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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase
Lara Pfaff1, Jian Gao2,3, Zhishuai Li2,4
1Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany.
ACS Catalysis
|August 15, 2022
Summary
Thermophilic polyester hydrolases (PES-H) enable plastic recycling. Engineered PES-H1 variants show enhanced polyethylene terephthalate (PET) hydrolysis, improving biocatalytic recycling efficiency for industrial applications.
Area of Science:
- Biocatalysis
- Protein Engineering
- Polymer Science
Background:
- Thermophilic polyester hydrolases (PES-H) are crucial for biocatalytic recycling of polyethylene terephthalate (PET).
- Increasing demand for efficient PET hydrolases necessitates further enzyme development.
- PET is a widely used synthetic polyester in packaging and textiles.
Purpose of the Study:
- To elucidate the structural basis of PET hydrolysis by PES-H enzymes.
- To engineer enhanced PES-H variants for improved PET degradation.
- To understand the impact of substrate binding and product inhibition on enzyme mechanism.
Main Methods:
- High-resolution crystal structure determination of PES-H1 and PES-H2.
- Co-crystallization with PET substrate analogues.
- Molecular dynamics simulations for mechanistic insights.
- Site-directed mutagenesis of key residues.
- Enzymatic activity assays on PET films and waste.
Main Results:
- Structural analysis revealed substrate binding modes and product inhibition mechanisms.
- Mutagenesis of PES-H1 yielded the L92F/Q94Y variant with 2.3-3.4 fold improved hydrolytic activity.
- The R204C/S250C variant showed increased thermostability (6.4 °C higher melting temperature).
- The L92F/Q94Y variant demonstrated 2.2-fold higher efficiency on low-crystallinity PET compared to LCC ICCG.
Conclusions:
- Structural insights guide the engineering of highly active PET hydrolases.
- Engineered PES-H1 variants show significant potential for industrial PET recycling.
- Enhanced enzyme activity and stability are key for effective biocatalytic plastic degradation.
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