Isolation, Identification, and Culture Optimization of a Novel Glycinonitrile-Hydrolyzing Fungus—Fusarium oxysporum H3

Gong, Jin‐Song; Lü, Zhen-Ming; Shi, Jian; Dou, Wenfang; Xu, Huaxi; Zhou, Zhemin; Xu, Zhenghong

doi:10.1007/s12010-011-9312-1

Cited by 19 publications

(7 citation statements)

References 42 publications

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“…NH 3 determination is usually performed using the phenol-hypochlorite reaction [86], as well as the CoCl 2 -based [87], Nessler [88], and Indophenol methods [89]. Mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, liquid and gas chromatography are often used as the quantitation methods for substrate and product assays [90].…”

Section: Introductionmentioning

confidence: 99%

“…The total nitrilase activity of F. solani O1, in the presence of 2-cyanopyridine, can be enhanced by a hundred fold compared with other inducers [111]. However, our experiments demonstrated that caprolactam was also a useful inducer for fungal nitrilase from the Fusarium genus [86]. Detailed work on caprolactam induction was attempted to further improve the fungal nitrilase formation.…”

Section: Introductionmentioning

confidence: 99%

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Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

et al. 2012

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Over the past decades, nitrilases have drawn considerable attention because of their application in nitrile degradation as prominent biocatalysts. Nitrilases are derived from bacteria, filamentous fungi, yeasts, and plants. In-depth investigations on their natural sources function mechanisms, enzyme structure, screening pathways, and biocatalytic properties have been conducted. Moreover, the immobilization, purification, gene cloning and modifications of nitrilase have been dwelt upon. Some nitrilases are used commercially as biofactories for carboxylic acids production, waste treatment, and surface modification. This critical review summarizes the current status of nitrilase research, and discusses a number of challenges and significant attempts in its further development. Nitrilase is a significant and promising biocatalyst for catalytic applications.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

et al. 2012

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“…And the reaction was terminated by the addition of 2 M HCl (0.1 mL). The enzyme activity was determined by measuring the generated ammonia in the reaction mixture [10]. This assay is based on the phenol–hypochlorite method.…”

Section: Methodsmentioning

confidence: 99%

“…So far, several nitrilases of different origins have been obtained and investigated in depth. Nitrilases have been mainly found in bacteria belonging to genera Bacillus , Klebsiella , Alcaligenes , Acinetobacter , Nocardia , Pseudomonas , and Rhodococcus [7], fungi belonging to genera Fusarium and Aspergillus [9], [10], plants belonging to genera Arabidopsis , Brassica , Lupinus , and Nicotiana [11], and animals [12]. With regard to catalytic mechanism of nitrilase, it was speculated that the carbon atom of the cyano group in nitrile molecule was attacked by a thiol group in cysteine residue of the catalytic triad, forming a tetrahedral thiomidate intermediate.…”

Section: Introductionmentioning

confidence: 99%

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

Gong

Zhu

et al. 2012

PLoS ONE

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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.

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“…Thus far, the relevant studies have mainly focused on bacterial nitrilase and reports on nitrilase from filamentous fungi are relatively limited . In 1977, Harper isolated a fungal strain of Fusarium solani IMI196840 using benzonitrile as the sole nitrogen and carbon source.…”

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus

Gong

Lü

et al. 2013

J. Basic Microbiol

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Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only <5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

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Isolation, Identification, and Culture Optimization of a Novel Glycinonitrile-Hydrolyzing Fungus—Fusarium oxysporum H3

Cited by 19 publications

References 42 publications

Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

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

Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus

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