• 대한전기학회논문지:전기물성ㆍ응용부문C
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• 제54권5호
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• pp.227-231
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• 2005

We have developed a microsystem with a capillary electrophoresis (CE) and an electrochemical detector (ECD). The microfabricated CE-ECD systems are adequate for a disposable type and the characteristics are optimized for an application to the electrochemical detection. The system was realized with polydimethylsiloxane (PDMS)-glass chip and indium tin oxide electrode. The injection and separation channels (80 um wide$\ast$40 um deep) were produced by moulding a PDMS against a microfabricated master with relatively simple and inexpensive methods. A CE-ECD systems were fabricated on the same substrate with the same fabrication procedure. The surface of PDMS layer and ITO-coated glass layer was treated with UV-Ozone to improve bonding strength and to enhance the effect of electroosmotic flow. For comparing the performance of the ITO electrodes with the gold electrodes, gold electrode microchip was fabricated with the same dimension. The running buffer was prepared by 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) titrated to PH 6.5 using 0.1 N NaOH. We measured olectropherograms for the testing analytes consisted of catechol and dopamine with the different concentrations of 1 mM and 0.1 mM, respectively. The measured current peaks of dopamine and catechol are proportional to their concentrations. For comparing the performance of the ITO electrodes with the gold electrodes, electropherograms was measured for CE-ECD device with gold electrodes under the same conditions. Except for the base current level, the performances including sensitivity, stability, and resolution of CE-ECD microchip with ITO electrode are almost the same compared with gold electrode CE-ECD device. The disposable CE/ECD system showed similar results with the previously reported expensive system in the limit of detection and peak skew. When we are using disposable microchips, it is possible to avoid polishing electrode and reconditioning.

• 한국토양비료학회지
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• 제33권4호
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• pp.253-260
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• 2000

Agrobacterium tumefaciens에 존재하는 Ti 플라스미드의 virulence(vir)유전자들은 상처난 식물세포에서 분비되는 페놀화합물에 의해서 발현이 유도된다. 본 연구에서는 3종류의 A. tumefaciens들을 대상으로 8종의 페놀화합물들 중에서 vir유전자의 발현에 영향을 미치는 페놀 화합물들의 종류와 이들 균주에서 발현되는 vir유전자의 활성을 조사하였다. A. tumefaciens MW102에 존재하는 vir유전자는 4-hydroxyacetophenone, phenol, catechol, resorcinol과 vanillin등 5종류의 페놀화합물들에 의해서 높게 발현된 반면, 다른 A. tumefaciens Mw105와 Mw108의 vir유전자들은 이들 페놀화합물들에 의해서 매우 낮게 발현되거나 또는 발현되지 않았다. 또한 A. tumefaciens Mw102는 A. tumefaciens Mw105와 Mw108의 vir유전자를 매우 높게 발현시키는 acetosyringene에 의해서는 매우 낮게 발현되었다. 따라서 vir유전자의 발현을 유도시키는 능력은 Ti 플라스미드들의 종류와 페놀화합물들의 종류에 따라서 서로 다르다는 결과를 얻었다. 결과적으로 vir유전자 유도능력의 차이는 vir A 유전자에서 발현되는 sensor단백질의 차이 때문일 것으로 사료된다.

• 미생물학회지
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• 제54권3호
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• pp.266-271
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• 2018

플라스미드가 제거된 Acinetobacter sp. B-W 균주의 돌연변이체를 글루탐산을 유일한 탄소 원과 질소원으로 함유한 배지에 $28^{\circ}C$에서 배양한 결과 글루탐산으로부터의 시드로포어 생산이 억제되었다. B-W 원 균주의 20 kb 플라스미드를 가진 형질 전환체 대장균 $DH5{\alpha}$는 같은 조건의 배지에서 시드로포어를 생산하는 것으로 조사되었다. 그러나 $36^{\circ}C$에서는 형질 전환체 대장균 $DH5{\alpha}$의 시드로포어 생산이 강하게 억제되었으며, 돌연변이체 B-W 균주는 $28^{\circ}C$에서와 마찬가지로 $36^{\circ}C$에서도 시드로포어를 생산하지 못하였다. 형질 전환체로부터 생산된 시드로포어의 종류는 원 균주 B-W와 마찬가지로 Arnow 시험 결과 카테콜 형으로 조사되었으며, $10{\mu}M\;FeCl_3$를 첨가한 배지에서는 시드로포어 생산이 완전히 억제되었다. 형질전환체로부터 생산된 시드로포어의 TLC상에서의 Rf값은 butanol-acetic acid-water (12:3:5) 용매상에서 0.32로 원 균주 B-W에서 생산된 시드로포어와 같았다. 위와 같은 실험 결과로 글루탐산으로부터 생산된 시드로포어의 생합성에 관여하는 유전자들이 20 kb 플라스미드 상에 있는 것으로 추정되었다.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.66-71
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• 2005

Doxorubicin is a clinically important anticancer polyketide compound that is typically produced by Streptomyces peucetius var. caesius. To improve doxorubicin productivity by S. peucetius, a doxorubicin pathway-specific regulatory gene, dnrI, was cloned into a high-copy-number plasmid containing a catechol promoter system. The S. peucetius containing the recombinant plasmid exhibited approximately 9.5-fold higher doxorubicin productivity compared with the wild-type S. peucetius. The doxorubicin productivity by this recombinant S. peucetius strain was further improved through the optimization of culture media composition. Based on the Fractional Factorial Design (FFD), cornstarch, $K_2HPO_4$, and $MgSO_4$ were identified to be the key factors influencing doxorubicin productivity. The Response Surface Method (RSM) results based on 20 independent culture conditions with varying amounts of key factors predicted the highest theoretical doxorubicin productivity of 11.1 mg/l with corn starch of 46.33 g/l, $K_2HPO_4$ of 4.63 g/l, and $MgSO_4$ of 9.26 g/l. The doxorubicin productivity of the recombinant S. peucetius strain with the RSM-based optimized culture condition was experimentally verified to be 11.46 mg/l, which was approximately 30.8-fold higher productivity compared with the wild-type S. peucetius without culture media optimization.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.27-30
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• 2002

Ralstonia eutrupha JMP134 is a well-known soil bacterium which can metabolite diverse aromatic compounds and xenobiotics, such as phenol, 2,4-dichlorophenoxy acetic acid (2, 4-D), and trichloroethylene (TCE), etc. Phenol is degraded through chromosomally encoded phenol degradation pathway. Phenol is first metabolized into catechol by a multicomponent phenol hydroxylase, which is further metabolized to TCA cycle intermediates via a meta-cleavage pathway. The nucleotide sequences of the genes for the phenol hydroxylase have previously been determined, and found to composed of eight genes phlKLMNOPRX in an operon structure. The phlR, whose gene product is a NtrC-like transcriptional activator, was found to be located at the internal region of the structural genes, which is not the case in most bacteria where the regulatory genes lie near the structural genes. In addition to this regulatory gene, we found other regulatory genes, the phlA and phlR2, downstream of the phlX. These genes were found to be overlapped and hence likely to be co-transcribed. The protein similarity analysis has revealed that the PhlA belongs to the GntR family, which are known to be negative regulators, whereas the PhlR2 shares high homology with the NtrC-type family of transcriptional activators like the PhlR. Disruption of the phlA by insertional mutation has led to the constitutive expression of the activity of phenol hydroxylase in JMP134, indicating that PhlA is a negative regulator. Possible regulatory mechanisms of phenol metabolism in R. eutropha JMP134 has been discussed.

• 미생물학회지
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• 제53권2호
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• pp.97-102
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• 2017

포도당과 글루탐산을 함유한 배지에서 시드로포어인 2, 3-dihydroxybenzoic acid (DHB)를 생산하는 Acinetobacter sp. B-W 균주를 글루탐산을 유일한 탄소원과 질소원으로 함유한 배지에 배양한 결과, 상등액에서 2, 3-DHB가 아닌 카테콜 형의 시드로포어를 생산하는 것으로 조사되었다. 글루탐산의 농도는 3%에서 시드로포어 생산이 최고로 나타났으며, 3% 보다 높은 농도에서는 감소하는 것으로 조사되었다. 글루탐산을 유일한 탄소원과 질소원으로 함유한 배지에서 자란 균주 B-W는 배양 온도 $28^{\circ}C$에서는 시드로포어를 생산하지만 $36^{\circ}C$에서는 생산하지 않았다. 또한 $10{\mu}M\;FeCl_3$를첨가한 배지에서는 시드로포어 생산이 완전히 억제되었다. 글루탐산 배지에서 생산된 균주 B-W의 시드로포어는 TLC 전개 용매 butanol: acetic acid: water =12:3:5에서 Rf치가 0.32로 나타나 Rf치가 0.82인 2, 3-DHB와는 다른 것으로 조사되었으며, 또한 항산화 활성도 없는 것으로 나타나 항산화 활성을 가진 2, 3-DHB와는 다른 시드로포어인 것으로 추정되었다.

• 한국식품영양학회:학술대회논문집
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• 한국식품영양학회 2001년도 동계 학술심포지움
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• pp.126-126
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• 2001

The Rhus verniciflua contains alkly(en)-catechol type allergens with a saiurated or unsaturated alkly chain of 15 or 17 carbon atoms. It has been recognized as an extremely active allergen causing skin reactions similar In poison ivy. The allergic contact dermatitis induced by the urushiol is known to be mediated be T lymphocytes whicht specifically recognize the hepten urushiol. Therefore. direct use of this plant as a medicinal purpose might imply a considerable hazard in Korea. In this study, using the established method for the detoxification from the stem bark of Rhus verniciflua, an strong antioxidant substance was isolated and characterized DPPH (diphenypricryl hydrazyl) assay measures hydrogen atom-donating activity and hence provides a measure of free radical scavenging antioxidant activity. DPPH, a purple-colored stable free radical, is reduced to yellow-colored diphenylpicryl hydrazine by antioxidants to deducing agents. Antioxidative effects of the water extract from RV were measured by DPPH assay. Twenty microliters of the extract was added to 1ml of 100mM DPPH solution in ethanol The mixture was shaken and left to stand for 10min at room temperature. The crude water extracts was purified by using HPLC method with a DEAE (anionic type), CN, ODS column. The purified compound remained stable at pH 3.0-6,0, but unstable above pH 6.5. It was stable heat at 10$0^{\circ}C$ for 4 hours, but still had about 80% of residual activity after treatment at 10$0^{\circ}C$ for 5 hours. The elemental composition of the HR-EI mass spectrum at m/z 170.02 was estimated the empirical formula as $C_{7}$ $H_{6}$ $O_{5}$. $C_{10}$ $H_4$ $O_2$N$_1$, $C_{5}$ $H_4$ $O_4$N$_3$, $C_{8}$$H_2O$$_1$N$_4$. In antimicrobial test, no inhibition was observed against Gram-positive and negative bacteria. This compound was stronger than that of commercial antioxidant by DPPH test, such as BHT, BHC at the same concentration (20$\mu$g/ml).ml).

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2001년도 추계학술대회
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• pp.16-25
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• 2001

A genomic library of biphenyl-degrading strain Comamonas sp. SMN4 was constructed by using the cosmid vector pWE15 and introduced into Escherichia coli. Of 1,000 recombinant clones tested, two clones that expressed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity were found (named pNB 1 and pNB2). From pNB1 clone, subclone pNA210, demonstrated 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, is isolated. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO, BphC) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the Comamonas sp. SMN4 gene bphC, which encodes 23DBDO, was cloned into a plasmid pQE30. The His-tagged 23DBDO produced by a recombinant Escherichia coli, SG 13009 (pREP4)(pNPC), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 23DBDO construction was active. SDS-PAGE analysis of the purified active 23DBDO gave a single band of 32 kDa; this is in agreement with the size of the bphC coding region. The 23DBDO exhibited maximum activity at pH 9.0. The CD data for the pHs, showed that this enzyme had a typical a-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. This structure maintained up to pH 10.5. However, this high stable folding strucure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The result of CD spectra observed with pH effects on 23DBDO activity, suggested that charge transition by pH change have affected change of conformational structure for 23DBDO catalytic reaction. The $K_m$ for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7 $\mu$M, 24 $\mu$M, 50 mM and 625 $\mu$M.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.643-650
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• 2000

A new aniline-utilizing microorganism, strain HY1 obtained from an orchard soil, was characterized by using the BIOLOG system, an analysis of the total cellular fatty acids, and a 16S rDNA sequence. Strain HY1 was identified as a Burkholderia species, and was designated Burkholderia sp. HY1. GC and HPLC analyses revealed that Burkholderia sp. HY1 was able to degrade aniline to produce catechol, which was subsequently converted to cis,cis-muconic acid through an ortho-ring fission pathway under aerobic conditions. Strain HY1 exhibited a drastic reduction in the rate of aniline degradation when glucose was added to the aniline media. However, the addition of peptone or nitrate to the aniline media dramatically accelerated the rate of aniline degradation. A fatty acid analysis showed that strain HY1 was able to produce lipids 16:0 2OH, and 11 methyl 18:1 ${\omega}7c$ approximately 3.7-, 2.2-, and 6-fold more, respectively, when grown on aniline media than when grown on TSA. An analysison the alignment of a 1,435 bp fragment. A phylogenetic analysis of the 16S rDNA sequence based on a 1,420 bp multi-alignment sowed of the 16s rDNA sequence revealed that strain HY1 was very closely related to Burkholderia graminis with 95% similarity based that strain HY1 was placed among three major clonal types of $\beta$-Proteobacteria, including Burkholderia graminis, Burkholderia phenazinium, and Burkholderia glathei. The sequence GAT(C or G)${\b{G}}$, which is highly conserved in several locations in the 16S rDNA gene among the major clonal type strains of $\beta$-Proteobacteria, was frequently replaced with GAT(C or G)${\b{A}}$ in the 16S rDNA sequence from strain HY1.

• 한국토양비료학회지
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• 제44권4호
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• pp.650-656
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• 2011

Lysobacter antibioticus HS124 isolated from pepper rhizosphere soil produced catechol type siderophore. Purified siderophore by Diaion HP-20 and silica gel column chromatography showed several hydroxyl functional groups adjacent to benzene rings by analysis of $^1H$ NMR spectroscopy. The strain HS124 showed different activities to suppress Phytophthora capsici with different concentrations of exogenous Fe (III) in minimal medium where antifungal activity with $100{\mu}M$ Fe (III) was approximately 1.5 times higher than in absence of Fe (III). Bacterial population in this Fe (III)-amended medium was also highest with $8.9{\times}10^8\;CFU\;ml^{-1}$ which also corresponded to the strongest siderophore activity. When grown in rich medium (minimal medium with N, $P_2O_5K_2O$ and glucose), HS124 exhibited approximately 2 times stronger antifungal activity compared to minimal medium. In pot trials, treatments of bacterial culture grown in rich medium with (C1) or without (C2) $100{\mu}M$ Fe (III) exhibited a high protection of pepper plants from disease, compared to medium only with (M1) or without (M2) $100{\mu}M$ Fe (III). Especially, treatment C1 showed the best disease control effect of about 70 %. Thus, the strain HS124 should be recommended as a potential biocontrol agent against P. capsici in pepper.