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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Combining Signal Peptide Optimization with Directed Evolution to Enhance Chitosanase Activity.

Yao Chen1, Min Chen1, Xinli Lin1

  • 1Taizhou Key Laboratory of Biomass Functional Materials Development and Application, School of Life Science, Taizhou University, Taizhou 318000, China.

Journal of Agricultural and Food Chemistry
|December 17, 2025
PubMed
Summary
This summary is machine-generated.

Directed evolution enhanced chitosanase performance by engineering its signal peptide for better screening. This led to the M8 variant with 10-fold higher activity and improved substrate affinity.

Keywords:
chitooligosaccharidechitosanasedirected evolutionsignal peptide optimization

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Area of Science:

  • Enzymology
  • Protein Engineering
  • Biotechnology

Background:

  • Directed evolution is key for enzyme improvement but often limited by screening.
  • Chitosanase enzymes are crucial for degrading chitosan, with applications in various industries.
  • Efficient screening is vital for identifying high-performance enzyme variants.

Purpose of the Study:

  • To develop an efficient screening system for directed evolution of chitosanase.
  • To engineer the signal peptide of Bacillus subtilis chitosanase (Csn168) to improve screening.
  • To discover novel chitosanase variants with enhanced catalytic activity and substrate affinity.

Main Methods:

  • Site-saturation mutagenesis of the Csn168 signal peptide to attenuate secretion.
  • Iterative directed evolution coupled with hydrolytic zone screening.
  • Biochemical characterization and molecular dynamics simulations of enzyme variants.

Main Results:

  • Engineered signal peptide enabled high-resolution screening based on hydrolytic zone formation.
  • Identified the M8 variant with a 10-fold increase in catalytic activity and 28 mm hydrolytic zone in 33 h.
  • M8 variant exhibited enhanced substrate affinity (Km reduced from 25.56 to 11.75 g/L) and faster catalytic turnover.

Conclusions:

  • Coupling signal peptide engineering with directed evolution is an effective strategy for rapid discovery of high-performance enzymes.
  • The engineered M8 variant shows significant potential for industrial applications requiring efficient chitosan degradation.
  • This approach provides a robust platform for optimizing other enzyme families through directed evolution.