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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
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Sortase-Mediated High-Throughput Screening Platform for Directed Enzyme Evolution.

Zhi Zou1,2, Diana M Mate1, Kristin Rübsam1

  • 1DWI-Leibniz-Institute for Interactive Materials , Forckenbeckstraße 50 , 52074 Aachen , Germany.

ACS Combinatorial Science
|January 25, 2018
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Summary
This summary is machine-generated.

A new SortEvolve platform enhances enzyme evolution using sortase-catalyzed ligations. This high-throughput screening method improves enzyme activity and minimizes background noise for directed evolution campaigns.

Keywords:
CueOLCISrtAanchor peptidedirected evolutionlaccase

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Area of Science:

  • Biocatalysis and biotechnology
  • Protein engineering
  • Enzyme evolution

Background:

  • Sortase-catalyzed ligations are vital for site-specific protein and peptide conjugation.
  • Directed evolution requires efficient high-throughput screening (HTS) platforms.
  • Existing HTS methods can suffer from high background noise, limiting their effectiveness.

Purpose of the Study:

  • To develop a general HTS platform, SortEvolve, for directed enzyme evolution.
  • To improve the activity of sortase A and a laccase (CueO).
  • To validate SortEvolve's efficacy in minimizing background activity.

Main Methods:

  • SortEvolve platform utilizing sortase-catalyzed ligations and a semipurification process.
  • Site-saturation mutagenesis (SSM) libraries of sortase A (Sa-SrtA) and CueO laccase.
  • Screening and rescreening of enzyme variants in 96-well microtiter plates (MTP).

Main Results:

  • Identification of improved Sa-SrtA and CueO variants.
  • A Sa-SrtA variant showed a 22-fold increase in catalytic efficiency.
  • A CueO variant exhibited a 103-fold improvement in catalytic efficiency.
  • SortEvolve reduced background noise by 20-fold for CueO evolution.

Conclusions:

  • SortEvolve is a versatile HTS platform for directed enzyme evolution.
  • The semipurification process effectively minimizes background activity.
  • SortEvolve enables successful directed evolution campaigns for enzymes like sortases and laccases.