title: Development of a highly sensitive assay for enzyme-mediated reductive degradation of polychlorinated dibenzo-p-dioxin.
abstract: The degradation of 2-chloro-4,5-O-(4'-methyl-7', 8'-diphenyl)ether (CMDPE), an analog of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), mediated by Geobacillus sp. UZO 3 cell-free extract was monitored. Ethyl acetate extracts of a complete reaction mixture incubated at 65°C for 18 h were analyzed either by thin layer chromatography (TLC) fractionation coupled with spectrometric detection or by gas chromatography-mass spectrometry (GC-MS). The reaction product 4-methylumbelliferone (4MU) was successfully isolated by TLC and visualized by a transilluminator at 450 nm. The 4MU, 4-chlorophenol, and reaction intermediate 6-chlorophenoxy-4-methylumbelliferone were all successfully detected by GC-MS. The presence of these compounds suggest that Geobacillus sp. UZO 3 cell-free extract also catalyzes the reductive cleavage of the diaryl ether bonds of CMDPE in a similar mechanism previously reported in 2,7-DCDD. In the present study, the authors describe a simple and highly sensitive fluorescent assay for a new dioxin degrading enzyme(s).
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Masaya
authors.initials: M
authors.lastname: Nakamura
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Otsuka
authors.forename: Nao
authors.initials: N
authors.lastname: Suzuki
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ohyama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawakami
authors.forename: Kanna
authors.initials: K
authors.lastname: Sato
authors.forename: Shinya
authors.initials: S
authors.lastname: Kajita
authors.forename: Shojiro
authors.initials: S
authors.lastname: Hishiyama
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takahashi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Katayama
mesh_terms: D002384:Catalysis; D002474:Cell-Free System; D005591:Chemical Fractionation; D002733:Chlorophenols / Q000302:isolation & purification; D002855:Chromatography, Thin Layer; D004147:Dioxins / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D056506:Geobacillus / Q000201:enzymology*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000302:isolation & purification
chemical_list: D002733:Chlorophenols; D004147:Dioxins; C029107:4-chlorophenol; C000925:2,7-dichlorodibenzo-4-dioxin; D006923:Hymecromone; C503656:dichlorodibenzo-p-dioxin