Molecular Origin of the Enhanced PET Degradation Activity of LCC-ICCG Revealed by Computational Modeling

Rational design of IsPETase variant toward efficient poly(ethylene terephthalate) ambient degradation.

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.

Temperature-Dependent Reorganization of Conformational Dynamics and Interaction Networks Underlies Thermostability in PET-Degrading Enzymes

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics and a major contributor to global plastic pollution because of its high resistance to degradation. Enzymatic degradation by PET hydrolases (PETase) has emerged as a sustainable strategy for PET recycling; however, the limited thermostability of wild-type PETase restricts its industrial application. To elucidate the molecular basis underlying the different thermal behaviors of PET hydrolases, long-timescale molecular dynamics simulations were performed on WT-PETase, FAST-PETase, and the thermostable cutinase variant LCC-ICCG at 30 °C, 50 °C, and 70 °C. Comparative analyses integrating structural stability, residue flexibility, rigidity networks, free energy landscapes, and neural relational inference models revealed that FAST-PETase and LCC-ICCG exhibited enhanced conformational stability and reduced structural flexibility compared with WT-PETase, particularly under elevated temperatures. The improved thermostability was associated with more compact free energy landscapes, strengthened residue interaction networks, and better preservation of the catalytic architecture during thermal perturbation. These results suggest that an optimal balance between structural rigidity and conformational flexibility is critical for maintaining enzyme stability at elevated temperatures. Overall, this study provides molecular-level insights into the structural determinants of PETase thermostability and offers a theoretical framework for the rational engineering of efficient and heat-resistant plastic-degrading enzymes.

Structure-Guided Terminal Modifications Enhance the Efficiency of PET Degradation

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.

Mechanistic insights into modulation of productive substrate accessibility for efficient PET depolymerization

Polyethylene terephthalate (PET) hydrolases have been extensively studied for their potential applications in plastic degradation. However, the structural and mechanistic factors that limit their catalytic efficiency are not yet fully understood. Here, we identify the protruding, surface-exposed C-terminal loop (SEC-loop) in Cryptosporangium aurantiacum PETase (CaPETase) that negatively impacts enzymatic activity by restricting productive access of enzyme to PET. Loop replacement experiments show the non-protruding SEC-loop enhances PET depolymerization rates, despite being ~25 Å from the active site. Kinetic and adsorption studies indicate the non-protruding SEC-loop promotes productive PET access to the enzyme without affecting binding affinity. To further assess the broader applicability of this strategy across diverse PETases, SEC-loop replaced variants of representative PETases are characterized through kinetic and adsorption analyses. We show an engineering strategy focused on modulating enzyme accessibility rather than simply modifying the catalytic site, in rational enzyme design aimed at improving PET degradation efficiency. Polyethylene terephthalate (PET) hydrolases have been extensively studied for their applications in plastic degradation, but the structural and mechanistic factors that limit their catalytic efficiency are not yet fully understood. Here, the authors identify the protruding, surface-exposed C-terminal loop (SEC-loop) in Cryptosporangium aurantiacum PETase that negatively impacts enzymatic activity by restricting productive access of enzyme to PET substrates.

Decoupled Molecular Motifs and Supramolecular Dimers for Highly Efficient FDCA Synthesis from Neutral Biomass.

Solar-driven biomass valorization is pivotal for defossilizing the chemical industry. The oxidation of abundant, low-cost 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA)─a key monomer for next-generation bioplastics─is a long-sought goal yet hampered by sluggish kinetics, poor selectivity, and alkaline dependency. Here, spatially decoupled catalytic sites are engineered on two-dimensional carbon nitride (CN): covalently grafted cyanamide (CA) motifs at the edges and π-π stacked J-type nickel phthalocyanine (NiPc) dimers on the planes. This design features spatiotemporally cascaded charge transfer and dual-site catalysis, achieving 54- and 160-fold enhancements in the HMF conversion rate and H2 evolution rate, respectively, versus pristine CN, during HMF reforming in pure water. The FDCA production rate reaches 2.14 mmol g-1 h-1 with 98.2% selectivity, outperforming benchmark systems. Fundamentally, CA motifs steer an ultrafast hole-initiated selective HMF oxidation with a hole transfer rate of 2.4 × 1010 s-1 (an order of magnitude faster than CN). The resulting long-lived electrons are extracted by the bottom-layer NiPc and transferred via its single Ni atom to the top-layer single Ni atom for proton reduction, with an electron transfer rate of 7.4 × 104 s-1. The asymmetric charge kinetics suppresses charge recombination, yielding a charge transfer efficiency of 98.9%.

Bioprocessing and Biomass Technology