Fungal His-Tagged Nitrilase from Gibberella intermedia: Gene Cloning, Heterologous Expression and Biochemical Properties

Abstract

BackgroundNitrilase is an important member of the nitrilase superfamiliy. It has attracted substantial interest from academia and industry for its function of converting nitriles directly into the corresponding carboxylic acids in recent years. Thus nitrilase has played a crucial role in production of commercial carboxylic acids in chemical industry and detoxification of nitrile-contaminated wastes. However, conventional studies mainly focused on the bacterial nitrilase and the potential of fungal nitrilase has been far from being fully explored. Research on fungal nitrilase gene expression will advance our understanding for its biological function of fungal nitrilase in nitrile hydrolysis.Methodology/Principal FindingsA fungal nitrilase gene from Gibberella intermedia was cloned through reverse transcription-PCR. The open reading frame consisted of 963 bp and potentially encoded a protein of 320 amino acid residues with a theoretical molecular mass of 35.94 kDa. Furthermore, the catalytic triad (Glu-45, Lys-127, and Cys-162) was proposed and confirmed by site-directed mutagenesis. The encoding gene was expressed in Escherichia coli Rosetta-gami (DE3) and the recombinant protein with His6-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity at 45°C and pH 7.8. This nitrilase was specific towards aliphatic and aromatic nitriles. The kinetic parameters V max and K m for 3-cyanopyridine were determined to be 0.81 µmol/min·mg and 12.11 mM through Hanes-Woolf plot, respectively. 3-Cyanopyridine (100 mM) could be thoroughly hydrolyzed into nicotinic acid within 10 min using the recombinant strain with the release of about 3% nicotinamide and no substrate was detected.Conclusions/SignificanceIn the present study, a fungal nitrilase was cloned from the cDNA sequence of G. intermedia and successfully expressed in E. coli Rosetta-gami (DE3). The recombinant strain displayed good 3-cyanopyridine degradation efficiency and wide substrate spectrum. This fungal nitrilase might be a potential candidate for industrial applications in carboxylic acids production.

Genetic and Functional Diversity of Nitrilases in Agaricomycotina

“…Recently, more than 10 new NLases in fungi of the division Ascomycota were characterized [14,15,16]. These enzymes could be roughly classified into aromatic NLases with a preference for (hetero) aromatic nitriles and arylacetoNLases with a preference for phenylacetonitrile (PAN) or mandelonitrile (MN).…”

Therapeutic Hypothermia Reduces Oxidative Damage and Alters Antioxidant Defenses after Cardiac Arrest

“…MTH was found to decrease frontal cortex MDA and carbonyl levels 24 h after ROSC in a porcine model of CA [11]. …”

“…Oxidative stress has been implicated as a crucial factor in organ damage and hemodynamic dysfunction during PCAS, and, in this setting, MTH may be a suitable treatment option to reduce oxidative stress. MTH was found to decrease frontal cortex MDA and carbonyl levels 24 h after ROSC in a porcine model of CA [ 11 ].…”

“…Mild therapeutic hypothermia (MTH) has been the only clinical treatment applied to reduce post-CA injury, and clinical and animal model studies have supported the benefits of MTH for improving brain recovery [ 9 – 11 ]. MTH also prevents damage to other tissues, including the myocardium, liver, and kidney [ 12 ].…”

“…MTH also prevents damage to other tissues, including the myocardium, liver, and kidney [ 12 ]. Damage protection by MTH is reinforced by studies demonstrating reduced oxidative damage after treatment [ 11 – 13 ]. Hypothermia may also be involved in preventing acidosis and lactate clearance, thereby restoring normal metabolism [ 14 , 15 ].…”