{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T01:46:10Z","timestamp":1791164770483,"version":"4.1.0"},"reference-count":40,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2004,6,1]],"date-time":"2004-06-01T00:00:00Z","timestamp":1086048000000},"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":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2004,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            Directed evolution of toluene\n            <jats:italic>ortho<\/jats:italic>\n            -monooxygenase (TOM) of\n            <jats:italic>Burkholderia cepacia<\/jats:italic>\n            G4 previously created the hydroxylase \u03b1-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with\n            <jats:italic>Escherichia coli<\/jats:italic>\n            TG1\/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (\n            <jats:italic>V<\/jats:italic>\n            <jats:sub>max<\/jats:sub>\n            of 9.3 versus 4.5 nmol\u2009\u00b7\u2009min\n            <jats:sup>\u22121<\/jats:sup>\n            \u2009\u00b7\u2009mg of protein\n            <jats:sup>\u22121<\/jats:sup>\n            and unchanged\n            <jats:italic>\n              K\n              <jats:sub>m<\/jats:sub>\n            <\/jats:italic>\n            ), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 \u00b1 0.07 versus 1.30 \u00b1 0.06 nmol\u2009\u00b7\u2009min\n            <jats:sup>\u22121<\/jats:sup>\n            \u2009\u00b7\u2009mg of protein\n            <jats:sup>\u22121<\/jats:sup>\n            [mean \u00b1 standard deviation]) at 91 \u03bcM and has a substantial decrease in regiospecificity, since\n            <jats:italic>o<\/jats:italic>\n            -cresol (50%),\n            <jats:italic>m<\/jats:italic>\n            -cresol (25%), and\n            <jats:italic>p<\/jats:italic>\n            -cresol (25%) are formed, in contrast to the 98%\n            <jats:italic>o<\/jats:italic>\n            -cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (\n            <jats:italic>V<\/jats:italic>\n            <jats:sub>max<\/jats:sub>\n            of 2.61 versus 0.95 nmol\u2009\u00b7\u2009min\n            <jats:sup>\u22121<\/jats:sup>\n            \u2009\u00b7\u2009mg of protein\n            <jats:sup>\u22121<\/jats:sup>\n            and unchanged\n            <jats:italic>\n              K\n              <jats:sub>m<\/jats:sub>\n            <\/jats:italic>\n            ) and chloride release (0.034 \u00b1 0.002 versus 0.012 \u00b1 0.001 nmol\u2009\u00b7\u2009min\n            <jats:sup>\u22121<\/jats:sup>\n            \u2009\u00b7\u2009mg of protein\n            <jats:sup>\u22121<\/jats:sup>\n            ). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 \u00b1 0.3 versus 1.30 \u00b1 0.06 nmol\u2009\u00b7\u2009min\n            <jats:sup>\u22121<\/jats:sup>\n            \u2009\u00b7\u2009mg of protein\n            <jats:sup>\u22121<\/jats:sup>\n            ). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with\n            <jats:italic>o<\/jats:italic>\n            -cresol (28%),\n            <jats:italic>m<\/jats:italic>\n            -cresol (18%), and\n            <jats:italic>p<\/jats:italic>\n            -cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.\n          <\/jats:p>","DOI":"10.1128\/aem.70.6.3246-3252.2004","type":"journal-article","created":{"date-parts":[[2004,6,7]],"date-time":"2004-06-07T23:03:20Z","timestamp":1086649400000},"page":"3246-3252","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":70,"title":["Saturation Mutagenesis of Toluene\n            <i>ortho<\/i>\n            -Monooxygenase of\n            <i>Burkholderia cepacia<\/i>\n            G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation"],"prefix":"10.1128","volume":"70","author":[{"given":"Lingyun","family":"Rui","sequence":"first","affiliation":[{"name":"Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3222"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Young Man","family":"Kwon","sequence":"additional","affiliation":[{"name":"Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3222"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayelet","family":"Fishman","sequence":"additional","affiliation":[{"name":"Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3222"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenneth F.","family":"Reardon","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Colorado State University, Fort Collins, Colorado 80523-1370"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas K.","family":"Wood","sequence":"additional","affiliation":[{"name":"Departments of Chemical Engineering and Molecular and Cell 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","first-page":"211","volume":"66","year":"2000","unstructured":"Bylina, E. 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