest a possibility of using the recombinant S.cerevisiae anchored with bacterial CGTase on the cell surface as a whole-cell biocatalyst for the production of cyclodextrin.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.673-679
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• 2003

Glutamine and urea, abundant in body fluids or plasma, yield net ammonium ions upon hydrolysis by ${\gamma}-glutamyl$ transpeptidase (${\gamma}-GTP$) and urease, respectively, and these two enzymes are largely produced from Helicobacter pylori. To investigate bacterial potential of ammonium production, we first quantified those in whole-cell systems and found that the relative ratio of their amounts varied greatly, especially with pH values and the cell's aging. During the H. pylori cultivation, the ratio appeared to be inversely proportional to each other, showing a progressive increase of the ${\gamma}-GTP$ with decreasing of the urease. Under the urease-defective conditions due to low pH or coccoids, the bacterial cells still possessed a considerable amount of ${\gamma}-GTP$, which was found exclusively in the external compartment, therefore, the cell's ammonium production was found to be solely dependent upon glutamine, and the external ammonium concentration was constant without any contribution of urea concentration. Such ammonium constancy would definitely have an adverse effect on the host, because of its absolute requirement for vacuolar degeneration by H. pylori VacA, maximized at approximately 10 mM $NH_4Cl$. It was also found that, by using the metal-saturated membrane vesicles, ammonium ions were likely to be involved in the pH-dependent cation-flux across the H. pylori membrane, where the role of ${\gamma}-GTP$ in ammonium homeostasis around cells was suggested, especially under the hostile conditions against H. pylori.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1174-1178
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• 2011

Exopolysaccharides (EPSs) are microbial polysaccharides that are released outside of the bacterial cell wall. There have been few studies on EPS-producing lactic acid bacteria that can enhance macrophage activity and the underlying signaling mechanism for cytokine expression. In the current study, EPS-overproducing Lactobacillus (L.) paracasei KB28 was isolated from kimchi and cultivated in conditioned media containing glucose, sucrose, and lactose. The whole bacterial cells were obtained with their EPS being attached, and the cytokine-inducing activities of these cells were investigated. Gas chromatography analysis showed the presence of glucose, galactose, mannose, xylose, arabinose, and rhamnose in EPS composition. EPS-producing L. paracasei KB28 induced the expression of tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-6, and IL-12 in mouse macrophages. This strain also caused the degradation of $I{\kappa}B{\alpha}$ and phosphorylation of the major MAPKs: Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2. The use of pharmacological inhibitors showed that different signaling pathways were involved in the induction of TNF-${\alpha}$, IL-6 and IL-12 by L. paracasei KB28. Our results provide information for a better understanding of the molecular mechanisms of the immunomodulatory effect of food-derived EPS-producing lactic acid bacteria.

• BMB Reports
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• v.28 no.5
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• pp.443-450
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• 1995

Cloned staphylococcal peptidoglycan hydrolase was used in determining the physiological characteristics of peptidoglycan hydrolase. This enzyme hydrolyzed the bacterial cell walls and released the N-terminal alanine, but not the reducing groups. This cloned gene product was localized in the cytoplasm of transformed Escherichia coli. Activity gels indicated the enzyme had an Mr of about 54,000, which was consistent with the deduced Mr from sequencing of the cloned gene. The activity bound to CM-cellulose but not DEAE-cellulose resin, indicating it as a basic protein. Enhanced enzyme activity in a low concentration of cations, and inhibited enzyme activity in a solution with dissolved phospholipids, suggested that the activity and the availability of this basic protein may be regulated between negatively charged and positively charged cellular molecules. The activity against boiled crude cell wall was much greater than against purifed cell wall, suggesting protein associated with crude cell wall may aid in the binding of the peptidoglycan hydrolase The cloned peptidoglycan hydrolase showed positive activity on whole cells of some lysostaphin-resistant coagulase-negative staphylococci. The cloned enzyme may be an alternative for lysostaphin for lysis of staphylococci.

• Journal of Microbiology and Biotechnology
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• v.30 no.3
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• pp.398-403
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• 2020

Rhamnose is a naturally occurring deoxysugar present as a glycogenic component of plant and microbial natural products. A recombinant mutant Escherichia coli strain was developed by overexpressing genes involved in the TDP-ʟ-rhamnose biosynthesis pathway of different bacterial strains and Saccharothrix espanaensis rhamnosyl transferase to conjugate intrinsic cytosolic TDP-ʟ-rhamnose with anthraquinones supplemented exogenously. Among the five anthraquinones (alizarin, emodin, chrysazin, anthrarufin, and quinizarin) tested, quinizarin was biotransformed into a rhamoside derivative with the highest conversion ratio by whole cells of engineered E. coli. The quinizarin glycoside was identified by various chromatographic and spectroscopic analyses. The anti-proliferative property of the newly synthesized rhamnoside, quinizarin-4-O-α-ʟ-rhamnoside, was assayed in various cancer cells.

• Journal of fish pathology
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• v.24 no.3
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• pp.189-195
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• 2011

Starry flounder, which are recently increasingly cultured in Korea, are known to highly vulnerable to Streptococcus parauberis infection. Five groups of starry flounder (n=30 for each group) were vaccinated with S. parauberis formalin-killed whole cells by intraperitoneal injection at a final concentration of 0, 0.01, 0.1, 1 and 10 mg $fish^{-1}$. Specific antibody production of 1 and 10 mg $fish^{-1}$ administered groups significantly increased at four weeks post immunization. All vaccinated groups showed higher survival rates than a control group when five groups of fish were challenged with S. parauberis at a dose of $1.14{\times}10^4$ cfu $fish^{-1}$ and $1.14{\times}10^2$ cfu $fish^{-1}$, respectively. In particular, 0.1 or higher concentrations of formalin killed bacterial cells are able to confer the fish high protection against S. parauberis infection.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.2
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• pp.481-489
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• 2005

Lonicerae Flos has antibacterial effects against Staphylococcus aureus, streptococci, pneumococci, Bacillus dysenterii, Salmonella typhi, and paratyphoid. It is an antiviral agent. The herb has a cytoprotective effect against $CCl_{4}-induced$ hepatic injury. It has antilipemic action, interfering with lipid absorption from the gut. Nowadays this herb is used mainly in the treatment of upper respiratory infections, such as tonsillitis and acute laryngitis. It is also used in the treatment of skin suppurations, such as carbuncles, and to treat viral conjunctivitis, influenza, pneumonia, and mastitis. Lonicerae Flos is dried flower buds of Lonicera japonica, L. hypoglauca, L. confusa, or L. dasystyla. But, for the most part, we use whole plant of Lonicera japonica, as a flower bud of it. And, little is known of the original copy of effects of whole plant, except for the 'Bon-Cho-Gang-Mok', which is written the effects of flower of Lonicera japonica are equal to effects of leaves and branch of it. The present study was conducted to evaluate the effect of flower and whole plant of Lonicera japonica on the regulatory mechanism of cytokines, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) for the immunological activities in Raw 264.7 cells. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, flower and whole plant of Lonicera japonica water extracts inhibited nitric oxide production in a dose-dependent manner and abrogated iNOS and COX-2. Flower and whole plant of Lonicera japonica water extract did not affect on cell viability. To investigate the mechanism by which flower and whole plant of Lonicera japonica water extract inhibits iNOS and COX-2 gene expression, we examined the on phosphorylation of inhibitor ${\kappa}B{\alpha}$ and assessed production of $TNF-{\alpha}$, $interleukin-1{\beta}$ $(IL-1{\beta})$ and interleukin-6 (IL-6). Results provided evidence that flower and whole plant of Lonicera japonica inhibited the production of $IL-1{\beta}$, IL-6 and activated the phosphorylation of inhibitor ${\kappa}B{\alpha}$ in Raw 264.7 cells activated with LPS. These findings suggest that flower and whole plant of Lonicera japonica can produce anti-inflammatory effect, which may play a role in adjunctive therapy in Gram-negative bacterial infections, respectively.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1835-1841
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• 2015

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

• Tuberculosis and Respiratory Diseases
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• v.53 no.5
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• pp.497-509
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• 2002

Background : The mechanisms through which cellular activation results in intracellular mycobacterial killing is only partially understood. However, in vitro studies of human immunity to Mycobacterium tuberculosis have been largely modeled on the work reported by Crowle, which is complicated by several factors. The whole blood culture is simple and allows the simultaneous analysis of the relationship between bacterial killing and the effect of effector cells and humoral factors. In this study, we attempted to determine the extent to which M. tuberculosis is killed in a human whole blood culture and to explore the role of the host and microbial factor in this process. Methods : The PPD positive subject were compared to the umbilical cord blood and patients with tuberculosis, diabetes and lung cancer. The culture is performed using heparinized whole blood diluted with a culture medium and infected with a low number of M. avium or M. tuberculosis $H_{37}Ra$ for 4 days by rotating the culture in a $37^{\circ}C$, 5% $CO_2$ incubator. In some experiments, methlprednisolone- or pentoxifyline were used to inhibit the immune response. To assess the role of the T-cell subsets, CD4+, CD8+ T-cells or both were removed from the blood using magnetic beads. The ${\Delta}$ log killing ratio was defined using a CFU assay as the difference in the log number of viable organisms in the completed culture compared to the inoculum. Results : 1. A trend was noted toward the improved killing of mycobacteria in PPD+ subjects comparing to the umbilical cord blood but there was no specific difference in the patients with tuberculosis, diabetes and lung cancer. 2. Methylprednisolone and pentoxifyline adversely affected the killing in the PPD+ subjects umbilical cord blood and patients with tuberculosis. 3. The deletion of CD4+ or CD8+ T-lymphocytes adversely affected the killing of M. avium and M. tuberculosis $H_{37}Ra$ by PPD+ subjects. Deletion of both cell types had an additive effect, particularly in M. tuberculosis $H_{37}Ra$. 4. A significantly improved mycobacterial killing was noted after chemotherapy in patients with tuberculosis and the ${\Delta}$ logKR continuously decreased in a 3 and 4 days of whole blood culture. Conclusion : The in vitro bactericidal assay by human whole blood culture model was settled using a CFU assay. However, the host immunity to M. tuberculosis was not apparent in the human whole blood culture bactericidal assay, and patients with tuberculosis showed markedly improved bacterial killing after anti-tuberculous chemotherapy compared to before. The simplicity of a whole blood culture facilitates its inclusion in a clinical trial and it may have a potential role as a surrogate marker in a TB vaccine trial.

• Fisheries and Aquatic Sciences
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• v.18 no.4
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• pp.379-385
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• 2015

We studied the effects of the proprietary prebiotic Immunogen$^{(R)}$ on the growth, hematology and gut microbiota of common carp fingerlings. A basal diet was formulated using common feed ingredients and supplemented with Immunogen$^{(R)}$ at concentrations of 0, 5, 10, 20 and $40g\;kg^{-1}$, each of which was tested experimentally on replicated groups of fish. The trials ran for 8 weeks. Common carp fingerlings with an initial weight of $4.82{\pm}0.05g$ were randomly distributed among the experimental tanks at a stocking density of 25 fish per tank. The experimental diets were provided thrice per day; on each occasion the fingerlings were given a weight of feed that amounted to 4% of fish biomass. At the end of the experimental period, we determined the growth performance, feed conversion ratio, hematological parameters, body composition and gut micro-flora parameters of the test fish. Inclusion of $5g\;kg^{-1}$Immunogen$^{(R)}$ in the diet significantly improved growth performance and feed utilization in comparison with controls. However, the whole-body composition of the fish was not significantly influenced by prebiotic inclusion. Inclusion of $5g\;kg^{-1}$ Immunogen$^{(R)}$ significantly increased the total bacterial and Lactobacillus counts in fish intestines, but these bacterial parameters were significantly negatively impacted by higher concentrations of the prebiotic. Red blood cells counts were increased by prebiotic dietary supplementation at concentrations of 5 and $10g\;kg^{-1}$ prebiotic. Glucose and cholesterol levels were elevated by administration of Immunogen$^{(R)}$. Thus, dietary supplementation with $5g\;kg^{-1}$ Immunogen$^{(R)}$ improved fingerling common carp growth performance and feed utilization, and beneficially influenced the gut microflora