• 생명과학회지
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• 제9권5호
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• pp.531-536
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• 1999

As an attempt to preserve resources in marine biological organisms, we first constructed a cDNA library from the red alga Porphyra yezoensis. The library construction method from P. yezoensis consists of three steps; those include protoplast presparation, RNA isolation, and phage library construction. Protoplast was prepared in order to remove much of the carbohydrate compounds which are characteristics of algal cell walls. Carbohydrate contamination in the purified RNA may inhibit further enzyme reactions, those carbohydrates should be removed. RNA samples prepared from protoplast still seemed to contain residual amount of carbohydrate because mRNA isolation with conventional method failed. We therefore developed a method with Poly ATtract mRNA isolation system. The constructed phage library was tested by analyzing cDNA insert in phage vector from randomly picked ten independent white plagues. All of the phages contained cDNA inserts with sizes ranging 0.5kb and 2.0kb.

• 약학회지
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• 제47권3호
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• pp.176-179
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• 2003

A library of unlimited number of novel lectins with diverse specificities has been previously generated by randomly mutating the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH). To establish the experimental environment capable of selecting high affinity mutant lectins in E. coli, phage display system was adapted. Carbohydrate binding capacity of two phagemid vectors, pComb3 and pComb8 displaying wild-type MAH lectin was assessed. Specific bindings of pComb3 and pComb8 phages expressing w.t. MAH to affinity-purified polyclonal anti-MAH antibody and to glycophorin was demonstrated. Both phages also showed strong hemagglutinating activity to intact but not sialidase-treated human erythrocytes, which is consistent to the specificity of native MAH. Taken together, two different phage display vectors successfully allowed the expression of active MAH as a fusion protein on the surface of bacteriophage, which will lead to preparation of unique plant lectins with high affinity toward a variety of carbohydrate chains.

• Asian-Australasian Journal of Animal Sciences
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• 제22권11호
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• pp.1555-1565
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• 2009

Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastix frontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five $\beta$-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis.

• 해양환경안전학회지
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• 제29권1호
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• pp.36-43
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• 2023

본 연구는 Tetraselmis suecica와 T. tetrathele의 영양물질 증진효과를 가져올 수 있는 배양시스템 구축을 위하여, 발광다이오드(LED)의 파장별(청색; 450 nm, 황색; 590 nm, 적색; 630 nm) 탄수화물, 단백질, 지질 함량을 측정하였다. 두 종 모두 단백질 비율(42~69%)이 가장 높았으며, 생장속도가 낮았던 황색파장에서 탄수화물, 단백질, 지질의 높은 함량을 보였고, 생장속도가 가장 높았던 적색파장에서는 낮은 함량을 보였다. 이러한 결과는 세포 분열 속도의 감소로 인해 단백질 합성과 함께 세포의 화학적 조성과 효소 활동에 변화를 주어 지질과 탄수화물 함량이 증가한 것으로 생각된다. 따라서, T. suecica와 T. tetrathele의 유용한 생화학적 물질의 증대를 위해 대수생장기 초기와 중기는 적색 LED 그리고 대수생장기 후기에는 황색 LED를 주사하는 2단계 LED 배양을 제안하였다.

• Bulletin of the Korean Chemical Society
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• 제25권1호
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• pp.63-68
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• 2004

Sugar-based gelator p-dodecanoyl-aminophenyl- ${\beta}$-D-aldopyranosides (1-3) have been shown to self-assemble in the presence of p-aminophenyl aldopyranosides. The hydrogel 1+4 showed the double-helical structure with 3-25 nm outer diameters, which is quite different from that of 1. The gel 2+5 revealed twisted ribbon structure with 30-50 nm in widths and a few micrometers of length whereas the gel 3+4 revealed the single and the bundled fiber structures. The difference in these gel supramolecular structures has successfully been transcribed into silica structures by sol-gel polymerization of tetraethoxysilane (TEOS), resulting in the doublehelical, the twisted-ribbon, the single and the multiple (lotus-shaped) hollow fiber structures. These results indicate that novel silica structures can be created by transcription of various superstructures formed in binary gels through the hydrogen-bonding interaction, and the amino group of the p-aminophenyl aldopyranosides acts as an efficient driving force to create novel silica nanotubes. Furthermore, electron energy-loss spectroscopy (ELLS) provided strong evidence for the inner hollow structure of the double-helical silica nanotube. This is a novel and successful example that a variety of new silica structures can be created using a library of carbohydrate gel fibers as their templates.

• Archives of Pharmacal Research
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• 제22권6호
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• pp.585-591
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• 1999

The linker which plays a role in connecting a polymer with a scaffold has become an important part n solid-phase reaction. To develop a new linker for alcohols and carbohydrates, dihydropyran moiety was selected in this study. New linker, 1-($4^{l},5^{l}$-dihydro-5H-pyranyl)-7-hydroxyheptan-3-one (5) was synthesized via four steps from $\delta$-valerolactone. This can be called as DDHP-linked Wang resin due to double dihy-dropyran rings. To the one pyran ring of new linker 5 was added Wang resin and other alcohols and carbohydrates as scaffolds were then added successfully to the another pyran ring. Carbohydrate and hydroxyl resins were connected via new linker in a 70% loading yield. The detachment of glucose moiety in the presence of PPTS (2 equiv.) in 1:1 n-buteanol/1,2-dichloroethane at $60^{\circ}C$ for 12 h was carried out quantitatively. When certain combinatorial chemical works are carried out using this dihydropyran linker, Wang resin itself can be recovered. Its fact is particularly very important in industry, because recovered resins can be recycled.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1077-1084
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• 2009

A genomic library was constructed to clone a xylanase gene (Mxyn10) from Demequina sp. JK4 isolated from a deep sea. Mxyn10 encoded a 471 residue protein with a calculated molecular mass of 49 kDa. This protein showed the highest sequence identity (70%) with the xylanase from Streptomyces lividans. Mxyn10 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10) and a carbohydrate-binding module (CBM) belonging to family 2. The optimum pH and temperature for enzymatic activity were pH 5.5 and $55^{\circ}C$, respectively. Mxyn10 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 3.5 to 10.0. The protein was not significantly affected by a variety of chemical reagents, including some compounds that usually inhibit the activity of other related enzymes. In addition, Mxyn10 showed activity on cellulose. These properties mark Mxyn10 as a potential enzyme for industrial application and saccharification processes essential for bioethanol production.

• 약학회지
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• 제50권1호
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• pp.33-39
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• 2006

We have previously reported the minimum criteria that can be applied to evaluate efficacy and safety of a DNA vaccine with use of Streptococcus pneumoniae DNA vaccine (SPDNA). The SPDNA was formulated by inserting the DNA sequences that are codons specific for the carbohydrate epitope in the capsule of S. penumoniae by phage display peptide library. Administration of the SPDNA into mice induced both humoral and cell-mediated immunities. The induction was protective even in the absence of CD4+ T lymphocyte in mice. Profiles of cytokine and isotyping of antibody displayed tendency of the Th1. In continuation of these studies, we examined if the efficacy of the SPNDA was provoked by the peptide recognized by codons specific for the capsule. Results showed that the peptide vaccine formulae (SPP) induced protective antibody in mice as did the SPDNA. Involvement of the cell-mediated immunity was also determined. Possible side effects of autoimmune diseases such as myositis and C3a production and tumor-formation were undetectable in mice given 7 times of SPDNA vaccination during entire of 92 days. Even after the frequent immunization, immunogenicity of the SPDNA was observed as determined for antibody production, suggesting that there was no immunotolerance provoked. All together, these examining factors would be applied to measurement of a DNA vaccine safety regarding the immunopathological aspect.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.518-524
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• 2010

A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and $60^{\circ}C$, respectively. The activity of Man26B was not affected by $Mg^{2+}$ and $Co^{2+}$, but was inhibited by $Hg^{2+},\;Ca^{2+},\;Cu^{2+},\;Mn^{2+},\;K^+,\;Na^+$, and $\beta$-mercaptoethanol, and slightly enhanced by $Pb^{2+}$ and $Zn^{2+}$. EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with $K_m,\;V_{max}$, and $k_{cat}$ values of 3.80 mg/ml, 91.70 ${\mu}mol$/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at $80^{\circ}C$ and $90^{\circ}C$ for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.

• 한국축산식품학회지
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• 제21권4호
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• pp.367-373
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• 2001

MFGM 당단백질의 하나인 PAS-7을 GPC 및 affinity chromatography에 의해 정제하여 2-AB로 형광 표식한 후, anion-exchange column 및 reversed-phase column을 이용해 5개의 성분을 분리하였다. 그 중 가장 상대량이 많은 성분 e에 대하여 RAAM2000을 이용한 당쇄구조 해석을 실시하여, 성분 e는 RAAM2000 GPC에 의하여 4개의 성분으로 분리되어 각각 calibration standard 12.10, 8.88, 5.84 및 4.86GU의 용출위치에 검출되었다. 이 용출위치와 당쇄구조는 livrary의 component-7457과 일치하며, 12.2GU의 크기로 분자량은 1788로 판단되며 library의 당쇄와 약 85%의 확률로 일치했다. 그 결과 성분 e의 당쇄구조는 환원말단에 $\alpha$1-6결합된 fucose를 1개 함유하며, core부분의 비환원말단에 N-acetyllactosamine branch를 2개 함유한 전형적인 biantennary 당쇄구조인 것으로 추축되어, 이전 HPLC, acetolysis, sequential exoglycosidase 소화, NMR분석에 의해 보고된 성분 7N1A의 구조와 일치함으로써, OGS RAAM2000을 이용한 PAS-7의 당쇄구조 해석의 유용성이 증명되었다.