Optimization of polyethylene terephthalate biodegradation using a self-assembled multi-enzyme cascade strategy

• Published in: Journal of hazardous materials 2024-09, Vol.476, p.134887, Article 134887
• Main Authors: Aer, Lizhu, Jiang, Qifa, Zhong, Linling, Si, Qiuyue, Liu, Xianghong, Pan, Yan, Feng, Juan, Zeng, Hongjuan, Tang, Lixia
• Format: Article
• Language: English
• Subjects: IsPETase; Multi-enzyme cascade; PET degradation; Surface display; TfCa
• ISSN: 0304-3894; 1873-3336; 1873-3336
• DOI: 10.1016/j.jhazmat.2024.134887

Although many efforts have been devoted to the modification of polyethylene terephthalate (PET) hydrolases for improving the efficiency of PET degradation, the catalytic performance of these enzymes at near-ambient temperatures remains a challenge. Herein, a multi-enzyme cascade system (PT-EC) was developed and validated by assembling three well-developed PETases, PETaseEHA, Fast-PETase, and Z1-PETase, respectively, together with carboxylesterase TfCa, and hydrophobic binding module CBM3a using scaffold proteins. The resulting PT-ECEHA, PT-ECFPE, PT-ECZPE all demonstrated outstanding PET degradation efficacy. Notably, PT-ECEHA exhibited a 16.5-fold increase in product release compared to PETaseEHA, and PT-ECZPE yielded the highest amount of product. Subsequently, PT-ECs were displayed on the surface of Escherichia coli, respectively, and their degradation efficiency toward three PET types was investigated. The displayed PT-ECEHA exhibited a 20-fold increase in degradation efficiency with PET film compared to the surface-displayed PETaseEHA. Remarkably, an almost linear increase in product release was observed for the displayed PT-ECZPE over a one-week degradation period, reaching 11.56 ± 0.64 mM after 7 days. TfCaI69W/L281Y evolved using a docking-based virtual screening strategy showed a further 2.5-fold increase in the product release of PET degradation. Collectively, these advantages of PT-EC demonstrated the potential of a multi-enzyme cascade system for PET bio-cycling. [Display omitted] •Multi-enzyme complexes (PT-ECs) exhibit efficient PET degradation at 40 ℃.•The activity of PT-ECEHA in degrading PET is 16.5-fold more than that of PETaseEHA.•Surface-displayed PT-ECEHA has 20-fold higher activity than displayed PETaseEHA.•Surface-displayed PT-EC enhances enzyme tolerance against organic solvent.•TfCaI69W/L281Y enhances the surface-displayed PT-EC enzyme activity by 2.5-fold.