• Genomics & Informatics
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• 제21권1호
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• pp.3.1-3.10
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• 2023

Characterization as well as prediction of the secondary and tertiary structure of hypothetical proteins from their amino acid sequences uploaded in databases by in silico approach are the critical issues in computational biology. Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), which is responsible for pneumonia alike diseases, possesses a wide range of proteins of which many are still uncharacterized. The current study was conducted to reveal the physicochemical characteristics and structures of an uncharacterized protein Q6S8D9_SARS of SARS-CoV. Following the common flowchart of characterizing a hypothetical protein, several sophisticated computerized tools e.g., ExPASy Protparam, CD Search, SOPMA, PSIPRED, HHpred, etc. were employed to discover the functions and structures of Q6S8D9_SARS. After delineating the secondary and tertiary structures of the protein, some quality evaluating tools e.g., PROCHECK, ProSA-web etc. were performed to assess the structures and later the active site was identified also by CASTp v.3.0. The protein contains more negatively charged residues than positively charged residues and a high aliphatic index value which make the protein more stable. The 2D and 3D structures modeled by several bioinformatics tools ensured that the proteins had domain in it which indicated it was functional protein having the ability to trouble host antiviral inflammatory cytokine and interferon production pathways. Moreover, active site was found in the protein where ligand could bind. The study was aimed to unveil the features and structures of an uncharacterized protein of SARS-CoV which can be a therapeutic target for development of vaccines against the virus. Further research are needed to accomplish the task.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 추계학술대회 및 정기총회
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• pp.9-9
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• 2014

Null mutants generated by targeted gene replacement are frequently used to reveal function of the genes in fungi. However, targeted gene deletions may be difficult to obtain or it may not be applicable, such as in the case of redundant or lethal genes. Constitutive expression system could be an alternative to avoid these difficulties and to provide new platform in fungal functional genomics research. Here we developed a novel platform for functional analysis genes in Magnaporthe oryzae by constitutive expression under a strong promoter. Employing a binary vector (pGOF1), carrying $EF1{\beta}$ promoter, we generated a total of 4,432 transformants by Agrobacterium tumefaciens-mediated transformation. We have analyzed a subset of 54 transformants that have the vector inserted in the promoter region of individual genes, at distances ranging from 44 to 1,479 bp. These transformants showed increased transcript levels of the genes that are found immediately adjacent to the vector, compared to those of wild type. Ten transformants showed higher levels of expression relative to the wild type not only in mycelial stage but also during infection-related development. Two transformants that T-DNA was inserted in the promotor regions of putative lethal genes, MoRPT4 and MoDBP5, showed decreased conidiation and pathogenicity, respectively. We also characterized two transformants that T-DNA was inserted in functionally redundant genes encoding alpha-glucosidase and alpha-mannosidase. These transformants also showed decreased mycelial growth and pathogenicity, implying successful application of this platform in functional analysis of the genes. Our data also demonstrated that comparative phenotypic analysis under over-expression and suppression of gene expression could prove a highly efficient system for functional analysis of the genes. Our over-expressed transformants library would be a valuable resource for functional characterization of the redundant or lethal genes in M. oryzae and this system may be applicable in other fungi.

• Journal of Microbiology
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• 제44권5호
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• pp.523-529
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• 2006

OSH3 is one of the seven yeast homologues of the oxysterol binding proteins (OSBPs) which have the major binding affinity to the oxysterols and function as regulator of cholesterol biosynthesis in mammals. Mutational analysis of OSH3 showed that OSH3 plays a regulatory role in the yeast-to-hyphal transition through its oxysterol-binding domain in Saccharomyces cerevisiae. The OSH3 gene was also identified in the pathogenic yeast Candida albicans. Deletion of OSH3 caused a defect in the filamentous growth, which is the major cause of the C. albicans pathogencity. The filamentation defect of the mutation in the MAPK-associated transcription factor, namely $cph1{\Delta}$ was suppressed by overexpression of OSH3. These findings suggest the regulatory roles of OSH3 in the yeast filamentous growth and the functional conservations of OSH3 in S. cerevisiae and C. albicans.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권3호
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• pp.155-162
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• 2002

The cyclic amidohydrolase family enzymes, which include allantoinase, dihydroorotase, dihydropyrimidinase and (phenyl)hydantoinase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in vivo. Each enzyme has been independently characterized, and thus well documented, but studies on the higher structural traits shared by members of this enzyme family are rare due to the lack of comparative study. Here, we report upon the expression in E. coli cells of maltose-binding protein (MBP)- and glutathione S-transferase (GST)-fused cyclic amidohydrolase family enzymes, facilitating also for both simple purification and high-level expression. Interestingly, the native quaternary structure of each enzyme was maintained even when fused with MBP and GST. We also found that in fusion proteins the favorable biochemical properties of family enzymes such as, their optimal pHs, specific activities and kinetic properties were conserved compared to the native enzymes. In addition, MBP-fused enzymes showed remarkable folding ability in-vitro. Our findings, therefore, suggest that a previously unrecognized trait of this family, namely the ability to functional fusion with some other protein but yet to retain innate properties, is conserved. We described here the structural and evolutionary implications of the properties in this family enzyme.

• Food Science and Biotechnology
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• 제15권5호
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• pp.704-709
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• 2006

The mucilage production and tyrosine content in soy milk cake (SMC) fermented by Bacillus firmus NA-1, Bacillus subtilis GT-D, and B. subtilis KU-A was improved by fortification with 10% defatted soybean flour. The fibrinolytic activity and consistency of the SMC were drastically increased by solid-state fermentation for 1 day. However, the consistency of the fermented SMC gradually decreased during fermentation for 3 days. Furthermore, the tyrosine content of the freeze-dried powder of SMC fermented by three Bacillus sp. was 9 times higher than that of unfermented SMC. The soybean proteins, including the 7S and 11S subunits, were partially digested during alkaline fermentation, producing lower molecular-weight peptides. The fibrinolytic enzyme produced in SMC fermented by B. firmus NA-l and B. subtilis KU-A exhibited higher thermal stability than that of B. subtilis GT-D fermentation. The powder obtained from B. subtilis GT-D fermentation had an ${\alpha}$-amylase activity and lower consistency compared to those of B. firmus NA-1 and B. subtilis KU-A. In addition, this powder contained 6.3% moisture content, 27% crude protein content and 9 units of fibrinolytic activity and proteolytic activity.

• Asian-Australasian Journal of Animal Sciences
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• 제18권12호
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• pp.1684-1690
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• 2005

Using mRNA differential display technique, we investigated differential gene expression in hybrids relative to their parents in a diallel cross involving four chicken breeds in order to provide an insight into the molecular basis of heterosis in chicken. The results indicated that there was extensive differential gene expression between chicken F1 hybrids and their parents which was classified into four kinds of patterns as following: (1) bands only detected in hybrid F1; (2) bands only absent in hybrid F1; (3) bands only detected in parent P1 or P2; (4) bands absent in parent P1 or P2. Forty-two differentially expressed cDNAs were cloned and sequenced, and their expression patterns were confirmed by Reverse-Northern dot blot. Sequence analysis and database searches revealed that genes showed differential expression between hybrid and parents were regulatory and functional genes involved in metabolism, mRNA splicing, transcriptional regulation, cell cycles and protein modification. These results indicated that hybridization between two parents can cause changes in expression of a variety of genes. In conclusion, that the altered pattern of gene expression in hybrids may be responsible for heterosis in chickens.

• Bulletin of the Korean Chemical Society
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• 제31권2호
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• pp.275-280
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• 2010

Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus bacterium that is associated with several human diseases, including endocarditis, meningitis, osteomyelitis, subcutaneous abscesses and periodontal diseases. A full-length Omp1-like protein gene from A. actinomycetemcomitans was cloned into a pQE30 vector and overexpressed in Escherichia coli BL21(DE3) cells. The protein revealed sequence homologies to Seventeen kilodalton proteins (Skp) from Pasteurella multocida and E. coli that have been characterized as periplasmic chaperones. This soluble Omp1-like protein was successfully purified to homogeneity for further folding and functional studies. The purity, identity, and conformation of the protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, fluorescence spectroscopic, and differential scanning calorimetric studies. We showed that the protein formed an oligomer larger than a tetramer. We found, further, that it is comprised of mostly $\alpha$-helices and boasts high thermal stability.

• 한국안전학회지
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• 제30권1호
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• pp.34-39
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• 2015

The feasibility of recycling wasted printed circuit board (PCB) is investigated by preparing PCB added flame retardant composites filled with either unsaturated polyester or polyurethane. In order to improve electroconductive properties, copper powder was added into the composites, which results also in improving their antistatic properties. The prepared composite samples showed a binding between the polymer fillers observed by a scanning microscope. The sample group using unsaturated polyester is elastomeric that led to appreciable elongation and elasticity. In case of polyurethane, the tensile strength increased proportionally as increase of the amount of PCB powder. The composite materials can be utilized as antistatic composite materials, since the surface resistivity result showed increase of the electroconductive properties by adding Cu. The flammability of the samples is not satisfactory according to UL-94 vertical test. However, the flame retardant properties were improved by adding PCB power. This study, therefore, showed that it is feasible to fabricate polymer composite materials and improve the material characteristics by adding PCB powder, which can replace existing additives used for the preparation of polymer composite materials and can reduce the environment contamination by recycling the wasted PCB.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.681-686
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• 2015

The purpose of this study was to find a cold-adapted and surfactant-stable alginate lyase as a candidate for biotechnological and industrial applications. The gene for a new alginate lyase, AlyL1, from Agarivorans sp. L11 was cloned and expressed in Escherichia coli. The recombinant AlyL1 was most active at 40℃ (1,370 U/mg). It was a cold-adapted alginate lyase, which showed 54.5% and 72.1% of maximum activity at 15℃ and 20℃, respectively. AlyL1 was an alkaliphilic enzyme and most active at pH 8.6. In addition, it showed high stability in the presence of various surfactants at a high concentration (from 0.1% to 1% (w/v)). AlyL1 was an endo-type alginate lyase that degraded both polyM and polyG blocks, yielding disaccharides and trisaccharides as the main products. This is the first report of the cloning and functional expression of a cold-adapted and surfactant-stable alginate lyase. AlyL1 might be an interesting candidate for biotechnological and industrial applications.

• 한국해양바이오학회지
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• 제7권1호
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• pp.19-27
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• 2015

The objective of the current study was to evaluate the inhibitory and antioxidant activities of powdered katsuobushi (dried bonito) protein hydrolysates and their corresponding fractions. The powdered katsuobushi (dried bonito) hydrolysates were obtained by enzymatic hydrolysis using Alcalase, ${\alpha}$-chymotrypsin, Neutrase, pepsin, papain, and trypsin. The antioxidant efficacy of the respective hydrolysates were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, superoxide, and alkyl radical-scavenging activities. Among the hydrolysates, the peptic-derived hydrolysate exhibited the highest antioxidant activity compared to other enzymatic hydrolysates. Therefore, the peptic-derived hydrolysate was further analyzed, and was found to contain an active peptide with an amino acid sequence identified as Pro-Met-Pro-Leu-Asn-Ser-Cys (756 Da). The purified peptides from powdered katsuobushi (dried bonito) had an $EC_{50}$ value of $105.82{\mu}M$, and exhibited an inhibitory effect against DNA oxidation induced by hydroxyl radicals. Taken together, these results suggests that powdered katsuobushi (dried bonito) could be used as a natural antioxidant in functional foods and prevent oxidation reactions in food processing.