Purification, characterization, and crystal structure of YhdA‐type azoreductase from Bacillus velezensis

Abstract

Azoreductases are being extensively investigated for their ability to initiate degradation of recalcitrant azo dyes through reduction of azo bonds. There is great interest in studying their diversity, structure, and function to facilitate better understanding and effective application. Current study reports azoreductase enzyme from Bacillus velezensis, which showed 69.5% identity to the Bacillus subtilis azoreductase YhdA. The enzyme was homotetrameric and molecular weight of each subunit was 20 kDa. It decolorized azo dyes with different structures. The Vmax for decolorization of congo red, methyl orange and methyl red was 14.7, 28.6, and 77.9 nmol/min/mg, respectively. The enzyme contained FMN as cofactor and used NADPH as the favored co‐substrate. It was oxygen‐insensitive, but the presence of reducing agents enhanced its activity, which is a new finding. The azoreductase expression in B. velezensis was found to be unaffected by addition of azo dyes, although azo dyes are known to induce azoreductase expression in few organisms. The enzyme was thermostable with melting temperature of 89.5°C and functioned in wide temperature range. Further, the enzyme was crystallized and its structure was solved. The structural basis of its functional attributes is discussed. In our knowledge, this is the first report on characterization of azoreductase enzyme from B. velezensis.

Enzyme-Responsive COF-Based Thiol-Targeting Nanoinhibitor for Curing Bacterial Infections

“…In contrast to heat-inactivated intracellular extracts, the activity of the enzyme was enhanced in the range of 2−4-fold in the presence of methyl red (MR) dyes, which was in accordance with documentary views that azoreductase largely exists in various quaternary forms, from dimers of Enterococcus faecalis and E. coli to tetramers of Bacillus subtilis and S. aureus. 33 The above results validate the drug release characteristic of Ag-TA-CON@EBS@PEG in response to bacteria. To our hypothesis, this characteristic of bacteria could be employed to develop bacteria-induced antimicrobial treatments.…”

Degradation of sulphonated mono and di-azo dye as the sole carbon source inSerratia marcescens: Insights from combined wet and dry lab analysis

“…In Bacillus sp. AzoR in monomeric form, FMN has been found to be bound in the loop pocket [15,59]. These insights also lead to the idea that AzoR in Serratia sp.…”

“…Lys85, Val130, Thr131, and Arg33 form hydrophobic interaction with oxygen atoms of FMN while Ser86 formed a hydrophobic interaction with the carbon atom of FMN. The pattern of FMN binding was compared to Bacillus valezensis [59], and common interacting residues were Arg and Thr (although hydrophobic interactions instead of hydrogen bonding were observed). Hydrogen bond interactions of the Chromobacterium violaceum [61],…”

Bioremediation of Reactive Dyes by Bacillus megaterium and Bacillus velezensis

“…Bacillus velezensis is known for its possession of azoreductases which are used to decolourise azo dyes of different molecular structures through enzymatic action 26,27 . It was also reported that B. velezensis contains a biopolymer that was isolated and used to decolourise azo dyes by bio-flocculation, with 91% efficiency 28 .…”