Molecular Cloning and Characterization of the Gene Coding for the Aerobic Azoreductase from Xenophilus azovorans KF46F

Abstract

The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.Azo dyes are characterized by the presence of one or more azo groups (-NAN-). They are the largest and most versatile class of dyes, and more than half of the annually produced dyes (estimated for 1994 worldwide as about 1 million tons) are azo dyes. Presumably more than 2,000 different azo dyes are currently used for the dyeing of various materials such as textiles, leather, plastics, cosmetics, and food (2,9,11,36,50).Azo dyes are generally considered to be xenobiotic compounds which are rather recalcitrant against biodegradative processes in conventional sewage treatment systems (33,40). Nevertheless, during the last years it has been demonstrated that several microorganisms are able to transform azo dyes to noncolored products or even mineralize them completely under certain environmental conditions. There are numerous reports which describe the reductive cleavage of azo dyes under anaerobic conditions which result in the decolorization of azo dyes. These reactions usually occur with rather low specific activities but are extremely unspecific with regard to the organisms involved and the dyes converted. In these unspecific anaerobic processes very often low-molecular-weight redox mediators (e.g., flavins or quinones) are involved (10,22,24,35,42).Some aerobic biotransformations of azo dyes by fungi and bacteria are also known. Thus, various lignolytic fungi were shown to decolorize azo dyes using ligninases, manganese peroxidases, or laccases (8). Only very few aerobic bacteria which can grow with azo compounds have been described. The ability of bacteria to grow with simple carboxylated azo compounds as the sole source of carbon and energy was shown first by Overney (32) who isolated a "Flavobacterium" which was able to grow aerobically with the model compound 4,4Ј-dicarboxyazobenzene. In subsequent work, it was shown that 4,4Ј-dicarboxyazobenzene-degrading mixed bacterial cultures could be adapted after prolonged continuous cultivation for several hundreds of generations under nonsterile conditions to t...

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

“…Most of these enzymes are shown to be ineffective in vivo (Blumel et al, 2002;Russ et al, 2000). Limited transport of dyes across the cell membrane and high reactivity of many of the ortho aminohydroxy aromatics that are formed after the reductive cleavage of the azo dyes restrict the reaction of azoreductases (Grundmann, 1979;Kudlich et al, 1999).…”

“…The monomer and dimer structures of the azoreductases determine their substrate specificity and thermostability (Brissos et al, 2014). Gene sequences have been determined for both dimeric azoreductases (Matsumoto et al, 2010;Nakanishi et al, 2001) and monomeric enzymes (Blumel & Stolz, 2003;Blumel et al, 2002). Dimeric azoreductases are thought to be more thermostable (Ooi et al, 2012) and these enzymes also show broad substrate specificity for azo dye reduction .…”

“…In most cases, the genes encoding aerobic flavin-dependent azoreductases have been cloned from a variety of bacteria including Bacillus sp. OY1-2 (Suzuki et al, 2001), Escherichia coli (Nakanishi et al, 2001), Xenophilus azovorans KF46F (Blumel et al, 2002), Pigmentiphaga kullae K24 (Blumel & Stolz, 2003), Enterococcus faecalis (Chen et al, 2004), Geobacillus stearothermophilus . All flavo-enzymes by definition contain a covalently or non-covalently bound flavin as a functional cofactor.…”

“…Two monomeric flavin-free azoreductases from X. azovorans KF46F (Blumel et al, 2002;Zimmermann et al, 1982) and P. kullae K24 (Blumel & Stolz, 2003) have been described. The deduced protein sequences from these cloned genes did not show significant homologies with flavin-dependent azoreductases and probably both types of reductases evolved from different origins (Chen et al, 2004).…”

COD reduction of textile effluent in three-phase fluidized bed bioreactor using Pseudomonas aureofaciens and Escherichia coli

“…Researchers have worked on Pseudomonas spp. (Vimala et al 2015;Sriram et al 2013;Blumel et al 2002), Escherichia coli (Sriram et al 2013;Vimala et al 2015) and Bacillus spp. (Shah et al 2013;Sriram et al 2013;Blumel et al 2002) for degradation of textile wastewater in an aerobic mode.…”