• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.808-817
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• 2011

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed ${\alpha}$- helices and ${\beta}$-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of $50^{\circ}C$ with specific activities against Avicel and p-nitrophenyl-${\beta}$-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2018.05a
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• pp.588-591
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• 2018

Baculovirus was originally isolated from the alfalfa looper and contains a 134-kbp genome with 154 open reading frames (ORF). The major capsid protein VP39 together with some minor proteins forms the nucleocapsid ($21nm{\times}260nm$) that encloses the DNA with p6.9 protein. They are double-stranded, circular, supercoiled DNA molecules in a rod-shaped capsid. Wild-type baculoviruses exhibit both lytic and occluded life cycles that develop independently throughout the three phases of virus replication. Recombinant baculoviruses can transfer their vectors and express their recombinant proteins in a wide range of mammalian cell types. Especially, inclusion of a dominant selectable marker in these baculoviral vectors can express diverse recombinant genes in many cells. Baculoviral vectors were reconstructed with cytomegalovirus (CMV) promoter,uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), protein transduction domain (PTD) gene and so on. These reconstructed vectors were infected into various cell and cell lines. We performed transfection and expression of these recombinant vectors comparison with other control vectors. From this study, we knew that transfection and expression of these recombinant vectors have higher efficacy than any control vector. This work was supported by a grant from Mid-Career Researcher Program(NRF-2016R1A2B4016552) through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).

• Journal of the Society of Naval Architects of Korea
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• v.39 no.3
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• pp.18-27
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• 2002

In order to improve the accuracy of the solution near the boundary in an analysis of viscous flow around an arbitrary boundary which move and be deformed using an Eulerian concept, a level-set based grid deformation method is introduced to concentrate grid points near the boundary. This paper presents a new monitor function which can easily control the level of the concentration of grid points along the boundary. Computations for steady flow around a semi-circular cylinder mounted on the bottom of the flow domain were carried out to check the improvement of the solution using the adaptive grid system with an immersed boundary method. The present numerical results show a good agreement with the solutions obtained by a body fitted grid system and more accurate solutions than those computed with non-adaptive grid system. For the validation of mechanical usefulness of the present method, an expanded analysis of flow around multi-body fixed in the flow domain was carried out. Finally, the present moving adaptive grid method was applied to a two-dimensional bubble rise problem. The computed results show well adapted grid points around the boundary of the bubble at every time and a good agreement with the result calculated by fixed grid system.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.775-784
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• 2017

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase ($CcXyn-{\Delta}5C$) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but $CcXyn-{\Delta}5C$ contained less ${\alpha}-helices$ (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature ($45^{\circ}C$) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and $CcXyn-{\Delta}5C$, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, $CcXyn-{\Delta}5C$ exhibited a much lower $K_m$ value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of $CcXyn-{\Delta}5C$ was 2.4-times higher than that of CcXyn. These properties make $CcXyn-{\Delta}5C$ a good model for the structure-function study of $({\alpha}/{\beta})_8$-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

• BMB Reports
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• v.40 no.2
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• pp.163-171
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• 2007

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry $\delta$- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, $Trp^{243}$, $Phe^{246}$, $Tyr^{249}$ and $Phe^{264}$, in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at $Tyr^{249}$ and $Phe^{264}$ still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of $Tyr^{249}$ and $Phe^{264}$ within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.

• Bulletin of the Society of Naval Architects of Korea
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• v.22 no.3
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• pp.9-18
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• 1985

The numerical calculation for solving boundary-value problem related to potential flows with a free surface is carried out by application of the localized finite element method. Only forced motion of 2-D body in infinitely deep fluid is considered, although this schemes is equally applicable to any first order time-harmonic problems of similar nature. The infinite domain of the fluid is separated into the inner flow field and the outer flow field with common inter-surface boundary. The finite element method is applied to obtain the solution in the inner flow field and the Green functions are utilized to represent the solution in the outer flow field. At the inter-surface boundary, the continuity of the value of potential and the normal derivative of the potential(i.e. matching condition) is conserved. The present method has better computational efficiency than the previous LFEM and the integral equation method of Frank. This enhanced computational efficiency is presumably due to the fact that the present method gives a symmetric coefficient matrix and requires less computational time in calculating the influence coefficient matrix of Green function than the integral equation method. And the irregular frequency desen't exist because the uniqueness of the solution is assured by the such that the exact free surface condition is satisfied on the boundary of the localized finite element region(i.e. inner region). As an example of the above method, the hydrodynamic forces for the circular cylinder and the rectangular cylinders are calculated. In the computed results, the small number of singularity distribution segments($3{\sim}6$) give good result relative to Ursell's and Vugts'.

• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

• The Journal of Korean Institute of Communications and Information Sciences
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• v.27 no.1B
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• pp.66-78
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• 2002

In this paper, we propose a new symbol offset estimation technique for an orthogonal frequency-division multiplexing (OFDM)-based wireless LAN. When both inter-symbol interference (ISI) and inter-channel interference (ICI) do not exist in an OFDM symbol, symbol offsets cause circular shifts in the estimated channel impulse response (CIR) by the amount of symbol offset. Also, the power delay profile of a typical multipath wireless channel can be modeled by exponentially decaying function, and most energy of multipath channel is concentrated at the beginning part of the CIR. Based on these properties, the proposed symbol offset estimation technique estimates the CIR, which is circularly shifted by the amount of symbol offset, and then calculates the partial mean power from the estimated impulse response by using a moving window with a finite length. And, symbol offset can be estimated from the index of a moving window having the maximal partial mean power. The proposed technique can reduce noise effect in the process of the CIR estimation, and remove ISI and ICI using repetitive training symbol structure in time-domain for minimum training overhead. The performances of the proposed symbol offset estimation technique in typical indoor channels are demonstrated by computer simulation.

• Tunnel and Underground Space
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• v.27 no.5
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• pp.303-311
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• 2017

The correlation analysis between the results obtained from DFN flow model and equivalent continuum flow model were conducted on total of 72 DFN blocks having various fracture geometry and domain size. A strong linear relation seems to exist between the two approaches under condition that normalized relative error for continuum behavior (ER) is less than 0.2, and the results from both methods are found to almost identical. To explore the field applicability of equivalent continuum flow model in DFN media, a total of 48 numerical schemes related to inflow of underground circular openings were implemented under various DFN configurations. The equivalent continuum flow model in DFN media with a constant hydraulic aperture was evaluated as valid. However, as the anisotropy increases due to variation of the hydraulic aperture, the results are likely to be overestimated compare to the DFN flow model.

• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.662-667
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• 2020

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.