• Journal of Life Science
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• v.29 no.12
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• pp.1408-1414
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• 2019

Enzymes are widely used in industrial applications such as detergents, food, feed production, pharmaceuticals and medical applications and major contributors to clean industrial products and processes. To screen, identify, and characterize the enzymes the zymography techniques are routinely used. The zymography technique is a simple, sensitive, and quantifiable technique that is widely used to detect functional enzymes following electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The method is a versatile two-stage technique involving protein separation by electrophoresis followed by the detection of enzyme activity in polyacrylamide gels under non-reducing conditions. It is based on SDS-polyacrylamide gel (PAG) copolymerization with substrates, which are degraded by the hydrolytic enzymes restored in enzyme reaction buffer after the electrophoretic separation. Any kind of biological sample can be applied and analyzed on zymography, including culture supernatants of microbes, plants extracts, blood, tissue culture fluids, enzymes in foods extracts and metaproteome. The advantage of zymography is that it is possible to directly detect the protein with activity on the electrophoretic gel as well as confirm the activity at the nanogram level. Thus, this zymography technology can be applied in various fields. However, these advantages are rather disadvantageous and can often lead to experimental errors. In this review, the advantages, disadvantages, and problem solving of zymography technique are described.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.118-124
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• 2009

Esterase EM2L8 gene isolated from deep sea sediment was expressed in Escherichia coli BL21 (DE3) and the esterase activity of the cell-free extract was assayed using p-nitrophenyl butyrate-spectrophotometric method. Its optimum temperature was $40-45^{\circ}C$ and 45% activity of the maximum activity was retained at $15^{\circ}C$. The activation energy at $15-45^{\circ}C$ was calculated to be 4.9 kcal/mol showing that esterase EM2L8 was a typical cold-adapted enzyme. Enzyme activity was maintained for 6 h and 4 weeks at $30^{\circ}C$ and $4^{\circ}C$, respectively. When each ethanol, methanol, and acetone was added to the reaction mixture to 15% concentration, enzyme activity was maintained. In the case of DMSO, enzyme activity was kept up to 40% concentration. (S)-4-Chloro-3-hydroxy butyric acid is a chiral intermediate for the synthesis of Atorvastatin, a hyperlipemia drug. When esterase EM2L8 (40 U) was added to buffer solution (1.2 mL, pH 9.0) containing ethyl-(R,S)-4-chloro-3-hydroxybutyrate (38 mM), it was hydrolyzed into 4-chloro-3-hydroxy butyric acid with a rate of $6.8\;{\mu}mole/h$. The enzyme hydrolyzed (S)-substrate more rapidly than (R)-substrate. When conversion yield was 80%, e.e.s value was 40%. When DMSO was added, hydrolysis rate increased to $10.4\;{\mu}mole/h$. The plots of conversion yield vs e.e.s in the presence or absence of DMSO were almost same, implying that the reaction enantioselectivity was not changed by the addition of DMSO. Taken together, esterase EM2L8 had high activity and stability at low temperatures as well as in various organic solvents/aqueous solutions. These properties suggested that it could be used as a biocatalyst in the synthesis of useful pharmaceuticals.

• Geomechanics and Engineering
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• v.12 no.5
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• pp.803-813
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• 2017

This study discussed the possible optimization of enzyme-mediated calcite precipitation (EMCP) as a soil-improvement technique. Magnesium chloride was added to the injection solution to delay the reaction rate and to improve the homogenous distribution of precipitated minerals within soil sample. Soil specimens were prepared in 1-m PVC cylinders and treated with the obtained solutions composed of urease, urea, calcium, and magnesium chloride, and the mineral distribution within the sand specimens was examined. The effects of the precipitated minerals on the mechanical and hydraulic properties were evaluated by unconfined compressive strength (UCS) and permeability tests, respectively. The addition of magnesium was found to be effective in delaying the reaction rate by more than one hour. The uniform distribution of the precipitated minerals within a 1-m sand column was obtained when 0.1 mol/L and 0.4 mol/L of magnesium and calcium, respectively, were injected. The strength increased gradually as the mineral content was further increased. The permeability test results showed that the hydraulic conductivity was approximately constant in the presence of a 6% mineral mass. Thus, it was revealed that it is possible to control the strength of treated sand by adjusting the amount of precipitated minerals.

• Journal of Environmental Health Sciences
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• v.22 no.1
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• pp.1-4
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• 1996

I have conducted two testings to find out which water is better for drinking water. First, I made 20 mM L-DOPA solutions by solving L-DOPA (3,4-Dihydroxyphenylalanine) in tap water, Waters' mineral water and reverse osmotic water. Then I measured activities after adding Tyrosinase (purifide enzyme, step 3), which was extracted from Salanum melongena(mad apple), in each L-DOPA solution. Second, I solved 0.1, 0.5 and 0.9% salt in each 20 mM L-DOPA distilled water to measure activity of each salt solution. The results of the testings are as follows: 1. 10 minutes after adding Salanum melongena(mad apple) tyrosinase in each L-DOPA solution, activity of Waters' mineral water was 0.867 tap water 0.777 and reverse osmotic water 0.742. 2. Activity of Waters' mineral water was higher than that of tap water by 10.4% and higher then reverse osmotic by 14.4%. 3. Activity of Waters' mineral water was much higher than that of 0.9% salt water by 41.8%. 4. The optimum pH of Salanum melongena (mad apple) tyrosinase is 9.0. Most enzymes working in the human metabolism are alkaline and body fluids' pH also alkaline. In conclusion, an alkaline water is believed better than an acidic water for drinking.

• BMB Reports
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• v.32 no.2
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• pp.203-209
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• 1999

Three-week grown Arabidopsis thaliana leaves were wounded by cutting whole leaves with a razor blade into pieces (about$3\;mm\;{\times}\;3\;mm$) submerged in various solutions, and incubated in a growth chamber for 24 h. We measured and compared activities of several enzymes such as phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), thioredoxin, thioredoxin reductase, thioltransferase, glutathione reductase, and $NADP^+$ -malate dehydrogenase. PAL activity was decreased in $HgCl_2$-, $CdCl_2$-, and glyphosate-treated leaf slices, and could not be detected after treatment with $CdCl_2$. TAL activity was found to be maximal in the $CdCl_2$-treated leaf slices. Activity of thioredoxin, a small protein known as a cofactor of ribonucleotide reductase and a regulator of photosynthesis, was significantly increased in the $CdCl_2$-treated leaf slices, while thioredoxin reductase activity was maximal in the $HgCl_2$-treated leaf slices. Thioltransferase and glutathione reductase activities were significantly decreased in the $HgCl_2$-treated leaf slices. $NADP^+$ -malate dehydrogenase activity remained relatively constant after the chemical treatments. Our results strongly indicate that sulfhydryl-related and phenylpropanoid-synthesizing enzyme activities are affected by chemical treatments such as hydrogen peroxide, heavy metals, and glyphosate.

• Biomedical Science Letters
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• v.12 no.4
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• pp.443-450
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• 2006

To demonstrate their possibilities as an enhanced vaccine delivery system, protein-loaded Poly lactide glycolide copolymer (PLGA) microspheres were prepared with different physical characteristics. Ethyl acetate (EA) solvent extraction process was employed to prepare microspheres and the effects of process parameters on drug release properties were evaluated. The biodeuadability of microspheres was also evaluated by the pH change and GPC (Gel permeation chromatography). Primary IgG antibody responses in BALB/c mice were compared with protein saline solutions as negative controls and adsorbed alum suspensions as positive controls after single subcutaneous injection for in vivo studies. The microspheres showed a erosion with a highly porous structure and did not keep their spherical shape at 45 days and this result could be confirmed by GPC. In vitro release of proteinous drug showed initial burst effect in all batches of microspheres, followed by gradual release over the next 4 weeks. PLGA microspheres were degraded until 45 days and the secondary structure of OVA was not affected by the preparation method. Enzyme-linked immunosorbent assays demonstrated that the single subcutaneous administrations of OVA-loaded PLGA microspheres induced enhanced serum IgG antibody response in comparison to negative and positive controls. These results demonstrated that microspheres providing the controlled release of antigens might be useful in advanced vaccine formulations for the parenteral carrier system.

• Preventive Nutrition and Food Science
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• v.6 no.2
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• pp.91-95
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• 2001

Competitive indirect enzyme linked immunosorbent assay (Ci-ELISA) was used to obtain the preliminary data for the detection of irradiated beef. Ci-ELISA was individually formatted with polyclonal antibodies produced from 2 kinds of bovine proteins, myosin and bovine serum albumin (BSA). Beef round, loin and tender loin were vacuum-packaged and subdivided into 3 groups of 1) irradiation; 2) irradiation and chilled at 4$^{\circ}C$ for 7 day; 3) irradiation and frozen at 2$0^{\circ}C$ for 2 months to observe the changes under different storage and/or distribution conditions. Irradiation was performed at 3, 5 and 7 kGy. Protein solutions prepared from the sample were tested by formatted Ci-ELISA. Detected concentrations of myosin and BSA decreased with the increased irradiation dose in all samples with different reduction rates. Myosin was more susceptible to freezing than BSA. Samples irradiated at 5 kGy or above could be differentiated from non-irradiated ones by Ci-ELISA. These results indicate that immunological assay can be used as a detection method for irradiated beef.

• Korean Journal of Environmental Agriculture
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• v.20 no.5
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• pp.340-345
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• 2001

High rates of nitrogen fertilization dangerously increase the nitrate content of vegetable crops, and the accumulation of nitrate in edible crops is undesirable because of potential risks to human health. Micronutrient solution containing Cu, Mn, Mo, Zn was tested for the suppression of nitrate accumulation in lettuce grown in pots treated with Mg fertilizer under a greenhouse condition. The micronutrient solution was sprayed on leaves at 3 and 4 weeks after transplanting of 20-day old seedlings. Plants were harvested after 5-week growth, and yield, contents of chlorophyll, sugar, micronutrient and nitrate, and also nitrate reductase activity were measured. Fresh weight of lettuce was significantly increased by the application of Mg and micronutrients, and the effect was the most significant in the Mg+micronutrient treatment. Also contents of chlorophyll and micronutrients were higher in the plants of micronutrient treatments. Contents of nitrate were reduced by about 14-18% in lettuce with Mg and/or micronutrient applications. Compared to the plants of control treatment, nitrate reductase activity was also higher in those plants treated with micronutrients, and in the treatment of Mg+micronutrients the enzyme activity was six times as high as that of control treatment. Although the effect of mineral nutrients on the suppression of nitrate accumulation in lettuce was relatively small in this study, an appropriate supply of mineral nutrients could be one of the solutions for the nitrate accumulation in vegetables.

• Korean Journal of Plant Resources
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• v.20 no.4
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• pp.348-352
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• 2007

To investigate the effects of soil pH on plants, the seedlings of french marigold (Tagetes patula L.) was transplanted into the soils acidified with $H_{2}SO_{4}$ solutions (pH 5.3, 4.5, 3.9, 3.5). The level of malondialdehyde was significantly increased by soil acidification. As the pH levels decreased from 5.3 to 3.5, the contents of dehydroascorbate and oxidized glutathione of the plant were significantly increased. The antioxidative enzyme activities of the plant affected by soil acidification were increased as the pH decreased.

• The Plant Pathology Journal
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• v.17 no.2
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• pp.88-93
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• 2001

Oxidative stress is one of the major limiting factor in plant productivity. Reactive oxygens species (ROS) generated during metabolic processes damage cellular functions and consequently lead to disease, senescence and cell death. Plants have evolved an efficient defense system by which the ROS is scavenged by antioxidant enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). Attempts to reduce oxidative damages under the stress conditions have included the manipulation of 갠 scavenging enzymes by gene transfer technology. Increased SOD activities of transgenic plants lead to increased resistance against oxidative stresses derived from methyl viologen (MV), and from photooxidative damage caused by high light and low temperature. Transgenic tobacco plants overexpressing APX showed reduced damage following either MV treatment of photooxidative treatment. Overexpression of glutathion reductase (GR) leads to increase in pool of ascorbate and GSH, known as small antioxidant molecules. These results indicate through overexpression of enzymes involved in ROS-scavenging could maintain or improve the plant productivities under environment stress condition. In this study, the rational approaches to develop stress-tolerant plants by gene manipulation of antioxidant enzymes will be introduced to provide solutions for the global food and environmental problems in the $21^\textrm{st}$ century.