Community genomics: Isolation, characterization and expression of gene coding for azoreductase

Abstract

Soil collected from banks of the Khari-cut canal (Vatva, Ahmedabad, Gujarat, India), contaminated by industrial wastes, was used as inoculum for developing consortium enriched with dye degrading and/or tolerating bacteria. The consortium V9 was able to decolourize 100 ppm of Reactive Violet 5 within 24 h at 37 °C. The amplified azoreductase gene had a length of 537 bp containing an ORF of 178 amino acids. BLASTn and BLASTx analyses showed 97% and 98% identity to sequences of azoreductase gene of Rhodobacter sphaeroides and Bacillus cereus G9241, respectively. The azoreductase gene was cloned in expression vectors and its in vitro activity was optimized. Co-factor NADPH.Na4 (1 mM) was essential to obtain 85–90% dye degradation by cloned azoreductase gene. Escherichia coli BL21(DE3) clone PET1 was able to degrade 90% of dye within 7 min. Conversely, only 20–30% of total dye was degraded with cell extracts of control strains after 3 h.