Development of a highly sensitive assay for enzyme-mediated reductive degradation of polychlorinated dibenzo-<i>p</i>-dioxin

Suzuki, Yuzo; Nakamura, Masaya; Otsuka, Yuichiro; Suzuki, Nao; Ohyama, Keisuke; Kawakami, Takeshi; Sato, Kanna; Kajita, Shinya; Hishiyama, Shojiro; Takahashi, Atsushi; Katayama, Yoshihiro

doi:10.1002/etc.1775

Cited by 4 publications

(3 citation statements)

References 20 publications

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“…In the present study, we developed a new on‐line assay of G6PDH enzyme based on TLC. As one of the oldest chromatographic methods, TLC has been applied for analysis of enzyme reactions in a few off‐line (or off‐plate) studies recently, in which enzyme reactions are carried out in a sample vial and TLC technique is used only for separation and detection of the products and unreacted substrates . Alternatively, several interesting studies have reported qualitative on‐plate inhibition assays based on TLC technique .…”

Section: Introductionmentioning

confidence: 99%

On-plate enzyme and inhibition assay of glucose-6-phosphate dehydrogenase using thin-layer chromatography

et al. 2015

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We performed on-plate enzyme and inhibition assays of glucose 6-phosphate dehydrogenase using thin-layer chromatography. The assays were accomplished based on different retardation factors of the substrates, enzyme, and products. All the necessary steps were integrated on-plate in one developing process, including substrate/enzyme mixing, reaction starting, and quenching as well as product separation. In order to quantitatively measure the enzyme reaction, the developed plate was then densitometrically evaluated to determine the peak area of the product. Rapid and high-throughput assays were achieved by loading different substrate spots and/or enzyme (and inhibition) spots in different tracks on the plate. The on-plate enzyme assay could be finished in a developing time of only 4 min, with good track-to-track and plate-to-plate repeatability. Moreover, we determined the Km values of the enzyme reaction and Ki values of the inhibition (Pb(2+) Cd(2+) and Cu(2+) as inhibitors), as well as the corresponding kinetics using the on-plate assay. Taken together, our method expanded the application of thin-layer chromatography in enzyme assays, and it could be potentially used in research fields for rapid and quantitative measurement of enzyme activity and inhibition.

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Section: Introductionmentioning

confidence: 99%

On-plate enzyme and inhibition assay of glucose-6-phosphate dehydrogenase using thin-layer chromatography

et al. 2015

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“…[4][5][6] Because of its widespread use, it is necessary to have an indepth study of its properties and develop appropriate methods for determining hymecromone. There have been some methods reported for the determination of hymecromone, such as high-performance liquid chromatography, 7 GC-MS 8 and electrochemical methods. [9][10][11] Comparing among these reported literature studies, electrochemical methods have some advantages in the determination of hymecromone, such as high sensitivity, easy operation, low cost and so on.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical characters of hymecromone at the graphene modified electrode and its analytical application

Wang

et al. 2015

Anal. Methods

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“…Transformants in the gene library of G. thermodenitrificans UZO 3 were screened for degradation activity using 2,7,-dichlorodibenzo-pdioxin (2,7-DCDD) as substrate. The enzymatic activity was evaluated by the detection of 4',5-dichloro-2hydroxydiphenyl ether (DCDE), a degradation intermediate (Suzuki et al, 2011(Suzuki et al, , 2012. As a result, we have cloned the gene for dioxin reductive ethrase dreE.…”

Section: Cloning and Sequence Of The Gene For Dioxin Reductive Ethera...mentioning

confidence: 99%

Cloning and sequencing of the gene encoding the enzyme for the reductive cleavage of diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Geobacillus thermodenitrificans UZO 3

Suzuki¹,

Nakamura²,

Otsuka³

et al.

Global NEST International Conference on Environmental Science &Amp; Technology

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We have previously reported that a cell-free extract prepared from Geobacillus thermodenitrificans UZO 3 reductively cleaves diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), a dioxin with the highest toxicity, in a sequential fashion producing 3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE) as the intermediate, and 3,4-dichlorophenol (DCP) as the final reaction product (1). The detection of TCDE implicated the discovery of an unprecedented dioxin-degrading enzyme that reductively cleaves the diaryl ether bonds. In this study, we report the cloning and sequencing of the dioxin reductive etherase gene dreE which codes for the 2,3,7,8-TCDD-degrading enzyme. We showed that dreE was expressed in Escherichia coli and that the product of the expression could reductively cleave diaryl ether bonds of 2,3,7,8-TCDD to produce TCDE. Furthermore, we established that the amino acid sequence encoded by dreE was homologous to an enzyme with yet unknown function that is encoded by a gene located in the riboflavin (vitamin B2) biosynthesis operon in Bacillus subtilis. We also showed that the amino acid sequence possesses a coenzyme A (CoA) binding site that is conserved in the N-acyltransferase superfamily. For the first time, the degradation of 2,3,7,8-TCDD at the molecular level using a enzyme of bacterial origin has been demonstrated.

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Development of a highly sensitive assay for enzyme-mediated reductive degradation of polychlorinated dibenzo-p-dioxin

Cited by 4 publications

References 20 publications

On-plate enzyme and inhibition assay of glucose-6-phosphate dehydrogenase using thin-layer chromatography

On-plate enzyme and inhibition assay of glucose-6-phosphate dehydrogenase using thin-layer chromatography

Electrochemical characters of hymecromone at the graphene modified electrode and its analytical application

Cloning and sequencing of the gene encoding the enzyme for the reductive cleavage of diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Geobacillus thermodenitrificans UZO 3

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