Basile Lacy J, Willson Richard C, Sewell B Trevor, Benedik Michael J
Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.
Appl Microbiol Biotechnol. 2008 Sep;80(3):427-35. doi: 10.1007/s00253-008-1559-2. Epub 2008 Jun 28.
A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.
已知多种真菌物种可通过氰化物水合酶的作用降解氰化物，氰化物水合酶是腈水解酶的一个特殊亚类，可将氰化物水解为甲酰胺。在本文中，我们报道了来自粗糙脉孢菌和构巢曲霉的两种此前未知且未表征的氰化物水合酶。在克隆了来自粗糙脉孢菌、构巢曲霉、玉米赤霉和高粱球腔菌的四种氰化物水合酶的基因，并在其N端带有六聚组氨酸纯化标签后，制备了它们的重组形式，在大肠杆菌中表达，并用固定化金属亲和层析进行纯化。根据它们的相对比活性、pH活性谱、热稳定性以及修复银和铜电镀槽中受氰化物污染废水的能力对这些酶进行了比较。尽管所有四种酶都相似，但粗糙脉孢菌的氰化物水合酶（CHT）具有最高的热稳定性和最宽的pH范围（活性>50%）。在两种重金属存在的情况下，粗糙脉孢菌也表现出最高的氰化物降解速率。构巢曲霉的CHT在所评估的四种真菌腈水解酶中具有最高的反应速率。这些数据将有助于确定在含氰废物生物修复中可能使用这些酶的优化程序。与已知的植物致病真菌类似，粗糙脉孢菌和构巢曲霉在KCN存在下生长时都会被诱导表达CHT。
