• Title/Summary/Keyword: nitroreductase

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Characterization of NAD(P)H-nitroreductase Purified from the TNT-degrading Bacterium, Stenotrophomonas sp. OK-5 (폭약 TNT 분해세균 Stenotrophomonas sp. OK-5에서 분리된 NAD(P)H-nitroreductase의 정제 및 특성 연구)

  • Ho, Eun-Mi;Cheon, Jae-U;Gang, Hyeong-Il;O, Gye-Heon
    • Korean Journal of Microbiology
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    • v.39 no.4
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    • pp.223-229
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    • 2003
  • The purpose of this work was to perform the characterization of NAD(P)H-nitroreductase isolated from Stenotrophomonas sp. OK-5 capable of degrading 2,4,6-trinitrotoluene (TNT). Initially, NADP(H)-nitroreductase by a series of purification processes including ammonium sulfate precipitation, DEAE-sepharose, andQ-sepharose was prepared. From samples harvested from fraction collector, three different fractions (I, II & III)having the enzyme activity of NAD(P)H-itroreductase were detected. Specific activities of three fractions I, II,and III of NAD(P)H-nitroreductase were determined to approximately 5.06 unit/mg, 4.95 unit/mg and 4.86 unit/mg, and concentrated to 10.5, 9.8, and 8.9-fold compared to crude extract, respectively. Among these three fractions,the fraction I of NAD(P)H-nitroreductase demonstrated the highest specific activity in this experiment. Several factors affecting on the enzyme activity of NAD(P)H-nitroreductase (fractions I, II & III) were investigated.The optimum temperature of all NAD(P)H-nitroreductase (fractions I, II & III) was 30oC, and the optimal pH was approximately 7.5. Metal ions such as Ag+, Cu2+, Hg2+ inhibited approximately 80% enzyme activity of all NAD(P)H-nitroreductase, and the enzyme activities were decreased about 30-40% inhibition in the presence of Mn2+ or Ca2+. However, Fe3+ showed stimulatory effect on the enzyme activity. The molecular weights of NAD(P)H-nitroreductase (fractions I, II & III) were measured about 27 kDa on the SDS-PAGE.

Biological Treatment of TNT-containing Wastewater (pink water) by Stenotrophomonas maltophilia OK-5, and RT-PCR Quantification of the Nitroreductase (pnrB) Gene (Stenotrophomonas maltophilia OK-5에 의한 TNT 함유폐수 (pink water)의 생물학적 처리 와 Nitroreductase (pnrB) 유전자의 RT-PCR 정량화)

  • Cho, Su-Hee;Cho, Yun-Seok;Oh, Kye-Heon
    • KSBB Journal
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    • v.24 no.6
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    • pp.556-562
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    • 2009
  • The biological treatment of TNT-containing wastewater, known commonly as pink water, was investigated using a stirred tank reactor with Stenotrophomonas maltophilia OK-5 bacterial culture. S. maltophilia OK-5 exhibited effective degradation of TNT contained in pink water, completely degrading TNT (100 mg/L) within 6 days of incubation. The dark-red brown color derived from Hydride-Meisenheimer complex became more pronounced during the incubation period, which was determined quantitatively. High-pressure liquid chromatography was used to measure residual TNT, which also resolved the metabolic intermediates (i.e., 2,4-dinitrotoluene, 2,6-dinitrotoluene and 2,4-dinitro-6-hydroxytoluene). Gas chromatography-mass spectrometry was used to verify these intermediates. Quantification of the nitroreductase (pnrB) gene isolated from S. maltophilia OK-5 growing in pink water was performed with real-time PCR. The amount of pnrB gene copies increased to $10^3$-fold after 5 days of incubation time.

Characterization of Nitroreductase Purified from TNT-degrading Bacterium, Pseudomonas sp. HK-6. (폭약 TNT를 분해하는 세균인 Pseudomonas SP. HK-6에서 분리정제된 Nitroreductase의 특성연구)

  • 호은미;강형일;오계헌
    • Microbiology and Biotechnology Letters
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    • v.32 no.3
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    • pp.230-237
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    • 2004
  • In this study nitroreductase from Pseudomonas sp. HK-6 capable of degrading 2,4,6-trinitrotoluene (TNT) was characterized. Through a series of purification process including ammonium sulfate precipitation, DEAE-sepharose, and Q-sepharose, three different fractions I, II, and III having the enzyme activity of NTRs whose molecular weights were approximately 27 kDa were detected in fractions from HK-6 cells. Specific activity of the three fractions were approximately 4.85 unit/mg, 5.47 unit/mg, and 5.01 unit/mg, and concentrated to 9.0-, 10.1-, and 9.3-fold compared to crude extract, respectively. The optimal pH and temperature for the three NTR fractions were approximately 7.5 and $30^{\circ}C$, respectively. Metal ions, $Ag^{+}$ , $Cu^{ 2+}$, $Hg^{2+}$ inhibited approximately 70% of enzymes activities of all NTR, while $Fe^{2+}$ did not stimulate or inhibit the activities. Monitoring the effect of chemicals on the enzyme activity revealed that those NTR fractions lost enzyme activity in presence of $\beta$-mercaptoethanol, but were a little influenced by dithiothreitol, EDTA and NaCl. The three NTR fractions demonstrated enzyme activities for nitrobenzene and RDX as well as TNT.

Physiological and Molecular Characterization of NAD(P)H-Nitroreductase from Stenotrophomonas sp. OK-5 (Stenotrophomonas sp. OK-5에서 분리한 NAD(P)H-Nitroreductase의 생리학적 및 분자생학적 특성 연구)

  • Ho Eun-Mi;Kahng Hyung-Yeel;Oh Kye-Heon
    • Korean Journal of Microbiology
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    • v.40 no.3
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    • pp.183-188
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    • 2004
  • Stenotrophomonas sp. OK-5 capable of degrading TNT has been found to have three nitroreductase fractions designated as NTR fractions I, II, and III. NTR in a previous study. This study was attempted to reveal physiological and molecular characteristics of NTR fractions I, II, and III in strain OK-5. Several chemicals (e.g., EDTA, NaCl, dithiothreitol, $\beta$-mercaptoethanol) were tested for their effect on enzyme activity of NTRs, demonstrating that enzyme activities of NTR fractions I, II, and III from OK-5 were inhibited in the presence of $\beta$-mercaptoethanol. Substrate specificity test showed that NTR fractions I, II, and III all have over 70% enzyme activities for nitrobenzene or RDX as a substrate. N-terminal amino acid sequence of NTR fraction I from Stenotrophomonas sp. OK-5 was $^1MSDLLNADAVVQLFRTARDS^20$ and exhibited 70% sequence homology with that of NTR from Xanthomonas campestris. NTR I gene from Stenotrophomonas sp. OK-5 (SmOK5nrI) shared extensive sequence homology in deduced amino acid sequence of PCR product with NTRs from Xanthomonas campestris (81 %), X. axonopodis (75%), Streptomyces avermitilis(30%), whereas they had low homology with that from P. putida KT2440 (pnrB) (16%).

Effect of Bifidobacterium longum HY8001 Administration on Human Fecal Bacterial Enzymes and Microflora (Bifidobacterium longum HY8001의 섭취가 사람의 장내세균층 및 장내세균 효소에 미치는 영향)

  • 이완규;이상명;배형석;백영진
    • Microbiology and Biotechnology Letters
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    • v.27 no.4
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    • pp.267-272
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    • 1999
  • The effects of Bifidobacterium longum HY8001 supplement intake on the fecal microflora and fecal bacterial enzyme activity were studied in ten healthy human volunteers, before, during and after intake (respectively for 3 weeks). During intake of B. longum HY8001 supplement, fecal, $\beta$-glucuronidase and nitroreductase activities significantly decreased 44.6%(p<0.005) and 32.3%(p<0.01), respectively. Although numbers of major bacterial groups of fecal microflora were not affected by B. longum HY8001 intake for 3 weeks, the number of Bifidobacterium was significantly increased (p<0.05). This result indicates that intake of B. longum HY8001 might be potentially beneficial for the prevention and inhibition of colon cancer and improvement of human intestinal microflora composition.

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Simultaneous Utilization of Two Different Pathways in Degradation of 2,4,6-Trinitrotoluene by White Rot Fungus Irpex lacteus

  • 김현영;송홍규
    • Korean Journal of Microbiology
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    • v.38 no.4
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    • pp.250-250
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    • 2002
  • This study confirmed that white rot fungus Irpex lacteus was able to metabolize 2,4,6-trinitrotoluene (TNT) with two different initial transformations. In one metabolic pathway of TNT a nitro group was removed from the aromatic ring of TNT. Hydride-Meisenheimer complexes of TNT (H/sup -/-TNT), colored dark redo were confirmed as the intermediate in this transformation by comparison with the synthetic compounds. 2,4-Dinitrotoluene as a following metabolic product was detected, and nitrite produced by denitration of $H^-$-TNT supported this transformation. In the other TNT pathway, nitro groups in TNT were successively reduced to amino groups via hydroxylamines. Hydroxylamino-dinitrotoluenes and amino-dinitrotoluenes were identified as the intermediates. The activity of a membrane-associated aromatic nitroreductase was detected in the cell-free extract of I. lacteus. This enzyme catalyzed the nitro group reduction of TNT with NADPH as a cofactor, Enzyme activity was not observed in the presence of molecular oxygen.

Simultaneous Utilization of Two Different Pathways in Degradation of 2,4,6-Trinitrotoluene by White Rot Fungus Irpex lacteus

  • Kim, Hyoun-Young;Song, Hong-Gyu
    • Journal of Microbiology
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    • v.38 no.4
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    • pp.250-254
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    • 2000
  • This study confirmed that white rot fungus Irpex lacteus was able to metabolize 2,4,6-trinitrotoluene (TNT) with two different initial transformations. In one metabolic pathway of TNT a nitro group was removed from the aromatic ring of TNT. Hydride-Meisenheimer complexes of TNT (H$\^$-/-TNT), colored dark redo were confirmed as the intermediate in this transformation by comparison with the synthetic compounds. 2,4-Dinitrotoluene as a following metabolic product was detected, and nitrite produced by denitration of H$\^$-/-TNT supported this transformation. In the other TNT pathway, nitro groups in TNT were successively reduced to amino groups via hydroxylamines. Hydroxylamino-dinitrotoluenes and amino-dinitrotoluenes were identified as the intermediates. The activity of a membrane-associated aromatic nitroreductase was detected in the cell-free extract of I. lacteus. This enzyme catalyzed the nitro group reduction of TNT with NADPH as a cofactor, Enzyme activity was not observed in the presence of molecular oxygen.

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Biological Effect of Metronidazole Resistance in Helicobacter Pylori (Helicobacter pylori에서 metronidazole 내성이 미치는 생물학적 영향)

  • Lee Gin Hyug;Lee Sun-Mi;Nam Won-Hee;Kim Eun Sil;Kim Jin Ho;Kang Ho Young;Jeong Jin-Yong
    • Journal of Life Science
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    • v.15 no.6 s.73
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    • pp.955-960
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    • 2005
  • Resistance to metronidazole, a key component of therapies against Helicobacter pylori, is common in clinical isolates. Resistance generally requires inactivation of rdxA (HP0954), and sometimes also frxA (HP0642), two related nitroreductase genes. Here we studied the effect of resistance to metronidazole on fitness of the gastric pathogen H. pylori. The effect of metronidazole resistance for H. pylori in culture was assessed first by looking at colonies formed by freshly constructed mutant derivatives of H. pylori strain 26695. Mutations resulting in metronidazole resistance caused premature death of H.pylori in stationary phase, but had no significant effect on early exponential growth. The effect of nitroreductase deficiencies on fitness in vivo was tested by infecting C57BL/6 mice with 1:1 mixtures of SS1 wild type and its isogenic metronidazole resistant derivatives. Inactivation of rdxA caused an inability to colonize mice in SS1 H. pylori strain. Derivatives of a metronidazole resistant strain that survived better in stationary phase, although remaining metronidazole resistant, could again colonize mice. In conclusion, metronidazole resistance diminishes H. pylori's fitness, but their costs can be suppressed by additional mutation.