azoreductase gene, cloning from soil collected from banks of the Khari-cut canal (Vatva, Ahmedabad, Gujarat, India), contaminated by industrial wastes, V9 consortium, phylogenetic analysis and tree, subcloning in Escherichia coli strain DH10B and recombinant expression in Escherichia coli strain BL21(DE3)
expressed in Escherichia coli BL21(DE3)pLysS using a phage T7-promotor system, no in vivo activity in recombinant strains due to missing azo dye uptake system
functional recombinant expression of the chimeric gene GFA, in which both GDH stop codon and the sequence encoding AZR signal peptide are removed, in Escherichia coli strain BL21 from plasmid pET28a(+)
gene azoA, subcloning in Escherichia coli strain DH5alpha, and constitutive recombinant expression in Pseudomonas fluorescens strain PfO-1 using vector pBBRMCS2 leading to 2fold increased azoreductase activity, coexpression of Mycobacterium vaccae strain N10 NAD+-dependent formate dehydrogenase gene. FDH results in a 3.5-4fold increased dye decolorization, thus developing an in vivo NADH regeneration system using formate as an electron donor. Overexpression of both genes azoA and fdh via transcription fusion under plac and/or pT7 promoters in Escherichia coli strain BL21 (DE3) and Pseudomonas fluorescens strain PfO-1 further increases the RV5R decolorization in formate constituted buffered conditions compared to only azoA or fdh overexpression
gene azoR, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) from three different vectors pET32a, pET28a and pET20bI. pET32a contains an N-terminal Trx-tag encoding for a 109-amino-acid highly soluble polypeptide thioredoxin, TrxA, the pET28a is an original vector without special fusion protein, while pET20bI (similar to pET22b) contains an N-terminal pelB signal sequence for potential periplasmic localization resulting in recombinant enzymes of molecular weight of 40.4 kDa, 26.2 kDa and 25.8 kDa, respectively. Recombinant AzrS_32 is soluble by 98%, AzrS_28 by 90%, while AzrS_20 is mostly accumulated as inclusion bodies, that have no activity, although pelB leads the target protein to the periplasmic space, where there is a more favorable environment for folding and disulfide bond formation. By contrast, the protein amount of AzrS_28 (i.e. 278 mg/l) is slightly higher than that of AzrS_32 (i.e. 253 mg/l). Therefore, the plasmid pET28a is considered as the ideal vector for production of AzrS