Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Institute of Applied Biochemistry and the Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):401-8. doi: 10.1016/j.bbrc.2014.05.127. Epub 2014 Jun 2.
Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, is the key enzyme for the production of amides in industries. However, the poor stability of this enzyme under the reaction conditions is a drawback of its industrial application. In this study, we aimed to improve the stability of NHase (PpNHase) from Pseudomonas putida NRRL-18668 using a homologous protein fragment swapping strategy. One thermophilic NHase fragment from Comamonas testosteroni 5-MGAM-4D and two fragments from Pseudonocardia thermophila JCM3095 were selected to swap the corresponding fragments of PpNHase. Seven chimeric NHases were designed using STAR (site targeted amino recombination) software and molecular dynamics to determine the crossover sites for fragment recombination. All constructed chimeric NHases showed 1.4- to 3.5-fold enhancement in thermostability and six of them become more tolerant to high-concentration product. Notably, one of these NHases, 3AB, exhibited a 1.4±0.05-fold increase in activity compared to the wild-type PpNHase. Circular dichroism spectrum analysis and homology modeling revealed that the 3AB slightly differed in secondary structure from wild-type PpNHase. The 3AB constructed in this study is useful for further industrial application, and the method for designing the chimeric protein using homologous protein fragment swapping without a decrease in activity may be a strategy to improve the stability of other enzymes.
腈水合酶（NHase）能够催化腈转化为酰胺，是工业生产酰胺的关键酶。然而，该酶在反应条件下的稳定性差，限制了其工业应用。在本研究中，我们旨在使用同源蛋白片段交换策略来提高来自恶臭假单胞菌（Pseudomonas putida NRRL-18668）的 NHase（PpNHase）的稳定性。从高温单胞菌（Comamonas testosteroni 5-MGAM-4D）中选择一个嗜热 NHase 片段和来自嗜热放线菌（Pseudonocardia thermophila JCM3095）的两个片段来交换 PpNHase 的相应片段。使用 STAR（site targeted amino recombination）软件和分子动力学设计了七个嵌合 NHase，以确定片段重组的交叉点。所有构建的嵌合 NHase 都表现出 1.4 到 3.5 倍的热稳定性增强，其中六种对高浓度产物更耐受。值得注意的是，这些 NHase 中的一种，3AB，与野生型 PpNHase 相比，活性提高了 1.4±0.05 倍。圆二色光谱分析和同源建模表明，3AB 的二级结构与野生型 PpNHase 略有不同。本研究构建的 3AB 可进一步用于工业应用，而不降低活性的使用同源蛋白片段交换设计嵌合蛋白的方法可能是提高其他酶稳定性的策略。
