Porter Joanne L, Boon Priscilla L S, Murray Tracy P, Huber Thomas, Collyer Charles A, Ollis David L
Research School of Chemistry, Australian National University , Canberra, Australian Capital Territory 2601, Australia.
ACS Chem Biol. 2015 Feb 20;10(2):611-21. doi: 10.1021/cb500809f. Epub 2014 Dec 5.
The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.
酶能够从其天然功能中进行改造并设计成专门用于工业或商业应用的能力，对于推动酶技术超越当前状态至关重要。定向进化是一种强大的工具，可用于改造具有改进的物理和催化特性的酶，并且可用于进化那些缺乏结构信息可能会阻碍合理设计应用的酶。在本研究中，我们以二烯内酯水解酶的形式采用通用且多样的α/β水解酶折叠框架，并通过总共14轮筛选的三轮独特连续进化对其进行进化，以生成一系列酶变体。天然酶对乙酸对硝基苯酯具有较低水平的混杂活性，但对较大的对硝基苯酯几乎检测不到活性。使用乙酸对硝基苯酯作为进化中间体，我们已生成了特异性和催化活性发生改变的变体，其对较大的非生理性对硝基苯酯底物的活性比天然酶高3个数量级。由于采用了多维筛选方法，几个变体还具有更高的稳定性。晶体结构分析和底物对接显示了在进化过程中酶活性位点如何作为突变的直接或间接结果而发生变化。
