Characterization and Evolutionary Study of Fungal Nitrate Reductase Through Bioinformatics and Partial Gene Amplification from Aspergillus niger PKA16 employing Degenerate Primers.

Nitrate contamination in water sources creates major health risks that primarily affect infants by causing methemoglobinemia ("blue baby syndrome") while also leading to congenital defects and cancer development. The human body absorbs nitrates mainly through drinking contaminated water. Enzyme nitrate reductase (NR) produced by microorganisms, functions as a key factor in nitrate detoxification. A partial NR gene (GenBank accession: MN833805) from Aspergillus niger PKA16 (KY907172.1) was amplified by employing degenerate primers in this research. The primer sequences were designed based on conserved protein motifs and orthologous diversity analysis of 399 NR protein sequences spanning 127 fungal genera. The NR proteins exhibited an extensive range which demonstrated extensive intra- and interspecies diversity. The multiple conserved domains included nine motifs which remained consistent despite the observed sequence variability. Two highly conserved sequences RLTGKHPFN and PDHGYPLRLV were validated through degenerate-PCR which demonstrated their effectiveness for partial NR gene detection and amplification. In the present study, the developed degenerate primers enable researchers to detect and amplify NR genes from majority of known and unknown fungal strains including those identified through metagenomic studies also. This research establishes fundamental principles for using biotechnology to amplify bioremediatory enzyme nitrate reductase from fungal origin to clean up water and food that contains nitrates, to reduce the risk of 'blue baby' disease and cancer.