Based on ab initio calculations at the MP2(FULL)/6-31+G**//RHF/6-31G** level, we compare the energetic and mechanistic features of a model reaction for catalytic action of Δ?-3-ketosteroid isomerase (KSL,E.C.5.3,3.1) with those of a corresponding nonenzymatic reaction in aqueous solution. The results show that the two catalytic acid residues,Tyr14 and Asp99, can lower the free energy of activation by 8.6kcal/mol, which is in good agreement with the experimentally predicted~9 kcal/mol,contribution of electrophilic catalyses to the whole enzymatic rate enhancement. The dienolate intermediate formed by proton transfer from the substrate carbon acid to the catalytic base residue (Asp38) ins predicted to be stabilized by 12.0 kcal/mol in the enzymatic reaction, making its formation thermodynamically favorable. It has been argued that enzymes catalyzing the reactions of carbon acids should resolve the thermodynamic problem of stabilizing the enolate intermediate as well as the kinetic porblem of lowering the free energy of activation for porton abstraction. We find that KSI can successfully overcome the thermodynamic difficulty ingerent in the nonenzymatic reaction through the electrophilic catalyses of the two acid residues. Owing to the stabilization of dienolate intermediate, the reketonization step could influence the overall reaction rate more significantly in the KSI- catalyzed reaction than in the nonenzymatic reaction, further supporting the previous experimental findings. However, the electrophilic catalyses alone cannot account for the whole catalygic capability (12-13 kcal/mol), confiming the earlier experimental implications for the invement of additional catalytic components. The present computational study indicates clearly how catalytic residues of KSI resolve the fundamental problems associated with the entropic penalty for forming the rate-limiting transition state and its destabilization in the bulk solvation environment.
Soil actinomycetes of 703 strains were isolated from 25 sampling points in Cheju Island using 4 different media. Arginine glycerol salts agar containing soil extract was found to be the best medium for the isolation of soil actinomycetes. Soil samples from pasture land showed higher population and diversity of the actinomycetes than those from citrus field, forest, island, hill or valley. When the antibacterial activity of the 526 isolates was tested against three bacterial strains, Escherichia coli, Staphylococcus aureus and Pseudomonas solanacearum the frequency of the isolates with antibacterial activity varied much depending upon the media used for isolation and cultivation. BL106Ba, one of the 10 isolates that showed antibacterial activity against all the above 3 test strains, was chosen based upon the pH and heat stability of its antibacterial metabolites, and was identified as Streptomyces sp. based upon its cultural, morphological and physiological characteristics. The partially purified white crystalline substance obtained from the culture supematant of BL1063a through cation exchange chromatography(AG MP-50) and three times consecutive gel filtration(Sephadex G-10) showed high antimicrobial activity against gram positive and negative bacteria, but low activity against yeasts. The partially purified substance was found to contain at least four different compounds with antibacterial activity by both thin layer chromatography and high performance liquid chromatography.
Journal of the Korean Society of Food Science and Nutrition
/
v.17
no.2
/
pp.149-157
/
1988
This study was carried out to prepare the flavoring substance using sardine for instant soup, and to examine the taste compounds and storage stability of the product. In preparation of product, raw sardine are gutted, boiled for 10 minutes and smoked 3 times to $9{\sim}10%$ moisture content at $80^{\circ}C$ for 8 hours. The smoked-dried sardine meat were followed to be 50 mesh of particle size. The powdered-dried sardine were mixed 4.0% sugar, 20.0% table salt, 3.0% monosodium glutamate, 0.2% black pepper, 0.2% garlic powder and 0.2% onion powder, Finally the powdered instant soup product were vacuum packed in a laminated film(PET/A1 foil/CPP) bag, and then stored at room temperature for 120 days. The effect of smoking on enhancing flavor and on preventing lipid oxidation of product during storage were observed. From the chemical analysis and omission test, the principal taste compounds of product were IMP, 478.2mg/l00g; free amino acids such as glutamic acid, histidine, arginine, phenylalaine 3292.5mg/l00g; non-volatile organic acids such as lactic acid, ${\alpha}-ketoglutaric$ acid, 712.2mg/l00g; total creatinine 409.0mg/100g, and small amount of betaine, TMAO. Fatty acid composition of product were mainly consisted of polyenoic acids such as 20:5, 22:6, followed by saturated acids, monoenoic acid. The major fatty acid were 16:0, 16:1, 18:1, 20:5 and 22:6. From the results of sensory evaluation and chemical experiments during storage, the vacuum packed product were good condition for preserving the quality during storage for 120 days. We may conclude that the quality of present product was not inferior to that of seasoning powder of anchovy on the market, and it can be commercialized as a flavoring substance in preparing soup and broth.
An adenine-guanine doubless and $\beta$-alanine requiring mutant, D-1550-40, which had been derived from Brevibacterium ammoniagenes ATCC 6872, produced a copious amount of xanthosine-5'-monophosphate (XMP). The optimum concentration of adenine and guanine for maximal accumulation of XMP was about 75 ml/l and 100 ml/l for growth. Concentrations higher than 100 mg/l of adenine and guanine inhibited cell growth and XMP accumulation strongly. The inhibition, however, could be recovered by adding $100{\mu}g$ of biotin per liter or 0.3% of casamino acids to the culture solution. High concentrations of phosphate and magnesium salts (1.0 to 1.5%(w/v) in media) were found to be indispensable for XMP accumulation, and the presence of manganese in the culture medium stimulated both growth of cells and accumulation of XMP leaving 5'-inosinic acid unaffected. The maximal accumulation of XMP reached to 60.5 mg/l after 4 days of fermentation which had been started with a medium containing 100 mg of adenine-guanine, 5 mg of $MnSO_4{\cdot}H_2O$ and $100{\mu}g$ of biotin per liter. The specific XMP synthesis(mg of XMP/mg of cells) was increased with the increase of the cell growth rate.
This study an analyzes the effects of the P/S ratio of dietary lipids and antioxidant vitamin supplements on serum lipids level and fatty acid profile, the degree of lipid peroxidation, and the antioxidant enzyme activities in the liver of rats treated with 7,12-dimethylbenz($\alpha$) anthracene(DMBA). P/S ratio of dietary lipids was made into 0.5, 1 and 2 by mixing palm oil, soybean oil, sesame oil and perilla oil at 10%(w/w) fat level and n-6/n-3 ratio was fixed to 4. Antioxidant vitamin of $\alpha$-tocopherol or $\beta$-carotene was supplemented in addition to vitamin mixture which was given at 1 % of the standard diet. female Sprague-Dawley strain rats, about 60 days old, were divided into three groups(LP : low P/S ratio(0.5), MP : medium P/S ratio (1.0), HP , high P/S ratio(2.0)) and each group was sub-divided into three groups(S ; standard, T ; tocopherol supplemented, C : carotene supplemented): Two weeks after feeding experimental diets, all groups were treated with a single dose of DMBA(2mg/100g BW) by gastric intubation and fed experimental diet for 9 week. The results were as follows ; 1) Serum total cholesterol(TC) level was not significantly influenced by diet but tended to be lower in HP groups compared to LP and MP groups. Triglyceride level was the highest in LP groups and the lowest in $\alpha$-tocopherol supplemented groups. 2) Thiobarbituric acid reactive substance(TBARS) level, representing lipid peroxidation in hepatic microsome, tended to be increased as the unsaturation of dietary lipids increases. $\alpha$-Tocopherol supplement significantly decreased TBARS level. 3) The activities of superoxide dismutase(SOD) and glutathione peroxidase(GSHPx) in hepatic cytosol showed the tendency to be high with increasing P/S ratio of dietary lipids. SOD activity was not significantly influenced by antioxidant vitamin, but GSHPx activity was significantly increased in $\alpha$-tocopherol supplemented groups. In summary, high polyunsaturated fat diet was effective on reducing the serum level of total cholesterol and triglyceride, while it increased unsaturation and peroxidizability of serum fatty acid. With increasing P/S ratio of dietary lipids, lipid peroxidation was increased in the liver and antioxidant enzyme system was induced to inhibit lipid peroxidation against oxidative damage. $\alpha$-Tocopherol supplement was effective in lowering lipid peoxidation, but $\beta$-carotene supplement did not exhibit antioxidant effect. (Korean J Nutrition 31(5) 906~913, 1998)
The effects of different concentrations (0, 0.3, 0.6 and 0.9%) of fish feed additives (Hanbangchun and Olyukchun) utilizing effective microorganisms and herb medicine on activity of liver function were examined in olive flounder, Paralichthys olivaceus, Moreover, we investigated the characteristics of the additives. Total number of microorganisms (Lactic acid bacteria, Bacillus subtilis, Saccharomyces cerevisiae, Photosynthetic bacteria and Azotobactor) in the additives was 5.6${\times}$10$^{8}$ CFU/g in the Hanbangchun and 3.0${\times}$10$^{8}$ CFU/g in the Olyukchun. Levels of three typical pathological microorgamisms (Edwardsiella tarda, Vibrio anguillarum and Streptococcus sp.) in moist pellets (MP) were significantly decreased by the additives in a concentration-dependent way. Hepatosomatic index of fish in the 0.3% group was significantly increased. Total serum protein was increased in all the groups containing additives, but the protein content in liver was higher in the control group. Higher activities of catalase and superoxide dismutase which are involved in physiological defense mechanisms were found in the dietary groups containing 0.3% and 0.6%, respectively. These results suggest that the additives, Hanbangchun and Olyukchun, can increase tolerance of olive flounder against stress and hypoxic conditions by increasing activities of body antioxidant enzymes.
Objective: Dairy cattle nutrient requirement systems acknowledge amino acid (AAs) requirements in aggregate as metabolizable protein (MP) and assume fixed efficiencies of MP used for milk protein. Regulation of mammary protein synthesis may be associated with AA input and milk protein output. The aim of this study was to evaluate the effect of nanoemulsified methionine and cysteine on the in-vitro expression of milk protein (casein) in bovine mammary epithelial cells (MAC-T cells). Methods: Methionine and cysteine were nonionized using Lipoid S 75 by high-speed homogenizer. The nanoemulsified AA particle size and polydispersity index were determined by dynamic light scattering correlation spectroscopy using a high-performance particle sizer instrument. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cytotoxicity effect of AAs with and without nanoionization at various concentrations (100 to $500{\mu}g/mL$) in mammary epithelial cells. MAC-T cells were subjected to 100% of free AA and nanoemulsified AA concentration in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12) for the analysis of milk protein (casein) expression by the quantitative reverse transcription polymerase chain reaction method. Results: The AA-treated cells showed that cell viability tended to decrease (80%) in proportion to the concentration before nanogenesis, but cell viability increased as much as 90% after nanogenesis. The analysis of the expression of genetic markers related to milk protein indicated that; ${\alpha}_{s2}$-casein increased 2-fold, ${\kappa}$-casein increased 5-fold, and the amount of unchanged ${\beta}$-casein expression was nearly doubled in the nanoemulsified methionine-treated group when compared with the free-nanoemulsified methionine-supplemented group. On the contrary, the non-emulsified cysteine-administered group showed higher expression of genetic markers related to milk protein ${\alpha}_{s2}$-casein, ${\kappa}$-casein, and ${\beta}$-casein, but all the genetic markers related to milk protein decreased significantly after nanoemulsification. Conclusion: Detailed knowledge of factors, such nanogenesis of methionine, associated with increasing cysteine and decreasing production of genetic markers related to milk protein (casein) will help guide future recommendations to producers for maximizing milk yield with a high level of milk protein casein.
The activity changes and biochemical properties of ${\beta}$-gal in tomato fruits during ripening were investigated. The total activity was increased during ripening and three isoenzymes (${\beta}$-gal I, II and III) were purified through DEAE Sephadex A-50 and Sephadex G-100 column chromatography. The activities of ${\beta}$gal isoenzymes (${\beta}$-gal I, II and III) during ripening were 69.8, 31.8 and 170.0 units in mature green phase, while those were 48.7, 88.4 and 136.8 units in Red phase, respectively. As the ripening proceeded the activities of ${\beta}$-gal I and III were some what decreased but the activity of ${\beta}$-gal II was incresed more than 2.8 fold. The optimum pH of ${\beta}$-gal I, II and III were 3.9, 4.2 and 3.9 and the optimum temperature of those were $60^{\circ}C$, $56^{\circ}C$ and $60^{\circ}C$, respectively. All isoenzymes were stable at pH 3.6~6.0 and lost their activity about 50% when it heated at $55^{\circ}C$ for 5 minute. $Mg^{{+}{+}}$-activated the three isoenzymes but $Ca^{{+}{+}}$ and SDS inhibited about 30~40%. $Hg^{{+}{+}}$ inhibited completely. The km value of ${\beta}$-gal I, II and III was 0.36mM, 0.63mM and 0.45mM, reaction rate was rapidly increased until the concentration of substrate was $6.0{\times}10^{-5}M$.
The $H_2Ge=Ge:$ and its derivatives ($X_2Ge=Ge:$, X = H, Me, F, Cl, Br, Ph, Ar${\ldots}{\ldots}$) is a new species. Its cycloaddition reactions is a new area for the study of germylene chemistry. The mechanism of the cycloaddition reaction between singlet state Cl2Ge=Ge: and formaldehyde has been investigated with CCSD(T)//MP2/$6-31G^*$ method. From the potential energy profile, it could be predicted that the reaction has only one dominant reaction pathway. The reaction rule presented is that the two reactants first form a fourmembered Ge-heterocyclic ring germylene through the [2+2] cycloaddition reaction. Because of the 4p unoccupied orbital of Ge: atom in the four-membered Ge-heterocyclic ring germylene and the ${\pi}$ orbital of formaldehyde forming a ${\pi}{\rightarrow}p$ donor-acceptor bond, the four-membered Ge-heterocyclic ring germylene further combines with formaldehyde to form an intermediate. Because the Ge: atom in intermediate hybridizes to an $sp^3$ hybrid orbital after transition state, then, intermediate isomerizes to a spiro-Ge-heterocyclic ring compound via a transition state. The research result indicates the laws of cycloaddition reaction between $H_2Ge=Ge:$ and formaldehyde, and laid the theory foundation of the cycloaddition reaction between $H_2Ge=Ge:$ and its derivatives ($X_2Ge=Ge:$, X = H, Me, F, Cl, Br, Ph, Ar${\ldots}{\ldots}$) and asymmetric ${\pi}$-bonded compounds, which is significant for the synthesis of small-ring and spiro-Ge-heterocyclic compounds. The study extends research area and enriches the research content of germylene chemistry.
The purpose of this study was to investigate the radiation protection effect of blueberries. The experimental animals used in this study were 8-week-old 21 SD male rats weighed 280-300 g. The animals were set to a normal group (A), a 5 Gy control group (B), and a 5 Gy experimental group (C) of seven rats each, and (50 mg/kg/day) of physiological saline solution of blueberries were orally administered twice a day with an oral dose of (200 mg/kg/day) for seven days and 5 Gy of radiation was irradiated in the case of groups B and C. As a result, it was identified that there was significance in white blood cells in this study (p<0.000). There was no significant difference in red blood cells or platelets. When examined in detail, among white blood cells (WBC), neutrocytes were found to be significantly different among the three groups: normal, control, and experimental groups (p<0.004). Lymphocytes were also found to be statistically significantly different among the three groups (p<0.000). Monocytes were not found to be statistically significantly different (p<0.483). When red blood cells (RBC) were examined, hemoglobin (HGB) was not found to be statistically significant different among the three groups (p<0.291). Hematocrit (HCT) was not found to be statistically significantly different among the three groups, either (p<0.564). Mean corpuscular volume (MCV) was found to be statistically significantly different among the three groups (p<0.001). Mean corpuscular hemoglobin (MCH) was also found to be statistically significantly among the three groups (p<0.028). Mean corpuscular hemoglobin concentration (MCHC) was found to be statistically significantly different among the three groups(p<0.020). Red blood cell distribution width (RDW) was not found to be statistically significantly different among the three groups (p<0.09). When platelets (PLT) were examined in detail, mean platelet volume (MpV) was found to be statistically significantly different among the three groups (MpV) (p<0.04). In conclusion, based on this study, blueberries are considered to have radiation protection effects.
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