• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2002.10a
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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.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.

• Fire Science and Engineering
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• v.30 no.1
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• pp.138-148
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• 2016

A firefighting simulation was developed in order to assess the physical work capacity of firefighters. The simulation consisted of eight common firefighting tasks, including walking with radiant heat for 3 min while wearing full personal protective equipment (PPE). Nine professional firefighters performed the test a total of three times with a 5 min rest interval between each session. The entire series of tests took approximately 30 min to complete ($381{\pm}30s$). Rectal temperatures were found to increase from $37.4{\pm}0.3^{\circ}C$ to $38.5{\pm}0.4^{\circ}C$, while heart rates were found to increase from $92{\pm}18bpm$ to $185{\pm}13bpm$ during testing. Time to completion of the test drills and non-modified physical fitness values showed a significantly negative correlation (r = -0.728, p < 0.05). Firefighters who had longer periods of firefighting service had longer duration time and also recorded higher scores using an integrated scoring system of time to completion (TTC) and physiological strain index (PhSI). The results indicated that the determination of TTC alone can be a misrepresentation of capability, as it neglects to account for accumulated heat strain. The simulated firefighting test provided a useful insight into physical fitness level, but also the comprehensive work capacity of the firefighters when assessed based on TTC and PhSI.

• KSBB Journal
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• v.18 no.3
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• pp.174-179
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• 2003

The bacterium NFQ-1 capable of utilizing quinoline (2,3-benzopyridine) as the sole source of carbon, nitrogen and energy was enriched and isolated from soil samples of dead coal pit areas. Strain NFQ-1 was identified as Pseudomonas nitroreducens NFQ-1 by BIOLOG system, and assigned to Pseudomonas sp. NFO-1. Pseudomonas sp. NFQ-1 was used with the concentration range of 1 to 10 mM quinoline. Strain NFQ-1 could degrade 2.5 mM quinoline within 9 hours of incubation. Initial pH 8.0 in the culture was reduced to 6.8, and eventually 7.0 as the incubation was proceeding. 2-Hydroxyquinoline, the first intermediate of the degradative pathway, accumulated transiently in the growth medium. The highest concentration of quinoline (15 mM) in this work inhibited cell growth and quinoline degradation. Pseudomonas sp. NFQ-1 was able to utilize various quinoline derivatives and aromatic compounds including 2-hydroxyquinoline, p-comaric acid, benzoic acid, p-cresol, p-hydroxybenzoate, protocatechuic acid, and catechol. The specific activity of catechol oxygenases was determined to approximately 184.7 unit/㎎ for catechol 1.2-dioxygenase and 33.19 unit/㎎ for catechol 2,3-dioxygenase, respectively. As the result, it showed that strain NFQ-1 degraded quinoline via mainly orthp-cleavage pathway, and in partial meta-cleavage pathway.

• Proceedings of the Korean Vacuum Society Conference
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• 2011.08a
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• pp.293-294
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• 2011

It is known that semiconductor quantum-dot (QD) heterostructures have superior zero-dimensional quantum confinement, and they have been successfully applied to semiconductor laser diodes (QDLDs) for optical communication and infrared photodetectors (QDIPs) for thermal images [1]. The self-assembled QDs are normally formed at Stranski-Krastanov (S-K) growth mode utilizing the accumulated strain due to lattice-mismatch existing at heterointerfaces between QDs and cap layers. In order to increase the areal density and the number of stacks of QDs, recently, sub-monolayer (SML)-thick QDs (SQDs) with reduced strain were tried by equivalent thicknesses thinner than a wetting layer (WL) existing in conventional QDs (CQDs) by S-K mode. Despite that it is very different from CQDs with a well-defined WL, the SQD structure has been successfully applied to QDIP[2]. In this study, optical characteristics are investigated by using photoluminescence (PL) spectra taken from self-assembled InAs/GaAs QDs whose coverage are changing from submonolayer to a few monolayers. The QD structures were grown by using molecular beam epitaxy (MBE) on semi-insulating GaAs (100) substrates, and formed at a substrate temperature of 480$^{\circ}C$ followed by covering GaAs cap layer at 590$^{\circ}C$. We prepared six 10-period-stacked QD samples with different InAs coverages and thicknesses of GaAs spacer layers. In the QD coverage below WL thickness (~1.7 ML), the majority of SQDs with no WL coexisted with a small amount of CQDs with a WL, and multi-peak spectra changed to a single peak profile. A transition from SQDs to CQDs was found before and after a WL formation, and the sublevel of SQDs peaking at (1.32${\pm}$0.1) eV was much closer to the GaAs bandedge than that of CQDs (~1.2 eV). These revealed that QDs with no WL could be formed by near-ML coverage in InAs/GaAs system, and single-mode SQDs could be achieved by 1.5 ML just below WL that a strain field was entirely uniform.

• Applied Biological Chemistry
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• v.43 no.3
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• pp.163-168
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• 2000

To remove phosphate accumulated in the soil and water, Acinetobacter lwoffi PO8 possessing a high ability to accumulate phosphate was isolated from a active sludge. Bacterium was cultured in the liquid medium containing $150\;{\mu}g/mL$ of phosphate at $30^{\circ}C$ in different culture conditions to examine intracellular phosphate uptake. The initial pH in the range of $7.5{\sim}8.5$ was effective on the growth and phosphate uptake of the strain. Glycerol and arabinose used as a carbon sources showed 93 and 91% the phsphate uptake, respectively. Among the nitrogen sources, ammonium salt such as $NH_4NO_3$ and $(NH_4)_2SO_4$ was effectively utilized on the phosphate uptake compared with amino compounds. The rate of phosphate uptake of $NH_4NO_3$, and $(NH_4)_2SO_4$, was 95 and 96%, respectively The growth and Phosphate uptake ability in the strain were significantly promoted when metal ions were added in the medium; $Co^{2+}$, however, was not utilized by the strain. The capacity of phosphate uptake was enhanced to $10{\sim}20%$ when arginine, methionine, or lysine was added. Using $^{32}P$ to examine the uptake Pattern of intracellular phosphate, experiment result showed that polyphosphate was largely found in the fraction of intracellular inorganic phosphate of Acinetobacter lwoffi PO8.

• Applied Biological Chemistry
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• v.13 no.2
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• pp.111-129
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• 1970

As a series of studies on the nucleic acids and their related substances 210 samples were collected from 76 places such as farm soil, compost of heap, nuruk and meju to obtain microbial strains which produce 5'-phosphodiesterase. From these samples total of 758 strains were isolated by the use of dilution pour plate method. For all isolated strains primary screening of the productivity of RNA depolymerase was performed and useful strains with regard to 5'-phosphodiesterase productivities were identified. For these useful strains optimum condition, the effect of various compounds on the activity of 5'-phosphodiesterase, and the optimum condition for enzyme reaction were discussed. The quantitative of 5'-mononucleotides produced by the action of 5'-phosphodiesterase was performed using anion-exchange column chromatography and their identified was done by paper chromatography, thinlayer chromatography, ultra violet spectrophotometry, and characteristic color reaction using carbazole and schiff's reagent. (1) Penicillium citreo-viride PO 2-11 and Streptomyces aureus SOA 4-21 from soil were identified as a potent 5'-phosphodiesterase producing strains. (2) Optimum culture conditions for Penicillium citreo-viride PO 2-11 strain isolated were found to be pH 5.0 and $30^{\circ}C$, and the optimum conditions for enzyme action of 5'-phosphodiesterase were pH 4.2 and $60^{\circ}C$. Best carbon source for the production of 5'-phosphodiesterase was found to be sucrose and ammonium nitrate for nitrogen source. Addition of 0.01% corn steep liquor or yeast extract exhibited 20% increase in the amount of 5'-phosphodiesterase production compared to the control. 5'-phosphodiesterase produced by this strain was activated by $Mg^{++},\;Ca^{++},\;Zn^{++},\;Mn^{++}$ and was inhibited by EDTA, citrate, $Cu^{++},\;CO^{++}$. 5'-phosphodiesterase produced 5'-mononucleotide from RNA at a rate of 65.81%, and among the 5'-mononucleotides accumulated 5'-GMP only was found to have flavorous and the strain was also found lack of 5'-AMP deaminase. Productivity of flavorous 5'-GMP was found to be 186.7mg per gram of RNA. (3) Optimum culture canditions for the isolated Streptomyces aureus SOA 4-21 strain were pH 7.0 and $28^{\circ}C$, and the optimum conditions for the action of 5'-phosphodiesterase were pH 7.3 and $50^{\circ}C$. The best carbon source for 5'-phosphodiesterase production was found to be glucose and that of nitrogen was asparagine. Addition of 0.01% yeast extract exhibited increased productivity of 5'-phosphodiesterase by 40% compared to the non-added control. 5'-phosphodiesterase produced by this strain was activated by $Ca^{++},\;Zn^{++},\;Mn^{++}$ and was inhibited by citrate, EDTA, $Cu^{++}$. It was also found that the strain produce 5'-AMP deaminase in addition to 5'-phosphodiesterase. For this reason although decomposition rate was 63.58% the accumulation of 5'-AMP, 5'-CMP, 5'-GMP and 5'-UMP occurred by the breakdown of RNA. In the course of these reaction 5'-AMP deaminase converted 60% of 5'-AMP thus produced into 5'-IMP and flavorous 5'-mono nucleotide production was significantly increased by this strain over the above mentioned one. Production rates were found to be 171.8mg per grain of RNA for 5'-IMP and 148.2mg per gram of RNA for 5'-GMP, respectively.

• Microbiology and Biotechnology Letters
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• v.9 no.2
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• pp.59-64
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• 1981

The cultural conditions and the intra cellular accumulation of cadmium was studied using a cadmium tolerant yeast strain B-7 which had been isolated from activated sludge collected from a zinc mining area. The organism was able to grow in a medium containing 3,000 $\mu\textrm{g}$/$m\ell$ of cadmium-ion. (C $d^{++}$) Optimum conditions for the growth of the organisms were 20~22$^{\circ}C$ and pH 5.0~8.0 under aerobic condition. The maximum cadmium accumulation was observed when the organism was grown at pH 6.0. The growth of B-7 was not affected by the addition of a silicone-based antifoamer, which stimulated the intra cellular accumulation of cadmium. The intra cellular cadmium accumulation started after the cell ceased to grow. One gram of cells accumulated 34.17mg of cadmium when the organism was grown in a medium containing 500 $\mu\textrm{g}$/$m\ell$ of cadmium and 0.2%, v/v silicone-based antifoamer at 28$^{\circ}C$ for 48 hours with shaking. About 73 % of the accumulated heavy metal by the organism was found in the cytoplasm.m.

• Microbiology and Biotechnology Letters
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• v.8 no.3
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• pp.173-180
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• 1980

A source of D-xylose was required for the enhanced production of D-glucose isomerase of Streptomyces sp. strain K-17. D-glucose supported the luxuriant growth of the organism as well as D-xylose, but D-glucose isomerase activity was hardly detected in the D-glucose-grown cells. When the D-glucose-grown cells were incubated aerobically for a few hours in 0.5% xylose solution in 0.05 M phosphate buffer, pH 7.0, it was found that inductive formation of D-glucose isomerase occurred in the cells without multiplication. In the non-growth phase of cells the inductive formation of D-glucose isomerase occurred because a source of nitrogen for the synthesis of enzymes was obtained from turnover of protein accumulated in cells. D-ribose, L-arabinose, D-glucose, D-mannose, citrate, succinate and tartrate could not induce the formation of D-glucose isomerase, but D-xylose could induce. Inductinn of D-glucose isomerase was repressed by D-glucose and its catabolites : glycerol, succinate and citrate. Inductive formation of the enzymes in the non-growth phase was stimulated by $Ba^{2+}$, $Mg^{2+}$ and $Co^{2+}$, and inhibited by C $u^{2+}$, C $d^{2+}$, A $g^{+}$and H $g^{2+}$. The synthesis of enzymes in the induction system composed of 0.5% xylose solution was disrupted by actinomycin D, streptomycin, chloramphenicol, kanamycin, tetracycline, p-chloromercuribenzo ate, arsenate and 2, 4-dinitrophenol, but not disrupted by mitomycin C and penicillin G.icillin G.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.79-83
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• 2011

Over-application of nitrogen fertilizer keeps increasing the salinity in the soils of greenhouse in domestic agriculture. In order to remove the excess amounts of soil nitrate, soil microorganisms which have high capacity of nitrate uptake were isolated from the upland soils and their nitrate uptake activities were measured. Strain GS2 was able to remove 50 mM nitrate within 12 h. After sequence comparison analysis of 16S rRNA gene, the strain was identified and named as Bacillus sp. GS2. When the growth and nitrate uptake activities were measured, maximal values were obtained at $30-40^{\circ}C$ and $37^{\circ}C$, respectively; however, both were optimal at pH 6-8. In the media containing 50 mM nitrate, Bacillus sp. GS2 removed 43 mM nitrate which is corresponding to 86% removal. Similar amounts of nitrate removal were observed at the nitrate concentrations up to 300 mM, showing a saturation in nitrate uptake at concentrations above 50 mM. These results imply that Bacillus sp. GS2 can be a good candidate for the microbial remediation of accumulated environmental nitrate because of its excellent growth and nitrate uptake activity.