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Title: TCE degradation by toluene/benzene monooxygenase of Pseudomonas aeruginosa JI104 and Escherichia coli recombinant

Abstract

Pseudomonas aeruginosa JI104 incorporates more than three degradation pathways for aromatic compounds such as benzene, toluene, and xylene. A dioxygenase and two monooxygenases were cloned in Escherichia coli XL1-Blue. The dioxygenase yielding cis-toluene dihydrodiol and one of the monooxygenases producing o-cresol from toluene did not exhibit conspicuous activity in trichloroethylene (TCE) oxygenation, although DNA sequencing proved that the former enzyme was an isozyme of toluene dioxygenase of the known TCE decomposer P.putida F1. The other toluene/benzene monooxygenase that could generate o-, m-, and p-cresol simultaneously from toluene showed TCE oxygenation activity resulting in TCE decomposition in E. coli. The activity was inhibited competitively by toluene, ethylbenzene, and o- and m-xylene: their inhibition constants were greater than those of propylbenzene and p-xylene. When the E. coli recombinant harboring the monooxygenase was induced by isopropyl {beta}-D-thiogalactopyranoside (IPTG) and incubated in the absence of toluene, TCE degradation activity decreased during incubation, compared to that with toluene. Toluene probably controlled the lifetime of the enzyme.

Authors:
 [1];  [2]
  1. National Univ. of Yokohama (Japan). Div. of Bioengineering
  2. Univ. of Tokyo (Japan). Dept. of Biochemistry and Biotechnology
Publication Date:
OSTI Identifier:
474277
Report Number(s):
CONF-950483-
ISBN 1-57477-005-5; TRN: IM9723%%167
Resource Type:
Conference
Resource Relation:
Conference: 3. international in situ and on-site bioreclamation symposium, San Diego, CA (United States), 24-27 Apr 1995; Other Information: PBD: 1995; Related Information: Is Part Of Bioremediation of chlorinated solvents; Hinchee, R.E.; Leeson, A. [eds.] [Battelle Memorial Inst., Columbus, OH (United States)]; Semprini, L. [ed.] [Oregon State Univ., Corvallis, OR (United States)]; PB: 348 p.; Bioremediation, Volume 3(4)
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; BIODEGRADATION; REMEDIAL ACTION; CHLORINATED ALIPHATIC HYDROCARBONS; BENZENE; TOLUENE; XYLENES; IN-SITU PROCESSING; BIOLOGICAL PATHWAYS; OXYGENASES; METABOLISM; ENZYME INHIBITORS

Citation Formats

Koizumi, Junichi, and Kitayama, Atsushi. TCE degradation by toluene/benzene monooxygenase of Pseudomonas aeruginosa JI104 and Escherichia coli recombinant. United States: N. p., 1995. Web.
Koizumi, Junichi, & Kitayama, Atsushi. TCE degradation by toluene/benzene monooxygenase of Pseudomonas aeruginosa JI104 and Escherichia coli recombinant. United States.
Koizumi, Junichi, and Kitayama, Atsushi. 1995. "TCE degradation by toluene/benzene monooxygenase of Pseudomonas aeruginosa JI104 and Escherichia coli recombinant". United States. doi:.
@article{osti_474277,
title = {TCE degradation by toluene/benzene monooxygenase of Pseudomonas aeruginosa JI104 and Escherichia coli recombinant},
author = {Koizumi, Junichi and Kitayama, Atsushi},
abstractNote = {Pseudomonas aeruginosa JI104 incorporates more than three degradation pathways for aromatic compounds such as benzene, toluene, and xylene. A dioxygenase and two monooxygenases were cloned in Escherichia coli XL1-Blue. The dioxygenase yielding cis-toluene dihydrodiol and one of the monooxygenases producing o-cresol from toluene did not exhibit conspicuous activity in trichloroethylene (TCE) oxygenation, although DNA sequencing proved that the former enzyme was an isozyme of toluene dioxygenase of the known TCE decomposer P.putida F1. The other toluene/benzene monooxygenase that could generate o-, m-, and p-cresol simultaneously from toluene showed TCE oxygenation activity resulting in TCE decomposition in E. coli. The activity was inhibited competitively by toluene, ethylbenzene, and o- and m-xylene: their inhibition constants were greater than those of propylbenzene and p-xylene. When the E. coli recombinant harboring the monooxygenase was induced by isopropyl {beta}-D-thiogalactopyranoside (IPTG) and incubated in the absence of toluene, TCE degradation activity decreased during incubation, compared to that with toluene. Toluene probably controlled the lifetime of the enzyme.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 1995,
month =
}

Conference:
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  • Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate ofmore » trichloroethylene degradation which decreased over time.« less
  • There exists a need for research, at all levels, tailored to address the needs of smaller, and possibly lesser developed, communities. One such area for research is the simultaneous disinfection and detoxification of a mildly contaminated water source. While photosensitization has been studied for disinfection, most of the work with regard to microorganisms has been done in the medical field. Research was conducted to provide preliminary determination of the efficacy of two dyes, methylene blue and rose bengal, for simultaneous photosensitized disinfection and detoxification. Specifically, the destruction of the aromatic hydrocarbons benzene and toluene, and the bacteria Escherichia coli, weremore » investigated.« less
  • Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, strain JS150 was also found to carry genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen ({sup 18}O{sub 2}) incorporation experiments using Pseudomonas aeruginosa strains carrying the cloned genes confirmed toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Encoding the toluene-2-monooxygenase and regulatory gene product was localized in two regionsmore » of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined, revealing six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. All the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomo-KR1 and Burkholderia (Pseudomonas) picketti PKO1. The relationship found between the phenol hydroxlases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest. 58 refs., 8 figs., 1 tab.« less
  • Burkholderia cepacia PR1{sub 23}(TOM{sub 23C}), expressing constitutively the TCE-degrading enzyme toluene ortho-monooxygenase (Tom), was immobilized on SIRAN{trademark} glass beads in a biofilter for the degradation and mineralization of gas-phase trichloroethylene (TCE). To interpret the experimental results, a mathematical model has been developed which includes axial dispersion, convection, film mass-transfer, and biodegradation coupled with deactivation of the TCE-degrading enzyme. Parameters used for numerical simulation were determined from either independent experiments or values reported in the literature. The model was compared with the experimental data, and there was good agreement between the predicted and measured TCE breakthrough curves. The simulations indicated thatmore » TCE degradation in the biofilter was not limited by mass transfer of TCE or oxygen from the gas phase to the liquid/biofilm phase (biodegradation limits), and predicts that improving the specific TCE degradation rates of bacteria will not significantly enhance long-term biofilter performance. The most important factors for prolonging the performance of biofilter are increasing the amount of active biomass and the transformation capacity enhancing resistance to TCE metabolism.« less
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