• Journal of Life Science
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• v.20 no.6
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• pp.845-852
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• 2010

Our previous study showed that lungs infected by Pseudomonas, a gram-negative bacteria, produce prostaglandin $D_2$ ($PGD_2$) and prostaglandin $E_2$ ($PGE_2$), the two major prostanoids generated by cyclooxygenase-2 (COX-2), and that the ratio of $PGD_2$ and $PGE_2$ can affect the outcome of the bacterial lung infection. In this study, we sought to uncover the mechanism that determines the ratio of $PGD_2$ and $PGE_2$ produced in lung inflammation. When treated with lipopolysaccharide (LPS), primary alveolar macrophages, extracted from mouse lung, more $PGE_2$ was produced than $PGD_2$, whereas MH-S, a murine alveolar macrophage cell line, produced more $PGD_2$ than $PGE_2$ in a similar experiment. Western blot analyses showed that the kinetics of COX-2 expression in both cell types is similar and epigenetic silencing of COX-2 expression did not affect expressions of lipocalin-PGD synthase (L-PGDS) and PGE synthase (mPGES-1), major enzymes synthesizing $PGD_2$ and $PGE_2$ in inflammation, respectively, indicating no effect of COX-2 on expressions of the two enzymes. Expressions of L-PGDS and mPGES-1 were also similar in both cell types, suggesting no effect of the two key enzymes in determining the ratio of $PGD_2$ and $PGE_2$ in these cells. A single intraperitoneal injection of LPS to C57BL/6 mice induced COX-2 expression and, similar to alveolar macrophages, produced more $PGE_2$ than $PGD_2$ in the lung. These results suggest that the differential expressions of $PGD_2$ and $PGE_2$ in the lung reflect those in alveolar macrophages and may not be directly determined by the enzymes responsible for $PGD_2$ and $PGE_2$ synthesis.

• Microbiology and Biotechnology Letters
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• v.39 no.1
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• pp.9-19
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• 2011

The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ${\Delta}$pyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ${\Delta}$pyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP-binding protein, and cell division protein FtsZ were overproduced in the PyrR-deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.66-74
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• 2012

The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and ${\beta}$-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 ${\mu}g$/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 ${\mu}g$/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 ${\mu}g$/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of $^{15}N$ into particle-associated microbial N ($^{15}N$-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) $^{15}N$-PAMN at 4 h only, but EFE on AS increased (p<0.001) $^{15}N$-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) $^{15}N$-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.23 no.2
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• pp.109-124
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• 1990

A rapid processing method for fish sauce of high quality stability and favorable flavor was investigated using mackerel waste as starting material. The chopped waste was homogenized with water and hydrolyzed by commercial proteolytic enzymes such as Complex enzyme-2000($2.18\cdot10^4$ U/g solid, Pacific Chem. Co.) and Alcalase ($1.94\cdot10^4$ U/g solid, Novo) in a cylindrical vessel with 4 baffles and 6-bladed turbine impeller. Optimal pH and temperature for the hydrolysis with Complex enzyme-2000 were 8.0 and $50^{\circ}C$, and those with Alcalase were 9.0 and $55^{\circ}C$. In both cases, the reasonabe amount of added water and enzyme concentration based on the waste weight were $40\%,\;3\%$ and hydrolyzing time was 100 min. Thermal treatment of the hydrolysate with $6\%$ of invert sugar for 2 hours at $90^{\circ}C$ was adequated to inactivation of the enzymes and pasteurization of the hydrolysate. Flavor, taste and color of the hydrolysate were improved during the thermal treatment in which the browning reaction products might participate and result in antioxidative and bactericidal effects. Combined use of $0.005\%$ of Caryophylli flos with $6\%$ of invert sugar was also effective for the improvement of taste. Yield of the fish sauce based on the total nitrogen of the raw waste was $93.7\~94.9\%$, and $87.6\~87.9\%$ of the total nitrogen in the fish sauce was in the from of amino nitrogen. The pH, salinity and histamine content of the fish sauce prepared with $15\%$ of table salt were $6.1\~6.2$, $14.0\~14.5\%$ and less than $10mg\%$, respectively. The fish sauce was stable on bacterial growth during the storage of 60 days at $26\pm3^{\circ}C$ and the quality was also maintained.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.23 no.2
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• pp.125-136
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• 1990

To develope a rapid processing method for fish sauce, processing conditions of fish sauce from sardine waste was investigated. The chopped waste was homogenized and hydrolyzed by commercial proteolytic enzymes such as Complex enzyme-2000($2.18\cdot10^4$ U/g solid) and Alcalase($1.94\cdot10^4$ U/g solid) in a cylindrical vessel with 4 baffles and 6-bladed turbine impeller. Optimal temperature for the case of hydrolysis with Complex enzyme-2000 was 50 and that with Alcalase was $55^{\circ}C$. In both cases, the reasonable pH, amount of water for homo-genization, enzyme concentration and hydrolyzing time were 8.0, $40\%$ (W/W), $3\%$ and 100 min, respectively. Heating of the filtrated hydrolysate for 2 hours at $90^{\circ}C$ with $6\%$ of invert sugar was suitable for pasteurization of the hydrolysate and inactivation of enzymes. Flavor, taste and color of the hydrolysate was improved during the thermal treatment in which the browning reaction products might participate and result in antioxidative and bactericidal effects. Combined use of $0.005\%$ of Caryophylli flos with invert sugar was also effective for the improvement of taste. Yield of the fish sauce based on the total nitrogen in the raw sardine waste was $91.2\~92.3\%$ and $87.2\~87.8\%$ of the total nitrogen in the fish sauce was in the form of amino nitrogen. The pH, salinity and histamine content of the fish sauce prepared with $15\%$ of table salt were $6.1\~6.2$, $14.2\~14.4\%$ and less than $10mg\%$, respectively. The fish sauce was stable during the storage of 60 days at $26\pm3^{\circ}C$ on bacterial growth and its quality was also maintained.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.12
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• pp.1784-1795
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• 2017

Objective: The study examined the effect of intravenous administration of bacterial endotoxin-lipopolysaccharide (LPS) -on the nocturnal secretion of melatonin and on the expression of enzymes of the melatonin biosynthetic pathway in the pineal gland of ewes, taking into account two different photoperiodic conditions: short-night (SN; n = 12) and long-night (LN; n = 12). Methods: In both experiments, animals (n = 12) were randomly divided into two groups: control (n = 6) and LPS-treated (n = 6) one. Two hours after sunset, animals received an injection of LPS or saline. Blood samples were collected starting one hour after sunset and continuing for 3 hours after the treatment. The ewes were euthanized 3 hours after LPS/saline treatment. The concentration of hormones in plasma was assayed by radioimmunoassay. In the pineal gland, the content of serotonin and its metabolite was determined by HPLC; whereas the expression of examined genes and protein was assayed using real-time polymerase chain reaction and Western Blot, respectively. Results: Endotoxin administration lowered (p<0.05) levels of circulating melatonin in animals from LN photoperiod only during the first hour after treatment, while in ewes from SN photoperiod only in the third hour after the injection. Inflammation more substantially suppressed biosynthesis of melatonin in ewes from SN photoperiod, which were also characterised by lower (p<0.05) cortisol concentrations after LPS treatment compared with animals from LN photoperiod. In the pineal gland of ewes subjected to SN photoperiod, LPS reduced (p<0.05) serotonin content and the expression of melatonin biosynthetic pathway enzymes, such as tryptophan hydroxylase and arylalkylamine-N-acetyltransferase. Pineal activity may be disturbed by circulating LPS and proinflammatory cytokines because the expression of mRNAs encoding their corresponding receptors was determined in this gland. Conclusion: The present study showed that peripheral inflammation reduces the secretion of melatonin, but this effect may be influenced by the photoperiod.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.40-45
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• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• Korean Journal of Food Science and Technology
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• v.33 no.2
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• pp.226-230
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• 2001

High hydrostatic pressure was applied to Foxtail Millet Takju to investigate the effects of high pressure on inactivation of microorganisms and enzymes. Total bacteria, lactic acid bacteria and yeast in untreated Takju were $6.8{\times}10^7,\;1.3{\times}10^8\;and\;8.4{\times}10^7\;CFU/mL$, respectively. Total bacterial count in Takju reduced to $2.2{\times}10^5\;CFU/mL$, while lactic acid bacteria and yeast were sterilized completely when heated at $65^{\circ}C$ for 30 min. Lactic acid bacteria and yeast decreased with the increase of treatment pressure, and pressurization of 400 MPa for 10 min at room temperature sterilized completely the lactic acid bacteria and yeast in Takju. Total bacteria were not sterilized with pressurization of even 600 MPa at room temperature. Total bacteria were completely sterilized at $66^{\circ}C/400\;MPa/60\;min\;and\;66^{\circ}C/600\;MPa/10\;min$. Pressurization of Takju caused a partial inactivation of ${\alpha}-amylase$, and after pressurization at 600 MPa for 10 min at room temperature, 73.2% of the original activity remained. The activity of glucoamylase increased with the increase of treatment pressure. Treatment at $66^{\circ}C/400\;MPa/10\;min$ reduced the activity of ${\alpha}-amylase$ by 59.7% and glucoamylase by 20.5%. ${\alpha}-Amylase$ was inactivated to less than 1.2% of the original activity at $66^{\circ}C/600\;MPa/30\;min$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.8
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• pp.1221-1226
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• 2003

High hydrostatic Pressure was applied to Foxtail Millet Yakju to investigate the effects of high pressure on inactivation of microorganisms and enzymes. Total bacteria, lactic acid bacteria and yeast in untreated Yakju were $1.5{\times}$10$^4$,1.9${\times}$10$^4$ and 1.4${\times}$10$^4$ CFU/mL, respectively. Total bacterial count was reduced to 4.1${\times}$l0$^2$ CFU/mL, while lactic acid bacteria and yeast were sterilized completely in Yakju heated at $65^{\circ}C$ for 15 min. Lactic acid bacteria and yeast decreased greatly with the increase of treatment pressure, and were sterilized completely in Yakju treated at more than 300 ㎫ for 10 min/$25^{\circ}C$. Total bacteria were not completely sterilized with pressurization of even 600 ㎫ at room temperature and reduced to 2 log cycle even at $65^{\circ}C$. Total bacteria decreased by 2∼3 log cycle with the increase of treatment time from 10 to 60 min at $25^{\circ}C$/300 ㎫. Pressurization of Yakju caused a partial inactivation of $\alpha$ -amylase and glucosamylase, and the activities of $\alpha$ -amylase and glucoamylase decreased by 18.1% and 21.1%, respectively at $25^{\circ}C$/600 ㎫/10 min. Activities of $\alpha$ -amylase and glucoamylase decreased with the increase of temperature, and 22.2% and 32.1% of the original activity were remained with the treatment at $65^{\circ}C$/300 ㎫/10 min, respectively. Enzyme activities decreased slightly with the increase of treatment time at $65^{\circ}C$/300 ㎫.

• Korean Journal of Food Science and Technology
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• v.16 no.2
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• pp.211-217
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• 1984

To study affinity of proteolytic enzymes to soy proteins, the physicochemical and functional properties of enzymatically modified protein products, kinetic parameters and degree of hydrolysis were measured using trypsin, alcalase (serine type protease) and pronase. Bacterial alcalase and pronase showed much greater affinity to soy protein than animal intestinal trypsin. This effect was very significant when unheated soy isolate was used as a substrate. Specific activities of these enzymes decreased with the increment of substrate concentration (over 2.0%, w/v) when heat denatured soy protein was used as a substrate. However, the decrease in specific activity was negligible at substrate concentrations lower than 2.0%. Polyacrylamide gel electrophoretic results showed that the pattern of 2S protein band changed distinctly in alcalase hydrolysis as compared with those of trypsin and pronase. Protein solubilities of alcalase and pronase hydrolyzates increased by 25-30%, at their pI (pH 5.0) over the control. Virtually no change was observed in solubility by trypsin hydrolysis. Heat coagulability and calcium-tolerance of the protein increased by enzymatic hydrolysis. No clear tendency, however, was observed for emulsion properties, foam expansion and the amount of free -SH groups. The enzyme treatment considerably decreased foam stability.