The escalating crisis of polyethylene terephthalate (PET) waste has prompted extensive research into microbial degradation and value-added recycling. However, the lack of hosts with high PET degradation efficiency suitable for value-added recycling poses a significant challenge to achieving a circular economy. In this study, we investigated the influence of terephthalic acid (TPA) metabolic regulation in Ideonella sakaiensis on PET degradation, aiming to engineer a host with enhanced PET degradation capabilities. Our results revealed that I. sakaiensis with deficient TPA metabolism exhibited a significantly reduced PET degradation capability. Gene transcription analysis further revealed a correlation between the transcription levels of TPA metabolism-related genes and those of PET hydrolysis-related genes. By engineering I. sakaiensis to accelerate TPA metabolism, we achieved a 46.9% increase in PET degradation, with the weight loss of PET rising from 58.0 mg to 85.2 mg (83.6% weight loss of PET). This represents the highest level of microbial PET degradation achieved at room temperature to date. This newly discovered metabolic regulation and enhanced PET degradation in I. sakaiensis provide a novel perspective for improving microbial PET degradation and further increase the potential for achieving a closed-loop process that converts waste PET into high-value chemicals.
Frank Peprah Addai,Jiacong Wu,Feng Lin,Xinnan Ma,Juan Han,Yuelin Liu,Yang Zhou,Yun Wang
In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0–8.0 and temperatures 40–60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme–support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.
Extensive use of polyethylene terephthalate (PET) has brought about global environmental problems. A recently reported PET hydrolase (PETase) discovered from Ideonella sakaiensis showed high potential for degrading PET at moderate temperatures, but its activity and stability need further improvement for practical applications. Herein, we proposed to use α-synuclein (αS) as a fusion chaperone and created six PETase-αS fusion enzymes with linkers of different types and lengths. All the fusion enzymes exhibited improved enzymatic performance, presenting 1.5 to 2.6-fold higher activity towards bis-2(hydroxyethyl) terephthalate than PETase, as well as significantly increased stabilities. Fluorescence spectroscopy indicated that the chaperone fusion tightened the overall conformation and resulted in the opening of the substrate binding pocket, which led to the improved thermal stability and catalytic activity of the fusion enzymes. Remarkably, one of the fusion proteins, PETase-[(GS)(EK)] 10 -αS, showed 3.2 to 5.1 times higher PET degradation capability than PETase. The significantly boosted PET degradation performance was not only attributed to the enhanced enzymatic activity and stability, but also possibly due to the binding affinity of the fused αS domain for PET. These findings demonstrated that αS was an effective fusion chaperone for significantly enhancing the enzymatic performance of PETase.
危险品化学品经营许可证（带存储）