赖氨酸脱羧酶多聚体界面氨基酸对酶活性和最适pH影响规律的研究

Lysine decarboxylases (LDCs) often exist as oligomers, and they are not only involved in the cell growth and acid stress response, but also the key enzymes for bio-production of cadaverine (a building block for polyamides). The biosynthesis of cadaverine at high pH is important for minimizing the acids added during the reaction and reducing environmental pollution. However, LDCs will depolymerize at high pH which often leads to low activity. Till now, only few reports attempted to study the structure-function relationship of LDCs. Additionally, in most studies, the variants with improved activity in one pH but not the structure-activity relationship of LDCs in multiple pH solutions were reported. Moreover, only few amino acid substitutions were usually studied, resulting in the difficulties in gaining the general principles of structure-function relationship of LDCs. In this study, a LDC from Escherichia coli named CadA is chosen as the model enzyme. In order to investigate the effect of interfacial residues of CadA on its structure and function, 30 important interfacial amino acid positions are first selected and mutated, afterwards 20 improved/decreased variants as well as their structures and enzymatic characteristics could be obtained. This study will guide protein engineers to tailor the activities and optimal pH of LDCs or other structurally-similar enzymes.

赖氨酸脱羧酶通常以多聚体形式存在，该酶不仅在细胞生长和酸胁迫响应中起着重要的作用，还是生物法制备尼龙材料单体戊二胺的关键酶。戊二胺制备要在高pH条件下进行才能最大限度减少反应过程中pH中和的成本，减少污染，而高pH会导致多聚体解聚，酶活性下降。目前赖氨酸脱羧酶结构和功能之间关系的研究还不多，这些文献通常只关注一个pH条件下活性提高的突变体，而不报道多个pH条件下酶结构和活性的关系，且报道的突变体数目一般只有几个，难以得到该酶结构变化和功能之间的一般性规律。本项目以大肠杆菌赖氨酸脱羧酶为模式酶，从多聚体界面上选择30个关键的氨基酸位点，获得20个性能提高或降低的酶突变体，通过分析它们的结构变化和酶学性质数据，解析赖氨酸脱羧酶界面上残基与酶的结构和活性、最适pH之间的关系。本项目预期成果，可以为蛋白质工程技术理性改造/设计赖氨酸脱羧酶或结构相似的一类酶的活性和最适pH提供理论指导。

赖氨酸脱羧酶是生物法制备尼龙材料单体戊二胺的关键酶，其中来源于大肠杆菌的诱导型酶CadA是目前研究较多的赖氨酸脱羧酶。文献报道CadA在最适条件(pH约5.5)主要以十聚体存在，在碱性条件下会导致CadA十聚体解聚，酶活性降低，而戊二胺制备要在高pH条件下进行才能最大限度减少反应过程中pH中和的成本，减少污染。因此，研究CadA十聚体界面残基对酸性(例如最适pH 5.5)和碱性(例如催化终点pH 8.0)条件下CadA结构与功能的影响规律具有重要意义。本项目以CadA为模式酶，从十聚体界面上选择30个关键残基构建点饱和突变体库，采用建立的三轮高通量筛选策略筛选突变体库。将粗酶筛选获得的比活性提高的六个突变体进行纯化和活性评价，发现多数突变体的比活性提高不显著。接着我们采用理性设计方法设计了50个破坏CadA十聚体界面相互作用的突变体，通过测定粗酶活性，获得21个在pH 5.5或8.0条件下活性显著降低(下降50%以上)的突变体，它们的纯酶活性与粗酶活性趋势较为一致。使用活性电泳方法初步分析21个突变体纯酶的聚集状态，然后选择八个代表性突变体，使用凝胶过滤层析技术测定了它们在pH 5.5和8.0时的聚集状态分布，以及两种pH条件下的动力学参数。综合获得的突变体的酶学性质数据和结构变化信息，初步阐明了十聚体界面残基对CadA活性与结构的影响规律。另外，我们成功获得一个活性和热稳定性同时显著提高的CadA突变体，用于全细胞催化284 g/L赖氨酸时，戊二胺时空产率提高43%。项目研究成果为后续对赖氨酸脱羧酶的进一步分子改造奠定基础，同时相关筛选和酶设计策略也将为其他酶的分子改造提供借鉴。