Evolutionary Design of Enzyme Specificity and Chemistry

描述（由申请人提供）：分离具有定制物理和催化特性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，技术和医学影响的潜力。拟议研究的广泛，长期目标是开发实验框架，该框架将定义精确特异性和高活性新酶催化剂进化设计的科学和技术基础。本实验室最近的技术突破使首次在大文库中进行超高通量分析和选择酶功能成为可能，基于在E. coli细菌，并通过高速多色流式细胞术进行分选/分析。我们的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）不需要的催化活性。这种新技术将被利用来调查，在一个系统的方式，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的方面的演变;底物特异性和催化化学。特别是，我们将在两个互补的酶系统中发展新的特异性和催化化学，细菌丝氨酸蛋白酶OmpT，一种代表性的“专业”酶，（一种具有精确底物特异性的酶），和P. solani角质酶，a/13水解酶超家族的一个成员，是一种“通才”酶（一种与各种底物表现出广泛的酯水解反应性）。底线是，在拟议的研究之后，我们将首次有足够的系统数据来提供校准，以确定使用酶定向进化的底物特异性和催化化学的变化是可行的，以及酶随机化和底物选择的方法带来了最大的有益功能变化。

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.