indigo-carmine and naphthalene

Indigo is one of the oldest dyes manufactured chemically and is mostly used in textile, food, and pharmaceutical industries. However, owing to the environmental hazards posed by the chemical production, the present scenario in the field stipulates a biosynthesis alternative for indigo production. The present study describes an indigenously isolated naphthalene-degrading strain Pseudomonas sp. HOB1 producing a blue pigment when indole was added in the growth medium. This blue pigment was analyzed by high-pressure thin-layer chromatography and other spectroscopic techniques which revealed it to be the indigo dye. Pseudomonas sp. HOB1 showed ability to produce 246 mg indigo liter(-1) of the medium. The K (m) for the enzyme naphthalene dioxygenase which is involved in indigo formation is 0.3 mM, and V (max) was as high as 50 nmol min(-1) mg dry biomass(-1). The bacterial indigo dye was further successfully applied for dyeing cotton fabrics. The high indigo productivity of Pseudomonas sp. HOB1 using naphthalene as growth substrate and its applicability on cotton fabrics, therefore, stems the probability of using this culture for commercial indigo production.

Topics: Biodegradation, Environmental; Biological Assay; Chromatography, Thin Layer; Coloring Agents; Cotton Fiber; Dioxygenases; Hydrogen-Ion Concentration; Indigo Carmine; Indoles; Multienzyme Complexes; Naphthalenes; Oxygenases; Permeability; Pseudomonas; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Time Factors

In this study, a novel indigo-producing bacterial strain PP-2 was isolated from activated sludge. It was identified as Acinetobacter sp. according to phylogenetic similarity of 16S rRNA gene sequence. This isolate was able to produce indigo from indole by utilizing a wide range of aromatic hydrocarbons. The results of SDS-PAGE analysis showed that the enzyme system induced by phenol was more abundant than that induced by other aromatic hydrocarbons. And the effects of metal ions on indigo production were also investigated, which indicated that the activity of cells induced with phenol could be inhibited by 0.5mM Fe(3+). Response surface methodology (RSM) was applied to optimize the process of indigo bio-production. The results exhibited that the maximal yield was achieved with 157.92 mg L(-1) phenol and 205.32 mg L(-1) indole. Under the optimal conditions, the indigo yield and transformation efficiency of indole were 202.92 mg L(-1) and 88%, respectively.

Topics: Acinetobacter; Analysis of Variance; Autoradiography; Electrophoresis, Polyacrylamide Gel; Indigo Carmine; Indoles; Metals; Naphthalenes; Phenols; Phylogeny; Substrate Specificity

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. The maximum production of indigo was achieved at 25 degrees C using 2.5 mM indole when J26 was grown in the complex medium JPP, while indole concentrations higher than 4 mM proved toxic for cells. The maximum rate of indigo production was 0.56 nmol min(-1) mg dry biomass(-1), obtaining 75.5 microM of indigo after 8 h of incubation, while a maximal concentration (138.1 microM) of indigo was obtained after 20 h.

Topics: Argentina; Culture Media; Geologic Sediments; Indigo Carmine; Indoles; Naphthalenes; Pseudomonas; Temperature

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of cis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.

Topics: Amino Acid Substitution; Binding Sites; Biodegradation, Environmental; Biphenyl Compounds; Culture Media; Dioxygenases; Escherichia coli; Indigo Carmine; Indoles; Models, Molecular; Multienzyme Complexes; Mutagenesis, Site-Directed; Naphthalenes; Oxygenases; Phenanthrenes; Plasmids; Substrate Specificity

A wild-type naphthalene-degrading strain Pseudomonas putida RKJ1 and two recombinant strains each of Ps. putida and Escherichia coli carrying the genes for naphthalene degradation on a recombinant plasmid pRKJ3, produced indigo and indirubin pigments from indole. Naphthalene, salicylate and IPTG induced cells of naphthalene-degrading recombinant bacteria produced up to two times higher indigo compared with the uninduced cells. The maximum rates of indigo formation by Ps. putida RKJ1, Ps. putida RKJ5/pRKJ3, Ps. putida KT2442/pRKJ3, E. coli TB1/pRKJ3 and E. coli AB1157/pRKJ3 were 0.60, 0.80, 0.60, 1.20 and 1.50 nmol min-1 mg dry biomass-1, respectively, using indole as the substrate. The apparent Km values of indigo formation by these same bacteria were 0.22, 0.15, 0.10, 0. 21 and 0.20 mmol l-1, respectively, again using indole as the substrate. The present study revealed that E. coli AB1157 was the most efficient of the hosts tested for the expression of the plasmid encoded genes (pRKJ3) from the wild-type strain Ps. putida RKJ1. In addition, both recombinant E. coli strains were capable of producing indigo directly from nutrient medium.

Topics: Coloring Agents; DNA, Recombinant; Escherichia coli; Indigo Carmine; Indoles; Naphthalenes; Pseudomonas putida

Indigo is one of the brilliant blue dyes, which was used to be extracted from plants, but now synthesized chemically. Indigo is also produced by bacteria. In recent years, Ensley, B. D. (1983) and Mermod, N. (1986) reported a pathway producing indigo of bacteria. We are currently studying indigo formation by bacteria. Using Pseudomonas sp. S13 harboring naphthalene degradation plasmid as donor and E. coli as recipient, conjugates and transformants with the plasmid were obtained. The conjugates and transformants were not only able to grow in medium with naphthalene as the sole carbon and energy source, but also to synthesize indigo in Lennox medium. The conjugates and transformants were grown in Lennox medium at 30 degrees C for 48 hr then resulted in synthesis of indigo. The production of indigo is increased in the presence of tryptophan or indole. Indigo formation was enhanced if the bacteria was grown in a medium supplemented with either 0.1% of naphthalene or 1% of salicylic acid. The present work opens a field for the synthesis of dyes by microbes and it might shed light on the potential use in controlling environmental pollution by engineering bacteria.

Topics: Coloring Agents; Conjugation, Genetic; Escherichia coli; Indigo Carmine; Indoles; Naphthalenes; Plasmids; Pseudomonas; Transformation, Bacterial