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Molecular Cloning, Purification, and Characterization of a Cold-Adapted Esterase from Photobacterium sp. MA1-3

  • Kim, Young-Ok;Heo, Yu Li;Nam, Bo-Hye;Kim, Dong-Gyun;Jee, Young-Ju;Lee, Sang-Jun;An, Cheul-Min
    • Fisheries and Aquatic Sciences
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    • v.16 no.4
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    • pp.311-318
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    • 2013
  • The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme was a serine-esterase and was active against $C_2$, $C_4$, $C_8$ and $C_{10}$ p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and $30^{\circ}C$, respectively. Relative activity remained up to 45% even at $5^{\circ}C$ with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, $Zn^{2+}$, and $Hg^{2+}$ ions.

A Study of Efficacy of Physical Water Treatment Devices for Mineral Fouling Mitigation Using Artificial Hard Water (인공 경수를 이용한 미네랄 파울링 저감에 물리적 수처리 기기들의 효과에 관한 연구)

  • Pak, Bock Choon;Kim, Sun Do;Baek, Byung Joon;Lee, Dong Hwan
    • Transactions of the Korean Society of Mechanical Engineers B
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    • v.29 no.11 s.242
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    • pp.1229-1238
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    • 2005
  • The objective of the present study was to investigate the efficacy of physical water treatment (PWT) technologies using different catalytic materials and an electronic anti-fouling device in the mitigation of mineral fouling in a once-through flow system with mini-channel heat exchanger. Effects of flow velocity and water hardness on the effectiveness of PWT technologies were experimentally studied. The artificial water hardness varied from 5.0 to 10 mo1/m$^{3}$ as CaCO$_{3}$. For 10 mo1/m$^{3}$ solution, fouling resistance reduced by 13-40$\%$ depending on flow velocity and types of PWT devices. On the other hand, fouling resistance reduced by 21-29$\%$ depending on the PWT devices for 5 mo11m3 solutions. The PWT device using alloy of Cu and Zn as catalyst (CM2) was slightly more effective than the others. SEM photographs of scale produced from the 10 mol/m$^{3}$ solution at 1.0 m/s indicated that calcium carbonate scales without PWT devices were needle-shaped aragonite, which is sticky, dense and difficult to remove. Scales with the PWT devices showed a cluster of spherical or elliptic shape crystals. Both the heat transfer test results and SEM photographs strongly support the efficacy of PWT technologies using catalytic materials and an electronic anti-fouling device in the mitigation of mineral fouling.

A cold-active acidophilic endoglucanase of Paenibacillus sp. Y2 isolated from soil in an alpine region

  • Lee, Jae Pil;Seo, Gu-Won;An, Shin-Deuk;Kim, Hoon
    • Journal of Applied Biological Chemistry
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    • v.60 no.3
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    • pp.257-263
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    • 2017
  • A cellulolytic strain Y2 was isolated from soil obtained in the Canadian Alpine region. The isolate was identified as Paenibacillus sp. Y2 by 16S rRNA sequencing. When grown in LB medium supplemented with carboxymethyl-cellulose (CMC), CMCase production increased to 122.0% of that observed in LB without CMC. Culture supernatant was concentrated by ultrafiltration and 80% ammonium sulfate precipitates were separated by Hi-Trap Q and CHT-II chromatography. The purified enzyme (EG-PY2) showed a homogeneous single band and the molecular mass was estimated to be 38 kDa by SDS-PAGE. Optimum pH and temperature of the enzyme were 4.5 and $30^{\circ}C$, respectively. The half-life of enzyme activity at 50 was 140.7 min, but the enzyme was drastically inactivated within 5 min at $55^{\circ}C$. The enzyme was highly activated to 135.7 and 126.7% by 5.0 mM of $Cu^{2+}$ or $Mg^{2+}$ ions, respectively, and moderately activated by $Ba^{2+}$ and $Ca^{2+}$ ions, whereas it was inhibited to 76.8% by $Fe^{2+}$, and to ${\leq}50%$ by $Mn^{2+}$, $Co^{2+}$, $Zn^{2+}$, and EDTA. The enzyme was activated to 211.5% in the presence of 0.5 M of NaCl and greatly tolerant to 3.15M of NaCl. The enzyme showed 2.98 times higher ${\beta}$-glucanase activity than CMCase activity. Based on these results, it can be concluded that EG-PY2 is an acidophilic, cold-active, and halotolerant endoglucanase. The authors suggest it is considered to be useful for various industrial applications, such as, fruit juice clarification, acidic deinking processes, high-salt food processing, textile and pulp industries, and for biofuel production from seaweeds.

Carbon Monoxide Ameliorates 6-Hydroxydopamine-Induced Cell Death in C6 Glioma Cells

  • Moon, Hyewon;Jang, Jung-Hee;Jang, Tae Chang;Park, Gyu Hwan
    • Biomolecules & Therapeutics
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    • v.26 no.2
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    • pp.175-181
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    • 2018
  • Carbon monoxide (CO) is well-known as toxic gas and intrinsic signaling molecule such as neurotransmitter and blood vessel relaxant. Recently, it has been reported that low concentration of CO exerts therapeutic actions under various pathological conditions including liver failure, heart failure, gastric cancer, and cardiac arrest. However, little has been known about the effect of CO in neurodegenerative diseases like Parkinson's disease (PD). To test whether CO could exert a beneficial action during oxidative cell death in PD, we examined the effects of CO on 6-hydroxydopamine (6-OHDA)-induced cell death in C6 glioma cells. Treatment of CO-releasing molecule-2 (CORM-2) significantly attenuated 6-OHDA-induced apoptotic cell death in a dose-dependent manner. CORM-2 treatment decreased Bax/Bcl2 ratio and caspase-3 activity, which had been increased by 6-OHDA. CORM-2 increased phosphorylation of NF-E2-related factor 2 (Nrf2) which is a transcription factor regulating antioxidant proteins. Subsequently, CORM-2 also increased the expression of heme oxygenase-1 and superoxide dismutases (CuZnSOD and MnSOD), which were antioxidant enzymes regulated by Nrf2. These results suggest that CO released by CORM-2 treatment may have protective effects against oxidative cell death in PD through the potentiation of cellular adaptive survival responses via activation of Nrf2 and upregulation of heme oxygenase-1, leading to increasing antioxidant defense capacity.

The Contents Of Heavy Metals In Sediments From The Southeastern Coastal Area Of Korea (한국 남해동부연안 해저퇴적물중의 중금속 함량에 관한 연구)

  • Lee, Dong Soo;Han, Sang Joon
    • 한국해양학회지
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    • v.13 no.2
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    • pp.11-16
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    • 1978
  • The contents of heavy metals were studied on sediments collected from the southeastern coastal area of Korea.Concentrations of the elements showed Cu 14∼ 48ppm (av. 18ppm), Pb 14∼49ppm (av. 28ppm), Zn 79∼187ppm (av. 11ppm), Co 11∼ 15ppm (av. 13ppm), Ni 22∼32ppm (av. 29ppm), Fe 3.1∼5.3% (av. 4.3%) and Mn 333 ∼1010ppm (av. 499ppm). When these values are compared with those of the unpolluted near-shore sediments from other countries,there seems to be little environmental contamination of heavy metals in the sediments of the study area. However, from the fact that the nearer to harbor the stations are, the higher the contents of copper, lead and zinc are in the sea off Pusan Bay, it is suggested that heavy metals in the sediments are starting to show some contamination from man's activities.

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Selective Metal Ion Sensing of Bipyridine-Bisterpyridine containing Fluorescent Dyes (다중 피리딘 구조를 가지는 형광염료의 금속 이온 반응성에 대한 연구)

  • Zo, Hye Jin;Kim, Arong;Jeong, Sooyeon;Park, Jong S.
    • Textile Coloration and Finishing
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    • v.25 no.4
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    • pp.254-261
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    • 2013
  • In this study, we synthesized a new fluorescent polypyridyl dye 2 containing a 2,2'-bipyridine in the center and two 2,2':6',2"-terpyridines at both ends. When exposed to various metal ions, the dye 2 showed selective fluorescence responses. In the presence of $Cu^{2+}$ and $Ni^{2+}$, it exhibited a highly effective fluorescence quenching, leading to large $K_{sv}$ values of up to $10^5$. In response to most other metal ions including $Al^{3+}$, in contrast, its fluorescence changes little, showing a small Ksv value at $10^2$. Meanwhile, the compound 2 revealed a differentiated fluorescence response to $Zn^{2+}$, which is evidenced by a large red shift of > 100 nm. Such a red shift from the ion binding is attributed to the planarization of the bipyridyl unit extending the effective conjugation length in conjunction. A polypyridyl compound will find important usefulness in chemosensor application due to its selective binding to metal ions. Subsequent research concerned with modified derivatives is currently going on, as a way to provide high solubility even after metal-complexing.

An Investigation for Air Pollutants Emitted from Small-Scale Incinerators in Highway Service Area (고속도로 휴게소의 소형소각로에서 배출되는 대기오염물질 조사)

  • Jang, Young-Kee;Choi, Sang-Jin;Kim, Kwan;Hong, Min-Sun;Choi, Join-In;Moon, Su-Ho;Kim, Soon-Tae;Kim, Seung-Do
    • Journal of Korean Society for Atmospheric Environment
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    • v.18 no.6
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    • pp.539-546
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    • 2002
  • The physicochemical properties of solid wastes generated from seven highway service areas, four branch offices, and one construction site were analyzed in concert with air pollutants including heavy metals emitted from near-by small-scale incinerators. The amount of solid wastes generated from highway areas has been increasing with recent increases in the number of highways and passengers. Twelve incinerators examined in this study generally had capacity smaller than 100 kg/hr, most of which were equipped with cyclone for dust removal. It was seen that the concentrations of the gas-phase air pollutants (e.g., SO$_2$, NO$_{x}$, HCl and H$_2$S) were above the acceptable emission standards except one or two sites. CO concentrations at all incinerators were also higher due to incomplete combustion. In addition, particulate matters showed concentration six times higher at their maximum. The results of heavy metal analysis showed that the concentrations of Cu, Cd, and Ni satisfied the emission standards. whereas Pb at one site and Zn at five sites exceeded the standards. Cr measurement results indicated that 9 of 12 incinerators had higher values than the standard; especially one branch office showed nine times higher than normal concentration. In order to satisfy more stringent emission standards in the near future, it is necessary to install air pollution control system and to develop an intensified management plan.n.

Simultaneous Determination of Zinc, Cadmium, Lead and Copper in Tungsten Matrix by Differential Pulse Anodic Stripping Voltammetry (펄스차이 벗김전압전류법에 의한 텅스텐 중 아연, 카드뮴, 납 및 구리의 미량성분 동시분석)

  • Bae, Jun Ung;Lee, Seong Ho
    • Journal of the Korean Chemical Society
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    • v.38 no.2
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    • pp.146-150
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    • 1994
  • The simultaneous determination of Zn, Cd, Pb and Cu in 1.000%(w/v) tungsten matrix by differential pulse anodic stripping voltammetry at a hanging mercury drop electrode has been studied. Tartaric acid(pH=5.00) was used as a supporting electrolyte. Optimum analytical conditions were found that the deposition potential was -1.2 volt(vs. Ag/AgCl), the deposition time was 3 minutes. The linear concentration range of all trace metal ions in 1.000%(w/v) tungsten matrix were 10 to 50 ppb. And the detection limit(3${\sigma}$) of zinc, cadmium, lead and copper were 1.25, 1.02, 1.69, and 1.02 ppb respectively. This method was superior to the ICP-AES method which detection limits(3${\sigma}$) in 1.000%(w/v) tungsten matrix were 8.0, 5.0, 120 and 5 ppb respectively.

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A Novel Metalloprotease from the Wild Basidiomycete Mushroom Lepista nuda

  • Wu, Y.Y.;Wang, H.X.;Ng, T.B.
    • Journal of Microbiology and Biotechnology
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    • v.21 no.3
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    • pp.256-262
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    • 2011
  • A 20.9-kDa metalloprotease was isolated from dried fruiting bodies of the wild basidiomycete mushroom Lepista nuda. The N-terminal amino acid sequence of the protease was seen to be ATFVLTAATNTLFTA, thus displaying no similarity with the sequences of previously reported metalloproteases. The protease was purified using a procedure that entailed ion-exchange chromatography on CM-Cellulose, Q-Sepharose, and Mono S, and FPLC-gel filtration on Superdex 75. The protease functioned at an optimum pH of 7.0 and an optimum temperature of $50^{\circ}C$. It was also noted that the protease demonstrated a proteolytic activity of 1,756 U/mg toward casein. The $K_m$ of the purified protease toward casein was 6.36 mg/ml at a pH of 7.0 and with a temperature of $37^{\circ}C$, whereas the $V_{max}$ was 9.11 ${\mu}g\;ml^{-1}\;min^{-1}$. The activity of the protease was adversely affected by EDTA-2Na, suggesting that it is a metalloprotease. PMSF, EGTA, aprotinin, and leupeptin exerted no striking inhibitory effect. The activity of the protease was enhanced by $Fe^{2+}$, but was curtailed by $Cd^{2+}$, $Cu^{2+}$, $Hg^{2+}$, $Pb^{2+}$, $Zn^{2+}$, and $Fe^{2+}$ ions. The protease also exhibited inhibitory activity against HIV-1 reverse transcriptase with an $IC_{50}$ value of 4.00 ${\mu}M$. The $IC_{50}$ values toward hepatoma Hep G2 and leukemia L1210 cells in vitro were 4.99 ${\mu}M$ and 3.67 ${\mu}M$, respectively.

Characteristics of a Novel Acinetobacter sp. and Its Kinetics in Hexavalent Chromium Bioreduction

  • M., Narayani;K., Vidya Shetty
    • Journal of Microbiology and Biotechnology
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    • v.22 no.5
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    • pp.690-698
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    • 2012
  • Cr-B2, a Gram-negative hexavalent chromium [Cr(VI)] reducing bacteria, was isolated from the aerator water of an activated sludge process in the wastewater treatment facility of a dye and pigment based chemical industry. Cr-B2 exhibited a resistance for 1,100 mg/l Cr(VI) and, similarly, resistance against other heavy metal ions such as $Ni^{2+}$ (800 mg/l), $Cu^{2+}$ (600 mg/l), $Pb^{2+}$ (1,100 mg/l), $Cd^{2+}$ (350 mg/l), $ZN^{2+}$ (700 mg/l), and $Fe^{3+}$ (1,000 mg/l), and against selected antibiotics. Cr-B2 was observed to efficiently reduce 200 mg/l Cr(VI) completely in both nutrient and LB media, and could convert Cr(VI) to Cr(III) aerobically. Cr(VI) reduction kinetics followed allosteric enzyme kinetics. The $K_m$ values were found to be 43.11 mg/l for nutrient media and 38.05 mg/l for LB media. $V_{max}$ values of 13.17 mg/l/h and 12.53 mg/l/h were obtained for nutrient media and LB media, respectively, and the cooperativity coefficients (n) were found to be 8.47 and 3.49, respectively, indicating positive cooperativity in both cases. SEM analysis showed the formation of wrinkles and depressions in the cells when exposed to 800 mg/l Cr(VI) concentration. The organism was seen to exhibit pleomorphic behavior. Cr-B2 was identified on the basis of morphological, biochemical, and partial 16S rRNA gene sequencing chracterizations and found to be Acinetobacter sp.