• Title/Summary/Keyword: E-coli

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Cloning, Over-expression, and Characterization of YjgA, a Novel ppGpp-binding Protein

  • Gnanasekaran, Gopalsamy;Pan, SangO;Jung, Wontae;Jeong, Kwangjoon;Jeong, Jae-Ho;Rhee, Joon Haeng;Choy, Hyon E.;Jung, Che-Hun
    • Bulletin of the Korean Chemical Society
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    • v.34 no.8
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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.

Effect of ${\beta}$-Mannanase (CTCZYME$^{(R)}$) on the Growth of Young Calf (${\beta}$-Mannanase (CTCZYME$^{(R)}$) 첨가가 어린 송아지의 성장에 미치는 영향)

  • Lee, Se-Young;Lee, Sang-Moon;Kim, Jong-Hyeong;Ki, Kwang-Seok;Kim, Hyeon-Shup;Kam, Dong-Keun;Lee, Jae-Hwan;Lee, Jung-Jin;Bae, Gui-Seck;Seo, Seong-Won
    • Korean Journal of Agricultural Science
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    • v.37 no.2
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    • pp.239-243
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    • 2010
  • The objective of this study was to evaluate effects of supplementation of ${\beta}$-mannanase (CTCZYME$^{(R)}$, CTCBIO, Inc.) on feed intake, growth performance and fecal health of calves fed two levels (3% vs. 8%) of palm kernel meal (PKM). A total of nine Holstein calves were divided into three groups, and fed a conventional starter containing 3% PKM (CON), CON+0.1% CTCZYME$^{(R)}$ (TRT1), or a starter containing 8% PKM+0.1% CTCZYME$^{(R)}$ (TRT2). No clinical symptom of calves was observed through the trial. We did not find significant differences among the treatments on mean feed intake, growth performance, or fecal health during the four-week experimental period. Feed efficiency tended to be improved by adding CTCZYME$^{(R)}$ (0.46, 0.87 and 0.52 for CON, TRT1 and TRT2, respectively). Compared with CON (921 g/d and 786 g/d), TRT2 had lower feed intake (727 g/d) and average daily gain (ADG, 631 g/d) before weaning. However, feed intake (2300 g/d) and ADG (1012 g/d) were similar or even higher in TRT2 than CON (2269 g/d and 560 g/d) after weaning. This was probably due to the effect of a large amount of mannan-oligosaccharide released from PKM by ${\beta}$-mannanase. Salmonella was not detected any fecal samples. No significant difference was observed in the number of fecal E. coli or fecal properties including color, smell, and watery indexes among the treatments. We conclude that a calf starter containing 8% PKM with 0.1% CTCZYME$^{(R)}$ is comparable with a conventional starter in feed intake and growth performance of calf, which is beneficial in terms of reduction in feed cost.

A STUDY OF NI-RESISTANT BACTERIA ISOLATED FROM GINGIVAL CREVICULAR FLUID ON THE PATIENTS WEARING NI-CR ALLOY PROSTHESIS (IN TERMS OF MOLECULAR BIOLOGICAL ASPECTS) (니켈-크롬 합금 보철물 주위 치은 열구내에서 발견된 니켈 내성 균주에 관한 분자생물학적 연구)

  • Chae Young-Ah;Woo Yi-Hyung;Kwon Kung-Rock
    • The Journal of Korean Academy of Prosthodontics
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    • v.41 no.2
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    • pp.207-222
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    • 2003
  • As a material of metal-ceramic prosthesis, nickel as a form of Ni-Cr alloy has been used for many dental prostheses in many cases. However, several problems in use of the alloy have been revealed (ex ; tissue stimulation, skin allergy, hypersensitivity cytotoxicity and carcinogenecity). Little is known about nickel with respect to the relationship between Ni-prosthesis and gaining of Niresistance in oral microorganisms. The present study was undertaken to check whether use of Ni-prosthesis leads to occurrence of Ni-resistant microorganisms. So this study may suggest the possible relationships between the oral microorganisms and nickel-resistance in oral environment. Bacteria were isolated from the gingival crevicular fluid on the patients wearing Ni-Cr prosthesis. The isolated bacteria were tested fir their Ni-resistance in nickel containing media at different concentration from 3mM to 110mM. E. coli HB101 was used as control. The Ni-resistant bacteria were isolated and biochemically identified. The Ni-resistant bacteria were tested several biochemical, molecular-biological tests. Performed tests were : measuring the growth curve, antibiotic test, growth ability test in liquid media, isolation of the chromosome and plasmid, digestion of DNA by restriction enzyme, electrophoresis of chromosome and plasmid DNA, identification of Ni-resistant genes by the DNA hybridization. The results were as follows 1) The bacteria isolated from gingival crevicular fluid on the patients wearing Ni-Cr alloy prosthesis showed nickel-resistance. 2) The isolated microorganisms grew at nickel containing media of high concentrations (60mM-110mM). 3) Based on the biochemical tests, the isolated microorganisms were identified as Enterococcus faecalis(13 cases), Klebsiella pneumoniae(1 case) and Enterobacter gergoviae(1 case). 4) Enterococcus faecalis expressed not only nickel resistance but also the multi-drug resistance to several antibiotics ; chloramphenicol, kanamicin, streptomycin, lincomycin, clindamycin, However, all strain showed the sensitivity against the tetracycline. 5) DNA hybridization result suggests that there is no homology between the previously known gene of nickel resistance in Klebsiella pneumoniae and chromosomal DNA of Enterococcus faecalis.

Formulation and Quality Characteristics of Noni Beverages Mixed with Red Ginseng, Rubus Coreanus and Pomegranate Extracts (홍삼, 복분자, 석류를 첨가한 노니 혼합음료의 개발과 품질특성)

  • Kim, Eun-Mi
    • Culinary science and hospitality research
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    • v.17 no.1
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    • pp.259-269
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    • 2011
  • The purpose of this study was to investigate the physicochemical and sensory characteristics of Noni beverages added with 10% of water(Noni 90), 1% of red ginseng(NR), 4% of rubus coreanus extract(NR), 5% of pomegranate extract(NP), and 4% of rubus coreanus extract and 5% of pomegranate extract(NPR). Calories, protein and total fiber contents were increased in NR, NP and NPR. Total sugar was significantly increased in NG and NPR. Fat, cholesterol and saturated fatty acid were not detected. Ca and Na contents were increased in NG while Fe content was increased in NG, NR, and NPR pH was significantly increased in NG and NP. Sugar degree was significantly increased in NP and NPR. L and b values were decreased in NG, NP, and NR and a value was increased in NG and NR General bacteria count was 0/mL, and Coliform, E. Coli and 0157:H7 were gram(-) in all beverages. In sensory evaluation, color was increased in NPR while taste and overall quality were increased in NG, NR and NP. Sour taste, sweet taste, and flavor were not different between Noni and the mixed beverages. Texture was significantly increased in NP and NPR. Therefore, mixed Noni beverages added with red ginseng, rubus coreanus extract, and pomegranate extract were better than Noni beverage.

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Construction of web-based Database for Haliotis SNP (웹기반 전복류 (Haliotis) SNP 데이터베이스 구축)

  • Jeong, Ji-Eun;Lee, Jae-Bong;Kang, Se-Won;Baek, Moon-Ki;Han, Yeon-Soo;Choi, Tae-Jin;Kang, Jung-Ha;Lee, Yong-Seok
    • The Korean Journal of Malacology
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    • v.26 no.2
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    • pp.185-188
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    • 2010
  • The Web-based the genus Haliotis SNP database was constructed on the basis of Intel Server Platform ZSS130 dual Xeon 3.2 GHz cpu and Linux-based (Cent OS) operating system. Haliotis related sequences (2,830 nucleotide sequences, 9,102 EST sequences) were downloaded through NCBI taxonomy browser. In order to eliminate vector sequences, we conducted vector masking step using cross match software with vector sequence database. In addition, poly-A tails were removed using Trimmest software from EMBOSS package. The processed sequences were clustered and assembled by TGICL package (TIGR tools) equipped with CAP3 software. A web-based interface (Haliotis SNP Database, http://www.haliotis.or.kr) was developed to enable optimal use of the clustered assemblies. The Clustering Res. menu shows the contig sequences from the clustering, the alignment results and sequences from each cluster. And also we can compare any sequences with Haliotis related sequences in BLAST menu. The search menu is equipped with its own search engine so that it is possible to search all of the information in the database using the name of a gene, accession number and/or species name. Taken together, the Web-based SNP database for Haliotis will be valuable to develop SNPs of Haliotis in the future.

Inhibitory Effect of Prunus mune Extracts on Physiological Function of Food Spoilage microorganisms (매실추출물이 변패미생물의 생리기능에 미치는 영향)

  • Ha, Myung-Hee;Park, Woo-Po;Lee, Seung-Cheol;Heo, Ho-Jin;Oh, Byung-Tae;Cho, Sung-Hwan
    • Food Science and Preservation
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    • v.14 no.3
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    • pp.323-327
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    • 2007
  • Moderate consumption of maesil(Prunus mune) was associated with pharmaceutical and physiological effects on human health. The object of this study was to determine the inhibitory effects of Prunus mune extracts(PME) on food spoilage microorganisms. PME was found to have an antibacterial effect on Colletotrichum fragariae. The hydrophilic fractions of PME showed more effective inhibition than did the hydrophobic fractions. In addition, the hydrophilic fractions of PME seemed to inhibit(12-40%) metabolic enzymes related to energy production, including glucose-6-phosphate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and hexokinase. Our data suggest that hydrophilic components of PME might control food spoilage microorganisms because of suppression of membrane enzymatic function.

Effect of commercial sanitizers on microbial quality of fresh-cut iceberg lettuce during storage (세척용 시판 살균제 종류에 따른 신선편의 양상추의 저장 중 미생물 변화)

  • Hwang, Tae-Young
    • Food Science and Preservation
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    • v.24 no.6
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    • pp.827-833
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    • 2017
  • This study was investigated the effects of various commercial sanitizers on microbial characteristics in fresh-cut iceberg lettuce during storage. For screening sanitizer, lettuce was cut and dipped in chlorine water ($0.2ml{\cdot}L^{-1}$), solution of organic acids such as ascorbic acid, citric acid, acetic acid, mixture of ascorbic acid and acetic acid (1-6%), and solutions of commercial sanitizers such as Formula 4$^{TM}$ (1,3,4%), Fresh produce wash$^{TM}$ (1,3,4%), Cleancol$^{TM}$ (1%), Chitochol$^{TM}$ (1%) and Natural Ca$^{TM}$ (0.1%) for 3 min, respectively. Washing lettuce with selected sanitizers resulted in reduction of aerobic bacteria of more than 2 log CFU/g. Initial pH of lettuce was related with the pH of sanitizers. pH ranged from 4.7 to 6.1 in Formula 4 (4%, pH 1.7) and Natural Ca (0.1%, pH 12.0), respectively. Chlorine water showed consistent and significant inhibition effect in all of microorganisms except total coliform. Over 3% of Formula 4 and Fresh produce wash were found to have high bactericidal activity among sanitizers. The sanitizers of chlorine water, Fresh produce wash, Chitochol and Natural Ca were effective in reducing yeast and mould populations. As coliform and E. coli, Formula 4 (4%) showed the highest bactericidal activity. The bactericidal effect of commercial sanitizers during storage varied with the kinds and concentrations of tested sanitizers. Although inhibition effect was not showed during storage, these results suggest that commercial sanitizers could be an alternative to chlorine for washing fresh-cut produce.

Antihypertensive, Antimicrobial and Antifungal Activities of Buckwheat Hydrolysate (메밀 가수분해물의 항고혈압, 항균 및 항곰팡이 활성)

  • Do, Jeong-Ryong;Heo, In-Sook;Back, Su-Yeon;Yoon, Hye-Sook;Jo, Jin-Ho;Kim, Young-Myoung;Kim, Ki-Ju;Kim, Sang-Kyo
    • Korean Journal of Food Science and Technology
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    • v.38 no.2
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    • pp.268-272
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    • 2006
  • Antibacterial, antifungal, and Angiotensin-I-converting enzyme (ACE) inhibitory activities of buckwheat (Fagopyrum esculentum and F. tataricum) hydrolyzed by Viscozyme L and Alcalase 2.4 L were investigated. The Alcalase 2.4L-hydrolyzed buckwheat showed highest yield of 22.10-24.65%. F. esculentum hydrolysate treated with Viscozyme L from Salmonella typhimurium (clear zone: 3-4.7 mm) and Listeria monocytogenes (clear zone: 4-7.2 mm) showed highest antimicrobial activity among enzymes used. F. esculentum hydrolysate treated with Trichoderma reesei showed strongest antifungal activity among enzymes used (clear zone: 3.7-12 mm). Alcalase 2.4L-hydrolyzed F. esculentum and F. tataricum showed strong ACE inhibitory activities (61.19% and 94.48%, respectively).

Effect of Extraction Solvent on the Physiological Properties of Korean Pear Peel (Pyrus pyrifolia cv. Niitaka) (추출 용매를 달리한 한국산 배(Pyrus pyrifolia cv. Niitaka) 과피의 생리 활성)

  • Park, Ji-Su;Han, Inhwa
    • Korean Journal of Food Science and Technology
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    • v.47 no.2
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    • pp.254-260
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    • 2015
  • The effect of the extraction solvent on the physiological properties of the peel of the Korean pear (Pyrus pyrifolia cv. Niitaka) was evaluated. The total phenol content was highest in the 80%(wt) methanol extract, whereas flavonoid content was highest in the 80% ethanol extract. The antioxidant activities of the extracts were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging abilities, and their reducing power. The water and 80% methanol extracts of the pear peel had highest DPPH and ABTS radical scavenging activities, and reducing power, respectively. The inhibition of ${\alpha}$-glucosidase was highest in the 80% methanol extract, and alcohol dehydrogenase activity was highest in the water extract. All three extracts had similar antimicrobial activity. Because water, 80% ethanol, and 80% methanol extracts exhibited high activities in different assays of physiological properties, each solvent could be used for specific purposes.

Cloning and Functional Analysis of Gene Coding for S-Adenosyl-L-Methionine Synthetase from Streptomyces natalensis (Streptomyces natalensis로부터 S-adenosyl-L-methionine synthetase 유전자의 클로닝 및 기능분석)

  • Yoo, Dong-Min;Hwang, Yong-Il;Choi, Sun-Uk
    • Journal of Life Science
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    • v.21 no.1
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    • pp.96-101
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    • 2011
  • S-Adenosyl-L-methionine synthtase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. SAM plays important roles in the primary and secondary metabolism of cells. A metK encoding a SAM-s was searched from Streptomyces natalensis producing natamycin, a predominantly a strong antifungal agent, inhibiting the growth of both yeasts and molds and preventing the formation of aflatoxin in filamentous fungi. To obtain the metK of S. natalensis, PCR using primers designed from the two highly conserved regions for metK genes of Streptomyces strains was carried out, and an intact 1.2-kb metK gene of S. natalensis was cloned by genomic Southern hybridization with PCR product as a probe. To identify the function of the cloned metK gene, it was inserted into pSET152ET for its high expression in the Streptomyces strain, and then introduced into S. lividans TK24 as a host by transconjugation using E. coli ET12567(pUZ8002). The high expression of metK in S. lividans TK24 induced actinorhodin production on R5 solid medium, and its amount in R4 liquid medium was 10-fold higher than that by exconjugant including only pSET152ET.