Evolutionary Design of Enzyme Specificity and Chemistry

描述(申请人提供)：分离具有特定物理和催化性质的酶是现代蛋白质科学的基本推动力之一，具有深远的社会、技术和医学影响。拟议研究的广泛和长期目标是开发实验框架，该框架将为精确特定和高活性新型酶催化剂的进化设计确定科学和技术基础。我们实验室最近的技术突破使首次在大型文库中进行超高通量的酶功能分析和选择成为可能，这种分析和选择是基于大肠杆菌表面表达和高速、多色流式细胞术的分选/分析。我们新方法的一个关键特征是能够选择(和量化)所需的新活性，同时取消选择(和量化)不想要的催化活性。这项新技术将被用来系统地研究酶催化两个最重要的方面-底物专一性和催化化学-进化过程中突变率和突变方法的关键实验参数。特别是，我们将在两个互补的酶系统中进化出新的特异性和催化化学，细菌丝氨酸蛋白酶OmpT是一种代表性的“专家”酶(具有精确的底物专一性的酶)，而立枯草杆菌角质酶是a/13水解酶超家族的成员，是一种“通用型”酶(一种与各种底物表现出广泛的酯解反应的酶)。底线是，在拟议的研究之后，我们将第一次有足够的系统数据来提供校准，关于使用酶指导的进化在底物特异性和催化化学方面的哪些变化是可行的，以及哪些酶随机化和底物选择带来最大有益的功能变化。

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.