• 농약과학회지
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• 제15권4호
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• pp.485-489
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• 2011

Bacillus amyloliquefaciens strain EXTN-1 처리에 의해 생육촉진 효과와 함께 광범위한 식물 병 방제효과가 보고되었다. EXTN-1의 PGPR 효과는 생육초기에 PR-1a, PDF1.2 등의 저항성 관련 유전자 발현에 의한 oxidative burst의 증가나 SA, JA나 ethylene 대사에 의한 유도저항성의 발현에 기인한다. 이 연구의 목표는 B. amyloliquefaciens EXTN-1가 기존에 보고된 다른 작물의 경우에서와 마찬가지로 벼의 생육촉진이나 벼줄무늬잎마름병에 대한 저항성에 관여하는지를 확인하기 위해 수행되었다. 벼 종자를 B. amyloliquefaciens EXTN-1에 침지한 후 파종하였을 때 생육촉진 효과와 병에 대한 저항성 발현이 확인되었다. B. amyloliquefaciens EXTN-1을 처리한 30일묘에서 벼의 초장, 생물중, 뿌리길이는 무처리구에 비해 각각 12.6%, 9.8%, 16.0% 증가하여 PGPR 효과가 나타남을 확인할 수 있었다. RSV 접종구에서도 B. amyloliquefaciens EXTN-1 20일묘는 초장, 생물중, 뿌리길이는 무처리구에 비해 각각 12.6%, 9.8%, 16.0% 증가하였다. 유도저항성 발현효과는 감수성 품종에서 저항성 품종에서 상대적으로 뚜렷하게 나타났다.

• 미생물학회지
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• 제42권3호
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• pp.195-198
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• 2006

Anthrax lethal toxin은 탄저병의 치사원인이 되는 독소이며, Lethal toxin은 두 종류의 단백질 PA (Protective antigen)과 LF (lethal factor)로 구성되어 진다. PA는 세포표면의 수용체와 결합하여 LF를 세포질 안으로 이동시켜 주는 역할을 한다. LF는 금속 이온$(Zn^{2+})$ 의존적 단백질 가수분해 효소로써 MKKs[MAPK (mitogen-activated protein kinase) kinases] 집단 단백질의 아미노 말단 부분을 절단하여 대상 세포를 죽음으로 유도하는 것으로 알려져 있다. 본 연구에서는 LF에 대한 특성 분석 및 억제제 개발에 과한 연구를 위해 cell-based high-throughtput screens 개발에 선행되어야 하는 기초 자료를 마련하는데 그 목적이 있다. 이를 위하여 LF의 절단 대상이 되는 기질이 MEK1을 yeast내에서 동시 발현시켜 LF의 활성을 검증하였다. 먼저 효모(Saccharomyces cerevisiae)를 숙주로 하여 LF의 기질인 MEK1 발현 vector를 구축하였고, 구축된 발현 system을 기본을 LF 활성을 검증하고자 yeast에 형질전환하여 plasmid의 안전성 및 MEK1 유전자의 발현 및 LF에 의한 MEK1 아미노말단의 절단 부위를 확인하였다. 본 연구는 세포내 검증 system 도입의 기초적 자료를 제공하였으며, yeast내의 MEK1 발현은 탄저병의 저해제 선별 및 활성 측정 검증을 생체에서 고효율적이며, 안정적으로 할 수 있다는 가능성을 나타냈다.

• 대한본초학회지
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• 제30권5호
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• pp.59-65
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• 2015

Objectives : Fermentation of herbs has been known to be helpful in improving the immune systems and protecting body against disease. The present study was conducted to evaluate anti-inflammatory effects of the fermentation extracts (FE) consisting of soybean, red ginseng andCitrus UnshiuPeel in lipopolysaccharide (LPS)-activated Raw264.7 cells.Methods : FE were prepared by the fermentation withBacillus Subtilisand then by extraction with ethanol (95%; prepared by the fermentation process). Cell viability was measured by MTT assay. Nitric oxide (NO) production was measured in culture media by Griess assay. The expression of nuclear factor (NF)-κB and inhibitory kappa B alpha (IκBα) was determined by Western blot.Results : LPS-induced production of NO and PGE₂was dose-dependently decreased by the treatment of FE in Raw264.7 cells. These suppressive effects of FE on NO and PGE₂production were related to the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. FE inhibited LPS-induced production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-1βin a dose-dependent manner. Furthermore, FE inhibited the NF-κB signaling pathway through the prevention of LPS-induced degradation of IκBαin cytosol and the nuclear translocation of NF-κB.Conclusions : These findings suggest that FE could have anti-inflammatory effects on LPS-induced inflammatory responses in macrophages.

• Journal of Microbiology and Biotechnology
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• 제16권11호
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• pp.1799-1808
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• 2006

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the ${\alpha}$-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA ($P_{nisA}$) and a signal peptide-encoding sequence of usp45 ($SP_{usp45}$) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing $X-{\alpha}-gal$ and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The ${\alpha}-amylase$ gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of $P_{nisA}\;and\;SP_{usp45}$ in pIMA20, and the plasmid was integrated into the L. lactis chromosome. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.

• Journal of Microbiology and Biotechnology
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• 제25권3호
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• pp.386-392
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• 2015

A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the nonrecombinant background. The bEasyBm bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBm was transposed with pDualBac-EGFP, the resulting recombinant virus, EasyBm-EGFP, showed high levels of EGFP expression efficiency compared with that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, nonrecombinant backgrounds were not detected in unpurified EasyBm-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

• Food Science and Biotechnology
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• 제15권6호
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• pp.851-855
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• 2006

Acarviosine-glucose (AcvGlc) is an ${\alpha}$-glucosidase inhibitor and has similar inhibitory activity to acarbose in vitro. We synthesized AcvGlc by treating acarbose with Bacillus stearothermophilus maltogenic amylase and fed C57BL/6J and db/db mice with diets containing purified AcvGlc and acarbose for 1 week. AcvGlc (50 and 100 mg/100 g diet) significantly reduced plasma glucose and triglyceride levels in db/db mice by 42 and 51 %, respectively (p<0.0001). The hypoglycemic and hypotriglyceridemic effects of AcvGlc were slightly, but significantly, greater than those seen with acarbose treatment (p<0.0001) in C57BL/6J mice. In an oral glucose tolerance test, glucose tolerance was significantly improved at all time points (p<0.01). The expression of two novel glucose transporters (GLUTs), GLUT10 and GLUT12, were examined by Western blot analysis. GLUT10 was markedly increased in the db/db livers. After AcvGlc treatment, the expression of hepatic GLUT10 was decreased whereas intestinal GLUT12 was significantly increased in both strains of mice. Our results show that AcvGlc improves plasma lipid and glucose metabolism slightly more than acarbose. Regulation of hepatic GLUT10 and intestinal GLUT12 may be important in controlling blood glucose levels.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.1999-2009
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• 2017

The secretion efficiency of a protein in a Sec-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, namely, two synthetic Sec-type and three Bacillus licheniformis alpha-amylase-derived signal peptides, were compared for periplasmic expression of the human growth hormone (hGH) in E. coli. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for hGH secretion in E. coli, differ in their N-terminal positively charged residues and hydrophobic region lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing a specific signal peptide by using a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.

• 농약과학회지
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• 제2권1호
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• pp.91-96
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• 1998

모기에 독성을 보이는 cry11Aa 유전자를 발현시키기 위해 cyanobacteria와 E. coli에서 발현 될 수 있는 두 가지 상이한 벡터 (pCYASK5-1, pCYASK5-2)를 제작하였다. 구축한 두 벡터를 E. coli에 형질전환하여 cry11Aa 유전자의 발현을 SDS-polyacrylamide gel electrophoresis (PAGE)와 Western blot analysis을 통해 조사한 결과, pCYASK5-1와 pCYASK5-2으로 형질전환된 E. coli는 각각 72kDa과 64kDa 크기의 cry11Aa 유전자를 발현하였다. 형질전환체의 모기살충성을 조사한 결과, pCYASK5-1과 pCYASK5-2을 가지는 형질전환체는 빨간집 모기유충(Culex pipiens)에 대해 각각 93%, 89%의 치사율을 보였다.

• 대한수의학회지
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• 제52권2호
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• pp.89-97
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• 2012

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been tried to use for immunotherapy. The CWS of Mycobacterium bovis bacillus Calmette-Guerin (BCG-CWS) was studied as an universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccine. Although a variety of biological activities of BCG-CWS have been studied, the effects of BCG-CWS on spleen cells are not fully elucidated. Using MTT assay and trypan blue exclusion test, we found that BCG-CWS significantly enhanced the viability and proliferation of cells. Multiple clusters, indicating proliferation, were observed in BCG-CWS-treated spleen cells and surface marker staining assay revealed that BCG-CWS promoted the proliferation of $CD19^+$ B lymphocyte rather than $CD4^+$ or $CD8^+$ T lymphocyte. In addition, BCG-CWS up-regulated the expression of anti-apoptotic molecules such as bcl-2, bcl-xL. BCG-CWS increased the surface expression of CD25 and CD69 as well as IL-2 production of spleen cells, suggesting increased activation. Furthermore, BCG-CWS enhanced the antigen-specific cell proliferation and interferon-gamma production of spleen cells. Taken together, these results demonstrate the immunostimulatory effects of BCG-CWS on spleen cells via multiple mechanisms, providing valuable information to broaden the use of BCG-CWS in clinical and research settings.