Engineered Leaf-Branch Compost Cutinase with Enhanced PET Degradation Across a Broad Temperature Range

Area of Science:

• Biotechnology
• Polymer Science
• Enzymology

Background:

• Enzymatic recycling offers advantages over traditional poly(ethylene terephthalate) (PET) recycling methods.
• Current PET-degrading enzymes exhibit limited efficiency under industrial conditions.

Purpose of the Study:

• To engineer and identify novel leaf and branch compost cutinase (LCC) variants with enhanced poly(ethylene terephthalate) (PET) degrading capabilities.
• To evaluate the performance of engineered LCC variants under industrially relevant conditions.

Main Methods:

• Engineering of LCC variants through targeted amino acid substitutions.
• Assessing PET microparticle hydrolysis rates and conversion efficiencies at various temperatures (55°C, 62°C, 70°C).
• Utilizing pH stat for continuous monitoring of PET degradation and comparing with initial rate measurements.

Main Results:

• A quadruple LCC variant (H218S-F243T-D238C-S283C) achieved over 90% hydrolysis of 9% crystalline PET microparticles within 17 hours at 70°C.
• The variant demonstrated high catalytic efficiency, producing 15.6 g TPA L⁻¹ h⁻¹ at 70°C, with a melting temperature of 82.1°C.
• Initial rate measurements were found to be poor predictors of overall PET weight loss, highlighting limitations in current enzyme screening methodologies.

Conclusions:

• The engineered LCC variant represents a significant advancement in enzymatic PET degradation, showing high efficiency at elevated temperatures.
• Improved enzyme screening methodologies focusing on complete conversion are crucial for identifying highly effective PETase variants for industrial applications.
• This research paves the way for more sustainable and efficient enzymatic recycling of PET plastics.