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Next-generation DNA sequencing (NGS) has made it feasible to sequence large number of microbial genomes and advancements in computational biology have opened enormous opportunities to mine genome sequence data for novel genes and enzymes or their sources. In the present communication in silico mining of microbial genomes has been carried out to find novel sources of nitrilases. The sequences selected were analyzed for homology and considered for designing motifs. The manually designed motifs based on amino acid sequences of nitrilases were used to screen 2000 microbial genomes (translated to proteomes). This resulted in identification of one hundred thirty-eight putative/hypothetical sequences which could potentially code for nitrilase activity. In vitro validation of nine predicted sources of nitrilases was done for nitrile/cyanide hydrolyzing activity. Out of nine predicted nitrilases,  Gluconacetobacter diazotrophicus ,  Sphingopyxis alaskensis ,  Saccharomonospora viridis , and  Shimwellia blattae  were specific for aliphatic nitriles, whereas nitrilases from  Geodermatophilus obscurus ,  Nocardiopsis dassonvillei ,  Runella slithyformis , and  Streptomyces albus  possessed activity for aromatic nitriles.  Flavobacterium indicum  was specific towards potassium cyanide (KCN) which revealed the presence of nitrilase homolog, that is, cyanide dihydratase with no activity for either aliphatic, aromatic, or aryl nitriles. The present study reports the novel sources of nitrilases and cyanide dihydratase which were not reported hitherto by in silico or in vitro studies.

Mining of Microbial Genomes for the Novel Sources of Nitrilases