【作者】 刘义；

【导师】 周哲敏；

【作者基本信息】 江南大学 ， 发酵工程， 2014， 博士

【摘要】 腈水合酶（Nitrile hydratase,简写NHase, EC4.2.1.84）是一种能够催化腈类化合物水合生成相应酰胺的金属酶。目前国际上已经广泛应用第三代工业用菌玫瑰红红球菌Rhodococcus rhodochrous J1实现酰胺类物质的工业生产。此法具有选择性好，回收率高，副产物少，反应可在常温常压下进行，省去了产品提浓和丙烯腈回收等步骤的优势，使生产工艺过程得到简化。我国利用腈水合酶生产丙烯酰胺的生物技术虽然起步较晚，但发展很快。腈水合酶的广泛应用推动了其基础研究的发展。本研究室长期从事腈水合酶机理的研究，前期发现R. rhodochrous J1来源的腈水合酶金属离子摄取机理是一种称为“亚基自身交换”的新型蛋白质翻译后修饰体系。尽管如此，这种新型的蛋白质翻译后修饰体系是否具有普遍性还需要进一步探讨。然而，腈水合酶不稳定的酶学性质限制了体外研究酶分子翻译后修饰的机制，因此，提高其稳定性对进一步系统探讨酶催化的分子机制以及广泛应用于工业生产具有重要的意义。本论文以恶臭假单胞菌Pseudomonas putida NRRL-18668中腈水合酶为研究对象，系统探讨了其Co离子摄取机理，并对酶分子进行了热稳定性改造研究，主要结果如下：（1）利用N端原则在大肠杆菌中成功表达了P. putida腈水合酶调控蛋白P14K。P14K对腈水合酶的功能性表达是必要的，但在大肠杆菌中很难表达。利用N端原则，将P14K的N端的第二位Lys突变成Ala、截断N端前16个氨基酸后使新的N端第二位氨基酸是Gly、在N端融合strep标签，都能实现P14K在大肠杆菌中成功表达。此外，通过在P14K的N端融合pelB信号肽也可以提高P14K的稳定性。（2）P. putida菌中腈水合酶（α亚基àβ亚基à调控蛋白）的Co离子摄取机制遵循亚基自身交换机制。首先分离了调控蛋白复合物，发现该复合物是由P14K与腈水合酶自身α亚基形成的蛋白质三聚体α(P14K)2。设计表达了含有两种不同分子量α亚基（α和HisT7-α）的腈水合酶(HisT7-α)2β2和调控蛋白α(P14K)2，把纯化的不含Co离子的(HisT7-α)2β2与含Co离子的α(P14K)2混合4小时后发现，原来的腈水合酶(HisT7-α)2β2变成了α2β2，说明这个过程中两者的α亚基相互发生了交换。由此证明了P. putida菌中腈水合酶的Co离子摄取机制是遵循亚基自身交换机制的。本实验不仅说明新型蛋白质翻译后修饰亚基自身交换机制存在于P. putida腈水合酶中，结合本实验室以前的研究结果也说明亚基自身交换机制具有一定的普遍性。（3）P14K上C端Leu85-Ala144（C-domain）高度柔性带正电的结构特征在腈水合酶Co离子摄取的过程中具有重要作用。分子动力学模拟显示C-domain具有高度柔性的结构特征。突变该结构域氨基酸序列上保守的并与结构柔性相关的Gly86，发现腈水合酶只保留了野生酶10%的酶活。说明C-domain高度柔性的结构特征对于腈水合酶的激活有重要的作用。突变C-domain上的带正电的氨基酸为中性氨基酸导致腈水合酶失活，说明C-domain带正电的特征也同样对于腈水合酶的激活有重要的作用。同时发现这些突变体的Co离子含量很低。另外，量子化学计算结果显示腈水合酶摄取Co离子时需要克服203kcal×mol-1的能垒，结合上述实验结果和分析，我们认为C-domain高度柔性带正电的结构特征恰好就是为了克服这个能垒而辅助α亚基摄取Co离子的。（4）在腈水合酶β亚基N端融合自组装肽ELK16（SAP-NHase-2）能诱导腈水合酶形成活性包涵体。在腈水合酶β亚基C端融合ELK16（SAP-NHase-10）或是融合自组装肽EAK16（SAP-NHase-1）虽都不能诱导腈水合酶形成活性包涵体，但能提高腈水合酶的稳定性，其中SAP-NHase-1, SAP-NHase-2和SAP-NHase-10的热稳定性较原始酶分别提高了45%,30%和50%。更多还原

【Abstract】 Nitrile hydratase (NHase, EC4.2.1.84) is a metalloenzyme that can catalyze thehydration of a nitrile to the corresponding amide. It is widely used in the industrial application.Millions of acrylamide were produced by NHase. NHase catalysis possesses advantages suchas good selectivity, productivity, less side product, under common temperature and pressure,without product concentration and recycle of acrylnitrile and so on. A rapid improvement ofNHase catalysis was conducted in China.A wide range use of NHase induces its fundamental research. A long research of NHasecatalysis was conducted in our laboratory. Recently, a cobalt-containing NHase inRhodococcus rhodochrous J1, which uses a novel mode of post-translational maturation, wasdiscovered. This novel mode of post-translational maturation was called self-subunitswapping. This not only enhances the research of NHase catalysis but also raises theknowledge of people, which lays the foundation for new method. However, further study wasimperative.The mechanism of cobalt incorporation into NHase in Pseudomonas putidaNRRL-18668was discovered. The stability of NHase was also researched. The main resultswere conducted as follows:(1) P14K was successfully expressed in Escherichia coli. Nitrile hydratase (NHase)activators are essential for functional NHase biosynthesis. However, the activator P14K of thecobalt-containing NHase from P. putida is difficult to heterogeneously express in E. coli,which has retarded the clarification of the mechanism underlying the involvement of P14K inthe maturation of NHase. We here successfully expressed P14K through genetic modificationsaccording to N-end rule and analyze the mechanism underlying the instability of this protein.We found that mutation of the second N-terminal amino acid lysine to alanine or truncatingthe N-terminal16amino-acid sequence resulted in successful expression of P14K. Moreover,either addition of a strep tag alone (strep-P14K) or fusion of a pelB leader and srtep tagtogether (pelB-strep-P14K) at the N-terminus increased the expression of P14K.(2) The incorporation of cobalt into another type of Co-NHase, with a gene organizationof <α-subunit><β-subunit><activator protein>, was also dependent on self-subunit swapping.We successfully isolated a recombinant NHase activator protein (P14K) of P. putida. ThisP14K was found to form a complex α(P14K)2with the α-subunit of the NHase. Theincorporation of cobalt into the NHase of P. putida was confirmed to be dependent on theα-subunit substitution between cobalt-containing α(P14K)2and cobalt-free NHase. Theseresults expand on the general features of self-subunit swapping maturation..(3) The flexibility and positive charge of the C-terminal domain (C-domain) of theself-subunit swapping chaperone (P14K) of NHase from P. putida might play an importantrole in the cobalt incorporation for NHase activation. We first proposed a flexible C-domainfrom L85to A144using molecular dynamic simulation, and found that the C-domaintruncation and the P14K(G86I) mutation, which alter the C-domain flexibility, resulted invery low NHase activity. In addition, elimination of the positive charge in the C-domain also sharply affected NHase activity, and these mutants exhibited low cobalt content. Based on thestructural and energetic analyses, we proposed that the flexible, positively charged C-domainmost likely performs an external action that allows the cobalt-free NHase to overcome theenergetic barrier (203kcal×mol-1), resulting in a cobalt-containing NHase.(4) The thermo-stability of nitrile hydratase (NHase) was enhanced by fusing with two ofthe SAPs (EAK16and ELK16). When the ELK16was fused to the N-terminus of β-subunit,the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16fused NHasein the N-terminus of β-subunit (SAP-NHase-1) and ELK16fused NHase in the C-terminus ofβ-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the wild typeNHase, the thermal stability of SAP-NHase-1, SAP-NHase-2and SAP-NHase-10wereenhanced by45%,30%and50%, respectively.更多还原