Harnessing Protein Language Model for Structure-Based Discovery of Highly Efficient and Robust PET Hydrolases

Plastic waste, particularly polyethylene terephthalate (PET), presents significant environmental challenges, prompting extensive research into enzymatic biodegradation. Existing PET hydrolases are limited to a narrow sequence space and demonstrate insufficient performance for biodegradation. This study introduces a novel discovery pipeline that combines protein language models (PLMs) with a structural representation tree to identify enzymes based on structural similarity. Using the crystal structure of IsPETase as a template, we employed PLMs and a representation tree to efficiently search and cluster target proteins. Screening of candidate proteins was further refined using PLM-based assessments of solubility and thermal stability. Biochemical experiments showed that 14 of 34 candidates exhibited PET degradation activity across a temperature range of 30-60 {degrees}C. Notably, we identified a PET hydrolase -KbPETase, which possesses a melting temperature 32 {degrees}C higher than that of IsPETase and exhibits the highest PET degradation activity within 30-50 {degrees}C compared to other wild-type PETases. KbPETase also shows higher catalytic efficiency than FastPETase. X-ray crystallography and molecular dynamics simulations further revealed that KbPETase has a conserved catalytic domain and enhanced intramolecular interactions. This work develops a novel deep learning approach to discover natural PETases with enhanced functions.