The accumulation of poly(ethylene terephthalate) (PET) waste in the environment poses a severe ecological threat. While extensive research has focused on high-performance PET degradation by thermophilic enzymes, PET hydrolases are efficient under lower-temperature conditions, which would better align with green and energy-saving demands the energy-efficient centralized treatment of PET waste remains underexplored. Herein, based on our previously engineered mesophilic IsPETaseS121P/D186A, we performed rational design to improve its PET degradation activity at relatively low temperature. Through rational design methods including salt bridge construction and hydrophobic engineering, we obtained effective variant PADFL (IsPETaseS121P/D186A/N246D/Y87F/N233L), demonstrating an 8.37-fold activity of IsPETaseS121P/D186A in PET degradation efficiency (56.52-fold of IsPETase). Molecular dynamics (MD) simulations further revealed stronger PET binding affinity, enhanced hydrogen bonding network, and reduced acylation energy barrier. Overall, this work enhances the degradation activity of the PET hydrolase through energy-based rational design and obtained optimized variant PADFL, offering a promising candidate for future efficient PET degradation under mild temperature conditions.
Poly(ethylene terephthalate) (PET) hydrolases have emerged as promising biocatalysts for closed-loop plastic recycling. Among the most efficient enzymes reported to date, LCC-ICCG exhibits exceptional PET-depolymerization performance under industrially relevant conditions. However, the molecular basis for its superior activity relative to engineered PETases such as FAST-PETase and HotPETase remains incompletely understood. Here, we combine microsecond-scale molecular dynamics simulations, quantum mechanical cluster calculations, pre-reaction-state analysis, noncovalent-interaction mapping, and distortion/interaction activation strain analysis to compare LCC-ICCG with FAST-PETase and HotPETase. The simulations show that LCC-ICCG samples catalytically competent pre-reaction-state geometries more frequently, mainly because V212 reshapes the local environment around the scissile ester. This residue relieves steric congestion, supports weak C–H···O guided substrate preorganization, and reinforces both the Asp-His catalytic dyad and the W190-associated pocket architecture. Density functional theory calculations further indicate that this preorganized active site lowers the acylation barrier to 15.5 kcal/mol by reducing substrate distortion and strengthening transition-state interactions. High-temperature simulations show that LCC-ICCG better preserves near-attack geometries at 350 K, linking thermal robustness to sustained catalytic preorganization. Moreover, reciprocal I208V mutations in IsPETase-derived enzymes enrich pre-reaction-state populations, supporting the transferability of the V212-centered design principle. Overall, these results establish pre-reaction-state stabilization as a key determinant of PET-hydrolase efficiency and provide mechanistic design rules for engineering next-generation PET depolymerases.
The extensive use of polyethylene terephthalate (PET) has resulted in severe environmental pollution and ecological stress. Despite advances in PET recycling, current processes struggle to achieve high product value, as the complete conversion to terephthalic acid remains energetically demanding and economically inefficient. The catalytic promiscuity inherent in natural enzyme evolution holds great promise for providing novel candidates to accelerate PET biodegradation and upcycling. Herein, we report for the first time that the acyltransferase metA from Mycobacterium tuberculosis (MtMetA) catalyzes the conversion of bis(2-hydroxyethyl) terephthalate (BHET), an intermediate of PET hydrolysis, into the high-value monomer mono(2-hydroxyethyl) terephthalate (MHET). Guided by molecular dynamics (MD) simulations, we applied a catalytic barrier-minimization strategy to optimize the active-site environment of MtMetA, yielding engineered variants, notably ΔBarrier2 and ΔBarrier3. Specifically, ΔBarrier2 achieved a 3.1-fold increase in MHET yield, while ΔBarrier3 demonstrated a 3.2-fold enhancement in catalytic efficiency (kcat/KM) relative to the wild-type. The truncated MtMetA variants also exhibited enhanced robustness, showing improved thermostability (3.5-fold increase in residual activity at 60 °C for ΔBarrier3), as well as higher tolerance to metal ions and organic solvents. Specifically, ΔBarrier2 displayed a 7.2-fold increase in product yield in Ca2+-containing systems, while ΔBarrier3 retained 2.3-fold higher residual activity in the presence of 50 % (v/v) isopropanol. MD simulations revealed that an enlarged active pocket and a shortened nucleophilic attack distance synergistically govern the enhanced catalytic activity and robustness. This work expands the enzymatic toolbox for PET recycling and targeted BHET degradation, advancing sustainable plastic waste management through biocatalytic innovations.
Jie Qiao, Yibo Song, Nan Zhao et al.· Bioresource Technology· 0 citations
Poly(ethylene terephthalate) (PET) is a widely used plastic whose persistence and improper disposal pose serious environmental and health risks. In this study, three novel PET hydrolases TbPETase, AbPETase, and AfPETase were identified from Thermoanaerobacterales, Acidimicrobiales, and Actinokineospora fastidiosa, respectively. Among these, TbPETase exhibited the highest enzymatic activity and thermostability. Based on structural analysis, we performed semirational truncations targeting the intrinsically disordered N- and C-terminal regions of TbPETase, generating two improved variants ΔN36 and ΔC4. The double mutant, TbPETaseΔN36/ΔC4, demonstrated a 2.3-fold increase in overall enzymatic activity and a 2.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme, along with significantly enhanced thermal stability. Molecular dynamics simulations revealed that the removal of flexible terminal regions increased the overall structural rigidity of TbPETaseΔN36/ΔC4. This structural stabilization was associated with the formation of a hydrogen bond at T215 and a π–π stacking interaction at W193. In a 100 mL one-pot reaction system, the combination of TbPETaseΔN36/ΔC4 with an engineered BMHETase variant, BMHETase6M, achieved 81.2% degradation of semicrystalline PET powder at 60 °C over 60 h, yielding terephthalic acid as the major product. These findings demonstrate the potential of TbPETaseΔN36/ΔC4 as a highly efficient and industrially applicable biocatalyst for PET degradation.
Lin Zhang, Keyan Chen, Zhiwen Xi et al.· ACS Synthetic Biology· 0 citations
Postconsumer plastic waste has the potential to serve as a valuable feedstock in an integrated circular economy in which it can be interconverted between thermoplastic and thermoset materials. However, the development of cost-effective and sustainable recycling or upcycling methods is often constrained by harsh reaction conditions, limited catalyst performance, and overall low process efficiency. Here, we report the low-temperature (120°C) alcoholysis of polyethylene terephthalate (PET) that allows the application of functional, bioderivable alcohols in the depolymerization process. Terpenoids such as prenol proved highly effective, supporting both efficient depolymerization and repolymerization to PET. The unsaturated terpenolysis products were also used to prepare polymer networks via thiol-ene photochemistry and formulated into photocurable resins suitable for three-dimensional printing using digital light processing technology. The terpenoid-based networks were also able to be efficiently depolymerized, demonstrating the potential for loop-to-loop chemical recycling of PET/terpenoid materials. This work establishes low-temperature alcoholysis of PET as a practical strategy for advancing a broader circular PET-based materials economy.
M. Price, Adam H Redfearn, Steven T. G. Street et al.· Science Advances· 0 citations
Polybutylene adipate terephthalate (PBAT) is prone to incomplete degradation, leading to environmental pollution and carbon resource waste. Biodegradation and valorization of waste plastics are essential for addressing plastic pollution and promoting a circular economy. Enzymatic degradation offers advantages, but free enzymes suffer from low stability and poor recyclability. Here, surface display technology was used to construct a cutinase Tfcut-DM display system in Escherichia coli BL21(DE3). The results showed that surface display significantly enhanced the stability and reusability of Tfcut-DM. Compared with free enzymes, its thermostability and pH stability increased by 11.9-fold and 42.1-fold, respectively, and approximately 80% of initial activity was retained after seven reuse cycles. Under optimized conditions, the surface-displayed strain released 255.9 µM of terephthalic acid (TPA) from PBAT films over 5 days, with near-complete degradation. To enable TPA valorization , the tph operon was introduced to C. necator H16 (CnH16-tph) for TPA-to-PHB conversion. A co-culture system comprising the surface-displayed strain and CnH16-tph was established for proof-of-concept one-pot conversion of PBAT to PHB. PHB accumulation is indirectly attributed to the TPA derived from PBAT degradation. Under optimized conditions, the maximum PHB yield reached 0.91 g/L. While isotopic tracing would be required for definitive carbon flux assignment, this indirect evidence strongly suggests the successful conversion of PBAT-derived TPA to PHB. This study provides a novel approach for the green degradation and resource utilization of waste PBAT, facilitating the recycling of discarded resources.
Jiali Liu, Jie Yang, Xin Wang et al.· Bioresource Technology· 0 citations
Polyethylene terephthalate (PET) degradation is often limited by insufficient polymer depolymerization and the downstream conversion of hydrolysis products. In this study, Burkholderia cepacia ZY1 and Pseudomonas harudinis G1B were compared, and a two-strain consortium, YB2, was constructed at an optimal inoculation ratio of 4:5. ZY1 showed relatively stronger PET depolymerization-related activity, whereas G1B exhibited greater growth in bis(2-hydroxyethyl) terephthalate (BHET)- and mono(2-hydroxyethyl) terephthalate (MHET)-containing media and a trend toward faster BHET conversion. The hydrolytic activities of both strains were mainly cell associated, and both remained culturable, with stable relative proportions during 0-7 d of cocultivation. At pH 7.0 and 30 °C, YB2 achieved 4.30% ± 0.11% PET film mass loss after 3 d, significantly exceeding the individual strains. YB2 treatment caused surface erosion, ester-related structural changes, and increased residual PET crystallinity. The detection of BHET, MHET, and terephthalic acid (TPA) further supported PET depolymerization, while the release of ethylene glycol (EG) was also observed. YB2 converted 96.4% of BHET and 97.3% of MHET within 2 d and almost completely utilized TPA within 5 d. EG initially accumulated and then declined, suggesting possible subsequent microbial utilization. Overall, ZY1 and G1B exhibited overlapping functions with relative functional differences and potential complementarity, supporting the use of synthetic microbial consortia to enhance PET depolymerization and downstream product conversion.
Jiarong Qiu, Yufeng Jin, Liangqing Zhang et al.· Journal of Environmental Man...· 0 citations