indigo-carmine and 2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid

Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2'-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80°C, and for the decolorization of indigo carmine at pH 8.0 and 60°C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2h at 37°C. The apparent K(m) of the enzyme displayed on spores was 443±124 μM for ABTS with a V(max) of 150 ± 16 U/mg spores. Notably, 1mg of spores displaying B. subtilis laccase (3.4 × 10(2)U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2h. The spore reactor (0.5 g of spores corresponding to 1.7×10(5)U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60°C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.

Topics: Bacillus subtilis; Bacterial Proteins; Base Sequence; Benzothiazoles; Biodegradation, Environmental; Bioreactors; Biotechnology; Coloring Agents; DNA, Bacterial; Enzyme Stability; Enzymes, Immobilized; Genes, Bacterial; Indigo Carmine; Kinetics; Laccase; Recombinant Proteins; Spores, Bacterial; Substrate Specificity; Sulfonic Acids; Textile Industry; Waste Disposal, Fluid

Cerrena unicolor laccase was immobilized by adsorption and covalent bonds formation on silica-gel carriers, functionalized with different organosilanes and surface densities. The effects of protein concentration, pH value of the coupling mixture and the enzyme purity on immobilization efficiency of the best carrier, moderately modified (0.75 mmol/g carrier) with 3-aminopropyltriethoxysilane were investigated. Activity of the best biocatalysts, expressed in ABTS oxidation, was 4028 U/mL of the carrier or 3530 U/mg of bound protein. Properties of immobilized laccase were determined to find excellent thermal stability improvement; t(1/2) for freely suspended enzyme was 2-3 min at 80 degrees C, whereas after immobilization over 100 min. Kinetic experiments in both batch and packed-bed reactors gave only four times lower k(cat)/K(m) value than for the native enzyme. A packed-bed reactor with silica-gel-bound laccase beads appeared to be very efficient in ABTS oxidation and its exceptional potentials were shown in the continuous decolorization of indigo carmine for 18 days without loss in activity. This system offers perfect ability to degrade recalcitrant dyes, but we can also envisage its use, with ABTS acting as a mediator, in regeneration of nicotinamide cofactors.

Topics: Basidiomycota; Benzothiazoles; Biocatalysis; Biotechnology; Color; Enzyme Activation; Enzyme Stability; Enzymes, Immobilized; Gels; Indigo Carmine; Kinetics; Laccase; Oxidation-Reduction; Silicon Dioxide; Sulfonic Acids; Temperature; Time Factors

The plant Ligularia fischeri var. spiciformis Nakai, a well-known edible medicinal herb in Korea, has been used to treat maladies such as jaundice, scarlet fever, rheumatoid arthritis, and hepatic function failure. In this research, 4 major antioxidant compounds were detected from this plant's leaves using an on-line high-performance liquid chromatography (HPLC)-ABTS screening system, which can determine the antioxidant activity based on a decrease in absorbance at 734 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals (ABTS(*)). In order to isolate these active compounds, a preparative HPLC was applied and their chemical structures were identified as 5-O-caffeoylquinic acid (5-CQA), 3,4-di-O-caffeoylquinic acid (3,4-DCQA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), and 4,5-di-O-caffeoylquinic acid (4,5-DCQA) by ESI/MS(n) and (1)H NMR. These 4 isomers comprised over 10% of the dried leaves, with 3,5-DCQA being the most abundant compound. The radical scavenging activity of each isomer was also evaluated simultaneously through the on-line HPLC-ABTS method, which showed 94% antioxidant activity of the ethanol extract derived from caffeoylquinic acids. Among these isomers, 3,4-DCQA contained the most strong antioxidant activity while 3,5-DCQA accounted for the highest radical scavenging capacity due to having the highest content.

Topics: Antioxidants; Asteraceae; Benzothiazoles; Chlorogenic Acid; Chromatography, High Pressure Liquid; Drug Discovery; Indicators and Reagents; Magnetic Resonance Spectroscopy; Medicine, Korean Traditional; Molecular Structure; Monosaccharides; Plant Extracts; Plant Leaves; Quinic Acid; Spectrometry, Mass, Electrospray Ionization; Sulfonic Acids; Tandem Mass Spectrometry

The effects of ultrasound on 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonate) enzymatic oxidation by laccase (Trametes villosa) has been studied by means of cyclic voltammetry. The reaction was allowed to proceed in the presence of a piece of wool and the coloration depth of the wool fabric was measured by means of K/S. It was observed that cyclic voltammetry is influenced the dyeing process and higher K/S values were obtained when the cyclic voltammetry was combined with the ultrasonic irradiation. Moreover, the K/S value is the sum of the values obtained when the wool staining is done in just the presence of cyclic voltammetry or in just the presence of ultrasound. The results obtained on the indigo carmine decolourization gives information on the importance of controlling the amount of ABTS(+) formed during the ultrasonication process.

Topics: Animals; Benzothiazoles; Indigo Carmine; Laccase; Molecular Structure; Oxidation-Reduction; Staining and Labeling; Sulfonic Acids; Ultrasonics; Wool

The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498-amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 x 10(4) U/l) as determined with ABTS (2,2'-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed.

Topics: Anthraquinones; Benzothiazoles; Cloning, Molecular; DNA, Fungal; Evans Blue; Fungal Proteins; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Indigo Carmine; Indoles; Laccase; Molecular Sequence Data; Pichia; Polyporales; Recombinant Proteins; Sequence Analysis, DNA; Substrate Specificity; Sulfonic Acids