• Korean Journal of Microbiology
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• v.30 no.4
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• pp.299-304
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• 1992

An intracellular protease form cells of Pseudomonas carboxydovorans DSM 1227 grown on carbon monoxide was purified 57-fold in six steps to homogeneity with a yield of 4.3% using azocoll as a substrate. The molecular weight of the enzyme was determined to be 150,000. Sodium dodecyl sulfate-gel electrophoresis revealed the purified enzyme to be a dimer with two identical subunits of molecular weight 72,000. The enzyme was stimulated by $Mg^{2+}$ but was inhibited completely by $Cd^{2+}$ $Fe^{2+}$ $Hg^{2+}$, and $^Zn{2+}$ The enzyme activity was also inhibited by EDTA, EGTA, phenylmethylsulfonyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl)carbodiimide, iodoacetamide and dithiothereitol. The optimal pH and temperature for the enzyme reaction were found to be 7-8 and 50.deg.C, respectively. Casein and bovine serum albumin were hydrolyzed by the enzyme, but carbon monoxide dehydrogenase was not.

• Journal of the Korean Chemical Society
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• v.54 no.5
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• pp.567-572
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• 2010

5,10-Methenyltetrahydrofolate synthetase from chicken liver was purified through 30-70% ammonium sulfate fractionation, Q Sepharose Fast Flow anion exchange and Source 15Phe hydrophobic interaction chromatography. Specific activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 0.0085, 0.031, 0.80 and 1.27 U/mg, respectively. Purification fold activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 1, 3.7, 94.1 and 149.4, respectively. HPLC gel permeation chromatography and SDS-polyacrylamide electrophoresis experiments indicated that the enzyme is a monomeric protein with a molecular weight of 22.8 kDa. Km for 5-methyl THF and Mg-ATP were $7.1\;{\mu}M$ and $63\;{\mu}M$, respectively. Optimum temperature and pH were $30^{\circ}C$ and 6.0, respectively. The data for metal ion specificity and stoichiometry showed that the maximum activity was obtained with a 1:l. ratio of $Mg^{2+}$. The ATP and Km values increased in the order of MgATP, MgCTP, MgUTP and MgGTP, and the maximum activities also decreased in the same order, indicating MgATP as the most efficient substrate. The enzyme was chemically modified only by tetranitrometane and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, indicating that tyrosine and carboxylate are present in the active site.

• Membrane Journal
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• v.15 no.1
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• pp.8-14
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• 2005

The hyaluronic acid (HA) with excellent biocompatibility can be combined with the monomer polylactide with good biodegradability to produce biocompatible materials which can control the period of degradation in a human body. By freeze drying method, HA and the lactic acid, monomer of polylactide, or lactide, the ester dimer of polylactide, were crosslinked with crosslinking agent, l-ethyl-3-(3-dimethyl aminopropyl) carbodiimide. The analysis of infrared spectroscopy showed that the ester linkage was formed and the analysis of nuclear magnetic resonance (NMR) spectroscopy showed that the ester linkage was due to the reaction of lactic acid and HA. The conversion (6∼32%) and degree of crosslinking (4∼19%) increased but the selectivity was almost constant at 62% as the mole ratio of LA to HA increased from 1 to 10 in the crosslinking reaction. The brittleness became more pronounced and the rate of degradation became faster with more addition of lactic acid resulting from the higher ratio of LA to HA, and the swelling ratio was in the range of 500 to 2000%.

• Journal of Life Science
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• v.18 no.8
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• pp.1036-1041
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• 2008

Methenyltetrahydrofolate synthetase extract was obtained from mouse liver and purified via $30{\sim}70%$ ammonium sulfate fractionation, Fast Q anion exchange and phenyl agarose chromatography. HPLC gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a monomer with molecular weight of 23 kDa. Optimum temperature and pH were $35^{\circ}C$ and 6.5, respectively. The enzyme was chemically modified only by tetranitromethane and 1-ethyl-3- (3-dimethyl aminopropyl)-carbodiimide (EDC), indicating that tyrosine and carboxylate are in the active site. pH studies showed that 2 tyrosines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that 2 tyrosines bind to ATP and 5-formylTHFand a carboxylate acts as a general base.

• Membrane Journal
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• v.15 no.4
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• pp.281-288
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• 2005

The hyaluronic acid (HA) with excellent biocompatibility can be combined with lactide, the ester dimer of polylactide, with good biodegradability to produce biocompatible materials applicable to drug delivery system. By freeze drying method, HA and lactide were crosslinked with crosslinking agent, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide. Degree of lactide and EDC reaction was determined by the analysis of nuclear magnetic resonance (NMR) spectroscopy. The degree of lactide and EDC reaction increased and swelling ratio decreased as the mole ratio of lactide to HA or crosslinking agent concentration increased or reaction temperature decreased. The drug release experiment result from membranes having different degree of lactide reaction showed that drug release rate reduced in proportion to the degree of lactide reaction. The drug release experiment result from drugs having different hyrodphobicity showed that the more hydrophobic drug was released more slowly.

• Polymer(Korea)
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• v.28 no.2
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• pp.194-200
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• 2004

Porcine small intestine submucosa (SIS) has been widely used as a biomaterial without immunorejection responses. Crosslinked SIS sponges were characterized for the possibility of the bio-interactive wound dressings and tissue engineered scaffolds. SIS powders were dissolved in 3% acetic acid aqueous solution at 48hrs followed by pouring into mold and then fabricated by freeze-drying method. SIS sponge was prepared by crosslinked with 1-ethyl-(3-3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) solution (deionized water: ethanol=5:95) with 1-100mM concentration for 24 hrs and Iyophilized. SIS sponges were characterized by scanning electron microscopy, differential scanning calorimeter, and Fourier transform infrared spectrometer and were tested their porosity and water absorption ability. It was observed that the concentration of EDC might be exceeded 50 mM to get good physical characteristics. In conclusion, it seems that SIS sponge could be very useful for the applications of wound healing and tissue construction.

• Polymer(Korea)
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• v.29 no.6
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• pp.599-604
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• 2005

The hyaluronic acid (HA) with excellent biocompatibility has been combined with lactide, the ester dimer of polylactide, with good biodegradability to produce biocompatible materials which can control the period of degradation in a human body. By freeze frying method, HA and lactide were crosslinked with crosslinking agent, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Degree of lactide and EDC reaction was determined by the analysis of nuclear magnetic resonance spectroscopy. Both lactyl group and EDC conversion increased as the mole ratio of lactide to HA increased from 5 to 13. The membrane swelled less and became more brittle with the more addition of lactyl group resulting from the higher mole ratio of lactide to HA. Swelling ratio decreased and tensile modulus increased due to the more addition of lactyl group as the EDC concentration increased or reaction temperature decreased. Drug release experiment from various membranes with different degree of crosslinking showed that permeability decreased with increasing degree of crosslinking. The degradation became slower with the more addition of lactyl group. Mechanical property and degradation rate of the synthesized membrane were shown to be controlled through adjusting operation parameters such as mole ratio, temperature, and crosslinking agent concentration.

• Membrane Journal
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• v.19 no.4
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• pp.310-316
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• 2009

The degradation characteristics of cross-linked lactide/hyaluronic acid (LA/HA) membranes were investigated for purpose of applying to tissue engineering. The lactide/hyaluronic acid cross-linked with 1,3-butadiene diepoxide (BD) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) was degradated in deionized water in water bath at $37^{\circ}C$. As the LA/HA mole ratio or crosslinking agent concentration decreased, the degradation rate of the crosslinked membranes increased. In order to investigate the structure change of the membrane in the degradation process, the control sample and 3, 6, 9 days-degradated samples were analysed by the nuclear magnetic resonance spectroscopy. In case of the membranes crosslinked with EDC, the HA-EDC bonding structure was degradated slowly whereas the HA-LA bonding structure was degradated quickly and dissappeared completely after 6 days. In case of the membranes crosslinked with BD, all the crosslinked bonding structure degradated slowly. The HA-BD bonding structure maintained its original state about 89, 83% in case of 3, 6 days-degardated samples respectively whereas the HA-LA bonding structure maintained its original state about 83, 65%. The scanning electron microscopy of the degradated membranes showed that the pore density in the surface, and the structure in the surface and cross section, of the before and after-degradation membranes did not change greatly, so the membranes was shown to be applied to materials for tissue engineering.

• KSBB Journal
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• v.21 no.4
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• pp.255-259
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• 2006

The biodegradable hyaluronic acid(HA) membranes cross-linked with lactide using the crosslinking agent, 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide(EDC) were prepared as a potential biocompatible material for tissue engineering. HA membranes having different mechanical properties were synthesised by varying degree of the mole ratio of lactide to HA, EDC concentration, and crosslinking temperature. HA membranes were degradable in water solution and the degradation became slower with the increasing mole ratio of lactide to HA. HA membranes were sterilized using ethylene oxide gas and extracted with cell culture medium for 24 h at $37^{\circ}C$ and 200 rpm. Cytotoxicity of the extract was tested using NIH/3T3 mouse embryo fibroblast as a model cell. Growth inhibition was not observed in the extracts of HA membranes with the mole ratios of lactide to HA, 5 or 10, and 10% EDC concentration, however 11% of growth inhibition was observed in the extract with the mole ratio of 13. Growth inhibition was not observed in the extracts of HA membranes prepared with 5% EDC or 10% EDC and the mole ratio of lactide to HA, 10, however 12% of growth inhibition was observed in the extract with 20% EDC. Cytotoxicity was not observed in the extracts of HA membranes prepared at varying crosslinking temperatures, $15^{\circ}C,\;25^{\circ}C,\;and\;28^{\circ}C$ with the mole ratio of lactide to HA, 10 and 10% EDC.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.35 no.2
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• pp.39-45
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• 2003

This study was performed to develop the new type enzyme immobilization sheet from carboxymethylated refiner mechanical pulp (CRMP) substrate. Enzyme immobilization was attempted to couple carboxyl groups of CRMP with amino groups of the enzyme, trypsin, through the reaction of carbodiimide reagent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodimide (EDC ). Immobilization carrier, water insoluble CRMP fraction (CRMP-IS), was successfully reacted with the enzyme, formed peptide linkage like -CONH- at 1680$cm^{-1}$ / and new ester linkage like -COO$CH_3$, methylester at 1735$cm^{-1}$ /, and produced enzyme immobilized substrate (CRMP-IST). The enzyme immobilized handsheet was prepared by mixing the above chelated enzyme immobilized substrate(CRMP-IST) with kraft pulp by paper sheet machine like papermaking process. The sheet weight and strength were increased with increasing dosage of CRMP-IST, and decreased at more than 10％ mixing of CRMP-IST, but higher than the controls. Concerning activities of immobilized trypsin(CRMP-IST) sheet by caseinolysis, the teared-off sheet with shaking was shown higher enzyme activities than sheet shape without shaking. In conclusion, this enzyme immobilized sheet would be expected easy handling for practical application and reutilization.