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Ultra-high throughput screening for novel protease specificities.

Eirini Rousounelou1, Steven Schmitt1, Luzius Pestalozzi1

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|September 18, 2020
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Summary

This study presents a novel, highly parallelized screening method for enzyme variants. It uses density shifts caused by oxygen production from catalase to detect changes in protease activity in bacterial microcolonies.

Keywords:
BuoyancyDroplet-based microfluidicsEnzyme engineeringHigh-throughput screeningProtease specificityProteases

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Area of Science:

  • Biocatalysis and enzyme engineering
  • Microfluidics and high-throughput screening
  • Synthetic biology for enzyme evolution

Background:

  • Enzyme variants are crucial for developing biocatalysts for diverse applications.
  • Traditional screening methods for enzyme libraries are often limited by throughput and assay volume.
  • Arrayed screening methods have advanced but offer room for improvement in parallelization.

Purpose of the Study:

  • To introduce a novel, highly parallelized screening method for enzyme variants.
  • To link changes in protease substrate specificity to a detectable physical output.
  • To enable efficient evolution of biocatalysts with improved properties.

Main Methods:

  • Development of a density shift-based screening assay for enzyme variants.
  • In vivo linkage of protease substrate specificity to catalase reporter protein production.
  • Utilizing oxygen bubble generation by catalase to induce density shifts in microcolonies within polymeric droplets.
  • Employing droplet buoyancy for high-throughput screening of enzyme variants.

Main Results:

  • Demonstrated successful screening of protease variants based on density shifts.
  • Established a correlation between protease activity and catalase-induced oxygen production.
  • Showcased the potential for highly parallelized screening of enzyme libraries.
  • Validated the floating droplet assay for identifying enzyme variants with altered substrate specificity.

Conclusions:

  • The density shift assay offers a powerful, highly parallelized alternative to arrayed screening for enzyme evolution.
  • This method facilitates the discovery of enzyme variants with tailored substrate specificities.
  • The approach has broad applicability for directed evolution of enzymes in various fields.
  • This technique enhances the efficiency of biocatalyst development for industrial and medical applications.