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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Enhancing Chitosanase Activity via Directed Evolution and Its Molecular Mechanisms
Yao Chen1, Xiangyu Zi1,2, Min Chen1
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
|September 27, 2025
Summary
Directed evolution significantly enhanced chitosanase activity, yielding a variant M1 with 812-fold higher performance. This engineered enzyme efficiently produces chitooligosaccharides (COS) from fungal chitosan, offering a valuable industrial tool.
Area of Science:
- Enzymology and protein engineering
- Biotechnology and industrial microbiology
Background:
- Chitosanases are crucial enzymes for degrading chitosan into valuable chitooligosaccharides (COS).
- Optimizing chitosanase performance is essential for efficient and scalable COS production.
Purpose of the Study:
- To enhance the performance of a rationally designed chitosanase (mSsCsn46) through directed evolution.
- To develop a highly efficient enzymatic tool for industrial chitooligosaccharide production.
Main Methods:
- Directed evolution of chitosanase SsCsn46 fused with an OmpA signal peptide.
- Enzyme activity assays and characterization of the evolved variant (M1).
- Molecular dynamics simulations to elucidate structural changes and substrate-binding interactions.
Main Results:
- The evolved variant M1 exhibited an 812-fold increase in enzyme activity (1680 U/mg) compared to the parent enzyme.
- M1 produced 142 g/L of COS with 89.9% conversion within 3 hours via continuous substrate feeding.
- M1 rapidly converted fungal chitosan to COS with 95.1% conversion in just 30 minutes.
Conclusions:
- Directed evolution effectively remodeled chitosanase for significantly improved COS production efficiency.
- The engineered chitosanase M1 presents a potent enzymatic solution for industrial-scale COS manufacturing.
- Structural insights from molecular dynamics simulations explain the enhanced enzyme performance.
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