Computer-guided enzyme engineering of PET hydrolase mutants towards improved PET affinity

Area of Science:

• Biotechnology and Environmental Science
• Enzyme Engineering
• Protein Engineering

Background:

• Polyethylene Terephthalate (PET) pollution is a significant environmental concern.
• Enzymatic degradation using PETase offers a sustainable solution for PET recycling.
• Existing research often focuses on thermal stability, neglecting active-site engineering for catalytic efficiency.

Purpose of the Study:

• To explore active-site mutations for enhancing PETase catalytic efficiency.
• To utilize an in silico platform for high-throughput enzyme engineering.
• To identify specific mutations that improve PET degradation performance.

Main Methods:

• Employed the Gene Discovery and Enzyme Engineering (GDEE) platform for in silico protein engineering.
• Generated and screened thousands of PETase variants with a focus on active-site mutations.
• Evaluated variants using docking studies and ranked them based on binding affinity and catalytic geometry.
• Introduced the identified S238Y mutation into FAST-PETase (FP) and ThermoStable-PETase (TSP) scaffolds.

Main Results:

• Identified the S238Y mutation as a key enhancer of PET degradation.
• Achieved a 2.2-fold increase in degradation for FP and a 3.4-fold increase for TSP.
• FP S238Y showed a 14.8-fold increase in bulk activity, TSP S238Y a 25.8-fold increase.
• Observed enhanced catalytic efficiency and reduced enzyme inhibition with the S238Y mutation.

Conclusions:

• Targeted active-site engineering is a powerful strategy for developing efficient biocatalysts.
• The GDEE platform accelerates the discovery of improved enzymes for plastic waste remediation.
• The S238Y mutation represents a significant advancement in PETase-based plastic degradation.