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Properties of NAD (P) H Azoreductase from Alkaliphilic Red Bacteria Aquiflexum sp. DL6

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Abstract

Azoreductase plays a key role in bioremediation and biotransformation of azo dyes. It initializes the reduction of azo bond in azo dye metabolism under aerobic or anaerobic conditions. In the present study, we isolated an alkaliphilic red-colored Aquiflexum sp. DL6 bacterial strain and identified by 16S rRNA method. We report nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent azoreductase purified from Aquiflexum sp. DL6 by a combination of ammonium sulfate precipitation and chromatography methods. The azoreductase was purified up to 30-fold with 37 % recovery. The molecular weight was found to be 80 kDa. The optimum activity was observed at pH 7.4 and temperature 60 °C with amaranth azo dye as a substrate. The thermal stability of azoreductase was up to 80 °C. The azoreductase has shown a wide range of substrate specificity, including azo dyes and nitro aromatic compounds. Metal ions have no significant inhibitory action on azoreductase activity. The apparent K m and V max values for amaranth azo dye were 1.11 mM and 30.77 U/mg protein respectively. This NAD (P) H azoreductase represents the first azoreductase to be characterized from alkaliphilic bacteria.

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Abbreviations

NADH:

Nicotinamide adenine dinucleotide

NADPH:

Nicotinamide adenine dinucleotide phosphate

EDTA:

Ethylenediaminetetraacetic acid

DTT:

Dithiothreitol

FADH2 :

Flavin adenine dinucleotide (reduced)

FMNH2 :

Flavin mononucleotide (reduced)

DEAE:

Diethylaminoethyl

BLAST:

Basic local alignment search tool

PCR:

Polymerase chain reaction

dNTP:

Deoxyribonucleotide triphosphate

PBS:

Phosphate buffered saline

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Acknowledgments

Santosh Misal gratefully acknowledges the senior research fellowship from University Grants Commission India, and BCUD, University of Pune for funding to carry out this work.

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  1. Santosh A. Misal

    Present address: Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada

Authors and Affiliations

  1. Biochemistry Division, Department of Chemistry, University of Pune, Pune, 411 007, India

    Santosh A. Misal, Devendra P. Lingojwar & Kachru R. Gawai

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  1. Santosh A. Misal
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  2. Devendra P. Lingojwar
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  3. Kachru R. Gawai
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Correspondence to Kachru R. Gawai.

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Misal, S.A., Lingojwar, D.P. & Gawai, K.R. Properties of NAD (P) H Azoreductase from Alkaliphilic Red Bacteria Aquiflexum sp. DL6. Protein J 32, 601–608 (2013). https://doi.org/10.1007/s10930-013-9522-1

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