Engineering and Application of a Thermostable MHETase for PET Depolymerization

Area of Science:

• Biotechnology and Industrial Microbiology
• Enzyme Engineering and Biocatalysis
• Polymer Science and Recycling

Background:

• Enzymatic hydrolysis of poly-(ethylene terephthalate) (PET) produces mono-(2-hydroxyethyl) terephthalate (MHET).
• MHET accumulation complicates PET recycling processes.
• Existing PETases are often thermophilic, while the known MHETase from *Ideonella sakaiensis* is mesophilic, necessitating the development of thermostable MHETases.

Purpose of the Study:

• To characterize novel thermostable MHET-active enzymes for improved enzymatic PET recycling.
• To engineer enhanced MHETase activity and thermostability through structure-guided and evolution-informed design.

Main Methods:

• Screening for thermostable MHET-active enzymes using a hidden Markov model based on archaeal ferulic acid esterase PET46.
• Characterization of enzyme activity at 70 °C and determination of melting temperatures (Tm,app).
• Structure-based site saturation mutagenesis and evolution-informed design (EID) to improve enzyme performance.
• Bioreactor experiments using postconsumer PET waste to assess MHET reduction.

Main Results:

• Identified novel thermostable MHET-active enzymes with higher stability than PET46.
• MHT077 showed high MHETase activity and kinetic stability, with a Tm,app of 94.6 °C.
• Evolution-informed design yielded EV-MHT043-5 with comparable thermostability (Tm,app = 96.1 °C) and a 3-fold increase in MHETase activity.
• A combination variant, MHT077LFK, significantly reduced MHET accumulation in bioreactor experiments.

Conclusions:

• This study expands the repertoire of protein scaffolds for MHET hydrolysis in enzymatic PET recycling.
• The developed thermostable MHETases offer promising solutions for more efficient and industrially viable PET degradation.
• Engineered variants demonstrate enhanced activity and stability, crucial for practical application in plastic waste management.