Ultrahigh-throughput screening enables efficient single-round oxidase remodelling
Ultrahigh-throughput screening enables efficient single-round oxidase remodelling
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DOI：
10.1038/s41929-019-0340-5
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发表时间：
2019-09-01
期刊：
影响因子：
37.8
通讯作者：
Hilvert, Donald
中科院分区：
文献类型：
--
作者：
Debon, Aaron;Pott, Moritz;Hilvert, Donald
生物催化提供了一种潜在的可持续的化学制造方法。然而，将酶定制到工业过程通常是费力和耗时的，这限制了这种方法的广泛实施。高通量筛选方法可以加快寻找合适的催化剂，但往往受到需要标记底物的限制。因此，推广这些技术将大大扩大其影响。在这里，我们已经建立了一个通用的超高通量微流控检测，使功能氧化酶的分离，从含有多达10（7）个成员的库。增加的吞吐量超过流行的方法，导致完整的活性位点改造的环己胺氧化酶在一轮的定向进化。一个960倍的催化效率的增加提供了一个酶的野生型水平的活性为非天然底物，允许生物催化合成的空间要求严格的药物中间体与完整的立体控制。偶联酶测定是无标记的，并且可以容易地适应于重新设计任何氧化酶。
Biocatalysis provides a potentially sustainable means of chemical manufacturing. However, the tailoring of enzymes to industrial processes is often laborious and time consuming, which limits the broad implementation of this approach. High-throughput screening methods can expedite the search for suitable catalysts, but are often constrained by the need for labelled substrates. The generalization of such techniques would therefore significantly expand their impact. Here we have established a versatile ultrahigh-throughput microfluidic assay that enables isolation of functional oxidases from libraries that contain up to 10(7) members. The increased throughput over prevalent methods led to complete active-site remodelling of cyclohexylamine oxidase in one round of directed evolution. A 960-fold increase in catalytic efficiency afforded an enzyme with wild-type levels of activity for a non-natural substrate, allowing biocatalytic synthesis of a sterically demanding pharmaceutical intermediate with complete stereocontrol. The coupled enzyme assay is label free and can be easily adapted to re-engineer any oxidase.