Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets.
Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets.
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DOI：
10.1021/acscatal.3c01609
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发表时间：
2023-08-04
期刊：
影响因子：
12.9
通讯作者：
Hollfelder, Florian
中科院分区：
文献类型：
--
作者：
Ladeveze, Simon;Zurek, Paul J.;Kaminski, Tomasz S.;Emond, Stephane;Hollfelder, Florian
关键词：
酶的发现和定向进化是当代利用生物催化技术改进工业过程的两种主要途径。用于改进单酶反应或从头反应开发的催化剂的定制通常是复杂和繁琐的。筛选活动的成功依赖于可以采样的序列空间的分数，无论是用于进化特定的酶还是筛选宏基因组。基于微流体系统中产生的皮升液滴在油包水乳液中的体外区室化的超高通量筛选（uHTS）允许筛选速率>1 kHz（或>107/天）。以这种形式筛选催化生物技术上有价值的反应的碳水化合物活性酶（CAZymes）提出了额外的挑战，因为释放的碳水化合物难以以高通量监测。具有大的光学活性疏水离去基团的活化底物提供了通用的光学读数，但是糖的分子识别性质将通过掺入这种氟或发色团及其通常更高的反应性而改变，因为与天然底物相比具有较低pKa值的离去基团使得更可能观察到混杂反应。为了克服这些问题，我们设计了微滴测定，其中光学惰性碳水化合物产物通过特定级联变得可见：未标记底物的初级反应导致下游的光学信号。在皮升液滴规模下成功实施此类测定使我们能够通过吸光度测量检测葡萄糖、木糖、葡萄糖醛酸和阿拉伯糖作为糖苷水解酶降解复杂寡糖的最终产物。使uHTS用于筛选迄今为止难以捉摸的CAZyme反应成为可能，将为更快、更容易地开发用于生物稳定化的特异性和有效的生物催化剂绘制路线，通过挑战与天然底物而不是模型底物反应的催化剂来指导酶的发现。
Enzyme discovery and directed evolution are the two major contemporary approaches for the improvement of industrial processes by biocatalysis in various fields. Customization of catalysts for improvement of single enzyme reactions or de novo reaction development is often complex and tedious. The success of screening campaigns relies on the fraction of sequence space that can be sampled, whether for evolving a particular enzyme or screening metagenomes. Ultrahigh-throughput screening (uHTS) based on in vitro compartmentalization in water-in-oil emulsion of picoliter droplets generated in microfluidic systems allows screening rates >1 kHz (or >107 per day). Screening for carbohydrate-active enzymes (CAZymes) catalyzing biotechnologically valuable reactions in this format presents an additional challenge because the released carbohydrates are difficult to monitor in high throughput. Activated substrates with large optically active hydrophobic leaving groups provide a generic optical readout, but the molecular recognition properties of sugars will be altered by the incorporation of such fluoro- or chromophores and their typically higher reactivity, as leaving groups with lowered pKa values compared to native substrates make the observation of promiscuous reactions more likely. To overcome these issues, we designed microdroplet assays in which optically inactive carbohydrate products are made visible by specific cascades: the primary reaction of an unlabeled substrate leads to an optical signal downstream. Successfully implementing such assays at the picoliter droplet scale allowed us to detect glucose, xylose, glucuronic acid, and arabinose as final products of complex oligosaccharide degradation by glycoside hydrolases by absorbance measurements. Enabling the use of uHTS for screening CAZyme reactions that have been thus far elusive will chart a route toward faster and easier development of specific and efficient biocatalysts for biovalorization, directing enzyme discovery by challenging catalysts for reaction with natural rather than model substrates.
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DOI：
10.1016/s1387-2656(05)11004-7
发表时间：
2005-01-01
期刊：
BIOTECHNOLOGY ANNUAL REVIEW, VOL 11
影响因子：
--
作者：
Berridge, MV;Herst, PM;Tan, AS
通讯作者：
Tan, AS
影响因子：
16.6
作者：
Colin PY;Kintses B;Gielen F;Miton CM;Fischer G;Mohamed MF;Hyvönen M;Morgavi DP;Janssen DB;Hollfelder F
通讯作者：
Hollfelder F
影响因子：
2.3
作者：
Cohn, Corey A.;Pedigo, Christopher E.;Schoonen, Martin A. A.
通讯作者：
Schoonen, Martin A. A.
影响因子：
6.1
作者：
Baret, Jean-Christophe;Miller, Oliver J.;Griffiths, Andrew D.
通讯作者：
Griffiths, Andrew D.
影响因子：
5.4
作者：
Arab-Jaziri, Faten;Bissaro, Bastien;O'Donohue, Michael J.
通讯作者：
O'Donohue, Michael J.