Jul 2026· International Journal of Molecular Sciences· Vol 27, pp. 6531· 0 citations· 44 references
Medicine
Abstract
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.
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.
Introduction Elucidating how cold-active enzymes maintain efficient catalysis under low-temperature conditions remains a significant question in enzymology. Although enhanced conformational flexibility has been frequently associated with cold adaptation, flexibility alone cannot fully explain how catalytic precision and reproducibility are maintained. This suggests that an additional layer of structural organization is required. Methods In this study, a psychrophilic β-glucosidase (pBGL) from Pseudoalteromonas sp. BSw20308 and its mutant variant pBGL-S306P were examined through biochemical assays, molecular dynamics simulations, and dynamic interaction network analysis. Results The results indicate that pBGL preserves high catalytic efficiency at low temperature despite pronounced thermal instability, maintaining a rigid and conserved catalytic core. Notably, increased flexibility is spatially redistributed toward peripheral and interfacial regions rather than being globally amplified. Network analysis reveals that cold adaptation is associated with a distributed interaction network that connects these flexible regions to the catalytic core, constraining motions into coordinated dynamics that support catalysis. Discussion These findings suggest that cold adaptation in pBGL may involve not only enhanced flexibility but also network-mediated organization of conformational dynamics, providing a structural framework for understanding the activity–stability balance of cold-active enzymes.
Xian He, Mengting Liu, Xintong Li et al.· Frontiers in Microbiology· 1 citation
The industrial application of enzyme catalysts is often constrained by the trade-off between thermostability and catalytic activity. Here, a region-focused engineering strategy was applied to a thermophilic GH10 xylanase to simultaneously improve both properties. The strategy integrates qProtein-guided hydrophobic cluster design for scaffold stabilization and dynamic loop analysis for active-site optimization. The resulting triple mutant A206S-N209D-F130L exhibited substantially improved thermostability, with a 5.79 °C increase in melting temperature and an 18.8-fold extension of the half-life at 60 °C. Its optimum temperature increased from 60 to 70 °C, accompanied by a 129.4% enhancement in catalytic activity at 70 °C relatively to the wild type. Molecular dynamics simulations indicated that these mutations reshape the conformational energy landscape by stabilizing hydrophobic packing and modulating loop dynamics. This study provides a generalizable framework for simultaneously improving enzyme stability and catalytic performance.
Zhaoran Li, Zhixin Dou, Sha Zhao et al.· Journal of Agricultural and...· 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
Xylanases with high catalytic efficiency and environmental robustness are important for lignocellulosic biomass valorization, but many enzymes are rapidly inactivated under the alkaline and high-temperature conditions used in industrial processes. In this study, a computationally guided rational-design strategy was developed to improve the catalytic performance and stability of the alkaline xylanase BhS7Xyl. Constant-pH molecular dynamics, isothermal compressibility perturbation analysis, and ECNet-assisted fitness prediction were integrated to identify alkaline-sensitive and structurally unstable residues for engineering. The triple mutant H51R/D150N/E287K showed the best overall performance, with a specific activity of 1045.29 U/mg, representing a 3.73-fold increase compared with the wild type. Its melting temperature increased from 55.82 °C to 64.58 °C, while its half-life at pH 10.0 increased from 33.96 to 95.84 min. The thermal half-life at 75 °C was extended from 10.97 to 215.42 min, corresponding to a 19.64-fold improvement. Structural analyses suggested that the improved performance of H51R/D150N/E287K was associated with a more continuous xylohexaose-binding interface, increased hydrogen-bonding contacts, additional electrostatic/polar interactions, strengthened local interaction networks and enhanced dissipation of local thermal perturbation. Under optimized hydrolysis conditions, the triple mutant produced higher levels of xylose and xylooligosaccharides from standard xylan, corn cob xylan, and hardwood pulp xylan than the wild type. These work demonstrates that multi-shell electrostatic remodeling is a useful strategy for improving the activity, alkaline tolerance, and thermal stability of xylanase for xylooligosaccharide production.
Chun-Lin Tan, Xin Yu, Lanxi Sun et al.· International Journal of Bio...· 0 citations
Xylanases catalyze the hydrolysis of β-1,4-xylosidic linkages in xylan, a major component of plant cell walls, and are widely used in the food, feed, pulp and paper, and biofuel industries. GH11 xylanase from the hemicellulose-degrading bacterium Thermoanaerobacterium saccharolyticum (TsaGH11) exhibits high catalytic activity, making it an attractive enzyme for industrial applications. The flexibility of the thumb domain of TsaGH11 has been investigated under cryogenic and room temperature conditions; however, the substrate recognition mechanism of TsaGH11 at the optimal temperature is unknown. To better understand the molecular mechanism of substrate recognition, the high-resolution crystal structure of TsaGH11 was determined at 1.4 Å resolution. All-atom molecular dynamics simulations at 300, 320, 340, and 360 K revealed that increasing the temperature induced fluctuations in the substrate-recognizing thumb domain. At an optimal temperature of 340 K, the substrate-binding cleft of TsaGH11 predominantly adopted a closed conformation. However, the thumb domain exhibited larger fluctuations at 340 K than at other temperatures, sampling both open and closed conformations, suggesting that substrate recognition in TsaGH11 proceeds through a conformational selection-like mechanism. At 360 K, TsaGH11 unfolded partially at a site opposite the substrate-binding cleft, providing potential targets for protein engineering to improve its thermostability for industrial applications. These findings provide a better understanding of the molecular mechanism of TsaGH11 and offer valuable guidance for the rational engineering of GH11 xylanases for industrial applications.
Ki Hyun Nam· International Journal of Mol...· 0 citations