赤霉菌腈水解酶副反应腈水合作用的分子基础及其理性改造

Nitrile hydrolysis by microbial nitrilase was one of the most promising pathways for carboxylic acids synthesis, which has attracted substantial attention in the field of food and pharmaceuticals processing. Conventional studies mainly focused on the bacterial nitrilase and the potential of fungal nitrilase has been far from being fully explored. The latter showed higher catalytic activity and wider substrate spectrum. However, the fungal nitrilase also displayed nitrile hydratase activity, which led to the formation of amide by-product and severely limited its application in carboxylic acids synthesis. The present project used fungal nitrilase as the research objective. The structure determination was carried out and the possible molecular basis of nitrle hydration by Gibberella intermedia nitrilase was demonstrated in detail. Subsequently, molecular modification of fungal nitrilase was performed through rational design, site-directed mutagenesis and saturation mutagenesis. Finally, the procedures of molecular modification for elimination of amide formation were brought up and the mutant libraries of high nitrilase activities were constructed. This work would lay the foundation for the industrial application of fungal nitrilase.

微生物腈水解酶高效催化的腈水解反应是最具应用潜力的有机酸合成途径之一，在食品及医药领域备受关注。目前研究主要集中于细菌腈水解酶，而真菌腈水解酶则鲜有报道。后者具有更强的催化活力、更宽的底物谱等酶学特征，但同时表现出一定的腈水合酶活性导致酰胺副产物生成，严重影响了该酶在有机酸合成中的应用。本项目拟以申请人前期筛选获得的赤霉菌腈水解酶为研究对象，针对其腈水合副反应，开展腈水解酶结构分析，系统研究并阐明赤霉菌腈水解酶副反应腈水合作用可能的分子基础，在此基础上通过理性设计、定点突变、饱和突变的技术手段进行酶分子改造，提出其腈水合副反应消除的分子改造策略并构建高腈水解酶活性的突变体，为真菌腈水解酶的工业化应用潜力奠定基础。

腈水解酶能将腈类化合物中的腈基转化为羧基，在食品、医药及化工等领域具有重要的应用价值。传统研究主要关注细菌腈水解酶，对于真菌腈水解酶相关则鲜有报道。据文献报道真菌腈水解酶具有更强的催化活力、更宽的底物谱等酶学特征，但同时表现出一定的腈水合活性导致酰胺副产物生成，严重影响了该酶的深入应用。本项目以赤霉菌腈水解酶为研究对象，开展了以下四方面的研究工作：.1、采用多种PCR策略从G. intermedia CA3-1野生菌的RNA扩增获得了真菌腈水解酶的编码基因，在E. coli宿主中实现了重组表达；.2、从分子水平上探讨了导致腈水解酶副反应腈水合作用的分子基础，确立了G. intermedia腈水解酶128位异亮氨酸及161位天冬酰胺对于酰胺副产物生成及酶活具有显著影响；.3、通过基因工程手段对G. intermedia腈水解酶进行了分子改造，成功构建了酰胺降低和酶活提高显著的突变株；.4、以改造后的腈水解酶为催化剂进行了3-氰基吡啶的生物转化，最终在440 min转化时间内累积合成烟酸264 g/L，为目前国际报道最高水平。