Stabilization of enzymes by Proside

Proside 稳定酶

• 批准号: 5427223
• 负责人: Professor Dr. Franz-Xaver Schmid
• 金额: --
• 依托单位: Laboratorium für Biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5427223?language=en
• 关键词: Stabilization; enzymes; Proside

Stable enzymes are of central importance in biotechnology, and the understanding of the principles of protein stability is a major goal in structural biology. Previously we developed an in-vitro selection method for stabilized proteins, termed Proside. It links the resistance towards proteolysis of the protein to be stabilized with the infectivity of a filamentous phage, which is a selectable property. We propose to further optimize the Proside method and then to employ it for the thermodynamic stabilization of enzymes. We will start by using TEM-1 b-lactamase as a model enzyme, and then extend the studies to k-carrageenase, an enzyme that is used in the food industry. In the second part of this project we intend to use Proside to elucidate how modern (ba)8 barrel enzymes might have evolved after the duplication of primordial (ba)4 half barrels. We hope to gain insight into how the interaction surfaces and the energetic cooperativity between domains might nave been formed. To understand the results of these in-vitro selection experiments, many protein variants will be produced, and their functions and stabilities will be elucidated by biophysical techniques. Based on these analyses we will cooperate with the theoreticians of this program to ultimately understand the molecular basis of the observed evolutionary stabilizations.

稳定的酶在生物技术中至关重要，而对蛋白质稳定性原理的理解是结构生物学的主要目标。以前，我们开发了一种体外选择方法，稳定的蛋白质，称为Proside。它将待稳定的蛋白质对蛋白水解的抗性与丝状噬菌体的感染性联系起来，这是一种可选择的性质。我们建议进一步优化Proside方法，然后将其用于酶的热力学稳定。我们将以TEM-1 β-内酰胺酶作为模型酶开始，然后将研究扩展到食品工业中使用的k-角叉菜胶酶。在这个项目的第二部分，我们打算使用Proside来阐明现代（ba）8桶酶是如何在原始（ba）4半桶复制后进化的。我们希望能够深入了解相互作用表面和区域之间的能量协同性是如何形成的。为了理解这些体外选择实验的结果，将产生许多蛋白质变体，并通过生物物理技术阐明它们的功能和稳定性。基于这些分析，我们将与该计划的理论家合作，最终了解所观察到的进化稳定性的分子基础。