• 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.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1650-1655
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• 2009

The $\beta$-glucuronidase (GUS) gene from Lactobacillus brevis RO1 was cloned and expressed in Escherichia coli GMS407. The GUS gene was composed of 1,812 bp, encoding a 603-amino-acid protein belonging to glycosyl hydrolase family 2 with three conserved domains. The amino acid similarity was higher than 70% with the $\beta$-glucuronidases of various microorganisms, yet less than 58% with the $\beta$-glucuronidase of L. gasseri ADH. Overexpression and purification of the GUS was performed in $\beta$-glucuronidase-deficient E. coli GMS407. The purified GUS protein was 71 kDa and showed 1,284 U/mg of specific activity at optimum conditions of pH 5.0 and $37^{\circ}C$. At $37^{\circ}C$, the GUS remained stable for 80 min at pH values ranging from 5.0 to 8.0. The purified enzyme exhibited a half-life of 1 h at $60^{\circ}C$ and more than 2 h at $50^{\circ}C$. When the purified GUS was applied to transform baicalin and wogonoside into their corresponding aglycones, $150\;{\mu}M$ of baicalin and $125\;{\mu}M$ of wogonoside were completely transformed into baicalein and wogonin, respectively, within 3 h.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1108-1113
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• 2015

Cadaverine (1,5-diaminopentane) is an important industrial chemical with a wide range of applications. Although there have been many efforts to produce cadaverine through fermentation, there are not many reports of the direct cadaverine production from lysine using biotransformation. Whole-cell reactions were examined using a recombinant Escherichia coli strain overexpressing the E. coli MG1655 cadA gene, and various parameters were investigated for the whole-cell bioconversion of lysine to cadaverine. A high concentration of lysine resulted in the synthesis of pyridoxal-5'-phosphate (PLP) and it was found to be a critical control factor for the biotransformation of lysine to cadaverine. When 0.025 mM PLP and 1.75 M lysine in 500 mM sodium acetate buffer (pH6) were used, consumption of 91% lysine and conversion of about 80% lysine to cadaverine were successfully achieved.

• Journal of Microbiology and Biotechnology
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• 제22권7호
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• pp.990-999
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• 2012

The microbial biosynthesis of fatty acid of lipid metabolism, which can be used as precursors for the production of fuels of chemicals from renewable carbon sources, has attracted significant attention in recent years. The regulation of fatty acid biosynthesis pathways has been mainly studied in a model prokaryote, Escherichia coli. During the recent period, global regulation of fatty acid metabolic pathways has been demonstrated in another model prokaryote, Bacillus subtilis, as well as in Streptococcus pneumonia. The goal of this study was to increase the production of long-chain fatty acids by developing recombinant E. coli strains that were improved by an elongation cycle of fatty acid synthesis (FAS). The fabB, fabG, fabZ, and fabI genes, all homologous of E. coli, were induced to improve the enzymatic activities for the purpose of overexpressing components of the elongation cycle in the FAS pathway through metabolic engineering. The ${\beta}$-oxoacyl-ACP synthase enzyme catalyzed the addition of acyl-ACP to malonyl-ACP to generate ${\beta}$-oxoacyl-ACP. The enzyme encoded by the fabG gene converted ${\beta}$-oxoacyl-ACP to ${\beta}$-hydroxyacyl-ACP, the fabZ catalyzed the dehydration of ${\beta}$-3-hydroxyacyl-ACP to trans-2-acyl-ACP, and the fabI gene converted trans-2-acyl-ACP to acyl-ACP for long-chain fatty acids. In vivo productivity of total lipids and fatty acids was analyzed to confirm the changes and effects of the inserted genes in E. coli. As a result, lipid was increased 2.16-fold higher and hexadecanoic acid was produced 2.77-fold higher in E. coli JES1030, one of the developed recombinants through this study, than those from the wild-type E. coli.

• 한국식품영양과학회지
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• 제38권12호
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• pp.1649-1655
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• 2009

곰취(Ligularia fischeri, GC), 고추잎(Capsicum annuum L., GCY), 취나물(Aster scaber, CNM), 머위대(Petasites japonicus S. et Z. Max, MYD) 및 고구마순(Ipomoea batatas L. (Lam), GGM)과 같은 산채들의 물 추출물의 항산화능력을 평가하고 이들 동결건조 블록 물 추출물들의 항산화력과 비교하였다. 산채 물 추출물들과 그들의 동결건조 블록물 추출물들의 항산화력 측정은 2,2-diphenyl-1-picrylhydrazyl(DPPH) radical 소거작용, hydroxyl radical 소거작용 및 아질산염 소거작용과 같은 방법에 의해 알아보았다. 산채 물 추출물은 그들의 동결건조 물 추출물보다 총 페놀함량이 더 높았다. GC, GCY, CNM, MYD 그리고 GGM 물추출물들의 총 페놀 함량은 각각 $471.66{\pm}3.52\;{\mu}g/mg,\;141.33{\pm}2.51\;{\mu}g/mg,\;177.33{\pm}2.88\;{\mu}g/mg,\;238.66{\pm}9.50\;\mu}g/mg\;그리고\;122.67{\pm}3.51\;{\mu}g/mg$이었다. 1000 ppm GC, GCY, CNM 그리고 GGM 물 추출물의 DPPH radical 소거작용은 그들의 동결건조 블록 물 추출물보다 더 높았고, 1000 ppm CNM, GC, GCY, MYD 그리고 GGM의 물 추출물의 DPPH radical 소거작용은 각각 90.9%, 89.9%, 76.6%, 71.1% 그리고 57.4%였다. 10000 ppm GC, GCY, CNM, MYD 그리고 GGM 물 추출물들은 hydroxyl radical 소거작용을 각각 38.8%, 33.4%, 35.9%, 34.3% 그리고 33.8%까지 증가시켰고, GCY, CNM 그리고 GGM의 물 추출물은 동결건조 블록 물 추출물과 유사한 활성을 나타내었으나 GC와 MYD의 물 추출물이이들 동결건조 블록들의 물 추출물의 hydroxyl radical 소거작용보다 약간 더 영향력이 있었다. 산채 물 추출물들과 이들 동결건조 블록 물 추출물들은 실험된 모든 농도에서 DPPH radical 소거작용 및 hydroxyl radical 소거작용을 나타내었다. GC 물 추출물의 아질산염 소거작용은 현저하게 농도 의존적으로 증가하였고, GC 물 추출물의 아질산염 소거작용이 그것의 동결건조 블록 물 추출물의 아질산염 소거작용보다 높았다. 이상의 결과들로부터 동결건조블록이 산채와 비교하여 산채가 가지고 있는 항산화력을 유지하고 있다는 것을 알수 있었다.

• 한국식품영양과학회지
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• 제41권12호
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• pp.1649-1655
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• 2012

본 연구에서는 국내에서 재배한 나무딸기류 과일(blackberry, Korean raspberry, black raspberry, boysenberry, golden raspberry)을 손으로 으깨거나 녹즙기를 사용하여 추출하여 이 추출물의 항산화 성분 및 항산화능을 분석하고, in-vitro 모델을 이용하여 각 추출물의 NO 소거능과 암세포항증식 활성을 분석하였다. 과일추출물의 총 polyphenol과 flavonoid 함량은 각각 0.6~8.9 mg/g과 0.1~7.9 mg/g으로 그 종류에 따라 다양하였다. Black raspberry 추출물은 갈지않고 추출하여도 다른 나무딸기류 과일에 비하여 polyphenol과 flavonoid를 매우 많이 함유하고 있었으며, blackberry, Korean raspberry, golden raspberry를 갈아서 추출한 것이 으깨어서 추출한 것보다 polyphenol 함량과 항산화능이 유의적으로 높았다. 또한 나무딸기류 과일추출물의 항산화능은 총 polyphenol(R=0.995) 및 flavonoid(R=0.967) 함량이 높을수록 증가하는 상관관계가 있었다. 나무딸기류 과일추출물 모두 0.25 mg/mL 이상의 농도에서 유의적인 NO소거능을 보였으며, 과일을 으깨어 추출한 것과 갈아서 추출한 것 간에 차이는 없었다. 나무딸기류 과일추출물을 0.1, 0.25, 0.5 mg/mL의 농도로 HT-29와 KATO-3 암세포에 처리하였을 때 이들 세포의 증식을 각각 3~32%와 0~57%씩 억제시켰다. Blackberry와 Korean raspberry는 0.5 mg/mL 농도에서 갈아서 추출한 추출물이 으깬 것보다 유의적으로 HT-29 암세포 증식을 억제했으며, KATO-3 암세포에서는 과일을 으깨어 추출한 것과 갈아서 추출한 것 간에 차이가 없었다. 나무딸기류 과일추출물의 NO 소거능이 증가할수록 HT-29(R=0.602)와 KATO-3(R=0.498) 암세포 증식 억제효과도 증가하였다.

• 한국발생생물학회지:발생과생식
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• 제16권3호
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• pp.227-233
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• 2012

Circadian timing system plays a major role in a wide range of reproductive function. However it is plausible idea that other environmental and/or internal cue might be simultaneously participated in the optimal regulation of reproductive system. In the present study we extended the reverse feeding (RF) time regimen up to 8 weeks, then measured the general and reproductive indices of the animals. The animals of ad libitum feeding group (Control, CON) have free access to food for 4, 6 and 8 weeks, respectively. The day feeding animals (reverse feeding, RF group) have restricted access to food during daytime (09:00-18:00) for 4, 6 and 8 weeks, respectively. When the feeding schedules were over, key indices were measured. After 4 weeks and 8 weeks of feeding, body weights of animals were not significantly different. However, body weights of 6 weeks RF animals were significantly smaller than those of control animals (CON : RF = $333.46{\pm}12.71$ g : $289.91{\pm}8.31$ g, p<0.01). The blood glucose levels of 4 weeks RF animals were significantly decreased compared to the levels of control animals (CON : RF = $161.4{\pm}2.7$ mg/dL : $176.7{\pm}5$ mg/dL, p<0.01) while the levels of 6 weeks RF and 8 weeks RF animals were not different form those of control animals. Reproductive and non-reproductive tissue weights from 6 weeks RF group were significantly lowered than those from CON group (testis, CON : RF = $1.4714{\pm}0.0174$ g : $1.3724{\pm}0.0168$ g, p<0.001; epididymis, CON : RF = $0.3574{\pm}0.0059$ g : $0.3243{\pm}0.0068$ g, p<0.001; seminal vesicle, CON : RF = $0.1655{\pm}0.0068$ g : $0.1328{\pm}0.0054$ g, p<0.001; prostate, CON : RF = $0.3350{\pm}0.0231$ g : $0.2528{\pm}0.0143$ g, p<0.01). After 4 weeks and 8 weeks of reverse feeding, sperm counts in RF animals were markedly reduced than those in control animals[CON 4W : RF 4W = $121.17{\pm}9.96\;({\times}10^6)$ : $50.86{\pm}9\;({\times}10^6)$, p<0.001; CON 8W : RF 8W= $138.69{\pm}9.8\;({\times}10^6)$ : $108.94{\pm}4.22\;({\times}10^6)$, p<0.001]. Present study indicates that RF may induce an adaptable metabolic stress and cause impairment of androgen-dependent reproductive tissues. On-going longitudinal studies will allow a better understanding of the how does mealtime shift affect the reproductive function and exact nature of adaptation.

• BMB Reports
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• 제41권2호
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• pp.112-118
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• 2008

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 ${\mu}M$ methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.748-755
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• 2014

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

• Bulletin of the Korean Chemical Society
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• 제34권8호
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• pp.2419-2424
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• 2013

Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.