• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1299-1306
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• 2015

This study aims to investigate the mechanism of action of the cinnamon bark essential oil (CB), when used singly and also in combination with piperacillin, for its antimicrobial and synergistic activity against beta-lactamase TEM-1 plasmid-conferred Escherichia coli J53 R1. Viable count of bacteria for this combination of essential oil and antibiotic showed a complete killing profile at 20 h and further confirmed its synergistic effect by reducing the bacteria cell numbers. Analysis on the stability of treated cultures for cell membrane permeability by CB when tested against sodium dodecyl sulfate revealed that the bacterial cell membrane was disrupted by the essential oil. Scanning electron microscopy observation and bacterial surface charge measurement also revealed that CB causes irreversible membrane damage and reduces the bacterial surface charge. In addition, bioluminescence expression of Escherichia coli [pSB1075] and E. coli [pSB401] by CB showed reduction, indicating the possibility of the presence of quorum sensing (QS) inhibitors. Gas-chromatography and mass spectrometry of the essential oil of Cinnamomum verum showed that trans-cinnamaldehyde (72.81%), benzyl alcohol (12.5%), and eugenol (6.57%) were the major components in the essential oil. From this study, CB has the potential to reverse E. coli J53 R1 resistance to piperacillin through two pathways; modification in the permeability of the outer membrane or bacterial QS inhibition.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.325-330
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• 2007

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell and expression of N-terminal domain consisted of 1-498 amino acids (N-Rne) is sufficient to support normal cellular growth. By utilizing these properties of RNase E, we developed a genetic system to screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that lead to various phenotypes. Using this system, we identified three kinds of mutants. A mutant N-Rne containing amino acid substitution in the S1 domain (I6T) of the protein was not able to support survival of E. coli cells, and another mutant N-Rne with amino acid substitution at the position 488 (R488C) in the small domain enabled N-Rne to have an elevated ribonucleolytic activity, while amino acid substitution in the DNase I domain (N305D) only enabled N-Rne to support survival of E. roli cells when the mutant N-Rne was over-expressed. Analysis of copy number of ColEl-type plasmid revealed that effects of amino acid substitution on the ability of N-Rne to support cellular growth stemmed from their differential effects on the ribonucleolytic activity of N-Rne in the cell. These results imply that the genetic system developed in this study can be used to isolate mutant RNase E with various phenotypes, which would help to unveil a functional role of each subdomain of the protein in the regulation of RNA stability in E. coli.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.504-511
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• 2016

Lactulose, a synthetic disaccharide, has received increasing interest because of its role as a prebiotic that can increase the proliferation of Bifidobacterium and Lactobacillus spp. and enhance the absorption of calcium and magnesium. While the industrial production of lactulose is still mainly achieved by the chemical isomerization of lactose in alkaline media, this process has drawbacks including the need to remove catalysts and by-products, as well as high energy requirements. Recently, the use of cellobiose 2-epimerase (CE) has been considered an interesting alternative for industrial lactulose production. In this study, to develop a process for enzymatic lactulose production using CE, we screened improved mutant enzymes ($CS-H^RC^E$) from a library generated by an error-prone PCR technique. The thermostability of one mutant was enhanced, conferring stability up to $75^{\circ}C$, and its lactulose conversion yield was increased by 1.3-fold compared with that of wild-type CE. Using a recombinant Escherichia coli strain harboring a CS35 $H^RC^E$-expressing plasmid, we prepared cell beads immobilized on a Ca-alginate substrate and optimized their reaction conditions. In a batch reaction with 200 g/l lactose solution and the immobilized cell beads, lactose was converted into lactulose with a conversion yield of 43% in 2 h. In a repeated 38-plex batch reaction, the immobilized cell beads were relatively stable, and 80% of the original enzyme activity was retained after 4 cycles. In conclusion, we developed a reasonable method for lactulose production by immobilizing cells expressing thermostable CE. Further development is required to apply this approach at an industrial scale.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.223-227
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• 2010

RNase E plays a major role in the degradation and processing of a large number of RNA transcripts in Escherichia coli and forms the core component of the degradosome, a large protein complex involved in RNA metabolism. RraA and RraB are recently discovered protein inhibitors of RNase E and are evolutionarily conserved. In this study, we observed that, unlike RraA, overexpression of RraB did not rescue growth arrest of E. coli cells overexpressing RNase E. To examine whether this phenomenon stems from differential inhibitory effects of RraA and RraB on RNase E substrates, we analyzed three in vivo RNase E substrates. The results showed that RraA inhibited RNase E activity more efficiently than RraB on the degradation of RNA I, which controls the copy number of ColE1-type plasmid, and rpsO mRNA encoding ribosomal protein S15, while RraB was unable to inhibit the processing of pM1 RNA, a precursor of the RNA component of RNase P, by RNase E. Our results imply that RraB inhibits RNase E activity in a more substrate-dependent manner than RraA and this property of RraB may explain why overexpression of RraB could not rescue cells overexpressing RNase E from growth arrest.

• Proceedings of the Korean Society of Life Science Conference
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• 2001.06a
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• pp.67-86
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• 2001

A strain producing strongly fibrinolytic enzyme was isolated from soil and was identified to be Bacillus subtilis by biochemical and physiological characterization. The optimal culture conditions for the production of fibrinolytic enzyme was determined to be 1.0% tryptone, 1.5% soluble starch, 0.5% Peptone, 0.5% NaCl, $(NH_{4})_{3}PO_4.3H_{2}O, and MgSO_{4}.7H_{2}O.$ Initial pH and temperature were pH 8.0 and $30^{\circ}C$ , respectively, The highest enzyme production was observed at 30 hours of cultivation at $30^{\circ}C$ The fibrinolytic enzyme was purified to homogeneity by DEAE Sephadex A-50 ion exchange column chromatography, 70% ammonium sulfate precipitation, Sephadex G-200 and G-75 gel filtration column chromatography. The molecular weight of the purified enzyme was 28,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A gene encoding the fibrinolytic enzyme was cloned into a plasmid vector pBluescript, transforming E.coli XL-1 Blue. The clone was able to degrade fibrin, This indicated that the gene could encode a fibrinolytic enzyme. The nucleotide sequence of the 2.7 kb insert was determined in both direction. One open reading frame composed of 1023 nucleotides was found to be a potential protein coding region. There was the putative Shine-Dalgano sequence and TATA box upstream of the open reading frame. The homology search data in the genome database showed that both the 2.7 kb insert and 1 kb open reading frame carried no significance in the nucleotide sequence of known fibrinolytic enzyme from Bacillus serovars. The recombinant cell harboring the novel gene involved in fibrinolysis was subjected to protein purification. The molecular mass of the purified fibrinolytic enzyme was determined to be 31864 Dalton, which was highly in accordance with the molecular mass(33 kDa) of the fibrinolytic gene deduced from the insert. The fibrinolytic enzyme was Purified 50.5 folds to homogeneity in overall yield of 10.7% by DEAE Sephadex A-50 ion exchange, 85% ammonium sulfate precipitation, Sephadex G-50, Superdex 75 HR FPLC gel filtration. In conclusion, a novel fibrinolytic gene from Bacillus subtilis was identified and characterized by cloning a genomic library of Bacillus subtilis into pBleuscript. For the soybean fermented by this strain, it is found that there increased assistant protein about 20% compared to the soybean not fermented and increased about 30% according to amino acid analysis and, in particular, essential amino acid increased about 40%. When keeping this fermented soybean powder at room temperature for about 70days, it showed very high stability maintaining almost perfect activity and, therefore, it gave us great suggestion its possibility of development as a new functional food.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.18 no.12
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• pp.575-580
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• 2017

This study analyzed the effects of RGG box of hnRNP A1 on its subcellular localization and stabilization of hnRNP A1 over a three year period from October 2014. First, a 6R/K mutation in RGG box was generated, and pcDNA1-HA-hnRNP A1(6R/K) was constructed. The subcellular localization of hnRNP A1(6R/K) from the HeLa cells transfected with this plasmid DNA was analyzed by immunofluorescence microscopy. HA-hnRNP A1(6R/K) was found to exhibit nuclear and cytoplasmic fluorescence. The stability of hnRNP A1(6R/K) was checked by Western blot analysis using the expressed protein from the HeLa cells transfected with the pcDNA1-HA-hnRNP A1(6R/K). The results show that HA-hnRNP A1(6R/K) has a smaller size. These confirm that HA-hnRNP A1(6R/K) is localized both in the nuclear and cytoplasm, not because 6R/K mutation affects the nuclear localization of hnRNP A1, but because 6R/K mutation causes hnRNP A1(6R/K) to cleave at the mutation or near the mutation site. The cleaved protein fragment, which lacks the M9 domain (i.e. nuclear localization signal of hnRNP A1), did not exhibit nuclear fluorescence. This suggests that the arginines of RGG box in hnRNP A1 play an important role in stabilizing hnRNP A1. An analysis of the RNA-binding ability of hnRNP A1(6R/K) expressed and purified from bacteria will be a subsequent research project.