PROIMI   05436
PLANTA PILOTO DE PROCESOS INDUSTRIALES MICROBIOLOGICOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Rapid determination of aromatic ring hydroxylating dioxygenases (ARHDs) activity in J26 strain whole-cell
Autor/es:
RIVA MERCADAL JP; SIÑERIZ F; FERRERO MA
Lugar:
Universidad Nacional de Rosario
Reunión:
Congreso; V Congreso Argentino de Microbiologia General; 2008
Institución organizadora:
Sociedad Argentina de Microbiologia General
Resumen:
ARHDs catalyzes the initial oxidation step of such polycyclic aromatic hydrocarbons (PAHs) and are recognized as the major pathway by which these compounds are mineralized in the environment. Some methods for activity determination of cells expressing these enzymes are based on the UV spectrometric determination of the concentration of the substrate or reaction products, on measuring the oxygen consumption and on the radiometric determination of the non-volatile metabolites of [14C] substrates after their separation by thin-layer chromatography.             Cells containing the ARHDs enzymes are capable of producing indigo when cultured in the presence of indole. The reaction sequence involves cis-dihydroxylation of the heterocyclic ring followed by dehydration to yield indoxyl, which autooxidizes to indigo, a blue color measured spectrophotometrically by absorbance at 600 nm. Recently, a colorimetric method was used by Hae-jin Woo et al, where cells are washed with phosphate buffer solution pH 7.2 and reaction was initiated by addition of indole in N,N-dimethlylformamide. However, they quantified indoxyl by fluorescence at 470 nm instead of indigo formation (600 nm) because of interference by the big amount of suspended biomass used. J26 strain is a marine PAH-degrading bacterium harboring type-<i>nahAc</i> naphthalene dioxygenase genes. It is capable of grow in naphthalene and phenanthrene as sole carbon and energy sources. It was isolated based on its capability of producing a blue color after 24 hs when indole crystals were placed on the lid of petri dish after growing in minimal seawater medium SWYE.             In this work, we developed a sensitive and rapid technique for measuring naphthalene dioxygenase activity in J26 strain whole-cell. The initial rate of indigo formation was determined by plotting the increase in indigo absorbance as a function of time when cells in late exponential phase grew (OD<sub>600nm</sub>= 0.8) in presence of naphthalene were exposed to indole. Assays were performed in a 96-wells microarray multiplate by duplicate. Indigo formation was monitored spectrophotometrically at 600 nm over 3 hours against a control (resuspended cells without indole) and different indole (2.5 and 5 mM), and biomass amounts were tested in order to optimize the response. The best indigo production was reached when the biggest amount of biomass tested was treated with indole 2.5 mM. Nevertheless, when indole in the same concentration was added to not-concentrated cells we obtained little less sensitive but operationally easier and more reproducible responses.             We conclude that this work offer a fast and sensitive spectrophotometric method for determining ARHD activity by whole-cells based on the biotransformation of indole to indigo.