Abstract
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.
Highlights
Advancement in the DNA sequencing technologies has led to sequencing of large number of genomes and the enormous sequence data are available in the public domain
The fourthgeneration DNA sequencing has made it possible to sequence a bacterial genome within a few hours at a reasonably low cost [1,2,3,4]
Amino acid sequences less than 100 amino acids were considered to be as false positive (FP) and were discarded
Summary
Advancement in the DNA sequencing technologies has led to sequencing of large number of genomes and the enormous sequence data are available in the public domain. Microbial nitrilases are considered to be the most important enzymes in the nitrilase superfamily that find application in the synthesis of fine chemicals, production of some important acids, and drug intermediates and in green chemistry [9,10,11,12,13]. Besides their wide applications nitrilases are prone to certain limitations, for example, their inactivation or inhibition by the acidic product, extremes of pH, temperature, and organic solvent [14, 15]. The present communication focuses on in silico screening of publicly available bacterial genomes for nitrilase genes and in vitro validation of the predicted novel sources of nitrilases
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