Degradation of chloroanilines by toluene dioxygenase from Pseudomonas putida T57.
In this study, we investigated the ability of Pseudomonas putida toluene dioxygenase to oxidize chloroanilines. Toluene-induced P. putida T57 cells degraded 4-chloroaniline (4CA) more rapidly than toluene-non-induced cells, suggesting that toluene dioxygenase pathway was involved in 4CA degradation. Escherichia coli harboring P. putida T57 genes encoding toluene dioxygenase complex (todC1C2BA) showed 4CA degradation activity, demonstrating that toluene dioxygenase oxidizes 4CA. Thin-layer chromatography (TLC) and mass spectrometry (MS) analyses identified 4-chlorocatechol and 2-amino-5-chlorophenol as reaction products, suggesting that toluene dioxygenase catalyzes both 1,2- and 2,3-dioxygenation of 4CA. A plasmid containing the entire tod operon (todC1C2BADE) was introduced to P. putida T57 to enhance its ability to degrade 4CA. Resulting P. putida T57 (pHK-C1C2BADE) showed 250-fold higher 4CA degradation activity than P. putida T57 parental strain. P. putida T57 (pHK-C1C2BADE) degraded 2-chloroaniline (2CA), 3-chloroaniline (3CA), and 3,4-dichloroaniline (34DCA) as well as 4CA, but not 3,5-dichloroaniline (35DCA). The order of the degradation rate was: 4CA > 3CA > 2CA > 34DCA.
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
Tisana
T
Nitisakulkan
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
Shota
S
Oku
Biotechnical Development Center, MicroBiopharma Japan Co., Ltd., Nakaizumi, Iwata, Shizuoka 438-0078, Japan.
Daizo
D
Kudo
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
Yutaka
Y
Nakashimada
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
Takahisa
T
Tajima
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; Center of Excellence for Environmental and Hazardous Waste Management (EHWM), Chulalongkorn University, Bangkok 10330, Thailand.
Alisa S
AS
Vangnai
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Electronic address: jun@hiroshima-u.ac.jp.
Junichi
J
Kato
D000814:Aniline Compounds / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002733:Chlorophenols / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D018628:Gene Dosage; D009876:Operon / Q000235:genetics; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014050:Toluene / Q000494:pharmacology
D000814:Aniline Compounds; D001426:Bacterial Proteins; D002733:Chlorophenols; D004269:DNA, Bacterial; C014464:3,4-dichloroaniline; C099673:2-amino-5-chlorophenol; D014050:Toluene; C031392:3-chloroaniline; D010105:Oxygenases; C023172:toluene dioxygenase; C025257:2-chloroaniline; C037267:3,5-dichloroaniline; C004658:4-chloroaniline
Biodegradation; Chloroaniline; Oxidative deamination; Pseudomonas putida; Toluene dioxygenase