• 약학회지
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• 제41권3호
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• pp.359-364
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• 1997

Resistance gene mphK was cloned from Escherichia coli 209K strain which is highly resistant to erythromycin (EM). By using the cloned plasmid pGE64, E. coli NM522 was transformed. The comparison of macrolide-phosphotransferase K [MPH(K)] activity between E. coli 209K and E. coli NM522(pGE64) showed that the total enzyme activity of MN522(pGE64) was fifty-fole higher than that of 209K. To identify characteristics of MPH(K) more precisely. MPH(K) was isolated and purified from the NM522 (pGE64). The final purification f MPH(K) through several stages of purification process was 89 fole and the overall recovery was 11%. This enzyme was monomer with the molecular weight of 34 kDa and its isoelectric point (pI) was 5.0. The optimal pH and temperature for activity were 8.0 and $40^{\circ}C$, respectively.

• Molecules and Cells
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• 제26권6호
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• pp.616-620
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• 2008

Maintaining redox balance is one of the crucial requirements for a cell to endure stress from the outside. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) plays an important role in the ascorbate-glutathione cycle; one of the major ROS scavenging systems in most known biological systems. A cDNA clone of the DHAR gene from Oryza sativa (OsDHAR) was isolated and overexpressed in Escherichia coli BL21 (DE3) strain from the pET-28a(+) expression vector. The OsDHAR transformed E. coli cells showed significantly higher DHAR activity and a lower level of ROS than the E. coli cells transformed by an empty pET-28a(+) vector. Also, the DHAR-overexpressing E. coli strain was more tolerant to oxidant- and heavy metal-mediated stress conditions than the control E. coli strain. The results suggest that the overexpressed rice DHAR gene effectively functions in a prokaryotic system and provide protection to various oxidative stresses.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2060-2065
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• 2016

Characteristics of E. coli O157:H7-specific infection bacteriophages (O157 coliphages) and broad-host-range bacteriophages for other E. coli serotypes (broad-host coliphages) were compared. The burst sizes of the two groups ranged from 40 to 176 PFU/infected cell. Distributions of the virulence factors stx1, stx2, ehxA, and saa between the two groups were not differentiated. Broad-host-range coliphages showed lower stability at $70^{\circ}C$, in relation to O157 coliphages. However, O157 coliphages showed high acid and ethanol tolerance by reduction of only 22% and 11% phages, respectively, under pH 3 and 70% ethanol for 1 h exposure. Therefore, these results revealed that the O157 coliphages might be more stable under harsh environments, which might explain their effective infection of the acid-tolerant E. coli O157:H7.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.780-786
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• 2002

An immunosensor based on surface plasmon resonance (SPR) with a self-assembled protein G layer was developed for the detection of Escherichia coli O157:H7. A self-assembled protein C layer on a gold (Au) surface was fabricated by adsorbing the mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol at various molar ratios and by activating chemical binding between free amine (-$NH_2$) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) in series. The formation of a self-assembled protein G layer on an Au substrate and the binding of the antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of the self-assembled protein G layer on the Au substrate, monoclonal antibody (Mab) against E. coli O157:H7 which was immobilized on protein G, and bound E. coli O157:H7 extracts on Immobilized Mab against E. coii O157:H7 were performed by atomic force microscopy (AFM). The detection limit of the SPR-based immunosensor for E. coli O157:H7 was found to be about $10^4$ cells/ml.

• 대한수의학회지
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• 제23권2호
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• pp.173-178
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• 1983

This paper deals with the isolation of citrate-utilizing variants of Escherichia coli ($Cit^+$ E. coli) from the animals, their biochemical reactivity and antibiotic susceptibility, and whether the citrate utilizing ability is transmissible. One hundred arid twenty-three isolates of $Cit^+$ E. coli were obtained from cattle and pigs. but from other animals, no isolates were obtaied. Antibiotic susceptibility testing of $Cit^+$ E. coli was performed by the agar dilution method, using the following 9 antibiotics, chloramphenicol(CP), tetracycline(TC), streptomycine(SM), kanamycin(KM), colistin(CL), gentamicin(GM), cephaloridine(CR), aminobenzycillin and nalidixic acid(NA). All the variants tested were susceptible to KM, CL, GM, CR and NA. Of all the variants, 80(65%) were resistant to the drugs tested and resistance to TC and SM was most frequent. The transmission of the ability to utilize citrate on Simmons citrate agar at $37^{\circ}C$ was demonstrated in 78(67.8%) out of the 115 $Cit^+$ E. coli. There were no significant difference in the transfer rates of citrate utilizing ability between resistant and susceptible variants to above 9 drugs. Of 123 isolates, 8 were lost their citrate utilizing ability, spontaneously.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1357-1363
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• 2008

The $P_{entC}$ promoter of the entCERA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the $P_{entC}$ promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the $P_{entC}$ promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding $\beta$-galactosidase. $\beta$-Galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level $\beta$-galactosidase expression by the $P_{entC}$ promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.

• 대한의생명과학회지
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• 제5권1호
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• pp.27-40
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• 1999

본 연구는 MarA와 함께 MarB가 AcrAB efflux pump를 목표로 하는지 여부, 그리고 항생제의 균체외 배출기능에 대하여 어떤 조절 작용이 있는지를 항생제 내성검사를 통하여 살펴보았다. 본 연구 결과는 MarB가 MarA와 함께 AcrAB/TolC efflux pump의 항생제 배출기 능을 억제적으로 조절한다는 것을 간접적으로 보여 주었으며, 한편 이미 알려진 대로 MarA는 acrRAB operon의 발현을 전사 수준에 서 positive regulation하므로, MarB는 AcrAB efflux pump의 기능을 억제적으로 조절한다는 것을 암시하고 있다. 그리고 MarA와 함께 MarB단백질의 작용 목표는 AcrAB efflux pump임을 간접적으로 보여주고 있다. 또한 MarB는 MarA와 함께 대장균의 다른 efflux system(들)의 항생제 배출기능에 대해서도 억제적으로 조절할 가능성이 높은 것으로 나타났다.

• 미생물학회지
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• 제38권4호
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• pp.241-246
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• 2002

Bacillus circulans의 endo-$\beta$-1,3-1,4-glucanase유전자를 발현 vector pQE30에 삽입시키고 E. coli Ml5에서 발현시켜 효소를 생산.정제하였다. 생산된 endo-$\beta$-1,3-1,4-glucanase는 nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography 과정을 거쳐 단일 단백질로 정제되었다. 정제된 효소의 분자량은 SDS-PAGE 전기영동법으로는 28 kDa이었다. 효소 최적 활성 pH와 온도는 각각 pH 6.8과 $60^{\circ}C$였다. 이 효소는 pH 5.5～7.5와$55^{\circ}C$ 이하의 온도에서 안정하였다. 또한 본 효소는 여러 가지 금속 이온에 의해 대부분의 활성이 억제되었고, 특히 $Hg^{2+}$에서는 강하게 효소 활성이 저해됨을 보였다. 유기 용매에 대한 활성은 10%의 methanol이나 ethanol, isopropanol, 1-butanol 에 대하여 모두 낮은 활성을 나타내었다.

• 융합정보논문지
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• 제11권6호
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• pp.198-205
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• 2021

본 배경 : 질염은 항생제를 복용하는 방식으로 치료를 하고 있으며, 이러한 항생제의 지속적인 치료는 내성을 유발할 수 있다. 연구 방법 : Lactic acid bacteria 2종에 에센셜 오일을 이용한 항균 활성을 보고자 한다. SE(Grapefruit Seed Extract), eucalyptus, tea tree, clove bud oil, cinnamon oil, lemongrass oil, thyme oil, ginger oil을 일정 비율로 넣어 배양 후, 병원성 미생물- E. coli, C. albicans와 Lactic acid bacteria은 균주에 맞는 배지를 사용하여 균 수를 측정하였다. 결과 : Essential oil 7종과 GSE가 병원성 미생물에 억제 효과가 있으며, 병원성균(E. coli, C. albicans)에 대한 Grapefruit seed extract(GSE)의 억제농도를 확인하였다, 병원성균은 억제하고 Lactic acid bacteria는 억제하지 않는 배합비도 확인하였다(GSE 농도가 200ppm에서 Eucalyptus globulus(EG) oil 50㎕와 Melaleuca alternifolia oil(Tea tree oil, TTO) 50㎕(pH 5.0, 5.5, 6.0)). 결론 : 본 실험에서 Essential oil은 다양한 항균 활성 가지고 있어 항생제 대안으로도 생각할 수 있으며, 장기 항생제 치료환자에 대한 보조 항균제로서도 유용할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제2권1호
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• pp.35-40
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• 1992

The influence of the nature of plasmids on fermentation parameters such as cell growth, cell viability, plasmid stability, and product formation has been investigated using E. coli M5248 and its recombinant derivatives M5248 [pBR322], M5248[pAS1], and M5248[pNKM21]. At a low temperature ($30^\circ{C}$), the cell growth, cell viability, and protein synthesis of the recombinants were nearly identical to those of the host cell. However, at high temperature ($42^\circ{C}$), in which transcription from the P_L$ promoter is derepressed, the recombinant cells showed decreased stability along with lower growth rates and cell viability. The ratio of total protein to cell mass was in the order of E. coli M5248>M5248[pBR322]>M5248[pAS1]>M5248[pNKM21]. It was found that transcription from the $P_L$ promoter adversely affect the plasmid maintenance and host cell metabolism even in the absence of the cloned-gene expression. Furthermore, profiles of ${\beta}$ activity were shown to vary with recombinant strains. E coli M5248[pBR322] showed highest ${\beta}-lactamase$ activity at $30^\circ{C}$, while at $42^\circ{C}\;{\beta}-lactamase$ activity was significantly reduced irrespective of the strains. The effect of the plasmid properties on plasmid-encoded gene expression has been further examined based on the relationship between $\{beta}-lactamase$ activity and plasmid-harboring cell numbers.