• Journal of Microbiology and Biotechnology
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• 제21권8호
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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.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2018년도 춘계학술대회
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• pp.588-591
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• 2018

Baculovirus는 원래 알팔파 루퍼 (looper)로부터 분리되었으며 154 개의 오픈 리딩 프레임 (ORF)을 가진 134-kbp 게놈을 포함하고 있다. 주요 캡시드 단백질 VP39는 약간의 단백질과 함께 p6.9 단백질로 DNA를 감싸는 뉴클레오 캡시드($21nm{\times}260nm$)로 형성된다. 그것들은 막대 모양의 캡시드 안에 이중 가닥의 고리 모양의 슈퍼 코일 DNA 분자이다. 야생형 baculovirus는 용균 및 폐색 된 생명주기를 모두 나타내며 바이러스 복제의 3 단계에 걸쳐 독립적으로 발달한다. 재조합 baculovirus는 광범위한 포유류 세포 유형에서 벡터를 전달하고 재조합 단백질을 발현 할 수 있다. 특히, 이들 baculovirus 벡터에 우세한 선별 마커를 포함시킴으로써 많은 세포에서 다양한 재조합 유전자를 발현시킬 수 있다. 본 연구의 배큘로 바이러스 벡터는 cytomegalovirus (CMV) 프로모터, uroplakin II promoter, polyhedron promoter, 수포 구내염 바이러스 G (VSVG), 녹색 형광 단백질 (EGFP), 단백질 전달 도메인 (PTD) 유전자 등으로 재구성되었다. 이러한 재구성 된 벡터를 다양한 세포 및 세포주에 감염시켰다. 우리는 다른 재조합 벡터와 비교하여 이러한 재조합 벡터의 전이 및 발현을 조사하는 수행하였다. 본 연구에서, 우리는 이 재조합 벡터의 형질 감염 및 발현이 어떤 대조군 벡터보다 더 높은 효능을 갖는다는 것을 알았다. 본 연구는 과학 기술부, 한국 정보 기술 진흥 기금 (MSIP)이 후원하는 한국 연구 재단 (NRF)을 통해 중견 연구원 프로그램 (NRF-2016R1A2B4016552)을 통해 지원되었다.

• 대한조선학회논문집
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• 제39권3호
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• pp.18-27
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• 2002

Eulerian개념을 사용한 격자계 내 임의의 경계면 주위 점성유동 해석에서, 운동하며 변형하는 경계면 근방 해의 정도를 향상시키기 위해서 격자생성시 경계면으로 격자점들을 집중시켜주는 레벨셋법에 바탕을 둔 격자변형법을 도입하였다. 본 연구에서는 격자점들을 경계면 근방으로 집중되는 정도를 용이하게 조절할 수 있도록 새로운 형태의 모니터함수를 제시하였다. 집중격자계를 사용함으로 얻어지는 향상된 해의 정도의 검증을 위하여 바닥에 고정된 반원 실린더 주위 정상유동에 대하여 가상경계법을 함께 사용하여 해석하였다. 수치계산결과는 물체적합 격자계를 사용해서 얻은 결과와 매우 잘 일치하였으며, 집중격자법을 사용하지 않은 해석결과보다 향상된 결과를 보여주었다. 수치계산의 또 다른 예제로서 다수의 고정된 물체주위 유동해석으로 확장 적용하여 공학적 유용성을 검증하였다. 마지막으로 이동 집중격자계의 생성법의 적용을 위해서 움직이면서 변형을 일으키는 2차원 기포상승문제를 해석하였다. 수치해석결과에서 격자점들은 매시간 기포의 변형에 맞추어 적합하게 집중된 형태를 잘 보여주었으며, 고정된 격자계를 사용한 결과와 잘 일치하였다.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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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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• 제40권2호
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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.

• 대한조선학회지
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• 제22권3호
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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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• 제28권12호
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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.

• 한국통신학회논문지
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• 제27권1B호
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• pp.66-78
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• 2002

본 논문에서는 OFDM(Orthogonal Frequency-Division Multiplexing) 기반의 무선 LAN 시스템을 위한 새로운 심볼 옵셋 추정기법을 제안한다. OFDM 심볼에 심볼간 간섭(Inter-Symbol Interference: ISI)과 채널간 간섭(Inter-Channel Interference: ICI)이 없는 경우 추정된 채널의 임펄스 응답은 심볼 옵셋만큼 순환 이동한다. 전형적인 다중경로 무선채널의 전력 지연 프로파일은 지수적 감소 함수로 모델링 할 수 있으며 대부분의 에너지가 임펄스 응답의 앞부분에 주로 집중된다. 제안된 기법은 이러한 성질을 이용하여 먼저 심볼 옵셋만큼 순환 이동된 다중 경로 채널의 임펄스 응답을 추정한 후 유한 길이의 이동 윈도우를 사용하여 임펄스 응답의 부분 평균 전력을 구한다. 그리고, 부분 평균 전력 중에서 최대값을 갖는 이동 윈도우의 인덱스로부터 심볼 옵셋을 추정한다. 제안된 기법은 임펄스 응답의 추정 과정에서 잡음의 영향을 감소시키고, ISI와 ICI를 제거시키며 최소의 오버헤드를 얻기 위해 반복적 구조의 훈련 신호를 사용한다. 실내 무선 채널 모델에서의 모의실험을 통해 제안된 기법의 성능을 검증한다.

• 터널과지하공간
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• 제27권5호
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• pp.303-311
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• 2017

본 연구는 다양한 기하학적 속성을 갖는 총 72개의 DFN 블록에 대하여 DFN 유동모델과 등가의 수리상수를 사용한 연속체 모델을 각각 적용하여 두 결과 간의 상관성을 분석하였다. DFN을 연속체로 가정한 이론적 블록수리전도도와 DFN 유동모델로 산정한 블록수리전도도 사이의 상대오차(ER)가 0.2 이하인 DFN 조건에서 두 접근법 사이에 강한 선형 관계를 이루며 두 결과가 거의 일치하는 것으로 평가되었다. DFN 매질에 대한 연속체 유동해석의 현장적용 가능성을 검토하기 위하여 다양한 DFN 조건을 갖는 지중 원형공동에서의 지하수 유입에 대한 모의 수치실험이 총 48회 수행되었다. 일정한 수리간극의 DFN 매질에 대한 등가연속체 유동모델은 유효한 것으로 평가되었지만 수리간극 변화로 인하여 이방성이 증대되면 DFN 유동모델에 의한 결과에 비하여 과대평가될 가능성이 높다.

• Journal of Microbiology and Biotechnology
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• 제30권5호
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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.