Optimization of indigo formation by a marine Pseudomonas strain

ISAAC P.; RIVA MERCADAL J.P.; FERRERO M.A.

Carlos Paz

Congreso; VI Congreso Argentino de Microbiología General SAMIGE; 2009

SAMIGE

Pseudomonas sp. J26 was isolated from intertidal sediment of Patagonia, Argentina, during a selective enrichment with naphthalene as a sole carbon and energy sources. It was selected as a dark blue colony by indole test, and was identified as Pseudomonas sp. closely related to P. plecoglossicida using biochemical tests and sequencing of 1400 pb of 16S rRNA gene (99.7 % similarity). Attempts at microbial bioindigo formation for industrial purposes started in 1983, when naphthalene dioxygenase genes from P. putida G7 were expressed in a recombinant E. coli strain that efficiently converted glucose to indole. After that, several oxidoreductase enzymes like monooxygenases and dioxygenases have been reported to yield indigo from many substrates. A technique developed to determine naphthalene dioxygenase (NDO) activity was optimized and used to study the biotransformation of indole to indigo by Pseudomonas sp. J26 whole cells. To provide an accurate indigo determination, a polypropylene vial scarification method was developed. Indigo formation reaction was started simultaneously in 30 polypropylene vials containing 0.5 ml of washed cells (OD=1.0) previously grown in JPP medium, by adding indole from a 100 mM indole in dimethylformamide (DMF) stock solution. Assays were performed by adding indole 0.25; 0.5; 1; 2.5; 4 and 5 mM, and samples were incubated at several temperatures within the mesophilic range. Tubes were collected over a 10 hours period. After that, reaction mixtures were centrifuged and supernatant were carefully pipetted off. The cell pellets were resuspended in 1 ml of DMF to ensure removal and dissolution of indigo associated to cells. The A600 of the supernatants were determined and the concentration of indigo extracted from J26 whole cell was calculated. Indigo formation was not detectable when indole concentrations lower than 1 mM were used, reaching a maximun at 2.5 mM. Activity dropped rapidly at higher concentrations, and was not detectable at 5 mM. The highest NDO activity was achieved at 25 °C according with previous data obtained for indigo production by different Pseudomonas strains. Finally, a maximum rate of indigo production (0.56 nmol min-1 dry mg biomass was achieved at 60 minutes from the beginning of the reaction and 75.5 µM indigo was produced in the next 8 hours. However, the maximal indole concentration (138.1 µM) was reached after 20 hours of incubation. In this work, we used a high-throughput and easy technique to determine NDO activity. In addition, indigo production was performed in a micro-batch culture from a whole cell system. To our knowledge, this is the first report of NDO activity measures using indole biotransformation and indigo production by a P. plecoglossicida related strain. Pseudomonas sp. J26 shows promise for bioindigo production with industrial purposes, avoiding the use of recombinant strains.