Engineering: β-sheet engineering enhances IsPETase for PET depolymerization

In a recent study published in Engineering, researchers have made significant strides in the enzymatic depolymerization of polyethylene terephthalate (PET) through β-sheet engineering of IsPETase. The team, led by Weidong Liu, Haifeng Liu and Leilei Zhu, systematically engineered the whole β-sheet regions in the core of IsPETase using a fluorescent high-throughput screening assay.

The researchers constructed 58 site saturation mutagenesis (SSM) libraries covering the β-sheet region of IsPETase. After screening approximately 12,000 clones and rescreening of 157 clones, they identified 21 beneficial amino acid substitutions. These substitutions were then iteratively recombined using sequential and K-means clustering algorithm-guided combination strategies. The best variant, DepoPETase β, exhibited a remarkable increase in melting temperature (T_m) by 22.9 °C compared to the wild type (WT). It also demonstrated superior depolymerization performance, completely depolymerizing 100.5 g of untreated post-consumer PET (pc-PET) in a liter-scale bioreactor at 50 °C within four days.

To understand the molecular mechanism behind DepoPETase β’s enhanced properties, the team solved its crystal structure and conducted molecular dynamics simulations. They found that the improved activity and thermostability were due to enhanced hydrogen bonds and salt bridges in the β-sheet region, a more tightly packed structure of the core sheets and surrounding helix, and improved binding of PET to the active sites. Notably, substitutions like D283R, F229Y, R280E, and N233K contributed to strengthening the hydrogen bonds and salt bridges in the outer β-sheets, while D186H and the conformational changes caused by F201I and V84L stabilized the inner β-sheets.

This research not only highlights the importance of engineering the β-sheet region in PET hydrolases but also presents DepoPETase β as a promising candidate for large-scale PET recycling. The findings could potentially revolutionize the way we approach PET waste management, offering a more sustainable solution to the environmental challenges posed by plastic waste. With further development, enzymatic depolymerization may become a key player in the circular economy of plastics.

The paper “β-sheet Engineering of IsPETase for PET Depolymerization,” authored by Songfeng Gao, Lixia Shi, Hongli Wei, Pi Liu, Wei Zhao, Lanyu Gong, Zijian Tan, Huanhuan Zhai, Weidong Liu, Haifeng Liu, Leilei Zhu. Full text of the open access paper: https://doi.org/10.1016/j.eng.2024.10.015. For more information about the Engineering, follow us on X (https://twitter.com/EngineeringJrnl) & like us on Facebook (https://www.facebook.com/EngineeringJrnl).