Applied Microbiology and Biotechnology

, Volume 102, Issue 21, pp 9171–9181 | Cite as

Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction

  • Hirokazu SuzukiEmail author
  • Tomoaki Abe
  • Katsumi Doi
  • Toshihisa Ohshima
Biotechnologically Relevant Enzymes and Proteins


Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.


Alkaliphile Azoreductase Bacillus Indigo Indigo carmine Polygonum 



The authors thank Aika Tanaka Kasuri Koubou (Fukuoka, Japan) for providing the polygonum alkaline liquor.

Funding information

This work was supported by an Innovative Research Program Award of the Japan Society for Bioscience, Biotechnology, and Agrochemistry and the Institute for Fermentation, Osaka, Japan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate SchoolKyushu UniversityFukuokaJapan
  2. 2.Department of Chemistry and Biotechnology, Graduate School of EngineeringTottori UniversityTottoriJapan
  3. 3.Centre for Research on Green Sustainable ChemistryTottori UniversityTottoriJapan
  4. 4.Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Graduate SchoolKyushu UniversityFukuokaJapan
  5. 5.Faculty of EngineeringOsaka Institute of TechnologyOsakaJapan

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