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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
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A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels.

Nina Lülsdorf1, Christian Pitzler, Michael Biggel

  • 1DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056 Aachen, Germany.

Chemical Communications (Cambridge, England)
|April 25, 2015
PubMed
Summary

A new high-throughput screening method using flow cytometry was developed to find better enzymes. This toolbox successfully identified improved variants for esterase, lipase, and cellulase with 1.3-7-fold increases in activity.

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Cell Biology

Background:

  • Enzyme discovery and engineering are crucial for various industrial applications.
  • High-throughput screening methods are needed to accelerate the identification of improved enzyme variants.
  • Current methods may lack the efficiency or scope for comprehensive enzyme characterization.

Purpose of the Study:

  • To develop and validate a high-throughput whole-cell flow cytometry screening toolbox.
  • To identify and characterize improved variants of hydrolases, including esterase, lipase, and cellulase.
  • To establish a novel screening principle based on coupled enzymatic reactions and fluorescent hydrogel formation.

Main Methods:

  • Development of a whole-cell flow cytometry screening platform.
  • Utilizing coupled enzymatic reactions with glucose derivatives for signal generation.
  • Employing a fluorescent hydrogel layer formation on the surface of E. coli cells as a readout.
  • Screening of enzyme variant libraries for esterase, lipase, and cellulase activities.

Main Results:

  • Successful validation of the developed flow cytometry screening toolbox.
  • Identification of enzyme variants with significantly improved hydrolase activity (1.3-7-fold increase).
  • Demonstration of the screening principle's efficacy in identifying superior enzyme variants.

Conclusions:

  • The developed high-throughput screening toolbox is effective for enzyme engineering.
  • This method enables the rapid discovery of enhanced hydrolase variants.
  • The novel screening principle offers a versatile approach for whole-cell enzyme screening.