• Biomolecules & Therapeutics
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• v.21 no.6
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• pp.487-492
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• 2013

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

• Journal of Plant Biotechnology
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• v.46 no.4
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• pp.338-345
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• 2019

The selection of a suitable solvent is very important when preparing an extract. However, the effect of ethanol solvent concentration in the extraction of sweet wormwood tea has not been reported. Thus, extracts were prepared from sweet wormwood tea using water and various ethanol concentrations, and the phenolic compounds, antioxidants and anti-enzyme activities of the extracts were analyzed. The phenolic acid and flavonoid components differed according to extraction solvent, which also resulted in different antioxidant and antienzyme activities. In particular, flavonoid rhamnetin was not extracted using 80% and 99.5% ethanol and was highest when 60% ethanol was used for extraction. In the case of chlorogenic acid, the highest extraction efficiency was obtained with 80% ethanol. These results suggest the need for research to increase specific extraction efficiency by targeting major compounds that affect physiological activity.

• Journal of Nutrition and Health
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• v.32 no.4
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• pp.353-368
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• 1999

This study was performed to investigate the effects of Korean native plant diets on lipid and cadmium (Cd) metabolisms and also antioxidative enzyme activities in rats. Seventy male Sprague-Dawley rats weighing 210.3$\pm$2.7g were blocked into ten groups according to body weight. Rats were raised for four weeks with diets containing 0 or 0.04%(w/w) cadmium chloride and 5%(w/w) plant powder-Ssook(Artemisia princeps var. orientalis HARA), Chamchwi(Aster scaber THUNB), Gomchwi(Lingularia fischeri TUBCZ). or Solbeerum (Portulaca oleracea LINNE). Food intake, weight gain, food efficiency ratio, and weights of liver, kidney, spleen and epididymal fat were significantly lower in Cd-exposed groups, food efficiency ratio, and weights of liver, kidney, spleen and epididymal fat were significanlty lower in Cd-exposed groups. Plasma total lipid level, liver total lipid, cholesterol and triglycerde concentrations, and fecal total lipid, cholesterol and triglyceride excretions were decreased by Cd administration. Total lipid and triglyceride concentration in plasma and liver were lower in Chamchwi groups together with increased fecal total lipid and triglyceride excretions. Cholesterol concentration in plasma and liver were lower in Gomchwi groups with increased fecal cholesterol excretions. Activities of red-blood-cell superoxide dismutase(SOD), and liver catalase, SOD and glutathione peroxidase (GSH-px) were decreased significantly by Cd administration. Thiobarbituric acid reactive substance(TBARS) concentrations of plasma and liver were increased by Cd administration. Thiobarbituric acid reactive substance(TBARS) concentration of plasma and liver were increased by Cd administration. Administration of plant diets decreased plasma and liver catalase, SOD and GSH-px activities in Cd-free diet groups and increased them in Cd-administered groups. Plasma and liver TBARS concentrations were decreased in animals fed plant diets, with Chamchwi showing the most effective antioxidative capacity. The concentration of Cd accumulated in blood and tissue decreased in Cd-exposed groups fed plant diets. Liver Cd concentration were lower in the Chamchwi and Gomchwi groups, and kidney Cd levels were lower in the Ssook and Chamchwi groups. Among them, Chamchwi was the most effective in Cd detoxification. In conclusion, Chamchwi and Gomchwi were effective in lowering tissue lipid levels, with Chamchwi having the greatest antioxidative and detoxifying effects.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.9
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• pp.1255-1261
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• 2013

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.

• KSBB Journal
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• v.31 no.2
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• pp.105-112
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• 2016

Ferritin is known to regulate iron metabolism and maintain iron in a variety of the eukaryotic organisms. The region encoding the mature ferritin (0.47 kb, H-type) of Periserrula leucophryna was amplified using the designed primers including restriction enzyme site and termination codon and subcloned in frame to the pRBAS secretion vector containing the signal sequence, RBS, and promoter of amylase gene (E. coli-Bacillus shuttle vector), resulting in recombinant pRBAS-PLF vector. Recombinant ferritin (18 kDa) was correctly processed and secreted from Bacillus subtilis LKS strain harboring the pRBAS-PLF vector and quantitatively analyzed by SDS-PAGE and western blot, respectively. Secretion of the ferritin was optimized by culture conditions (host, medium, temperature, nitrogen source) in 3 L batch culture and 5 L jar fermenter. Finally. the ferritin was largely produced using 50 L fermenter as the following conditions; at $30^{\circ}C$, 150 rpm, 1 vvm in Bacillus subtilis LKS using PY medium. The secreted ferritin was maximally measured (approximately 177.6 ug/ml) when the cell density reached to 14.4 at $OD_{600}$ (20 h incubation). The iron binding activity was confirmed by Perls' staining in 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in the culture broth after secretion. Biologically, the culture broth and powder type containing ferritin were tested for possibility as feed additive in chicken broiler. As a result, the ferritin stimulated the growth of chick broil and improved feed efficiency and production index.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1471-1480
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• 2010

An extremely thermostable xylanase gene, xynB, from the hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. The response surface methodology (RSM) was also applied to optimize the medium components for the production of XynB secreted by the recombinant K. lactis. The secretion level (102 mg/l) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/l, 16 U/ml) in the original medium (yeast extract, lactose, and peptone; YLP). The secretory efficiency of mature XynB was also improved when using the YLU medium. When the mRNA levels of 13 characterized secretion-related genes in the K. lactis cultured in YLP and YLU were detected using a semiquantitative RT-PCR method, the unfolded protein response (UPR)-related genes, including ero1, hac1, and kar2, were found to be up-regulated in the K. lactis cultured in YLU. Therefore, the nutrient ingredients, especially the nitrogen source, were shown to have a significant influence on the XynB secretory efficiency of the host K. lactis.

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.269-280
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• 2010

Lilium ${\times}$ formolongi was genetically engineered by Agrobacterium-mediated transformation with the plasmid pCrtZW-N8idi-crtEBIY, which contains seven enzyme genes under the regulation of the CaMV 35S promoter. In the transformants, ketocarotenoids were detected in both calli and leaves, which showed a strong orange color. In transgenic calli, the total amount of carotenoids [133.3 ${\mu}g/g$ fresh weight (FW)] was 26.1-fold higher than in wild-type calli. The chlorophyll content and photosynthetic efficiency in transgenic orange plantlets were significantly lowered; however, after several months of subculture, they had turned into plantlets with green leaves that showed significant increases in chlorophyll and photosynthetic efficiency. The total carotenoid contents in leaves of transgenic orange and green plantlets were quantified at 102.9 and 135.2 ${\mu}g/g$ FW, respectively, corresponding to 5.6- and 7.4-fold increases over the levels in the wild-type. Ketocarotenoids such as echinenone, canthaxanthin, 3'-hydroxyechinenone, 3-hydroxyechinenone, and astaxanthin were detected in both transgenic calli and orange leaves. A significant change in the type and composition of ketocarotenoids was observed during the transition from orange transgenic plantlets to green plantlets. Although 3'-hydroxyechinenone, 3-hydroxyechinenone, astaxanthin, and adonirubin were absent, and echinenone and canthaxanthin were present at lower levels, interestingly, the upregulation of carotenoid biosynthesis led to an increase in the total carotenoid concentration (+31.4%) in leaves of the transgenic green plantlets.

• Journal of Food Hygiene and Safety
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• v.6 no.3
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• pp.127-131
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• 1991

${\alpha}-amylase\;and\;{\beta}-amylase$ were extracted from barley and wheat malt, respectively. Their kinetic parameters on gultinous and nonglutinous rice starch were examined. During the germination of barley and wheat, the increaments of ATP levels were significant after 2-day germination and the levels were reduced after 5 days. The dry weights were decreased after 3 days. The activities of amylases were the highest for 6 days in the barley and wheat malt. As for ${\alpha}-amylase$, that the substrate affinity of barley malt on nonglutionous rice starch was greater than other cases. The $V_{max}$ values of ${\alpha}-amylase$ from wheat malt on either type of rice starch showed high, and from barley malt on nonglutinous rice starch were high. The ${\beta}-amylse$ from barley malt showed high substrate affinity on the glutinous rice starch, and $V_{max}$ value of the enzyme from wheat malt on glutinous rice starch was higher than other. The substrate efficiency ($V_{max}/K_{m}$) of ${\beta}-amylase$ on the non glutinous rice strach was better than other cases.

• Archives of Pharmacal Research
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• v.27 no.5
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• pp.570-575
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• 2004

2-Hydroxymuconic semialdehyde (2-HMS) dehydrogenase catalyzes the conversion of 2-HMS to 4-oxalocrotonate, which is a step in the meta cleavage pathway of aromatic hydrocarbons in bacteria. A tomC gene that encodes 2-HMS dehydrogenase of Burkholderia cepacia G4, a soil bacterium that can grow on toluene, cresol, phenol, or benzene, was overexpressed into E. coli HB 101, and its gene product was characterized in this study. 2-HMS dehydrogenase from B. cepacia G4 has a high catalytic efficiency in terms of V$_{max}$K$_{max}$ towards 2-hydroxy-5-methyl-muconic semialdehyde followed by 2-HMS but has a very low efficiency for 5-chloro-2-hydroxymuconic semialdehyde. However, the enzyme did not utilize 2-hydroxy-6-oxo-hepta 2,4-dienoic acid and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid as substrates. The molecular weight of 2-HMS dehydrogenase from B. cepacia G4 was predicted to be 52 kDa containing 485 amino acid residues from the nucleotide sequence of the tomC gene, and it exhibited the highest identity of 78% with the amino acid sequence of 2-HMS dehydrogenase that is encoded in the aphC gene of Comamonas testosteroni TA441. 2-HMS dehydrogenase from B. cepacia G4 showed a significant phylogenetic relationship not only with other 2-HMS dehydrogenases, but also with different dehydrogenases from evolutionarily distant organisms.sms.

• Proceedings of the KSR Conference
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• 2005.11a
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• pp.1171-1176
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• 2005

The latest technical development of steel plate girder railway bridge are developing in ways to maximize its durability of materials in use of high strength steel and efficiency of maintenance and management by the introduction of simplified and standardization ideas. In addition to this, it is also expected to reduce the cost of bridge construction and to simplify the process of bridge manufacturing. Referring to this, composite few plate girder railway bridge is highly recommendable that is very economical with the fine exterior. In this paper, it will analyse the variation of dynamic behavior of existing composite few plate girder railway bridge with ballast caused by modified Slab Track through interpretation of limited enzyme in order to obtain the existing data for improvement of Slab Track system from Ballast Track system. Consequently, it can help maximize economic efficiency and structural capability. As a results, although the natural frequency by modified Slab Track are decreased, it is hardly influencing on the safety of railway bridges. It is also evident in the case of slab deck with a reduced scale in comparison with Ballast Track. Therefore, it is expected to reduce the cost of a railway bridge plan. And, it can expect the synergistic effect of the ensure long term durability of bridge caused by decreased stresses of bottom flange due to reduced dead load. As a result, the analytical study are carried out to investigate the composite few plate girder railway bridge could be the optimal design method for the dynamic safety of a girder section.