Azoreductase activity of dye-decolorizing bacteria isolated from the human gut microbiota

Sara A Zahran,Amal E Ali,Ramy K Aziz,Abdelgawad M Hashem,Marwa Ali-Tammam

doi:10.1038/s41598-019-41894-8

Abstract

The gut microbiota enriches the human gene pool and contributes to xenobiotic metabolism. Microbial azoreductases modulate the reduction of azo-bonds, activating produgs and azo polymer-coated dosage forms, or degrading food additives. Here, we aimed to screen the healthy human gut microbiota for food colorant-reducing activity and to characterize factors modulating it. Four representative isolates from screened fecal samples were identified as E. coli (AZO-Ec), E. faecalis (AZO-Ef), E. avium (AZO-Ev) and B. cereus (AZO-Bc). Both AZO-Ef and AZO-Ev decolorized amaranth aerobically and microaerophilically while AZO-Ec and AZO-Bc had higher aerobic reduction rates. The isolates varied in their activities against different dyes, and the azo-reduction activity mostly followed zero-order reaction kinetics, with a few exceptions. Additionally, the isolates had different pH dependence, e.g., AZO-Ec was not affected by pH variation while AZO-Bc exhibited variable degradation kinetics at different pH levels. Cell-free extracts showed NADH-dependent enzymatic activities 14–19 times higher than extracellular fractions. FMN did not affect the reducing activity of AZO-Ef cell-free extract, whereas AZO-Ec, AZO-Ev and AZO-Bc had significantly higher reduction rates in its presence (P values = 0.02, 0.0001 and 0.02, respectively). Using Degenerate primers allowed the amplification of azoreductase genes, whose sequences were 98–99% similar to genes encoding FMN-dependent-NADH azoreductases.

Highlights

The human body is inhabited by trillions of microbes that play essential roles in human health[1]
After aerobic incubation of the fecal dilutions for 24 h on BHIS agar supplemented with amaranth, 43 morphologically distinct bacterial colonies were recovered, 17 of which were surrounded by clear zones indicating their azoreductase activity (Fig. S1A)
They were identified as E. coli, E. faecalis, E. avium and B. cereus (Table 1), and the sequences of their 16S rRNA genes were deposited in GenBank under accession numbers MG596978, MG596786, MH797010 and MG596976, respectively

Summary

Introduction

The human body is inhabited by trillions of microbes that play essential roles in human health[1]. This enables them to highly affect vital host biological processes such as biotransformation of orally administered natural products and xenobiotics[4,5,6,7] One such enzymatic activity performed by human gut microbes is the production of azoreductases, enzymes that degrade azodyes, extensively used in pharmaceuticals, food, cosmetic and textile industries. Oxygen-insensitive azoreductases have been previously reported, little information is available about their characteristics and substrate specificity; we chose to focus on them in this study Based on their cofactor dependency, azoreductases were mainly classified into two major classes: flavin-free enzymes[20], and flavin-dependent enzymes. Genes coding for oxygen-insensitive azoreductases were cloned from different organisms, such as Bacillus sp., E. coli, E. faecalis and Pseudomonas sp. proved to have specific substrates and optimum pH level[21,22,25,26,27,28]

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