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Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:Flavin Adenine Dinucleotide Oxidoreductase (TftC) of Burkholderia cepacia AC1100. Michelle R. Gisi, 2003.Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source . Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD) . Sequence analysis suggests that they are separate enzymes . The two proteins were separately produced in Escherichia coli, purified, and characterized . TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase . A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate . Kinetic and binding property analysis showed that FAD was a better substrate than FMN . TftD was a reduced FAD (FADH2)-utilizing monooxygenase, and FADH2 was supplied by TftC . It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product . TftD interacted with FADH2 and retarded its rapid oxidation by O2 . A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates . The reclassification of the two enzymes further supports the new discovery of FADH2-utilizing enzymes, which have homologues in the domains Bacteria and Archaea . Detection of Bacteria Carrying the stx2 Gene by In Situ Loop-Mediated Isothermal Amplification. Fumito Maruyama, 2003.A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described . Loop-mediated isothermal amplification (LAMP) was used to detect stxA2 in Escherichia coli O157:H7 cells . The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR . It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E . coli O157:H7 cells with an stxA2 gene . Higher-contrast images were obtained with this method than with in situ PCR .
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