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

Abstract

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 His6-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity in the range of 35-40 °C and pH 8.0. EDTA, Mg(2+), Zn(2+), Ca(2+), Fe(2+), Fe(3+) and Mn(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+) and Ni(2+) 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 μmol/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.

Thermoresponsive Gigaporous Microspheres Facilitate the Efficient Refolding of Recombinant Nitrilase Inclusion Bodies

“…Nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of various nitriles to corresponding carboxylic acids and ammonia, has been recognized as a high commercial-value biocatalyst because of its high efficiency and eco-friendly transformation. − In this regards, several nitrilases have been found in different sources, and some of these enzymes have been successfully applied in industries. − Recently, our laboratory has identified a novel nitrilase from Pannonibacter carbonis Q4.6, whose conservative triplet catalytic sit “Glu-Lys-Cys” has changed into “Glu-Ser-Cys.” To explore the properties of the enzyme, the gene encoding the nitrilase was cloned and expressed in Escherichia coli BL21 (DE3) . However, most of the expressed nitrilases formed inclusion bodies (IBs), which need to undergo refolding process to restore their bioactive structures.…”

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“…Nitrilase expressed in Rhodococous rhodochrous J1 showed the inhibitory effect of CuCl 2 whereas no influence of EDTA on the enzyme activity 20 . In Fusarium proliferatum, EDTA has enhanced the activity and NiCl 2 has inhibitory activity 28 . In certain studies, EDTA does not affect or very little influence on the activity of nitrilase protein indicating absence of metal ion or lack of requirement of metal ions as cofactors for enzyme catalysis 29,30 which is quite similar to the present findings.…”

<b><i>Clostridium thermocellum </i></b>Nitrilase Expression and Surface Display on <b><i>Bacillus subtilis</i></b> Spores

“…Several recent studies have examined nitrilase applications in chemical synthesis, with various nitrilase-producing organisms including bacteria, filamentous fungi, yeasts and plants being described [Molojwane et al, 2015;Rustler and Stolz, 2007;Sun et al, 2015;Yusuf et al, 2015]. Some of these microbial cell factories have been used for the commercial production of carboxylic acids on an industrial scale.…”