Mining Thermophile Genomes for New PETases with Exceptional Thermostabilities Using Sequence Similarity Networks

Area of Science:

• Biotechnology
• Enzymology
• Polymer Science

Background:

• Enzymatic hydrolysis of polyethylene terephthalate (PET) is crucial for a circular PET economy.
• Existing PET-degrading enzymes have limitations, and the full potential of the α/β hydrolase family remains unexplored.

Purpose of the Study:

• To identify novel PET-hydrolyzing enzymes (PETases) within the α/β hydrolase fold-5 subfamily.
• To prioritize enzymes from thermophiles for enhanced thermostability suitable for industrial applications.

Main Methods:

• Sequence similarity networks were used to identify potential PETases.
• Ten candidate enzymes with sequence similarity to LCC-PETase were selected.
• Seven enzymes were overexpressed, purified, and characterized in vitro for PET hydrolysis activity.

Main Results:

• Seven novel enzymes were successfully characterized, with three demonstrating the ability to hydrolyze PET films.
• The identified PETases exhibit high melting temperatures (Tm > 55°C) and retain activity after heat treatment.
• The crystal structure of one enzyme, AroC, revealed salt bridges contributing to thermostability and a conserved unique loop.

Conclusions:

• Novel thermostable PETases have been identified, expanding the toolkit for PET biodegradation.
• These enzymes show potential for engineering into efficient industrial biocatalysts for PET recycling.
• The findings pave the way for developing advanced enzymatic solutions for a circular PET economy.