• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.401-409
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• 2019

Heat-resistant microbial hosts are required for bioprocess development using high cell density cultivations at the industrial scale. We report that the thermotolerance of Escherichia coli can be enhanced by overexpressing ybeD, which was known to encode a hypothetical protein of unknown function. In the wild-type E. coli BL21(DE3), ybeD transcription level increased over five-fold when temperature was increased from $37^{\circ}C$ to either $42^{\circ}C$ or $46^{\circ}C$. To study the function of ybeD, a deletion strain and an overexpression strain were constructed. At $46^{\circ}C$, in comparison to the wild type, the ybeD-deletion reduced cell growth half-fold, and the ybeD-overexpression promoted cell growth over two-fold. The growth enhancement by ybeD-overexpression was much more pronounced at $46^{\circ}C$ than $37^{\circ}C$. The ybeD-overexpression was also effective in other E. coli strains of MG1655, W3110, DH10B, and BW25113. These findings reveal that ybeD gene plays an important role in enduring high-temperature stress, and that ybeD-overexpression can be a prospective strategy to develop thermotolerant microbial hosts.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.259-264
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• 1991

K.pneumoniae의 nif 유전자 발현에 미치는 glnA 산물 (GS)의 영향을 살펴보기 앞서 K.pneumoniae의 GS를 분리.정제하여 그 성질을 조사하였다. K.pneumoniae의 GS는 분자량 약 600,000의 12개 동일 subunit으로 구성되어져 이었으며 최적 반응 pH 및 온도는 각기 pH 7.0, $37^{\circ}C$ 였고 pH 5-8사이에서 활성의 변화가 크지 않았으며 $57^{\circ}C$에서 10분간 열처리 할 때 50의 활성 감소가 나타났다. 또 암모니아 농도의 변화에 따라 adenylylation-deadenylylation 기작에 의한 활성조절기작이 E.coli GS와 동일 했으며 anti-E.coli GS 항체를 이용한 immunodiffusion에서도 K.pneumoniae GS가 E.coli GS와 혈청적으로 매우 유사함이 드러났다.

• 대한의생명과학회지
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• 제2권2호
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• pp.231-240
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• 1996

인체 혈액 응고 9인자는 간에서 생성되며 461개의 아미노산으로 구성된 당 단백질이다. 따라서 인체 혈액 응고 9인자 cDNA를 찾기 위해 태아의 간(fetal liver) cDNA library를 PCR(Polymerase Chain reaction) 방법으로 screening하였으며, 그 결과 ATG개시 코돈으로부터 TAA종료 코돈까지 포함하는 1.4 kb의 9인자 cDNA를 찾았다. 또한 클론된 9인자 cDNA를 박테리아에서 발현시키기 위해 박테리아 발현 벡터인 pGEX-2T 플라스미드에 클로닝하므로써 pGEX-F9 플라스미드를 제조하였다. pGEX-F9로 형질전환된 E. coli에서 PGEX-F9의 발현을 유도하면 73 kDa 크기의 GST-factor9 융합 단백질이 다량생성되며 , 이 단백질이 혈액 응고 9인자 단백질을 함유하는 융합 단잭질임을 혈액 응고 9인자 항체를 이용한 Western blot으로 입증하였다. E. coli에서 발현된 GST-factor 9 융합 단백질은 전체 단백질의 약 20%를 차지하며 GST agarose bead를 이용한 one step purificarion 방법을 통해 GST-factor9 융합 단백질을 쉽게 분리 할 수 있다.

• 미생물학회지
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• 제26권1호
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• pp.6-12
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• 1988

Cellobiase ($\beta$-glucosidase) is an enzyme of the cellulase system in cellulolytic microor-ganisms. The chromosomal DNA fragment which include cellobiase gene of Cellulomonas biazotea was cloned in Eschericia coli via plasmid pBR 322 vector. Restriction enzyme Sal I was used to obtain adequate size of fragments from C. biazotea. chromosomal DNA. The transformant of E. coli HB101 with recombinant plasmid pBG101 showed cellobiase activity, which is not ordinary in E. coli HB101. The enzyme activity of the transformant was as of 20% lower than that of C. biazotea.

• BMB Reports
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• 제30권5호
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• pp.320-325
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• 1997

Thioredoxin is a multi-functional protein which is ubiquitous in microorganisms, animals and plants. Previously, expression of the E. coli thioredoxin gene was found to be negatively regulated by cAMP. In the present study, the effect of cAMP on two separate promoters of the E. coli thioredoxin gene was investigated. Cyclic AMP had a repressible effect on P1 and P1P2 promoter activity of the constructs. This effect was also observed in the cya strain. The P2 promoter construct gave very high -galactosidase activity, and its expression was not affected by exogenous cAMP. It was assumed that a cis-acting negative element, probably the cAMP-CRP binding site, might have been deleted in the P1 promoter construct. Repression of the thioredoxin gene expression by cAMP appeared to be independent of ppGpp.

• 한국미생물·생명공학회지
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• 제27권3호
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• pp.260-265
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• 1999

The structural $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis 7962 was cloned into plamid vector pKF18, which was designated as pKF-gal. Expression of the lacZ from L. lactis 7962 was found to be higher when cells were grown at 3$0^{\circ}C$ than 37$^{\circ}C$. Maximum $\beta$-galactosidase activity was obtained when E. coli/pKF-gal was cultivated for 6hr at 3$0^{\circ}C$ and for 3hr at 37$^{\circ}C$, and L. lactis 7962 was grown for 8hr at 3$0^{\circ}C$. Enzyme induction was achieved by the addition of lactose, galactose, or lactose+IPTG to growing culture. The addition of glucose had no effect on enzyme induction.

• KSBB Journal
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• 제13권2호
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• pp.168-173
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• 1998

Gamma irradiated and non-irradiated Agrimonia pilosa Ledebour were extracted by 70% ethanol. The combined effects of the Agrimonia pilosa Ledebour extract and NaCl on survival of Escherichia coli O157:H7 in tryptic soy broth were investigated. E. coli O157:H7 decreased ca 1 log cycle by the addition of 2% sample extract, and the anthbacterial activity was increased as the concentration of sample extract was increased. The irradiation effect of the sample on antibacterial activity was not observed. On the treatment of NaCl alone, E. coli O157:H7 was inactivated (ca 3~4 log cycle reduction within 48 hr) in more than 7% NaCl. The higher inactivation(ca 5 log cycle reduction within 48 hr) occurred in the presence of 2% sample extract and 5% NaCl than in the addition of each alone. The extracted antibacterial substance was stable in the pH range of 4.0 to 7.0, heat treatment at 121$^\circ C$ for 15 min, and freezing at -18$^\circ C$ and thawing at 37$^\circ C$. There fore, the sample extract, would substantially increase the food-safety in terms of E. coli O157:H7.

• 미생물학회지
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• 제29권2호
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• pp.80-84
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• 1991

We cloned and expressed hepatitis B viral core antigen (HBcAg) gene in E. coli using $P_{L}$ promoter system. For optimal expression of the gene, we undertook the studies on the effects of the distance between Shine-Dalgarno (SD) sequence and start codon, copy number of repressor gene, induction temperature, and the stability of the core antigen. The results demonstrated that the induction at 37.deg.C was more efficient than at 42.deg.C, and the 11 base pairs (bp) distance between SD sequence and start codon of HBcAg gene was more efficient than the 15 bp distance in E. coli. The copy number of cI857 repressor gene did not influence on the expression of HBcAg, and the expression level of HBcAg in mutant type (low protease activity) and wild type strains was almost the same. The produced core antigen appeared to be HBcAg not HBeAg judged by two different radioimmunoassat (RIA) kits. This result suggested that the antigen was stable in E. coli.i.

• Bulletin of the Korean Chemical Society
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• 제35권8호
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• pp.2460-2464
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• 2014

Recently studies indicate that the microcalorimetry is a suitable measurement for metabolic activities of organisms by recording the rate of heat outputs. In this work, we investigated the growth thermogenic curves of Escherichia coli (E. coli) affected by three kinds of Glycyrrhiza extracts by microcalorimetry. The power-time and exponential phase power-time curves of the E. coli growth at various concentrations of extracts were generated. The kinetic parameters such as the growth rate constants (C), maximum power outputs (Pm), peak times (Tm), and inhibition ratios were calculated and the relationships between Pm or Tm and C were established. Also, the clear correlations among the antimicrobial effects, Pm and C were obtained. The results revealed the Glycyrrhiza extracts had inhibitory activities towards E. coli while the Glycyrrhiza polysaccharides showed the most potent effects.

• 한국식품저장유통학회지
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• 제9권4호
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• pp.434-440
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• 2002

쇠고기의 위생적 품질을 개선하고자 신선육의 HACCP의 위해 요소로 작용하는 병원성 미생물인 E. coli O157:H7 CDF1, A. sobria CDF3, S. aureus CDF2를 도체표면에서 분리하여 이들의 성장특성을 검토하였다. 신선육의 도축처리단 계별 총균수는 세척 후가 세척 전 보다 $10^1$~ $10^2$/$\textrm{cm}^2$ 더 높았다. 냉각 후 총균수는 냉각 전보다 $10^1$~ $10^2$/$\textrm{cm}^2$ 감소하였고, 수송 후 도체표면의 총균수는 계절에 관계없이 증가하는 경향을 나타내었다. E. coli O157:H7 CDF1와 S. aureus CDF2 는 37$^{\circ}C$, A. sobria CDF3는 3$0^{\circ}C$에서 가장 성장이 좋았다. E. coli O157:H7 CDF1은 pH 4에서도 완만한 성장을 하였으나 A. sobria CDF3와 S. aureus CDF2는 성장이 관찰되지 않았다. E. coli O157:H7 CDF1은 NaCl 농도 6%에서도 성장이 관찰되었으며 A. sobria CDF3는 4%에서 성장이 억제되었고, 6%에서는 성장이 관찰되지 않았다. E. coli O157:H7 CDF1과 S. aureus CDF2 는 lactic acid 0.3% 첨가구가 무첨가구에 비해 뚜렷한 성장억제현상을 나타내었으며 0.5% 첨가구에서는 성장이 관찰되지 않았다. A. sobria CDF3은 lactic aicd 0.3%에서도 성장이 거의 관찰되지 않았다. E. coli O157:H7 CDF1의 경우 1$0^{\circ}C$에서는 3일 이후 급격히 성장하여 배양 전 기간 동안 양호한 성장률을 보였으나 4$^{\circ}C$에서는 성장을 거의 관찰 할 수 없었다. A. sobria CDF3는 4$^{\circ}C$에서 3일째부터 성장하였다. 분리균에 대한 열내성을 측정한 결과 E. coli O157:H7 CDF1은 6$0^{\circ}C$에서 3분 열처리, A. sobria CDF3는 6$0^{\circ}C$에서 3분, 7$0^{\circ}C$에서 2분간 열처리, S. aureus CDF2는 7$0^{\circ}C$에서 2분간 열처리시 이들 균주가 관찰되지 않았다.