• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• 대한화학회지
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• 제54권5호
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• pp.556-566
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• 2010

새로운 동적 분광 광도 측정법은 천연의 물 샘플에서 망간(II)의 측정을 위해 개발되었다. 그 방법은 620 nm에서 활성화제로서 nitrilotriacetic acid (NTA)를 사용하면서 $KIO_4$에 의한 갈로시아닌(Gallocyanin)의 산화와 함께 망간(II)의 촉매 효과에 기초했다. 최적조건은 갈로시아닌(Gallocyanin) $4.00{\times}1^{-5}\;M$, $KIO_4$ $1.00{\times}10^{-4}\;M$, NTA $1.00{\times}10^{-4}\;M$, pH = 3.50인 0.1 M HAc/NaAc 완충용액, 5분의 반응시간 그리고 $30^{\circ}C$의 온도에서 얻어졌다. 최적조건 하에서 제안된 방법은 $0.1\;-\;4.0\;ng\;mL^{-1}$의 범위에서 망간(II)의 측정을 허용했고 $0.025\;ng\;mL^{-1}$이하의 검출한계를 가지고 있다. 표준 망간(II) 용액을 측정하는 것에서 회수율은 98.5 - 102% 범위에 있다. 그리고 상대표준편차(RSD)는 0.76 - 1.25%의 범위에 있다. 새롭게 개발된 동적 방법은 약간의 주위의 물과 만족할 만한 결과를 갖는 JAC-0031의 공인된 표준 기준 강물 샘플 둘 다에서 망간(II)의 측정에 성공적으로 응용되었다. 또한, 얼마 안 되는 양이온과 음이온은 망간(II)의 측정을 방해한다. 다른 촉매-동적 방법과 기기적 방법과 비교했을 때, 제안된 동적 방법은 상당히 좋은 선택성과 감도, 낮은 가격, 저렴함, 낮은 검출한계와 신속함을 보인다. 상대적으로 낮은 염분을 갖는 진짜 물 샘플과 병으로 된 마시는 물, 차갑고 뜨거운 용천수, 호수, 강물 샘플 같은 복잡한 모체들에 적용하는 것은 쉽고 성공적으로 적용될 수 있다.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.784-788
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• 1995

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E. coli TG1 strain carrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni$^{++}$-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.

• 약학회지
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• 제46권1호
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• pp.24-29
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• 2002

Enoyl-Acyl Carrier Protein Reductase (Fabl) of bacteria is hem as an important target for new antibacterial drugs and plays a determinant role in completing cycles of elongation in type-H fatty acid synthase system. In this study, a fabI gene from Staphylococcus aureus 6538p cloned in pET-l4b vector and FabI protein was over-produced in Escherichaia coli BL2l (DE3). $NH_2$-terminal His-tagged FabI protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) metalaffinity chromatography Purified 6xHis-tagged FabI showed a catalytic activity on tram - 2 - octenoyl - N -acethlcysteamine by utilizing NADPH as a cofactor. For the discovery of new FabI inhibitors from chemical libraries, a target-oriented screening system using a 96-well plate was developed. About 10,000 chemical libraries from Korea Chemical Bank wore tested in this screening system, and 26 chemicals (0.25%) among them showed an inhibitory activity against FabI enzyme. This result showed that a new screening system can be used for the discovery of new FabI inhibitors.

• 산업기술연구
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• 제29권B호
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• pp.121-125
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• 2009

New cellulase genes, named as CelV2 and CelN1, respectively, were isolated from Erwinia carotovora ATCC15713 and expressed in E. coli. The CelV2 and CelN1 gene were PCR amplified with degenerated primers and PCR products were sequenced and expressed in E. coli. Two new cellulase genes showed 97% homologies with previously reported Erwinia cellulase genes. The recombinant cellulase were purified with Ni-NTA column chromatography and its enzymatic properties were characterized. The optimum temperature of two enzymes were about $50^{\circ}C$ degree and optimum pH were around pH7.0. The newly isolated celluase genes could be used for enhancing substrate range of alcohol-producing bacteria such as Zymomonas mobilis.

• 산업기술연구
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• 제37권1호
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• pp.16-20
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• 2017

A new putative laccase gene (soncotA) which show 78% homology with that from Bacillus licheniformis (liccotA) was isolated from draft genome sequence of Bacillus sonorensis KCTC 13918. A 1,545 bp of PCR product corresponding 514 amino acids was cloned into NdeI-NotI site of pET21c and expressed as soluble form in E. coli. About 59 kDa size of recombinant laccase was purified into homogenity by Ni-NTA column and laccase activity was confirmed by zymography. The enzymatic properties of recombinant laccase were characterized. The specific activity of B. sonorensis laccase was 0.033 fold lower than that of Bacillus licheniformis laccase. The finding of new laccase gene broadened the enzymatic diversity of Bacillus species laccases.

• Bulletin of the Korean Chemical Society
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• 제32권7호
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• pp.2405-2409
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• 2011

The psychrophilic bacteria Psychromonas arctica survives at subzero temperatures by having adapted several protective mechanisms against freezing and oxidative stresses. Many reactive oxygen species are likely generated in P. arctica as a result of reduced metabolic turnover rates. A previous study identified the pasod gene for superoxide dismutase from P. arctica using a series of PCR amplifications. Here, upon cloning into a His-tag fused plasmid, the sod gene from P. arctica (pasod) was successfully expressed by IPTG induction. His-tagged PaSOD was subsequently purified by $Ni^{2+}$-NTA affinity chromatography. The purified PaSOD exhibited a higher SOD activity than that of Escherichia coli (EcSOD) at all temperatures. The difference in activity between PaSOD and EcSOD becomes even more significant at 4$^{\circ}C$, indicating that PaSOD plays a functional role in the cold adaptation of P. arctica in the Arctic.

• Biotechnology and Bioprocess Engineering:BBE
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• 제2권2호
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• pp.86-89
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• 1997

Epidermal growth factor(EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector in which the gene product was supposed to be expressed with 6$\times$His tag for the subsequent purification. The recombinant mature EGF was expressed in M15[Rep4], an Escherichia coli host strain, in amount of 30-40% of total proteins pressent in E. coli extract by the addition of isopropylthio-$\beta$-galactopyranoside (IPTG). The recombinant EGF purified using a Ni2+-NTA affinity colume chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate proteins when murine NH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology and information for the production of recombinant EGF.

• Applied Microscopy
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• 제19권2호
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• pp.1-16
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• 1989

To investigate the research of the tumor seeker using liposomes with negatively stained. The fine structure and size in liposomes composed of PC, DSPC, DAPC and SM phospholipid were observed. And the yield of vesicle affected with sonication by mechanical force were determined. The results were as follows. 1. The stain of 2% UA are obtained a good resolution from electron microscopic observation to compare the negatively liposomes with PTA, AM and UA solution. 2. The fine structure of liposomes except DAPC, PC and CH alone samples could be observed from EM experiment of liposomes composed of PC, DSPC, DAPC and SM phospholipid. 3. The results of experiment from 10, 20, 40 and 60 times sonication with 30sec, prepared 0.1mM NTA-SM-liposomes were obtained unilamella vesicles from groups of multilamella vesicles of phospholipid. 4. About 50nm diameter liposomes obtained through membration filtration step to prepare homogenized liposomes.

• 분석과학
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• 제9권3호
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• pp.279-291
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• 1996

미량 금속이온을 선택적으로 분리, 농축 및 회수하기 위해 물리적 특성이 서로 다른 XAD-2, 4 및 16 다공성 수지에 2-(2-thiazolylazo)-p-cresol(TAC) alc 4-(2-thiazolylazo)orcinol(TAO)을 화학적으로 결합시켜 킬레이트 수지를 합성하고 뱃치법으로 U(VI) 등 10개 금속이온들의 흡착 특성을 조사 비교하였다. 킬레이트 수지의 합성률은 XAD-16-TAC형 수지는 0.60 mmol/g이었고 XAD-16-TAO형은 0.68mmol/g으로서 수지의 표면적이 큰 XAD-16형 수지가 XAD-2, 4형 수지보다 높은 합성률을 나타내었다. XAD-16-TAC와 XAD-16-TAO 킬레이트 수지는 1~5M 의 $HNO_3$, HCl 및 NaOH 등의 산과 염기성 용액에서 비교적 안정하였다. 그리고 U(VI) 이온을 10회 이상 반복적으로 흡착 및 탈착을 시킨 결과 연속적으로 재사용할 수 있는 안정한 내구성을 가지고 있음을 확인하였다. XAD-16-TAC 및 XAD-16-TAO형 수지에 대한 금속 이온의 최척 흡착조건과 흡착 특성을 조사한 결과는 금속이온이 킬레이트 수지에 흡착될 때 평형에 도달하는 시간은 약 1시간 정도였으며, XAD-16형 수지에서 흡착률이 가장 높았다. 그리고 금속이온들의 흡착률에 미치는 pH의 영향을 보면 U(VI)을 비롯한 대부분의 금속이온들은 pH 5~6 범위에서 최대로 흡착됨으로써 최척 pH 를 5로 정하였다. 또한 U(VI) 이온의 흡착에 미치는 공존이온의 영향을 보면 음이온인 $Cl^-$, $NO{_3}^-$, ${SO_4}^{2-}$, $CH_3COO^-$ 이온 및 $Na^+$, $K^+$, $Mg^{2+}$ 및 $Ca^{2+}$과 같은 양이온들은 흡착에 거의 영향을 미치지 않았다. 그러나 $CO{_3}^{2-}$는 $UO{_2}^{2+}$와 $[UO_2(CO_3)_3]^{4-}$의 안정한 착물을 형성하므로 U(VI) 이온의 흡착능을 크게 감소시켰다. 선택적인 특정 금속이온의 분리를 위하여 EDTA, CDTA 및 NTA 등과 같은 가리움제를 첨가하여 그 영향을 조사한 결과 NTA에 의한 가리움 효과가 10가지 혼합 금속이온 중 U(VI) 이온에 대하여 가장 큰 것으로 나타났다.