Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.

<h4>Background</h4>Nitrilase 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 crucia...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Jin-Song Gong, Heng Li, Xiao-Yan Zhu, Zhen-Ming Lu, Yan Wu, Jing-Song Shi, Zheng-Hong Xu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/638382a243394e8e93cc93213ad6d327
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:638382a243394e8e93cc93213ad6d327
record_format	dspace
spelling	oai:doaj.org-article:638382a243394e8e93cc93213ad6d3272021-11-18T08:06:47ZFungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.1932-620310.1371/journal.pone.0050622https://doaj.org/article/638382a243394e8e93cc93213ad6d3272012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23226336/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Nitrilase 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.<h4>Methodology/principal findings</h4>A 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 His(6)-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.<h4>Conclusions/significance</h4>In 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.Jin-Song GongHeng LiXiao-Yan ZhuZhen-Ming LuYan WuJing-Song ShiZheng-Hong XuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 11, p e50622 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jin-Song Gong Heng Li Xiao-Yan Zhu Zhen-Ming Lu Yan Wu Jing-Song Shi Zheng-Hong Xu Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.
description	<h4>Background</h4>Nitrilase 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.<h4>Methodology/principal findings</h4>A 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 His(6)-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.<h4>Conclusions/significance</h4>In 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.
format	article
author	Jin-Song Gong Heng Li Xiao-Yan Zhu Zhen-Ming Lu Yan Wu Jing-Song Shi Zheng-Hong Xu
author_facet	Jin-Song Gong Heng Li Xiao-Yan Zhu Zhen-Ming Lu Yan Wu Jing-Song Shi Zheng-Hong Xu
author_sort	Jin-Song Gong
title	Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.
title_short	Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.
title_full	Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.
title_fullStr	Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.
title_full_unstemmed	Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.
title_sort	fungal his-tagged nitrilase from gibberella intermedia: gene cloning, heterologous expression and biochemical properties.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/638382a243394e8e93cc93213ad6d327
work_keys_str_mv	AT jinsonggong fungalhistaggednitrilasefromgibberellaintermediagenecloningheterologousexpressionandbiochemicalproperties AT hengli fungalhistaggednitrilasefromgibberellaintermediagenecloningheterologousexpressionandbiochemicalproperties AT xiaoyanzhu fungalhistaggednitrilasefromgibberellaintermediagenecloningheterologousexpressionandbiochemicalproperties AT zhenminglu fungalhistaggednitrilasefromgibberellaintermediagenecloningheterologousexpressionandbiochemicalproperties AT yanwu fungalhistaggednitrilasefromgibberellaintermediagenecloningheterologousexpressionandbiochemicalproperties AT jingsongshi fungalhistaggednitrilasefromgibberellaintermediagenecloningheterologousexpressionandbiochemicalproperties AT zhenghongxu fungalhistaggednitrilasefromgibberellaintermediagenecloningheterologousexpressionandbiochemicalproperties
_version_	1718422231581720576

Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties.

Ejemplares similares