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A photoclick-based high-throughput screening for the directed evolution of decarboxylase OleT.
Chemistry - A European Journal ( IF 4.3 ) Pub Date : 2020-09-21 , DOI: 10.1002/chem.202003637
Ulrich Markel 1 , Pia Lanvers 1 , Daniel F Sauer 1 , Malte Wittwer 1 , Gaurao V Dhoke 1 , Mehdi D Davari 1 , Johannes Schiffels 1 , Ulrich Schwaneberg 1, 2
Affiliation  

Enzymatic oxidative decarboxylation is an up‐and‐coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof‐of‐principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co‐substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed‐up the directed evolution of OleT and other decarboxylases.

中文翻译:

脱羧酶 OleT 定向进化的基于 photoclick 的高通量筛选。

酶促氧化脱羧是一种新兴的反应,但缺乏用于脱羧酶定向进化的有效筛选方法。在这里,我们描述了一种用于检测脱羧产物的简单 photoclick 测定及其在脱羧酶 OleT 的原理验证定向进化研究中的应用。该测定与两种常用的 OleT 操作模式(直接使用过氧化氢作为酶的共底物或使用还原酶伴侣)兼容,饱和诱变文库的筛选确定了两种酶变体,将酶的底物偏好从长链脂肪酸转向苯乙烯衍生物。总的来说,这种光点击分析有望加速 OleT 和其他脱羧酶的定向进化。
更新日期:2020-09-21
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