腈水合酶是一类重要酶制剂，在催化合成丙烯酰胺等化学品中体现出无可替代的应用优势。然而当单独克隆腈水合酶的结构基因在E. coli细胞中异源表达时，重组蛋白没有表现出任何相应的水合活力。通过研究发现，腈水合酶基因在细菌基因组中通常以复杂的基因簇形式存在，而且某些基因区域对其异源功能表达至关重要，其中活化元件基因的影响较为明显，但是其调控功能和机制并不清楚。本项目将针对P. putida 5B腈水合酶的胞内生物合成过程进行研究，试图阐明基因簇中活化元件基因的调控机制。该酶代表另一类Co型腈水合酶，其结构基因的序列为αβ。我们将采用基因重组和体外再生策略，在E. coli细胞中重新构建该腈水合酶的生物合成过程，重点阐述活化元件基因的调控功能和机制。通过本项目的研究，期望为腈水合酶的胞内合成机理研究提供可参考价值，并为构建高效的异源表达系统和蛋白质定向进化奠定工作基础。

Nitrile hydratases (NHases) have considerable practical importance as biocatalysts for industrial production of acrylamide. However, attempts to heterogeneous expression by cloning the structural gene of NHases in E. coli were disappointing due to formation of inactive and insoluble forms. Recent studies found that the NHase gene always exists as a complex cluster, and some elements in the gene cluster were potentially essential for the active expression of NHase. In this project, the biosynthesis of NHase from Pseudomonas putida 5B will be studied. The order of structural gene is αβ, which is different to reported Co-type NHase from Rhodococcus rhodochrous J1. By gene recombination technology and renaturation in vitro, the biosynthesis process will be restructured in E. coli. Furthermore, the function and mechanism of activator gene in NHase gene cluster. The research may be helpful to design efficient expression system and directed evolution of the NHase.