Evolutionary Design of Enzyme Specificity and Chemistry

酶特异性和化学的进化设计

• 批准号: 6767842
• 负责人: BRENT L IVERSON
• 金额: $ 28.38万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6767842
• 关键词: catalyst; chemistry; endopeptidases; enzyme activity; enzyme mechanism; enzymes; flow cytometry; fluorescence resonance energy transfer; high performance liquid chromatography; protein structure function

DESCRIPTION (provided by applicant): The isolation of enzymes with tailored physical and catalytic properties is one of the fundamental thrusts of modern protein science, with the potential for profound societal, technological, and medical impact. The broad, long term objective of the proposed research is to develop the experimental framework that will define the scientific and technological foundation for evolutionary design of precisely specific and highly active new enzyme catalysts. Recent technological breakthroughs in our laboratory make possible the first ultra-high throughput analyses and selections of enzyme function in large libraries, based on expression on the surface of E. coli bacteria and sorting/analysis by high speed, multi-color flow cytometry. A key feature of our new approach is the ability to select for (and quantify) desired new activity, while simultaneously deselecting (and quantifying) unwanted catalytic activity. This new technology will be exploited to investigate, in a systematic manner, the key experimental parameters of mutation rate and mutation method during the evolution of the two most important aspects of enzyme catalysis; substrate specificity and catalytic chemistry. In particular, we will be evolving new specificity and catalytic chemistry in two complementary enzyme systems, the bacterial serine protease OmpT, a representative "specialist" enzyme, (an enzyme with precise substrate specificity), and P. solani cutinase, a member of the a/13 hydrolase superfamily that is a "generalist" enzyme (one that exhibits broad esterolytic reactivity with various substrates). The bottom line is that following the proposed studies, we will, for the first time, have enough systematic data to provide calibration with respect to what changes in substrate specificity and catalytic chemistry are feasible using enzyme directed evolution, and what approaches to enzyme randomization and substrate selection bring about maximum beneficial changes of function.

描述（由申请人提供）：具有量身定制的物理和催化特性的酶是现代蛋白质科学的基本推力之一，具有深远的社会，技术和医学影响。拟议的研究的广泛长期目标是开发实验框架，该框架将定义精确特定且高度活跃的新酶催化剂的进化设计的科学和技术基础。我们实验室中的最新技术突破使得基于大肠菌细菌表面的表达以及高速，多色流式细胞仪的分类/分析的大型酶功能的第一个超高吞吐量分析和选择。我们新方法的一个关键特征是能够选择（和量化）所需的新活动，同时取消选择（和量化）不需要的催化活性。这项新技术将被利用，以系统的方式研究突变率和突变方法的关键实验参数，在酶催化的两个最重要方面的进化过程中；底物特异性和催化化学。 In particular, we will be evolving new specificity and catalytic chemistry in two complementary enzyme systems, the bacterial serine protease OmpT, a representative "specialist" enzyme, (an enzyme with precise substrate specificity), and P. solani cutinase, a member of the a/13 hydrolase superfamily that is a "generalist" enzyme (one that exhibits broad esterolytic reactivity with various基材）。最重要的是，遵循拟议的研究，我们将首次拥有足够的系统数据来提供有关底物特异性和催化化学变化的校准，使用定向酶的进化是可行的，以及哪些酶随机化和底物选择方法的方法可带来最大的功能造成功能的最大收益变化。