• Title/Summary/Keyword: E. coli(K99)

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Cloning and Expression of a Chitinase Gene from Thermoactinomyces vulgaris KFB-C100

  • Yooh, Ho-Geun;Kim, Hee-Yun;Lim, Young-Hee;Cho, Hong-Yon
    • Journal of Microbiology and Biotechnology
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    • v.8 no.6
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    • pp.560-567
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    • 1998
  • We have found that Thermoactinomyces vulgaris KFB-Cl00 produces a chitinase. The optimum temperature and pH of the enzyme activity were $55^{\circ}C$ and 6.5. The enzyme was stable after heat treatment at $80^{\circ}C$ for 30 min and stable in acidic and basic conditions (PH 6.0~11.0). The thermostable endo-chitinase from Thermoactinomyces vulgaris KFB-C100 was cloned into the plasmid pBR322 by using E. coli DH5$\alpha$ as a host strain. The positive clone carrying a recombinant plasmid (PKCHI23) with a 4.1-kb fragment containing the chitinase gene was found. The recombinant plasmid was analyzed to determine the essential region for chitinase activity and obtained a 2.3-kb fragment, which was sub cloned into pTrc99A using the PstI and SalI sites to construct pTrc99A/pKCHI23-3. The resulting plasmid exerted high chitinase activity upon transformation of E. coli XL1-Blue cells. Chitinase was overproduced 14 times more in the clone cells than in the wild-type cells and the enzyme was purified to homogeneity. The purified enzyme showed the similar properties as the native chitinase from T. vulgaris in terms of molecular weight and substrate specificity. The catalytic action of the cloned enzyme was an endo type, producing chitobiose as a major reaction product.

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Characterization of Natural Antiseptic System Utilized Propolis and Herb Essential Oil (프로폴리스와 허브에센셜오일을 이용한 천연방부제형의 특성)

  • Jeong, Noh-Hee;Shin, Kwang-Jun
    • Journal of the Korean Applied Science and Technology
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    • v.24 no.2
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    • pp.99-108
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    • 2007
  • The cosmetic and toiletries are necessary health care & household for common life. However we need antiseptic which is effecting harmlessly to the human body. There are propolis, Lavender, Lemon, essential oil in the natural antiseptic materials. This work proceeded design Natural-antiseptic system with three materials as above-mentioned. Natural-antiseptic system was accomplished with propolis (2%), Lavender essential oil (0.3%), Lemon essential oil (0.3%) safety out of Polysorbate 20 (0.5%), Polysorbate 80 (0.5%), PEG (60) hydrogenated castor oil (0.45%), ethanol (5%). The antimicrobial test was experimented on E. coli and Bacillus stearothermophilus. In this antimicrobial test, we found that the effect of antisepsis against E. coli and Bacillus stearothermophilus with propolis 0.3%, Lavender essential oil 0.045% and Lemon essential oil 0.045% was improved. Therefore could expect Natural-antiseptic system product for moisturizing skin toner for face, nourishing essence and wet tissue for clean other things.

Year-round Monitoring of Verotoxin-producing Escherichia coli from Feces of Dairy Cattle

  • Kobayashi, Y.;El-Sawy, H.B.I.
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.5
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    • pp.789-794
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    • 2007
  • A PCR-aided monitoring of verotoxin-producing Escherichia coli (VTEC) was performed over the period of 12 months by using fresh feces collected monthly from 5 dairy cows that had been identified as VTEC carriers. The PCR products were confirmed to be verotoxin genes by Southern hybridization using a gene fragment of verotoxin 2 as a probe. Although seasonal variation of VTEC shedding seemed to depend on each cow, several factors may have influenced the frequency of detection. Shedding of VTEC tended to be reduced during grazing from the middle of May up to the beginning of October. Only one cow was positive for VTEC in August. Dry-off was also suggested to have a depressive effect on VTEC shedding, i.e. 3 of 4 dry cows showed no shedding of VTEC. Contrary to these factors, winter or indoor rearing tended to increase VTEC with only 5/24 samples being negative during the period from November to April. Total VFA concentration was higher (p<0.05) in VTEC-positive feces than in VTEC-negative feces, while fecal pH and VFA proportions were not different. Partial sequences of verotoxin genes from feces of 4 VTEC-positive cows were nearly identical (99-100%), suggesting that gut bacteria sharing the same gene were distributed among the cows. The present results indicate that grazing and dry-off could be factors which reduce VTEC shedding, while winter/indoor rearing may be a factor which increases the shedding, possibly through on-farm interactions.

Characterization of Lactobacillus acidophilus Isolated from Piglets and Chicken

  • Ahn, Y.T.;Lim, K.L.;Ryu, J.C.;Kang, D.K.;Ham, J.S.;Jang, Y.H.;Kim, H.U.
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.12
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    • pp.1790-1797
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    • 2002
  • Lactic acid bacteria were isolated from piglets and chicken and characterized. Lactic acid bacteria showing resistance to low pH and bile, adhesion to intestinal epithelium cells, and the inhibition of Escherichia coli and Salmonella spp. were identified as Lactobacillus acidophilus. L. acidophilus PF01 survived for 2 h in MRS broth adjusted to pH 2. L. acidophilus CF07 was less resistant than L. acidophilus PF01 to pH 2, but survived at pH 2.5 for 2 h. Both of isolates were able to grow in MRS broth containing 0.3% (w/v) bile, with L. acidophilus CF07 being more tolerant to bile than L. acidophilus PF01. L. acidophilus PF01 and CF07 adhered specifically to the duodenal and jejunal epithelium cells of piglet, and the cecal and duodenal epithelium cells of chicken, respectively. Both of isolates did not adhere to the epithelium cells of the various animal intestines from which they were isolated. When L. acidophilus was cultured with E. coli and Salmonella spp. in MRS broth, MRS broth containing 2% skim milk powder or modified tryptic soy broth at $37^{\circ}C$, L. acidophilus PF01 and CF07 inhibited the growths of E. coli K88 and K99, and S. enteritidis and S. typhimurium, respectively. Both of isolates were found to possess the essential characteristics of probiotic lactic acid bacteria for piglet and chicken.

Production of a Specific Yolk Antibody against Enterotoxigenic E. coli F41 Fimbrial Antigen (장독성대장균 F41 섬모항원에 대한 특이난황항체 생산)

  • Shin, S.O.;Kim, J.W.
    • Journal of Animal Science and Technology
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    • v.44 no.5
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    • pp.633-642
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    • 2002
  • Enteric colibacillosis has economically become an important disease of young animals as a result of increasing intensification of farrowing management. The objective of this experiment is to isolate fimbrial antigen from enterotoxigenic E. coli F41, to develop specific polyclonal IgY which can effectively neutralize or reduce the proliferation of pathogens in feed or living animal system, and to apply IgY technologies to animal industry. The results obtained were as follows: The molecular weight of the purified F41 antigen was 29,500 dalton on sodium dodecyl sulfate-polyacrylamide gels. Fimbrial antigen was confirmed by the western blot method. It was observed that after immunization the level of serum antibody titer of laying hen was shown in two weeks and gradually increased. The antibody titer in egg yolk appeared two weeks after it was shown in serum antibody. The titers of egg yolk antibody were gradually increased to the maximum level of 320,000 (antigen 50${\mu}g$/$m\ell$), 450,000 (antigen 200${\mu}g$/$m\ell$), and 320,000 (antigen 600${\mu}g$/$m\ell$). According to the results of specificity test by ELISA, the anti-F41 antibodies from chicken serum and egg yolk reacted only with ETEC F41 antigen. There was no cross reaction with other ETEC strains (K88, K99, and 987P). In vitro condition, as a result of antigen binding ability of yolk antibodies, bacterial concentration was rapidly decreased to $10^5$ CFU/$m\ell$ from $10^9$ CFU/$m\ell$ when 2${\sim}$4 mg/$m\ell$ of freeze dried WSF (water soluble fraction) was added.

Comparative Characterization on Bacterial Disinfection Using Trichloroisocyanuric Acid, Bromochlorodimethylhydantoin, and Calcium Hypochlorite (삼염화이소시아눌산, 브룸염화히단토인, 및 칼슘하이포클로라이트의 농도 및 접촉시간에 따른 미생물 사멸특성)

  • Oh, Se-Hyun;Yoo, Hyun-Duk;Lee, Han-Na;Kim, Eung-Soo
    • Microbiology and Biotechnology Letters
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    • v.35 no.4
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    • pp.334-338
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    • 2007
  • The disinfection effects of trichloroisocyanuric acid (TICA), calcium hypochlorite (CH), and Bromochlorodimethylhydantoin (BCDMH) on various bacteria in aqueous suspension were comparatively characterized at various concentrations and exposure times of each disinfectant. When various bacteria cells ($10^8\;CFU/ml$) including Escherichia coli, Pseudomonas aeroginosa, Enterococcus faecalis, Bacillus cereus, Legionella pneumophila, and Staphylococcus aureus were exposed with a solution containing 8 ppm each of TICA, a 99% of the initial cells were killed in 60 sec, 368 sec, 372 sec, 506 sec, 812 sec, and 909 sec, respectively. In addition, the minimum exposure time required to kill 99% of E. coli ($10^8\;CFU/ml$) with 8 ppm of each TICA, BCDMH and CH was measured at 60 sec, 114 sec, and 7,100 sec, respectively. These comparative studies demonstrate that disinfection efficacy is highly variable depending on microbial species as well as disinfectant type, concentration, and exposure time.

Treatment of Thickened Sewage Sludge by Using the Sea Waste Resource and the Radiation Technology (방사선과 폐수산자원을 이용한 하수 농축슬러지 처리에 관한 연구)

  • Yoo Dae-Hyun;Lee Jae-Kwang;Lee Myun-Joo;Kang Ho
    • Resources Recycling
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    • v.13 no.4
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    • pp.17-22
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    • 2004
  • This study was focused on the treatment of thickened sewage sludge. General bacteria and E. coli were disinfected over 99% and organic compounds were decomposed after irradiation. It was suggested that this process can be an alternative for digestion Process. The moisture content in sludge was decreased over 10% (w/w) and the coagulation of sludge particles was enhanced by irradiation at the dose of 15 kGy and addition of sea waste resource (star-fish) as a dewatering aid.

Cloning and High Expression of Nattokinase Gene from Bacillus subtilis BB-1 (Bacillus subtilis BB-1으로부터 나토키나아제 유전자 크로닝 및 대량발현)

  • Lee Young-Hoon;Lee Sung-Ho;Park Ki-Hoon;Choi Young-Ju;Jeong Yong-Kee;Gal Sang-Wan
    • Journal of Life Science
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    • v.16 no.2 s.75
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    • pp.274-281
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    • 2006
  • A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

Manufacturing and Characteristics of the Electrodeless UV Lamp for Disinfection of the Sewage Effluent (하수 방류수 살균소독을 위한 무전극 UV 램프의 제조 및 특성)

  • Shin, Dong Ho;Lee, Yong Taek
    • Applied Chemistry for Engineering
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    • v.16 no.4
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    • pp.570-575
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    • 2005
  • In this study, we have manufactured electrodeless ultraviolet lamp which has a long life and a high degree of efficiency than the existing electrode UV lamp used in sewage effluent sterilization disinfection. First, we investigated change of UV intensity and temperature of lamp by activation materials. The best results for the dose response experiments were 250 minutes stabilizing to UV intensity of $300{\mu}W/cm^2$ and surface temperature $200{\sim}250^{\circ}C$ in Hg/Ind's weight ratio 95/5. When electrodeless UV lamp emits light for prolonged hours, surface temperature of lamp increases. therefore, temperature change is studied using a duplex lamp for cooling in actual sewerage process. Also, manufactured electrodeless UV lamp showed sterilization efficiency of more than 99.9% as result that experiment manufactured electrodeless UV lamp by E-coli. for sterilization disinfection of sewage effluent.

Molecular Cloning and Nucleotide Sequence of Xylanase gene (xynT) from Bacillus alcalophilus AX2000. (Bacillus alcalophilus AX2000 유래 xylanase 유전자 (XynT)의 Cloning과 염기서열 분석)

  • Park Young-Seo
    • Journal of Life Science
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    • v.15 no.5 s.72
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    • pp.734-738
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    • 2005
  • A gene coding for xylanase from alkali-tolerant Bacillus alcalophilus AX2000 was cloned into Escherichia coli $DH5\alpha$ using pUC19. Among 2,000 transformants, one transformant showed clear zone on the detection agar plate containing oat-spells xylan. Its recombinant plasmid, named pXTY99, was found to carry 7.0 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynT) was determined, xynT gene was found to consist of 1,020 base-pair open reading frame coding for a poly-peptide of 340 amino acids with a deduced molecular weight of 40 kDa. The coding sequence was preceded by a putative ribosome binding site, and the transcription initiation signals. The deduced amino acid sequence of xylanase is similar to those of the xylanases from Bacillus sp. Nl37 and B. stearothermophilus 21 with $61\%$ and $59\%$ identical residues, respectively.