Cloning and functional characterization of nitrilase from Fusarium proliferatum AUF-2 for detoxification of nitriles

A fungal nitrilase gene from Fusarium proliferatum AUF-2 was cloned through reverse transcription-PCR. The open reading frame consisted of 903 bp and potentially encoded a protein of 301 amino acid residues with a theoretical molecular mass of 33.0 kDa. The encoding gene was expressed in Escherichia coli strain BL21 and the recombinant protein with His(6)-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity in the range of 35-40 A degrees C and pH 8.0. EDTA, Mg2+, Zn2+, Ca2+, Fe2+, Fe3+ and Mn2+ stimulated hydrolytic activity, whereas Cu2+, Co2+ and Ni2+ had inhibitory effect on nitrilase activity. Ag+ ions showed a strong inhibitory effect on the recombinant nitrilase activity. This nitrilase was specific towards aliphatic, heterocyclic and aromatic nitriles. The kinetic parameters V (max) and K (m) for benzonitrile substrate were determined to be 14.6 mu mol/min/mg protein and 1.55 mM, respectively. Homology modelling and molecular docking studies provided an insight into the substrate specificity and the proposed catalytic triad for recombinant nitrilase consisted of Glu-54, Lys-133 and Cys-175. This is the first report on the cloning and heterologous expression of nitrilase from Fusarium proliferatum.