• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1529-1536
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• 2006

We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-t1 (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -t1, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their $M_rs$ were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some $50{\mu}g$ of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at $10{\mu}l$ of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose ($10{\mu}l$) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-t1, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.721-727
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• 2007

Stability-enhanced mutants, H44, 11-94, 5A2-84, and F8, of L-threonine aldolase(L-TA) from Streptomyces coelicolor A3(2)(SCO1085) were isolated by an error-prone PCR followed by a high-throughput screening. Each of these mutant, had a single amino acid substitution: H177Y in the H44 mutant, A169T in the 11-94 mutant, D104N in the 5A2-84 mutant and F18I in the F8 mutant. The residual L-TA activity of the wild-type L-TA after a heat treatment for 20 min at $60^{\circ}C$ was only 10.6%. However, those in the stability-enhanced mutants were 85.7% for the H44 mutant, 58.6% for the F8 mutant, 62.1% for the 5A2-84 mutant, and 67.6% for the 11-94 mutant. Although the half-life of the wild-type L-TA at $63^{\circ}C$ was 1.3 min, those of the mutant L-TAs were longer: 14.6 min for the H44 mutant, 3.7 min for the 11-94 mutant, 5.8 min for the 5A2-84 mutant, and 5.0 min for the F8 mutant. The specific activity did not change in most of the mutants, but it was decreased by 45% in the case of mutant F8. When the aldol condensation of glycine and 3,4-dihydroxybenzaldehyde was studied by using whole cells of Escherichia coli containing the wild-type L-TA gene, L-threo-3,4-dihydroxyphenylserine(L-threo-DOPS) was successfully synthesized with a yield of 2.0 mg/ml after 20 repeated batch reactions for 100 h. However, the L-threo-DOPS synthesizing activity of the enzyme decreased with increased cycles of the batch reactions. Compared with the wild-type L-TA, H44 L-TA kept its L-threo-DOPS synthesizing activity almost constant during the 20 repeated batch reactions for 100 h, yielding 4.0 mg/ml of L-threo-DOPS. This result showed that H44 L-TA is more effective than the wild-type L-TA for the mass production of L-threo-DOPS.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1636-1643
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• 2012

Enzymatic pre-bleaching by modification of pulp fibers with xylanases is an attractive approach to reduce the consumption of toxic bleaching chemicals in the paper industry. In this study, an alkaliphilic endoxylanase gene was isolated from metagenomic DNA of a structurally stable thermophilic lignocellulose-degrading microbial consortium using amplification with conserved glycosyl hydrolase family 10 primers and subsequent genome walking. The full-length xylanase showed 78% sequence identity to an endo-${\beta}$-1,4-xylanase of Clostridium phytofermentans and was expressed in a mature form with an N-terminal His6 tag fusion in Escherichia coli. The recombinant xylanase Xyn3F was thermotolerant and alkaliphilic, working optimally at $65-70^{\circ}C$ with an optimal pH at 9-10 and retaining >80% activity at pH 9, $60^{\circ}C$ for 1 h. Xyn3F showed a $V_{max}$ of 2,327 IU/mg and $K_m$ of 3.5 mg/ml on birchwood xylan. Pre-bleaching of industrial eucalyptus pulp with no prior pH adjustment (pH 9) using Xyn3F at 50 IU/g dried pulp led to 4.5-5.1% increase in final pulp brightness and 90.4-102.4% increase in whiteness after a single-step hypochlorite bleaching over the untreated pulp, which allowed at least 20% decrease in hypochlorite consumption to achieve the same final bleaching indices. The alkaliphilic xylanase is promising for application in an environmentally friendly bleaching step of kraft and soda pulps with no requirement for pH adjustment, leading to improved economic feasibility of the process.

• 대한환경공학회지
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• 제32권10호
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• pp.979-985
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• 2010

본 연구는 서울시에 위치한 먹는물 약수터를 대상으로 분변오염 지표미생물인 장구균 검출 특성과 염기서열을 이용한 동정 및 반코마이신 내성 장구균 특성을 평가하였다. 먹는물 약수터의 장구균은 212건의 시료 중 42건(19.8%)에서 0~110 CFU/100 mL의 범위로 검출되었고 대장균의 검출빈도와 유사(t-검정, p-값 0.268, 유의수준 0.05)하여 지표미생물로서 적용가능성을 확인하였다. 16S rDNA 염기서열을 이용한 동정에서는 표본적으로 추출한 56개 검체에서 Enterococcus faecalis (24검체), E. faecium (10검체), E. casseliflavus (10검체), E. gallinarum (3검체), E. hirae (2검체), E. durans (2검체), E. sanguinicola(1검체) 순서로 나타났으며, 가장 높은 빈도로 검출되는 E. faecalis는 국내 병원환자의 임상검체에서 분리되는 우점종 분포와 유사하였다. 반코마이신 내성 장구균은 검체 시료 53검체 중 14(26.4%)검체에서 vanB 및 vanC1/C2 형이 각각 2와 12검체가 확인 되었다. 본 연구결과는 먹는물 약수터가 분변오염 등으로 인하여 장구균 검출 가능성이 있을 것으로 판단되어 소독 등 외부오염물질 유입 차단 및 저감대책이 필요할 것을 시사하였다.

• Journal of Animal Science and Technology
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• 제47권1호
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• pp.39-48
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• 2005

Outside muscle of pork ham were cut to cube(7 $\times$ 10 $\times$2 ern) and three Korea traditional seasonings such as soybean paste(Tl), garlic paste(T2), red pepper paste(T3) were seasoned by the proportions of meat to seasonings(1 : 1), respectively. The seasoned samples were fermented by fill into plastic box at 0 $\pm$ 1 $^{\circ}C$ for 10 days. And then, the fermented meat from each pack was vacuum-packaged and stored at 0 $\pm$ 1 $^{\circ}C$ for up to 9 weeks. pH and shear force were decreased during storage periods in all treatment groups and WHC was decreased with storage in T2. The saccarinity of T1 was increased and salinity increased during storage in all treatment groups. pH of T2 was increased than that of other treatments, while decreased saccarinity and shear force of in T2. The salinity were higher in the order of T1 > T2 > T3. Volatile basic nitrogen (VBN) value were increased with storage in all treatment groups. Thiobarbituric acid reactive substances (TSARS) value of Tl was increased with storage while it was decreased T2. Thiobarbituric acid reactive substances(TSARS) value was higher in the order of T1 > T3 > T2 at 9weeks of storage. Surface meat L' values of T1 was increased with storage and T3 decreased with storage whereas, surface meat a' values of T1 was decreased with storage, and T2 was increased with storage. Surface meat b' values of T3 was decreased with storage. Escherichia coli were decreased during storage periods in all treatment groups.

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.645-654
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• 1995

Porphyromonas endodontalis is a black-pigmented anaerobic Gram negative rod which is associated with endodontal infections. It has been isolated from infected dental root canals and submucous abscesses of endodontal origin. DNA probe is an available alternative, offering the direct detection of a specific microorganism. Nucleic-acid probes can be off different types: whole different: whole-genomic, cloned or oligonucleotide probes. Wholegenomic probes are the most sensitive because the entire genome is used for possible hybridization sites. However, as genetically similar species of bacteria are likely to be present in specimences, cross-reactions need to be considered. Cloned probes are isolated sequences of DNA that do not show cross-reactivity and are produced in quantity by cloning in a plasmid vector. Cloned probes can approach the sensitivity found with whole-genomic probes while avoiding known cross-reacting species. Porphyromonas endodontalis ATCC 35406 (serotype $O_1K_1$) was selected in this experiment to develop specific cloned DNA probes. EcoR I-digested genomic DNA fragments of P. endodontalis ATCC 35406 were cloned into pUC18 plasmid vector. From the E. coli transformed with the recombinant plasmid 4 clones were selected to be tested as specific DNA probes. Restriction-digested whole-genomic DNAs prepared from P. gingivalis 38(serotype a), W50(serotype b), A7A1-28(serotype C), P. intermedia 9336(serotype b), G8-9K-3(serotype C), P. endodontalis ATCC 35406(serotype $O_1K_1$), A. a Y4(serotype b), 75(serotype a), 67(serotype c), were each seperated on agarose gel electrophoresis, blotted on nylon membranes, and were hybridized with digoxigenin-dUTP labeled probe. The results were as follows: 1. Three clones of 1.6kb(probe e), 1.6kb(probe f), and 0.9kb(probe h) in size, were obtained. These clones were identified to be a part of the genomic DNA of P. endodontalis ATCC 35406 judging from their specific hybridization to the genomic DNA fragments of their own size on Southern blot. 2. The clones of 4.9kb(probe i) was identified to be a part of the genomic DNA of P. endodontalis ATCC 35406. but not to specific for itself. It was hybridized to P. gingivalis A7A1-28, P. intermedia G89K-3.

• Applied Biological Chemistry
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• 제39권3호
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• pp.195-199
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• 1996

$crp^{\ast}$ 유전자가 도입된 대장균 MK2001($crp^{{\ast}1}$}, cya::km)을 숙주로 사용하여 mal 유전자 발현을 촉진시키는 유전자의 하나인 sfs1(sugar fermentation stimulation)의 구조해석 결과에 의하면, 잠정적인 sfs1의 promoter 영역에는 CRP 단백질과의 결합영역으로 보이는 염기배열이 존재하였다. 본 실험에서는 sfs1 유전자의 cAMP-CRP에 의한 발현 조절을 확인하고자, lacZ 와의 융합 유전자를 작성하였다. 작성된 융합 유전자는 cya 결손주인 Tp2010에서 cAMP의 첨가에 의해 ${\beta}-galactosidase$ 활성이 크게 증가하였으며, Western blotting의 실험에서도 같은 결과를 나타냈다. in vivo에서 발현이 확인된 전사산물은 cAMP에 의해 전사 촉진이 일어났으며, CRP의 결합부위로 예상되는 DNA 영역은 cAMP가 존재하면 CRP 단백질과 결합하는 특성을 나타내었다. 이상의 결과로 보아, sfs1 유전자의 발현은 UMP-CRP에 의한 전사촉진 현상을 받는 것으로 나타났다.

• Journal of Applied Biological Chemistry
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• 제55권2호
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• pp.117-121
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• 2012

손바닥 선인장(Opuntia humifusa) 줄기 열수와 75% 에탄올 추출물의 일부 생리활성을 조사한 결과는 다음과 같다. DPPH 전자공여능은 열수 추출물에서 79.07%, 에탄올 추출물에서 82.54%를 보였다. 각 세포주에 대한 세포독성은 열수 추출물이 에탄올 추출물을 처리(100 ${\mu}g/well$)한 경우에서 보다 전반적으로 높게 나타났다. 열수 추출물은 HeLa와 AGS 세포주에서 50% 이상 세포 독성을 나타내었다. 항균 효과를 조사한 결과 그람 음성세균인 S. aureus KCCM 11593에 대해서는 물과 75% 에탄올 추출물에 있어서 항균효과를 나타내었으나 나머지 세균에 있어서는 항균 효과가 없었으며, 오히려 세포 생장효과가 있는 것으로 나타났다. 또한 NO 생성 저해 효과에 있어서는 물 추출물(34.31%)이 에탄올 추출물(25.59%)에서 보다 더 항염증효과가 높은 것으로 나타났다. 전구지방세포(3T3-L-1)의 분화에 미치는 영향에서는 처리 농도가 증가할수록 지방세포의 분화가 증가되어 물과 에탄올 추출물 모두 80과 100 ${\mu}g/mL$처리구 에서는 분화촉진제인 IBMX + dexamethasone의 처리구보다 다소 높은 분화정도를 보였다. 이와 같은 결과로 볼 때 천년초 줄기는 항염, 항균, 항산화, 항암효과에서 우수한 활성을 나타내어 앞으로 여러 가지 유용한 기능성 식품과 화장품 소재로의 개발을 기대할 수 있을 것으로 보인다.

• 대한수의학회지
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• 제42권1호
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• pp.45-54
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• 2002

The study was carried out to characterize the pharmacokinetics after intravenous (iv, 20 mg/kg) and oral (p.o. 100 mg/kg) administration as oxytetracycline (OTC) and tiamulin (TIA) mixture in swine and to determine interaction between OTC and TIA against various pig pathogenic bacteria. The antibacterial effects of OTC in combination with TIA in vitro showed synergistic effect against Salmonella typhimurium 1925, Pasteurella multocida Type A, P. multocida Type D, Krebsiella Pneumoniae 2001, K. Pneumoniae 1560, K. Pneumoniae 2208, Haemophillus pleuropneumonia S 2, and H. pleuropneumonia S 5, but against additive effect E. coli K88ab and S. choleraesuis on the basis of fractional inhibitory concentration (FIC) index. On the while, after i.v. and p.o. administration of OTC and TIA mixture, each OTC and TIA concentrations in plasma were fitted to an open two-compartment model. After i.v. administration of OTC-TIA mixture, the mean distribution half-life ($T_{1/2{\alpha}}$) of OTC and TIA in plasma showed 0.29 h and 0.17 h, and the mean elimination half-life ($T_{1/2{\beta}}$) of those was 4.36 h and 6.64 h, respectively. The mean volume of distribution at steady state ($Vd_{ss}$) of OTC and TIA was $0.85{\ell}/kg$ and $2.44{\ell}/kg$, respectively. After oral administration of OTC and TIA mixture, the mean maximal absorption concentrations ($C_{max}$) of OTC and TIA were $0.60{\mu}g/m{\ell}$ at 1.07 h ($T_{max}$) and $1.68{\mu}g/m{\ell}$ at 1.85 h ($T_{max}$), respectively. The mean elimination half-life ($T_{1/2{\beta}}$) of those showed 6.84 h and 6.36 h. In conclusion, we could suggest in this study that the combination of OTC and TIA may be recommended for the antibacterial therapy against polymicrobial infections, and both OTC and TIA showed large distribution to tissues and high $C_{max}$ after p.o. administration.