(Focused) Directed Evolution to Unterstand Relationships in alpha/beta-Hydrolase-Fold Enzymes

（重点）定向进化以了解 α/β-水解酶折叠酶中的关系

• 批准号: 29632332
• 负责人: Professor Dr. Uwe T. Bornscheuer
• 金额: --
• 依托单位: Arbeitskreis Biotechnologie und Enzymkatalyse
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2006
• 资助国家: 德国
• 起止时间: 2005-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/29632332?language=en
• 关键词: Focused; Directed; Evolution; Unterstand; Relationships

Esterases, epoxide hydrolases and haloalcohol dehalogenases have a highly conserved protein scaffold ((/(-hydrolase fold) with (apparently) only minor differences in key amino acid residues. However, changing conserved amino acids (i.e. the serine in the esterase to an aspartate to create epoxide hydrolase activity) does not lead to functional changes. In this proposal, a combination between rational protein design and directed evolution will be applied to create catalytically promiscuous activity into the esterase from Pseudomonas fluorescens (PFE). Preliminary experiments already confirmed that epoxide hydrolase activity could be gained by this approach. This will be extended by further rounds of directed evolution, back mutations and saturation mutagenesis. We expect to get a detailed picture of the mechanism and key residues required to confer the promiscous activity. This insight into the structure-function basis should not only extend our understanding how epoxide hydrolases work, but also provide information about a/ß-hydrolase function in general. The same concept will be applied to confer haloalcohol dehalogenase activity in PFE.

酯酶、环氧化物水解酶和卤代醇脱卤酶具有高度保守的蛋白质支架（（/（-水解酶折叠），（显然）在关键氨基酸残基中仅具有微小差异。然而，改变保守氨基酸（即酯酶中的丝氨酸变为天冬氨酸以产生环氧化物水解酶活性）不会导致功能变化。在这个建议中，合理的蛋白质设计和定向进化之间的组合将被应用到创建催化混杂活性的酯酶从荧光假单胞菌（PFE）。初步实验已经证实，环氧化物水解酶活性可以通过这种方法获得。这将通过进一步的定向进化、回复突变和饱和诱变来扩展。我们希望得到一个详细的图片的机制和关键残基所需的赋予混杂的活动。这种对结构-功能基础的洞察不仅应该扩展我们对环氧化物水解酶如何工作的理解，而且还提供了关于α-水解酶功能的一般信息。相同的概念将被应用于赋予PFE中的卤代醇脱卤酶活性。