Header

UZH-Logo

Maintenance Infos

Ultrahigh‐Throughput Screening of an Artificial Metalloenzyme using Double Emulsions


Vallapurackal, Jaicy; Stucki, Ariane; Liang, Alexandria Deliz; Klehr, Juliane; Dittrich, Petra S; Ward, Thomas R (2022). Ultrahigh‐Throughput Screening of an Artificial Metalloenzyme using Double Emulsions. Angewandte Chemie Internationale Edition, 61(48):e202207328.

Abstract

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.

Abstract

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.

Statistics

Citations

Dimensions.ai Metrics
2 citations in Web of Science®
3 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

17 downloads since deposited on 09 Jan 2023
6 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Scopus Subject Areas:Physical Sciences > Catalysis
Physical Sciences > General Chemistry
Uncontrolled Keywords:General Chemistry, Catalysis
Language:English
Date:25 November 2022
Deposited On:09 Jan 2023 08:14
Last Modified:29 Mar 2024 02:37
Publisher:Wiley-VCH Verlag
ISSN:1433-7851
OA Status:Green
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/anie.202207328
PubMed ID:36130864
Project Information:
  • : FunderSNF
  • : Grant ID200020_182046
  • : Project Title
  • : FunderEuropean Research Council
  • : Grant IDDrEAM—694424
  • : Project Title
  • : FunderEuropean Research Council
  • : Grant IDHybCell-681587
  • : Project Title
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)