Jeschek M, Panke S, Ward T R
ETH Zurich, Basel, Switzerland.
University of Basel, Basel, Switzerland.
Methods Enzymol. 2016;580:539-56. doi: 10.1016/bs.mie.2016.05.037. Epub 2016 Jun 15.
Artificial metalloenzymes represent an attractive means of combining state-of-the-art transition metal catalysis with the benefits of natural enzymes. Despite the tremendous recent progress in this field, current efforts toward the directed evolution of these hybrid biocatalysts mainly rely on the laborious, individual purification of protein variants rendering the throughput, and hence the outcome of these campaigns feeble. We have recently developed a screening platform for the directed evolution of artificial metalloenzymes based on the streptavidin-biotin technology in the periplasm of the Gram-negative bacterium Escherichia coli. This periplasmic compartmentalization strategy comprises a number of compelling advantages, in particular with respect to artificial metalloenzymes, which lead to a drastic increase in the throughput of screening campaigns and additionally are of unique value for future in vivo applications. Therefore, we highlight here the benefits of this strategy and intend to propose a generalized guideline for the development of novel transition metal-based biocatalysts by directed evolution in order to extend the natural enzymatic repertoire.
人工金属酶是一种将先进的过渡金属催化与天然酶的优势相结合的有吸引力的方法。尽管该领域最近取得了巨大进展，但目前对这些混合生物催化剂进行定向进化的努力主要依赖于对蛋白质变体进行费力的单独纯化，这使得通量以及这些研究的成果都很有限。我们最近基于革兰氏阴性细菌大肠杆菌周质中的链霉亲和素-生物素技术，开发了一个用于人工金属酶定向进化的筛选平台。这种周质区室化策略具有许多引人注目的优势，特别是对于人工金属酶而言，这导致筛选研究的通量大幅增加，并且对于未来的体内应用也具有独特价值。因此，我们在此强调该策略的优势，并打算提出一个通用指南，用于通过定向进化开发新型基于过渡金属的生物催化剂，以扩展天然酶的种类。
