• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1249-1253
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• 2007

A hyperthermostable endoglucanase from Pyrococcus horikoshii with the capability of hydrolyzing crystalline cellulose was analyzed. A protein engineering study was carried out to obtain a reduced-size mutant. Five amino acid residues at both the N- and C-terminus were found to be removable without any loss of activity or thermal stability. Site-directed mutagenesis was also performed on R102, N200, E201, H297, Y299, E342, and W377, residues possibly involved in the active center or in the recognition and binding of a cellulose substrate. The activity of the resulting mutants was considerably decreased, confirming that the mutated residues were all important for activity. A reduced-size enzyme, as active as the wild-type endoglucanase, was successfully obtained, plus the residues critical for its activity and specificity were confirmed. Consequently, an engineered enzyme with a reduced size was obtained, and the amino acids essential for activity were confirmed by site-directed mutagenesis and comparison with a known three-dimensional structure.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.70-70
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• 2003

Active sites and substrate bindings of 1-aminoxyclopropane-1-carboxylate oxidase (MD-ACO1) catalyzing the oxidative conversion of ACC to ethylene have been determined based on site-directed mutagenesis and comparative modeling methods. Molecular modeling based on the crystal structure of Isopenicillin N synthase (IPNS) provided MD-ACO1 structure. MD-ACO1 protein folds into a compact jelly roll shape, consisting of 9 ${\alpha}$-helices, 10 ${\beta}$-strands and several long loops. The MD-ACO1/ACC/Fe(II)/Ascorbate complex conformation was determined from automated docking program, AUTODOCK. The MD-ACO1/Fell complex model was consistent with well known binding motif information (HIS177-ASP179-HIS234). The cosubstrate, ascorbate is placed between iron binding pocket and Arg244 of MD-ACO1 enzyme, supporting the critical role of Arg244 for generating reaction product. These findings are strongly supported by previous biochemical data as well as site-directed mutagenesis data. The structure of enzyme/substrate suggests the structural mechanism for the biochemical role as well as substrate specificity of MD-ACO1 enzyme.

• Molecules and Cells
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• v.45 no.7
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• pp.495-501
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• 2022

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD⁺ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60℃ and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD⁺) were determined. The crystal structure represents that the subunit has more compact structure than homologs' structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

• The Transactions of The Korean Institute of Electrical Engineers
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• v.61 no.2
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• pp.270-275
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• 2012

This paper presents a portable and compact optical device which can conveniently be used to perform a functional analysis of human liver function. The proposed system employed red/green LEDs, as a light source, and CMOS image sensor, which is commonly used in cellular phones. With this system, several blood serum samples have been evaluated for liver functional analysis by measuring light absorption level through the blood serum samples depending on aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin concentration. The light absorption through the blood serum samples containing AST, ALT, or total bilirubin can provide their concentrations. The green light absorption is more sensitive to the concentration of AST or ALT, and the red light absorption is more sensitive to the total bilirubuin concentration. Additional calibration steps were performed by using a MATLAB program in order to eliminate the light scattering effects from the extraneous particles existing in each blood serum sample. From the blind test, three standard light intensity curves through each enzyme have been obtained and the enzyme concentration values have been compared to those obtained from a commercially available biochemistry analyzer (Toshiba 200 FR). The average percent difference in the obtained concentrations from two systems for AST, ALT, and total bilirubin concentration came out to be 7.79%, 7.98%. and 7.56%, respectively, with the adjusted coefficient of determination (R2) higher than 0.98. This system can possibly lead to a low-cost and simple system that can be used as a point-of-care (POC) system in a condition without advanced equipments.

• Journal of Electrochemical Science and Technology
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• v.8 no.2
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• pp.87-95
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• 2017

Enzymatic fuel cells are promising low cost, compact and flexible energy resources. The basis of enzymatic fuel cells is transfer of electron from enzyme to the electrode surface and vice versa. Electron transfer is done either by direct or mediated electron transfer (DET/MET), each one having its own advantages and disadvantages. In this study, the DET and MET of laccase-based biocathodes are compared with each other. The DET of laccase enzyme has been studied using two methods; assemble of needle-like carbon nanotubes (CNTs) on the electrode, and CNTs/Nafion polymer. MET of laccase enzyme also is done by use of ceramic electrode containing, ABTS (2,2'-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]) /sol-gel. Cyclic voltammetric results of DET showed a pair of well-defined redox peaks at $200{\mu}A$ and $170{\mu}A$ in a solution containing 5and $10{\mu}M$ o-dianisidine as a substrate for needle-like assembled CNTs and CNTs-Nafion composite respectively. In MET method using sol-gel/ABTS, the maximum redox peak was $14{\mu}A$ in the presence of 15 M solution o-dianisidine as substrate. The cyclic voltammetric results showed that laccase immobilization on needle-like assembled CNTs or CNTs-Nafion is more efficient than the sol-gel/ABTS electrode. Therefore, the expressed methods can be used to fabricate biocathode of biofuel cells or laccase based biosensors.

• Journal of Microbiology and Biotechnology
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• v.21 no.9
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• pp.960-967
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• 2011

Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a Plackett-Burman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature ($30^{\circ}C$), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.

• BMB Reports
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• v.34 no.1
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• pp.21-27
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• 2001

The structural stability of brain pyrydoxine-5'-phosphate (PNP) oxidase and the catalytic properties of the monomeric species were investigated. The unfolding of brain pyridoxine-5'-phosphate (PNP) oxidase by guanidine hydrochloride (GuHCl) was monitored by means of fluorescence and circular dichroism spectroscopy Reversible dissociation of the dimeric enzyme into subunits was attained by the addition of 2 M GuHCl. The perturbation of the secondary structure under the denaturation condition resulted in the release of the cofactor FMN. Separation of the processes of refolding and reassociation of the monomeric species was achieved by the immobilization method. Dimeric PNP oxidase was immobilized by the covalent attachment to Affi-gel 15 without any significant lass of its catalytic activity. Matrix-bound monomeric species were obtained from the reversible refolding processes. The matrix bound-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to the refolded dimeric enzyme. In addition, limited chymotrypsin digestion of the oxidase yields two fragments of 12 and 161 kDa with a concomitant increase of catalytic activity The catalytically active fragment was isolated by ion exchange chromatography and analyzed for association of two subunits using the FPLC gel filtration analysis. The retention time indicated that the catalytic fragment of 16 kDa behaves as a compact monomer. Taken together, these results are consistent with the hypothesis that the native quaternary structure of PNP oxidase is not a prerequisite for catalytic function, but it could play a role in the regulation.

• Journal of the Korean Society of Clothing and Textiles
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• v.27 no.1
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• pp.40-47
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• 2003

This paper discussed the assessment of hand of Tencel denim fabrics finished by enzymatic hydrolysis. The subjective hand and the preference of Tencel denim fabrics were evaluated using the developed scale. The factors affecting consumers taste for Tencel denim fabrics were analyzed by statistics using SAS program. Also, the effects of cellulase treatment on the properties of Tencel denim fabrics were evaluated by the subjective hand measurements. The results are as follows: As the weight loss increased. fabrics were evaluated as finer, smoother, softer, warmer, more refined (surface properties), more compact and weaker (durability), more flexible, flossier, lighter, softer, thinner (sense of weight), more elastic, and less wrinkly (shape recovery). Fabrics were evaluated to have the dry touch regardless to the rate of weight loss (moisture properties). Overall hand preference of Tencel denim fabrics was in the side of not preferred. Hand of Tencel fabrics seems not to appeal to Korean people. Color preferences were not significantly different among five groups. The correlations between subjective hand and preference showed that fine, smooth, flexible, warm, refined, loose, soft, dry touches were preferred in Tencel denim fabrics.

• Archives of Pharmacal Research
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• v.9 no.2
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• pp.87-91
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• 1986

In attempt to develop a drug delivery system using serum albumin microspheres, bovine serum albumin microspheres containing antitumar agent. Cytarabine, were prepared. The shape, surface characteristics, size distribution, behavior of in vivo distribution, drug release behavior, and degradation of albumin microsphers in animal liver issue homogenate and proteolytic enzyme were investigated. The shape of albumin microspheres was spherical and the surface was smooth and compact. The size distribution of the albumin microspheres was effected by dispertion forces during emulsification and albumin concentration. Distribution of albumin microspheres after imtravenous administration in rabbit was achieved immediately. In vitro, albumin microsphere matrix was so hard that it retained most of cytarabine except initial burst during the first 10 minutes, and the level of drug release during the initial burst was affected by heating temperature, drug/albumin microsphere matrix was so hard that it retained most of cytarabine except initial burst during the first 10 minutes, and the level of drug release during the initial burst was affected by heating temperature, drug/albumin concentration ratio and size distribution. After drug release test, the morphology of albumin microspheres was not changed. Albumin microsphere matrix was degraded by the animal liver issue homogenate and proteolytic enzyme. The degree of degradation was affected by heating temperature.

• Archives of Pharmacal Research
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• v.8 no.3
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• pp.159-168
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• 1985

In attempt to develop a drug delivery system using serum albumin microspheres, bovine serum albumin microspheres containing antitumor agent, cytarabine, were prepared. The shape, surface characteristics, size distribution, behavior of in vitro distribution, drug release behaior, and degradation of albumin microspheres in animal liver tissue homogenate and proteolytic enzyme were investigated. The shape of albumin microspheres was spherical and the surface was smooth and compact. The size distribution of the albumin microspheres was affected by dispersion forces during emulsification and albumin concentration. Distribution of albumin mirospheres after intravenous administration in rabbit was achieved immediately. In vitro, albumin microsphere matrix was so hard that it retained most of cytarabine except initial burst during the first 10 minutes, and the level of drug release during the initial burst was affected by heating temperature, drug/albumin concentration ratio and size distribution. After drug release test, the morphology of albumin micropheres was not changed. Albumin microsphere matrix was degraded by the rabbit liver tissue homogenate and proteolytic enzyme. The degree of degradation was affected by heating temperature.