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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
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Ultrahigh-Throughput Activity Engineering of Promiscuous Amidases through a Fluorescence-Activated Cell Sorting

Ina Somvilla1, Hannes Meinert1, Clemens Cziegler1

  • 1Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Greifswald 17487, Germany.

ACS Catalysis
|June 12, 2025
PubMed
Summary

We developed a fluorescence-activated cell sorting (FACS) assay for enzyme engineering. This method accelerates the directed evolution of amidases, enhancing their activity for biotechnological applications.

Keywords:
amidasebiocatalysisflow cytometryhigh-throughput screeningloop engineering

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Molecular Biology

Background:

  • Ultrahigh-throughput screening methods are crucial for enzyme directed evolution.
  • Flow cytometry enables the screening of millions of variants daily.
  • Amidase activity engineering requires robust detection methods.

Purpose of the Study:

  • To develop a fluorescence-activated cell sorting (FACS) assay for detecting and engineering amidase activity in whole cells.
  • To establish a stable genotype-phenotype linkage for assay readout.
  • To demonstrate the assay's utility by engineering an amidase from *Sphingomonas alpina* (SaAmd).

Main Methods:

  • Developed a FACS-based assay coupling coumarin hydrolysis to intracellular glutathione via coexpressed glutathione S-transferase.
  • Screened combinatorial libraries of *Sa*Amd with simultaneous randomization of multiple amino acid positions.
  • Utilized FACS to isolate and analyze enzyme variants with enhanced activity.

Main Results:

  • Engineered *Sa*Amd variants with proximal double mutations showing 5-fold improved activity against diverse amide substrates.
  • Observed coevolved promiscuous carbamate- and ester-hydrolyzing activities, up to 6-fold higher than wildtype.
  • Identified triple variants with distal mutations exhibiting up to 16-fold enhanced specific activity toward N-aryl amide and carbamate bonds.

Conclusions:

  • The developed FACS assay effectively enables the detection and engineering of amidase activity.
  • Engineered *Sa*Amd variants show significantly enhanced activity and broadened substrate specificity.
  • This assay holds great potential for accelerating enzyme engineering for applications in synthesis and environmental remediation.