Production Purification and Characterization of Nitrilase and its Role in Biotransformation

Abstract

Microbial biotransformation is exploited for converting pollutants like pharmaceuticals, hydrocarbons, and metals. Nitrilases (EC 3.5.5.1) efficiently hydrolyze nitriles, yielding carboxylic acids and ammonia. A nitrile-hydrolyzing bacterium, AGAB-2, was isolated from Amlakhadi river, Ankleshwar, Gujarat, through enrichment culture in a nitrile-supplemented mineral medium. The culture AGAB-2 was identified as Bacillus subtilis- AGAB-2 by 16s DNA. Nitrilase production was statistically optimized by PBD followed by the RSM approach which was validated by an AI tool and the maximum nitrilase production of 9832 ± 15.3 U/ml was achieved. The nitrilase enzyme was purified by size exclusion chromatography, and SDS PAGE analysis indicated a 29 KD monomer protein. Optimal activity and stability were observed at pH 7, 37ºC with 0.1 M SPB, exhibiting a half-life of 134.2 hours. It showed stability against various solvents but strong inhibition with Co+2, Hg+2, and Cu+2 at 100mM concentration. Kinetic parameters include a Vmax of 1603.84 Sec-1, Km of 2.8 mM, and catalytic efficiency of 572.8 S-1 mM-1 using acrylonitrile as a substrate. Sodium azide and Urea displayed non-competitive-uncompetitive inhibition, while hydroxylamine showed competitive inhibition with the nitrilase. The whole genome (accession number: JAPXFT0000000) for the isolate Bacillus Subtilis- AGAB-2 was evaluated which revealed presence of 4188 genes in total which include industrially important genes and, natural compounds (bacilysin and terpenes). A nitrilase superfamily gene ychX was discovered and used for homology modelling a docking study with 10 nitrile compounds which revealed amino acids present at active site. The chemoenzymatic transformation of the 8 nitrile compounds was attempted using nitrilase enzyme and, the maximum yield gt95 % was observed with acrylic acid. Bacillus subtilis AGAB-2 cells were immobilized using various metrices, with alginate proving the most efficient. Alginate immobilized cells showed high reusability and efficiency.