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Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration

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Abstract

Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new β-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution.

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Acknowledgements

This research was supported by the Slovenian Research Agency (J2-7018-0795) and the Slovenia–Austria 2007–2008 scientific and technological collaboration program.

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Authors and Affiliations

  1. Department of Textile Materials and Design, University of Maribor, Smetanova ul. 17, SI-2000, Maribor, Slovenia

    Mojca Božič & Vanja Kokol

  2. Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8010, Graz, Austria

    Sina Pricelius & Georg M. Guebitz

Authors
  1. Mojca Božič
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  2. Sina Pricelius
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  3. Georg M. Guebitz
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  4. Vanja Kokol
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Correspondence to Vanja Kokol.

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Božič, M., Pricelius, S., Guebitz, G.M. et al. Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration. Appl Microbiol Biotechnol 85, 563–571 (2010). https://doi.org/10.1007/s00253-009-2164-8

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  • DOI: https://doi.org/10.1007/s00253-009-2164-8

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