The rate limiting first step of the biodegradation pathway of recalcitrant azo dyes is their decolorization, which involves the reductive cleavage of the azo bond (-N=N-) catalysed by the enzyme azoreductase. Enterococcus sp. L2, a novel bacterial isolate obtained from a Reactive violet 5R decolorizing consortium, was found to decolorize a wide spectrum of azo dyes. The cloning and heterologous expression of azoA gene of Enterococcus sp. L2 encoding the NADH driven FMN dependent azoreductase in Escherichia coli DH5α and Pseudomonas fluorescens PfO-1 using the broad host range expression vector pBBRMCS2 resulted in IPTG inducible expression of azoA in the former strain and constitutive expression in the latter. The increased AzoA activity improved ∼2 fold decolorization as compared to the vector control. To further increase the efficiency of decolorization process, intracellular NADH pool was enhanced by the expression of Mycobacterium vaccae N10 NAD+ dependent formate dehydrogenase gene. FDH over-expression in E. coli and P. fluorescens PfO-1 resulted ∼3.5–4 fold increase dye decolorization, thus developing an in vivo NADH regeneration system using formate as an electron donor. Overexpressing both azoA and fdh via transcription fusion under plac and/or pT7 promoters in E. coli BL21 (DE3) and P. fluorescens PfO-1 further increased the RV5R decolorization in formate constituted buffered conditions compared to only azoA or fdh overexpression. This demonstrates a whole cell based efficient system for the azo dye decolorization.
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