• 유기물자원화
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• 제13권3호
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• pp.89-96
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• 2005

본 연구에서는 유기인 화합물인 Paraoxon의 분해를 위하여 재조합 대장균에서 세포내 간극(periplasmic space)으로 분비되는 organophosphorus hydrolase(OPH)의 생산에 대하여 고찰하였다. OPH 생산의 향상을 위하여 성장 배지에 첨가되는 최적의 조건은 1.0 mM isopropyl-${\beta}$-D-thiogalactopytanoside (IPTG), 0.25 mM $Co^{2+}$ 및 0.1 mM ethylenediamine tetraacetate (EDTA) 이었다. 이 조건에서 최대OPH 생산은 $174Unit/L{\cdot}OD$를 나타내었다. 또한 1 mM의 Paraoxon은 OPH에 의하여 완전히 분해되었다. 이러한 연구 결과는 토양 및 수계에 잔류하는 환경독성 유기인 화합물을 제거하는 bioremediation의 수수단으로 활용될 수 있음을 보여주었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.277-277
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• 2005

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.234-238
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• 2012

Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.

• 공업화학
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• 제18권5호
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• pp.501-505
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• 2007

본 연구에서는 동물세포에서 매우 높은 독성을 갖는 유기인 살충제 coumaphos를 생분해하기 위하여, 재조합 대장균으로부터 발현된 organophosphorus hydrolase (OPH)가 사용되었다. OPH 생산성을 향상시키고자, 균주의 배지에 비이온성계면활성제(Tween 20, PEG 1000)와 유기용매(glycerol, propanol, ethanol)에 대한 효과를 고찰하였다. 0.25% Tween 20과 0.5% glycerol를 배지에 첨가하였을 때, 최대의 OPH가 생산되었다. 초음파를 이용한 균주 파쇄 공정으로부터 생성된 OPH를 사용 하였을 때, 0.2, 0.5, 1.0, 2.0 mM coumaphos는 각각 100, 88, 84, 78%의 생분해효율을 나타내었다. 이 연구에서 얻어진 결과들은 coumaphos가 다양한 농도로 오염된 지역을 생물학적으로 처리할 수 있는 새로운 방법으로서 활용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제24권3호
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• pp.379-385
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• 2014

The purpose of this study was to compare the ability of an engineered Escherichia coli to degrade chlorpyrifos (Cp) using an organophosphorus hydrolase enzyme, encoded in both Flavobacterium sp. ATCC 27551 or Pseudomonas diminuta, by employing the Lpp-OmpA chimera and the N-terminal domain of the ice nucleation protein as anchoring motifs. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by both anchors on the outer membrane. This is the first report on the presentation of OPH on the cell surface by Lpp-OmpA under the control of the T7 promoter. The results showed cell growth in the presence of Cp as the sole source of energy, without growth inhibition, and with higher whole-cell activity for both cells harboring plasmids pENVO and pELMO, at approximately 10,342.85 and 10,857.14 U/mg, respectively. Noticeably, the protein displayed by pELMO was lower than the protein displayed by pENVO. It can be concluded that Lpp-OmpA can display less protein, but more functional OPH protein. These results highlight the high potential, of both engineered bacteria, for use in the bioremediation of pesticide-contaminated sources in the environment.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.946-951
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• 2008

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency ($V_{max}/K_m$) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.144-153
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• 2021

Organophosphorus nerve agents (OPNAs), including both G- and V-type nerve agents such as sarin, soman, tabun and VX, are extremely neurotoxic organophosphorus compounds. Catalytic bioscavengers capable of hydrolyzing OPNAs are under development because of the low protective effects and adverse side effects of chemical antidotes to OPNA poisoning. However, these bioscavengers have certain limitations for practical application, including low catalytic activity and narrow specificity. In this study, we generated a fusion-hybrid form of engineered recombinant human paraoxonase 1 (rePON1) and bacterial organophosphorus hydrolase (OPH), referred to as GV-hybrids, using a flexible linker to develop more promising catalytic bioscavengers against a broad range of OPNAs. These GV-hybrids were able to synergistically hydrolyze both G-type OPNA analogs (paraoxon: 1.7 ~ 193.7-fold, p-nitrophenyl diphenyl phosphate (PNPDPP): 2.3 ~ 33.0-fold and diisopropyl fluorophosphates (DFP): 1.4 ~ 22.8-fold) and V-type OPNA analogs (demeton-S-methyl (DSM): 1.9 ~ 34.6-fold and malathion: 1.1 ~ 4.2-fold above) better than their individual enzyme forms. Among the GV-hybrid clones, the GV7 clone showed remarkable improvements in the catalytic activity toward both G-type OPNA analogs (kcat/Km (106 M-1 min-1): 59.8 ± 0.06 (paraoxon), 5.2 ± 0.02 (PNPDPP) and 47.0 ± 6.0 (DFP)) and V-type OPNA analogs (kcat/Km (M-1 min-1): 504.3 ± 48.5 (DSM) and 1324.0 ± 47.5 (malathion)). In conclusion, we developed GV-hybrid forms of rePON1 and bacterial OPH mutants as effective and suitable catalytic bioscavengers to hydrolyze a broad range of OPNA analogs.

• 센서학회지
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• 제23권1호
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• pp.35-41
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• 2014

Microbes have been used extensively in various fields of researches and industries but has not been used widely for microfluidic biosensor applications because it is difficult to immobilize properly to a small space. Therefore, we developed a microbial immobilization method for microfluidic devices using single-walled nanotubes and dielectrophoretic force. Single-walled nanotubes and Escherichia coli were aligned between two cantilever electrodes by a positive dielectrophoretic force resulting in a film of single-walled nanotubes with attached Escherichia coli. The optimal condition of film formation without a cell lysis was investigated. Diameter of single-walled nanotubes and electric field (intensity and duration of application) had an effect on the cell viability. On the other hand, the cell concentration of the suspension did not affect the cell viability. Paraoxon was detected using single-walled nanotubes film with attached Escherichia coli that expressed organophosphorus hydrolase. This film which is suspended from the substrate showed faster response time than sensors that are not suspended from the substrate.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권6호
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• pp.436-440
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• 2000

Recently, we reported an improved technology for the degradation of organophosphate nerve agents using whole cells of genetically engineered Escherichia coli that anchored and displayed the enzyme organophosphorus hydrolase on the cell surface. In this paper we report the immobilization of these cells on highly porous sintered glass beads and the subsequent application of the immobilized cell in a continuous-flow packed bed bioreactor for the biodetoxification of a widely used insecticide, coumaphos.

• 유기물자원화
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• 제14권3호
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• pp.110-116
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• 2006

본 연구에서는 재조합 대장균으로 부터 Organophosphorus Hydrolase (OPH)를 이용하여 유기인 살충제 화합물인 paraoxon의 생분해 속도를 향상시켰다. OPH의 비 활성도 (Specific whole cell OPH Activity)를 증가시키기 위한 배지의 최적 조건은 초기 pH 8.5의 조절과 5.0 % acetone 첨가가 필요하다는 것을 알 수 있었다. 또한, 이 최적의 조건에서 498 Unit/L의 OPH가 생산될 때, 275 mg/L paraoxon은 반응 10분 동안 98% 생분해 효율을 나타내었고, 그 결과 생분해 속도를 $29.2mg/g{\cdot}min$까지 향상시킬 수 있었다. 이러한 실험 결과들은 지하수 또는 토양에 잔류하는 유기인 살충제를 빠른 시간 안에 효과적으로 생분해시키는 실질적인 생물 복원 기술로 사용될 수 있을 것이다.