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.
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 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.
Fe(II)/α-ketoglutarate (αKG)-dependent halogenases that catalyze site-selective C-H halogenation of free substrates without carrier proteins are attractive biocatalysts for diversifying pharmaceuticals and agrochemicals. However, their application remains limited by the narrow diversity of natural halogenases, poor stability, and restricted substrate scope. Protein stabilization is a common strategy to enhance mutational tolerance during enzyme engineering; however, Fe(II)/αKG-dependent halogenases are structurally closely related to hydroxylases, and consensus-based stabilizing mutations risk shifting activity toward competing hydroxylation. To address this challenge, a workflow was designed to improve structural stability while preserving substrate and product specificity by combining computational identification of substrate-recognition residues with Rosetta-based stabilization. This approach was applied to the l-lysine 4-chlorinase BesD from Streptomyces cattleya as a model enzyme. The resulting variants exhibited a T50 increase of more than 45 °C with no loss of substrate specificity or regioselective chlorination activity and served as stable seed enzymes for subsequent substrate scope expansion. This strategy, which systematically excludes substrate recognition- and/or reaction-selectively related residues from the mutation space to preserve native enzyme function, may provide a versatile platform for stabilizing enzymes without substantially compromising catalytic activity.
Teppei Niide, Keita Miyawaki, Hyuga Miyamoto et al.· ACS Chemical Biology· 0 citations
Thermostability is critical for the industrial applications of xylanase, including paper production, animal feed, and lignocellulosic biomass conversion. Here, we report that the rational introduction of surface-exposed glutamic acid (Glu) residues significantly enhances the thermostability of GH10 xylanases. Engineered variants of XT6, BhS7Xyl, and FXYN exhibited prolonged half-lives that were elevated by 2.5-, 1.5-, and 3-fold relative to their respective wild-type enzymes. The stabilization arises from strengthened conformational rigidity due to the formation of numerous new salt bridges. This strategy was further validated in two novel xylanases of Xyn466 and Xyn486 from Cellulomonas bogoriensis 69B4T. In contrast to disulfide bond engineering and ΔΔG-based engineering, surface Glu modification provides superior stabilization (Xyn466-9QE of 5.9-fold and Xyn486-11QE of 9.7-fold increased half-life at 60 °C) with lower mutational load. Our results provide a more efficient strategy with a higher success rate and lower activity trade-off for improving the thermostability of GH10 xylanases.
Jing Tian, Xueting Qu, Wen Huang et al.· Journal of Agricultural and...· 0 citations
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
d-Allulose 3-epimerase (DAEase) catalyzes d-fructose conversion to d-allulose, but the poor thermostability of Clostridium cellulolyticum H10 DAEase limits its industrial application. Here, we enhanced DAEase thermostability by targeting the subunit interface using PROSS-guided combinatorial engineering and spatial clustering. Candidate mutations were classified into interface core, interface-adjacent, and distal regions, followed by stepwise iterative combination. Two mutants, M5 and M6, retained WT-like activity but showed markedly improved thermostability. The Tm values of M5 and M6 increased by 11.4 and 12.4 °C, respectively, while their half-lives at 65 °C increased 3-fold and 12-fold. Structural analysis indicated that interface mutations promoted salt-bridge reconstruction, distal mutations stabilized monomers, and interface-adjacent mutations optimized the assembly microenvironment. This spatially coordinated strategy provides an effective approach for engineering thermostable multimeric enzymes.
Kaifan Qiu, Xingfei Li, Yuxiang Bai et al.· Journal of Agricultural and...· 0 citations