TY - JOUR
T1 - Efficient approach to enhance whole cell azo dye decolorization by heterologous overexpression of Enterococcus sp. L2 azoreductase (azoA) and Mycobacterium vaccae formate dehydrogenase (fdh) in different bacterial systems
AU - Rathod, Jagat
AU - Dhebar, Shivani
AU - Archana, G.
N1 - Funding Information:
Authors acknowledge financial support by Department of Biotechnology (DBT), Ministry of Science and Technology, New Delhi, India (Project No. BT/PR-6555-BCE/08/424/2005). JR is thankful to University Grants Commission, New Delhi, India for Research Fellowship in Sciences for Meritorious Students (UGC-RFSMS).
Publisher Copyright:
© 2017 Elsevier Ltd
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2017/10
Y1 - 2017/10
N2 - 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.
AB - 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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U2 - 10.1016/j.ibiod.2017.04.023
DO - 10.1016/j.ibiod.2017.04.023
M3 - Article
AN - SCOPUS:85018331563
VL - 124
SP - 91
EP - 100
JO - International Biodeterioration and Biodegradation
JF - International Biodeterioration and Biodegradation
SN - 0964-8305
ER -