Metagenomic bioprospecting of novel oxygen insensitive nitroreductase for degradation of nitro aromatic compounds

Abstract

Nitroreductases are enzymes that catalyse the reduction of nitro/nitroaromatic compounds. We have employed functional metagenomics, a culture independent technique, to isolate a novel nitroreductase. The metagenomic DNA was isolated directly from pharmaceutical industry effluent (rich in nitrocompounds), purified and size selected. Sequence unbiased fosmid library was constructed using pCC2FOS fosmid vector and subsequently transformed into E.coli EPI300- T1 cells by λ-phage expression. The host cells harbouring the metagenomic DNA were selected for oxygen insensitive nitroreductases, total of 26 colonies which showed positive for nitroreductase expression were selected for invitro assay. The 6 clones which showed maximum activity were selected, sequence of fosmid DNA was determined using Illumina Nextseq platform and reads were assembled. The BLAST analysis of assembled sequence detected the presence of a nitroreductase gene. The metagenome derived oxygen insensitive nitroreductase (NR1) was further cloned and expressed in expression host E.coli BL21. The optimal reaction temperature and pH for the activity of NR1 enzyme was found to be 40 °C and 8.0 respectively. NR1 is found to prefer NADPH over NADH as a cofactor for its reductase activity. Metal ions and enzyme inhibitors are found to have no effect on NR1 activity. Reductase activity of NR1 against various nitro/nitroaromatic compounds were tested and found to have maximum activity against 2,4-6- trinitrotoluene and 4-nitrophenol. NR1 was immobilized on Celite (545), the immobilized enzyme retained more than 50% of its initial activity for 20 cycles of reuse. The isolated nitroreductase needs to be further characterised for the potential application in degradation of nitrocompounds.