2-hydroxy-6-oxo-6-phenylhexa-2-4-dienoate and diphenyl

To understand the biochemical role of white rot fungus Trametes sp. SQ01 manganese peroxidase (MnP) towards 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoates (HOPDA)/HOPDA derivatives and to reveal the new catalytic features of MnP, white rot fungus Trametes sp. SQ01 MnP was extracted, and the purified enzymes were used in the oxidation of HOPDAs.. UV-vis spectrophotometry was used to study the transformation of 10 substituted HOPDAs by manganese peroxidase and measure the steady-state kinetics parameters of manganese peroxidase against parts of HOPDAs. The molecular structures of HOPDA and HOPDA oxidation product were analyzed by infrared spectroscopy.. Manganese peroxidase exhibited catalytic activity towards both HOPDA and halogenated HOPDA. Especially, our manganese peroxidase used 3,8,11-3Cl HOPDA as substrate, while biphenyl hydrolase (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) and Rhodococcus sp. R04 showed negligible activity towards this substrate. The steady-state kinetic analysis indicated that HOPDA displayed the lowest Km among 5 HOPDAs, the catalytic efficiency (Kcat/Km) of 3, 10-2F HOPDA was the highest. UV-visible spectroscopy analysis indicated that the maximum absorption of products of HOPDA showed blue-shift with increasing the reaction time in the visible region. Infrared analysis showed that MnP converted conjugated diene of HOPDA to monoethylenically, and cause hydroxyl on Cβ to disappear.. Manganese peroxidase can effectively degrade HOPDA and its derivatives. Such catalytic properties of manganese peroxidase provide a new strategy for successfully degrading biphenyl and its intermediate metabolites.

Topics: Biocatalysis; Biodegradation, Environmental; Biotransformation; Biphenyl Compounds; Fatty Acids, Unsaturated; Fungal Proteins; Kinetics; Molecular Structure; Peroxidases; Trametes

A new method for isolating targeted live bacterial cells was established with the use of cell sorting by flow cytometry (FCM) based on the fluorescence of the intermediate metabolite of biphenyl degradation. During biphenyl degradation, a PCB degrader, Comamonas testosteroni TK102, produces a meta-cleavage intermediate metabolite, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), which emits green fluorescence. HOPDA was produced from 2,3-dihydroxy biphenyl as a substrate, but it was not appropriate for labeling cells because it was released from the cells into the medium. When we used 4-n-butylbiphenyl and 4-n-heptylbiphenyl, we found that the cells produced and accumulated 2,3-dihydroxy intermediate metabolites. By the addition of synthesized 2,3-dihydroxy-4'-butylbiphenyl (2,3-DHBBP), we were able to label the cells with strong green fluorescence, suggesting the persistence of fluorescent intermediate metabolite in the cells by the introduction of the alkyl tail. 2,3-DHBBP was then used to label strain TK102 and the cells were sorted with FCM. The sorting efficiency of FCM was defined as the percentage of colony numbers per sorting events. Strain TK102 cells were successfully enriched by 4.1-fold from the mixture with environmental indigenous bacteria with a sorting efficiency of 7.3%. The method we present here serves as a basic technique for the specific and direct isolation of live bacterial cells which contain dioxygenases active on dihydroxylated aromatic compounds.

Topics: Animals; Bacteria; Biphenyl Compounds; Comamonas testosteroni; Dioxygenases; Fatty Acids, Unsaturated; Flow Cytometry; Fluorescence; Polychlorinated Biphenyls