{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T06:13:51Z","timestamp":1782886431201,"version":"3.54.5"},"reference-count":47,"publisher":"American Society for Microbiology","issue":"2","license":[{"start":{"date-parts":[[2002,1,15]],"date-time":"2002-01-15T00:00:00Z","timestamp":1011052800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Bacteriol"],"published-print":{"date-parts":[[2002,1,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene\n            <jats:italic>ortho<\/jats:italic>\n            -monooxygenase (TOM) of\n            <jats:italic>Burkholderia cepacia<\/jats:italic>\n            G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in\n            <jats:italic>Escherichia coli,<\/jats:italic>\n            the variant TOM-Green degraded TCE (2.5 \u00b1 0.3 versus 1.39 \u00b1 0.05 nmol\/min\/mg of protein), 1,1-dichloroethylene, and\n            <jats:italic>trans<\/jats:italic>\n            -dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 \u00b1 0.03 versus 0.029 \u00b1 0.004 nmol\/min\/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 \u00b1 0.03 nmol\/min\/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the \u03b1-subunit of the hydroxylase, which appears to act as a smaller \u201cgate\u201d to the diiron active center. This hypothesis was supported by the ability of\n            <jats:italic>E. coli<\/jats:italic>\n            expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.\n          <\/jats:p>","DOI":"10.1128\/jb.184.2.344-349.2002","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:58:18Z","timestamp":1027763898000},"page":"344-349","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":162,"title":["Directed Evolution of Toluene\n            <i>ortho<\/i>\n            -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation"],"prefix":"10.1128","volume":"184","author":[{"given":"Keith A.","family":"Canada","sequence":"first","affiliation":[{"name":"Departments of Chemical Engineering and Molecular &amp; Cellular Biology, University of Connecticut, Storrs, Connecticut 06269-3222"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sachiyo","family":"Iwashita","sequence":"additional","affiliation":[{"name":"Departments of Chemical Engineering and Molecular &amp; Cellular Biology, University of Connecticut, Storrs, Connecticut 06269-3222"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hojae","family":"Shim","sequence":"additional","affiliation":[{"name":"Departments of Chemical Engineering and Molecular &amp; Cellular Biology, University of Connecticut, Storrs, Connecticut 06269-3222"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas K.","family":"Wood","sequence":"additional","affiliation":[{"name":"Departments of Chemical Engineering and Molecular &amp; Cellular Biology, University of Connecticut, Storrs, Connecticut 06269-3222"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.64.10.3626-3632.1998"},{"key":"e_1_3_2_3_2","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1021\/es970498d","volume":"32","year":"1998","unstructured":"Bradley, P. 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