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首页> 外文期刊>Functional & integrative genomics >Cloning and functional characterization of nitrilase from Fusarium proliferatum AUF-2 for detoxification of nitriles
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Cloning and functional characterization of nitrilase from Fusarium proliferatum AUF-2 for detoxification of nitriles

机译：枯萎镰刀菌AUF-2中腈水解酶的克隆及功能表征

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摘要

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.
机译：通过逆转录-PCR克隆了来自镰刀菌AUF-2的真菌腈水解酶基因。开放阅读框由903 bp组成，并可能编码301个氨基酸残基的蛋白质，理论分子量为33.0 kDa。编码基因在大肠杆菌BL21菌株中表达，带有His（6）-tag的重组蛋白被纯化至电泳均一性。纯化的酶在35-40 A摄氏度和pH 8.0的范围内表现出最佳活性。 EDTA，Mg2 +，Zn2 +，Ca2 +，Fe2 +，Fe3 +和Mn2 +刺激了水解活性，而Cu2 +，Co2 +和Ni2 +对腈水解酶活性具有抑制作用。 Ag +离子对重组腈水解酶活性表现出强烈的抑制作用。该腈水解酶对脂族，杂环和芳族腈具有特异性。测定苄腈底物的动力学参数V（max）和K（m）分别为14.6μmol/ min / mg蛋白和1.55 mM。同源性建模和分子对接研究提供了对底物特异性的深入了解，拟议的重组腈水解酶催化三联体由Glu-54，Lys-133和Cys-175组成。这是关于枯萎镰刀菌腈水解酶的克隆和异源表达的首次报道。

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