• 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.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.759-767
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• 2017

Lactophoricin (LPcin), which is a part of proteose peptone isolated from bovine milk, is a cationic amphipathic ${\alpha}-helical$ antimicrobial peptide. Its truncated variants and mutated analogs were designed and their antimicrobial activities were evaluated by using various assays, like broth dilution methods and disk diffusion methods as well as hemolysis assay. Three analogs, LPcin-C8 (LPcin-YK1), LPcin-T2&6W (LPcin-YK2), and LPcin-T2&6W-C8 (LPcin-YK3), which showed better antibiotic activities than LPcin, were selected. Their secondary structures were also characterized by using CD spectropolarimetry. These three analogs of LPcin could be used as an alternative source of powerful antibacterial agents.

• 대한환경공학회지
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• 제31권2호
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• pp.147-152
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• 2009

본 연구에서는 톨루엔 계열 화합물로 오염된 환경에 대해 고정화한 유전자 재조합 균주 KG1206의 적용 가능성에 대해 조사하였다. 재조합 균주 KG1206은 직접 유도제인 m-toluate, benzoate 뿐만 아니라 톨루엔, 자일렌 이성질체가 간접 유도제로서 발광 활성을 나타낸다. 연구에 의해 결정된 고정화 프로토콜의 최적 조건은 다음과 같다: 균주 농도(1 : 1 (v/v)), 오염원 용액(인산염 완충액), 발광 측정에 필요한 비드 수(4개), 5가지 오염원에 대한 최대 발광 활성은 일반적으로 m-toluate > p-xylene > 톨루엔 > o-xylene > m-xylene 순으로 나타났다. 생물발광과 오염원 감소는 HPLC로 확인하였으며, 고정 균주에 의해 초기 5 mM m-toluate는 5시간 배양 후 약 48%의 감소율을 나타내었으며 계속 분해되는 경향이 관찰되었다. 알긴산 균주 고정화에 대한 본 연구 결과는 톨루엔 계열 화합물을 함유한 석유계 탄화수소에 오염된 특정 환경을 생물학적 모니터링에 유용한 방법으로 사용할 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제32권8호
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• pp.1041-1046
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• 2022

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDT^L59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDT^WT, respectively. Subsequently, both NDT^WT and NDT^L59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDT^L59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59^th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

• 대한환경공학회지
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• 제30권1호
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• pp.79-84
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• 2008

본 연구에서는 유전자 재조합 발광균주, Pseudomonas putida mt-2 KG1206,의 발광 촉진조건을 석유계 탄화수소 오염 지하수에 적용 가능성에 대해 조사하였다. 일반적으로 순수 유도제 오염원에 대한 발광 강도는 m-methyl benzyl alchohol > m-toluate > toluene > m-xylene > benzoate > p-xylene > o-xylene의 순으로 나타났다. 일반적으로 지하수에 오염된 유도제 오염원의 농도에 따라 발광 정도가 관찰되었다. 그러나 일부 지하수 시료의 경우에는 낮은 농도에 높은 발광과 같은 현상을 관찰할 수 있었다. 이러한 현상은 환경시료의 수질 복잡성에 따른 영향일 것이다. 선행연구에서 발광촉진제로 알려진 SL (sodium lactate)와 KNO$_3$에 의한 발광영향을 비교하였다. 모두 발광을 촉진하였지만, KNO$_3$가 조금 더 촉진력이 강한 것으로 조사되었다. 질소화합물(20 g KNO$_3$/L) 첨가군 시료의 발광균주는 첨가하지 않은 대조군에 비해 뚜렷하게 발광이 높게 나타났다. 따라서 일반적으로 낮은 유도제 오염원 함유 시료에 대해서는 발광촉진 조건이 지하수 오염원 검출 및 모니터링을 용이하게 할 수 있을 것이다. 또한 비슷한 특성을 가진 다양한 발광균주를 더욱 효율적으로 오염지역의 기초평가에 이용할 수 있는 조건이 될 수 있을 것이다.

• 대한환경공학회지
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• 제27권5호
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• pp.507-512
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• 2005

본 연구에서는 유전자 재조합 발광균주, Pseudomonas putida mt-2 KG1206을 이용하여 토양에 오염된 m-toluate 탐지 방법 및 적용 가능성에 대해 조사하였다. KG1206은 톨루엔 계열 화합물의 중요 중간 분해물질인 m-toluate 및 benzoate가 직접 생물 발광 유도제로 작용하며, 또한 톨루엔 계열 화합물들이 간접 유도제로서 발광 활성을 나타내었다. 토양에 오염된 유도제 오염원 검출을 위해 발광 균주 9.9 mL에 에탄올 추출물 0.1 mL을 첨가하여 조사하였다. 생물발광에 근거하여 작성된 m-toluate 검량선은 대략 $R^2>0.97$ 이상의 상관관계가 관찰되었다. 토양에 임의 오염된 m-toluate(직접 발광유도제)는 정립한 방법에 따라 발광활성에 근거하여 추측하였고, 기기분석치와 통계적으로 유의한 것으로 조사되었다. 본 연구 결과를 통해서 특정 화합물에 대해 발광을 생산하는 유전자 재조합 균주가 특정 오염원에 오염된 지역의 관리를 위한 수단으로 사용할 수 있는 가능성을 확인할 수 있었다.

• 한국지하수토양환경학회지:지하수토양환경
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• 제18권4호
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• pp.32-39
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• 2013

This research focused on the effects of heavy metals on the biomonitoring activity of genetically engineered bioluminescent bacteria, Pseudomonas putida mt-2 KG1206. KG1206 was exposed to single or binary mixtures of different heavy metals as well as soils contaminated with heavy metals. In case of single exposure with different inducer pollutant, the toxicity order was as followings : As(III) > Cd, As(V) >> Cu, Cr(VI). The toxic effects of the binary mixtures was compared to the expected effect based on a simple theory of probabilities. The interactive effects were mostly additive, while in few cases antgonistic and synergistic mode of action was observed for some concentration combinations. No considerable correlation was found between the total metal contents in soils and the bioluminescence activity of each sample. However, statistically significant differences (p = 0.0102) were observed between two groups, classified based on arsenite contamination. These results demonstrate the usage of recombinant bioluminescent microorganism in biomonitoring and the complex interactive effects of metals.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2002년도 학술발표대회
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.