• Journal of Microbiology and Biotechnology
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• v.29 no.2
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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).

• Journal of the Korean Chemical Society
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• v.54 no.5
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• pp.556-566
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• 2010

A new kinetic spectrophotometric method is developed for the measurement of Mn(II) in natural water samples. The method is based on the catalytic effect of Mn(II) with the oxidation of Gallocyanin by $KIO_4$ using nitrilotriacetic acid (NTA) as an activation reagent at 620 nm. The optimum conditions obtained are $4.00{\times}1^{-5}\;M$ Gallocyanin, $KIO_4$, $1.00{\times}10^{-4}\;M$ NTA, 0.1 M HAc/NaAc buffer of pH = 3.50, the reaction time of 5 min and the temperature of $30^{\circ}C$. Under the optimum conditions, the proposed method allows the measurement of Mn(II) in a range of $0.1\;-\;4.0\;ng\;mL^{-1}$ and with a detection limit of down to $0.025\;ng\;mL^{-1}$. The recovery efficiency in measuring the standard Mn(II) solution is in a range of 98.5 - 102%, and the RSD is in a range of 0.76 - 1.25%. The newly developed kinetic method has been successfully applied to the measurement of Mn(II) in both some environmental water samples and certified standard reference river water sample, JAC-0031 with satisfying results. Moreover, few cations and anions interfere with the measurement of Mn(II). Compared with the other catalytic-kinetic methods and instrumental methods, the proposed kinetic method shows fairly good selectivity and sensitivity, low cost, cheapness, low detection limit and rapidity. It can easily and successfully be applied to the real water samples with relatively low salt content and complex matrices such as bottled drinking water, cold and hot spring waters, lake water, river water samples.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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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.

• YAKHAK HOEJI
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• v.46 no.1
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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.

• Journal of Industrial Technology
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• v.29 no.B
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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.

• Journal of Industrial Technology
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• v.37 no.1
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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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• v.32 no.7
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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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• v.2 no.2
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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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• v.19 no.2
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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.

• Analytical Science and Technology
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• v.9 no.3
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• pp.279-291
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• 1996

The new chelating resins, XAD-2, 4, 16-TAC and XAD-2, 4, 16-TAO were synthesized by Amberlite XAD-2, XAD-4, and XAD-16 macroreticular resins with 2-(2-thiazolylazo)-p-cresol(TAC) and 4-(2-thiazolylazo)orcinol(TAO) as functional groups and were characterized by elemental analysis and FT-IR spectrometry. It was found that the content of functional group in chelating resin was 0.60mmol/g in XAD-16-TAC and 0.68mmol/g in XAD-16-TAO respectively. The chelating resins were stable in acidic and alkaline solution and can be reused over 10 times. The sorption behavior of some metalions to two chelating resins was investigated by batch method, which included batch equilibrium, effect of pH, coexisting ions and masking agent. For the optimum condition of sorption, the time required for equilibrium was about 1 hour and optimum pH was 5. In the presence of anions such as ${SO_4}^{2-}$ and $CH_3COO^-$, the sorption of U(VI) ion was slightly reduced but other anions such as $Cl^-$ and $NO{_3}^-$ revealed no interference effect. Also, sorption capacity of U(VI) ion was decreased by addition of $CO{_3}^{2-}$ ion because of complex formation of $[UO_2(CO_3)_3]^{4-}$, but alkali metals and alkali earth metals including Na(I), K(I), Mg(II), and Ca(II) were not affected for the sorption extent. Masking agent, NTA showed better separation efficiency of U(VI) ion from coexisting metal ions such as Th(IV), Zr(IV), Hf(IV), Cu(II), Cd(II), Pb(II), Ni(II), Zn(II) and Mn(II) than EDTA, CDTA.