Synergistic engineering PETase reveals loop-region mutations for enhanced catalytic activity and thermal stability

Abstract

Biodegradation of Polyethylene Terephthalate (PET) offer a strategic avenue for addressing the global plastic pollution crisis. However, the degradation performance of PET hydrolases remains a technological bottleneck for the industrial realization of plastic biodegradation. In this study, we employed an integrated strategy combining semi-rational design and directed evolution to discover new mutation sites (N114, N205, N233 and S269) that enhance the catalytic activity and thermostability of Ideonella sakaiensis PETase (IsPETase). Subsequently, through the combined design of newly discovered mutation sites, we screened the novel quadruple mutant (N114I/N205K/N233K/S269V, named QM-PETase-2), which exhibited a 4.9-fold increase in catalytic efficiency and a ΔT_m of +12.4 °C. Interestingly, the four newly discovered mutation sites are all located in the loop region of the enzyme structure, which might play a crucial role on the structural stability of enzyme. Also, molecular dynamics simulations revealed that the QM-PETase-2 exhibited a more stable structure and an expanded substrate-binding cleft, which would facilitate the binding of the polymer PET substrate. Especially, the newly quadruple mutation sites were introduced into three reported high-performance PETase mutants FAST-PETase, PA-PETase, and DepoPETase. The obtained combined mutants, named QMFAST-PETase, QMPA-PETase, and QM-DepoPETase, demonstrated higher activity and thermal stability, indicating that the newly discovered mutations are universal for improving the performance of PETase. This research would be helpful in guiding the optimization and development of PETase.

Graphical abstract

1. Introduction

Over the past 150 years, plastic polymers with different properties have been mass-produced and used [1], bringing convenience and causing severe white pollution. The natural mineralization process of plastics is slow, and the negative effects produced are considered irreversible [2]. Polyethylene terephthalate (PET) is a polyester compound formed by the dehydration and condensation of terephthalic acid (TPA) and ethylene glycol (EG) [3], which is difficult to degrade and cause great harm to the environment [4]. In recent years, scientists have found that many marine organisms have abnormal growth and development, and nearly 700 species of marine organisms have been detected to have PET microplastic particles in their bodies. These plastics are the main culprits of endocrine disorders, stunting, cell necrosis and damage to immune cells, and even with the enrichment of biological chains, PET fragments in these organisms can enter the human body and threaten human health, such as reducing the migration and proliferation of human bone marrow mesenchymal stem cells and endothelial progenitor cells [[5], [6], [7], [8]]. Traditional photooxidative degradation methods [9,10] are limited by the area exposed to sunlight [10]. However, traditional thermal degradation methods [11] are limited by high energy consumption, environmental changes and secondary pollution, so biodegradation of PET is positioned as a promising and sustainable alternative [[12], [13], [14], [15]].

Biodegradation of PET relies on members of the α/β-hydrolase family, including cutinases (EC 3.1.1.74) [[16], [17], [18], [19]] and lipases (EC 3.1.1.3) [20,21]. Among them, Ideonella sakaiensis-derived PETase (IsPETase) has attracted considerable attention due to its exceptional PET-degrading activity under mild conditions [22]. IsPETase cleaves PET through a conserved Ser–Asp–His catalytic triad, where S131, assisted by D177 and H208, forms an enzyme-substrate intermediate, followed by nucleophilic attack by water to break the ester bond [[23], [24], [25]]. However, IsPETase suffers from two major limitations: (i) the poor thermostability (T_m < 50 °C), which is far below the glass transition temperature (T_g = 70–80 °C) of PET [[26], [27], [28]], and (ii) the inadequate plastic degradation efficiency, especially in inefficient degradation of crystalline regions.

To overcome these limitations, protein engineering strategies have been widely applied to optimize IsPETase. Rational design approaches have primarily focused on modifying the active site. For instance, Son et al. [27] engineered ThermoPETase (S121E/D186H/R280A), which achieved a 14.0-fold increase in depolymerization efficiency via stabilization of the β6-β7 loop. Lu et al. [29] developed FAST-PETase (S121E/D186H/R224Q/N233K/R280A), capable of degrading 51 commercial PET products within a week. By contrast, non-rational approaches such as directed evolution have yielded unexpectedly potent mutants. Shi et al. [30] screened over 10,000 clones through three rounds of directed evolution and developed DepoPETase (T88I/D186H/D220 N/N233K/N246D/R260Y/S290P), with a 1407.0-fold activity improvement; notably, key mutations such as T88 and S290 are located >20 Å from the catalytic center. Ying et al. [31] reported PA-PETase (S121P/D186A), which showed a T_m increase of 24.1 °C and a 109.0-fold increase in depolymerization efficiency toward amorphous PET at 40 °C. Directed evolution has proven uniquely valuable in engineering enzymes for non-natural substrates, as exemplified by the success of Frances Arnold's group in evolving a biocatalyst for silicon-carbon bond cleavage [32].

Current engineering efforts face two critical challenges: (i) rational design is constrained by existing structural information and cannot efficiently probe long-range mutational effects, such as those observed in DepoPETase where T88 lies over 20 Å from the active site; and (ii) the compatibility of novel mutation sets with industrial-grade mutants remains largely unexplored. Integrating semi-rational design with directed evolution offers a promising solution. Semi-rational approaches can guide target selection through sequence conservation and phylogenetic analysis [33], and by coupling error-prone PCR with high-throughput screening, directed evolution offers a powerful and efficient strategy for constructing mutational libraries and rapidly identifying improved enzyme mutants [34].

In this study, we implemented an iterative optimization strategy (Fig. 1). First, based on sequence alignment, non-conserved residues N205 and S269 were identified and subjected to semi-saturation mutagenesis, yielding the double mutant N233K/N205K with a 3.5-fold increase in activity. Incorporation of S269Q and S269V resulted in a 4.4 and 4.2-fold increase in enzyme activity. Subsequent directed evolutionary screening led to the identification of N114, and the final quadruple mutant QM-PETase-1 (N233K/N205K/S269Q/N114I) and QM-PETase-2 (N233K/N205K/S269V/N114I) exhibited a 5.4 and 4.9-fold enhancement in catalytic efficiency, and QM-PETase-2 exhibited a 12.4 °C increase in T_m. Molecular dynamics simulations further revealed that this mutation, with the key mutation localized in the loop region, stabilized the overall structure, thereby explaining the enhanced catalytic performance. Integration of this mutation set into high-performance PETase mutants such as FAST-PETase, DepoPETase and PA-PETase yielded the novel Combined PETase mutants with stronger degradation capacity, respectively. These findings unveil a synergistic mechanism of multi-region mutation combinations of enzyme structures and establish a promising trajectory for the comprehensive engineering PETase for industrial deployment.

Fig. 1.

Mutants design and screening via a combination of semi-saturated mutations and error-prone PCR. (a) Workflow for mutants design. The phylogenetic tree of cutinase (EC 3.1.1.74), lipase (EC 3.1.1.3), and IsPETase (EC 3.1.1.101) is shown to guide iterative semisaturation mutations at sites N205 and S269. Error-prone PCR is then used to further mine and screen for beneficial mutation sites; (b) High-throughput screening for the mutants by the values of the characteristic absorption peaks of the PET degradation products MHET and TPA at A254 nm. The degradation activity of wild-type and mutant subjects was determined after 24 h of reaction with PET at 45 °C.

2. Materials and methods

2.1. Semi-saturated mutation sites determined by semi-rational design

By reviewing the literature, we organized and summarized to get the sites that have been mutated on IsPETase in the current published articles, patents and other studies, and removed its first 29 amino acid sequences to get the library of potential mutation sites that have not been studied. Here, removing the first 29 residues in the signal peptide region facilitates the soluble expression of IsPETase in Escherichia coli (E. coli). And then, based on the high homology between IsPETase and many thermophilic cutinases, they were analyzed by homologous sequence comparison, and the phylogenetic tree and conservativeness analysis were used in MEGA software, and the potential sites were selected for semi-saturation and mutation validation among the sites that were not highly conserved.

2.2. Acquisition of mutant libraries of IsPETase

The genes involved in this experiment, IsPETase (pET_28a(+)), FAST-PETase (pET_28a(+)), PA-PETase (pET_28a(+)), and DepoPETase (pET_28a(+)), were synthesized from Beijing Tsingke Biotech, and codon-optimized with E. coli as the host, encoding N-free end of the 29 amino acid sequence. For high-throughput screening, the pelB secretion signal peptide was introduced at the N-terminus. In addition, in order to obtain a better heat-resistant mutant, based on the previous studies, it was chosen to start from N233 K²⁵. Thus, the starting genotype for this study was determined as pelB-N233K. The pelB-N233K was constructed as a template for semi-saturation mutation and directed evolution. Semi-saturated mutations were performed at selected potential loci to obtain semi-saturation mutation libraries. Mutant libraries were obtained using the SYBR Real-Time Error-Prone PCR Kit (Tenzinga) (Table S1 and S2). The mutants obtained from the screening were added to FAST-PETase, PA-PETase and DepoPETase by targeted mutagenesis to obtain a combined mutation library.

2.3. Expression and purification in E. coli BL21(DE3)

The wild-type and constructed mutants were transformed into E. coli BL21 (DE3) cells and cultured in LB medium overnight (9–12 h) at 37 °C for primary culture. And then passaged cultured to OD₆₀₀ of 0.6–0.8, the medium was cooled to 16 °C and induced using IPTG with a final concentration of 0.1 mM at 16 °C, 180 r/min for 24 h. The secreted expression was collected using a high-speed centrifugation machine (40 min, 4 °C, 4200 r/min) to collect, secreted expressed IsPETase, the supernatant was taken, and intracellularly expressed IsPETase was taken as a precipitate. To the bacterial precipitate, 30–40 mL of lysis buffer (50 mM Na₂HPO_4, 100 mM NaCl, 10 mM imidazole, pH 7.5), 50 mg/L bacteriophage DNase, lysozyme, and one-hundredth of a percent PMSF (100 mM) were added and resuspended. Cells were broken by sonication on ice (350 W, 30 min), and the supernatant was collected by high-speed centrifugation (11500 rpm, 4 °C, 45 min). The supernatant was combined with Ni-NTA for 1 h and washed with 30 mL buffer (50 mM Na₂HPO₄, 100 mM NaCl, 80 mM imidazole, pH 7.5), then collected by elution with 30 mL of eluent (50 mM Na₂HPO₄, 100 mM NaCl, 250 mM imidazole, pH 7.5), and finally imidazole was replaced using an ultrafiltration tube. purified protein (1–2 mL) was obtained. The protein concentration was determined using a Nanodrop spectrophotometer at A₂₈₀ (measured at 280 nm = 1 mg protein/mL).

2.4. Enzymatic catalytic activity of PETase mutants

Take 50 μL of the secretion supernatant and add it to 550 μL of glycine (50 mM)-NaOH reaction buffer (pH 9.0), or prepare the reaction at 500 nM of pure enzyme addition, and replenish to 600 μL with glycine-NaOH reaction buffer. And Gf-PET film was used as the substrate, which was purchased from GoodFellow (product number: ES30-FM-000145). All measurements were conducted in triplicate (n = 3). After the reaction, measuring the absorbance of the supernatant near λ = 254 nm or use high performance liquid chromatograph (HPLC) to characterize the enzyme activity. The HPLC was equipped with a Welch Ultimate XB-C18 column (4.6 × 250 mm, 5 μm, Welch Materials, Shanghai, China), and an Agilent 1100 series liquid chromatograph at 25 °C. Mobile phase A (acetonitrile: distilled water = 3:2) and mobile phase B (0.1 % (v/v) formic acid) were used. (0.1 % (v/v) formic acid). The absorbance of both products, TPA and MHET, was 254 nm and were identified according to the retention time of the standard compounds. The retention time was 9 min ± for TPA and 12 min ± for MHET. The calibration curves were plotted based on the absorption peak area versus the concentration of the standard solution, and the R-squared value of the calibration curves was at least 0.99 (Fig. S1). The absorption peak area of the samples allowed for the calculation of the concentration of each product.

2.5. Half-life measurements of IsPETase and its mutants

The thermal stability of IsPETase and its mutants was assessed by determining their half-lives at different heat treatment temperatures. For the enzyme heat treatment temperature, the optimal reaction temperatures corresponding to the IsPETase mutants were selected. Specifically, the enzymes were thermally incubated at the following temperatures: IsPETase and QM-PETase-2 at 40 °C; PA-PETase and QM-PA-PETase at 50 °C; FAST-PETase, QM-FAST-PETase, DepoPETase, and QM-DepoPETase at 55 °C. Following thermal treatment, the reaction was performed under the same conditions as described in the previous section. Through plotting the percentage curve of residual activity against the duration of heat treatment, the half-life of IsPETase and its mutants can be estimated.

2.6. Observation of morphological changes on the surface of PET films using scanning electron microscopy (SEM)

The reaction conditions and system are the same as above. Substrate selection Gf-PET was cut into small 6-mm discs of diameter. The Gf-PET samples at the end of the reaction were washed and dried, and then placed in the sampling spot of a scanning electron microscope (Hitachi S-4800, Japan). The magnification was selected to be 2000× and 5000× , and the microscopic surface changes at relevant locations on the sample surface were observed.

2.7. Analytical method for measuring melting temperature (T_m) of PETases

To evaluate the thermostability of IsPETase and its mutants, differential scanning calorimetry (DSC) was used to determine their respective T_m. Purified protein samples were concentrated to roughly 2.0 mg/mL. Then 30 μL of concentrated protein solution (phosphate buffer pH 7.5) was placed in an aluminium Tzero pan and sealed with a Tzero hermetic lid (TA Instruments). T_m of the protein samples was analyzed by a DSC-214. A DSC procedure was used depending on the anticipated denaturation temperature of the protein. It heated from 30 °C to 90 °C at 10 °C/min. The denaturation temperature of the proteins was measured on the heating ramp trace as the midpoint value at half-height.

2.8. Molecular dynamic (MD) simulation of PETases

System Setup: The enzyme structure from Ideonella sakaiensis (PDB ID: 5XJH) was preprocessed by removing auxiliary molecules while preserving the primary catalytic chain. Protonation states of ionizable residues were assigned using the H++ web server (pH 7.4) (http://newbiophysics.cs.vt.edu/H++/index.php) with default ion accessibility parameters. The protein topology was constructed using the Amber ff19SB [35,36] force field, while the 3PET substrate was parameterized with GAFF2 [37] and RESP [38] charges derived from single-point DFT calculations at the B3LYP/def2-TZVP level using ORCA 5.0.4 package. Charge neutrality was achieved by incorporating Na⁺ or Cl⁻ counterions. The system was immersed in a TIP3P water box with a 16 Å buffer distance using periodic boundary conditions. The system first underwent two-stage energy minimization, initiating with 5,000 steepest descent iterations to resolve atomic clashes followed by 10,000-step conjugate gradient refinement for enhanced geometric convergence. Thermal equilibration proceeded through three discrete heating phases, elevating the temperature from 5 K to 318.15 K over 60 ps cumulative duration via Langevin dynamics (collision frequency 2 ps⁻¹) while maintaining 5 kcal/mol/Ų restraints on non-solvent heavy atoms. For the QM-DepoPETase mutant system, the target temperature was elevated to 328.15 K (55 °C) to probe its thermal adaptability, whereas all wild-type and remaining mutant systems maintained the 318.15 K regime. Isotropic pressure equilibration was subsequently achieved across three sequential 20 ps NPT cycles using Monte Carlo barostat protocol (1 bar target pressure), progressively reducing positional restraints from 5 to 0 kcal/mol/Ų. Finally, a production trajectory of 200 ns was acquired using unconstrained NPT dynamics under SHAKE-enabled bond constraints (2 fs timestep) [39] and periodic 9.0 Å cutoff for nonbonded interactions. All simulations were performed using the GPU-accelerated Amber 24 software, and trajectory analyses were conducted using the CPPTRAJ toolkit [40]. Specifically, molecular docking was carried out with AutoDock Vina [41], and subsequent interaction was analyzed with PyMOL [42].

3. Results and discussion

3.1. Design, construction and preliminary screening of IsPETase mutants

To enhance the catalytic performance of the IsPETase, we employed a strategy combining semi-rational design with directed evolution to carry out iterative mutagenesis and screening (Fig. 1a). Meanwhile, using the pelB signal peptide for secretory expression and leveraging the characteristic absorbance of hydrolysis products at 254 nm, we established an efficient expression and high-throughput mutant-screening strategy for efficient screening of mutants (Fig. S1).

In detail, phylogenetic analysis and multiple sequence alignment (Fig. S2) revealed that residues N205 and S269 are located in relatively flexible loop regions with low conservation, indicating their potential as engineering hotspots for enzyme stability. And based on the previously identified N233K mutant, we introduced a two-round semi-saturated mutation at residue N205 and S269. In the round 1 of mutation, the double mutant N233K/N205K was achieved, which have a significant improvement in catalytic efficiency. Subsequently, in the round 2 of mutation, we screened two triple mutants: N233K/N205K/S269Q and N233K/N205K/S269V, with the further improvement in catalytic efficiency (Fig. 1b). In parallel, a mutant library based on the obtained semi-saturated mutant was constructed and screened using the high-throughput platform, exploiting a newly mutation site of N114I. Ultimately, four beneficial mutation sites were recombined to generate two quadruple mutant N233K/N205K/S269Q/N114I and N233K/N205K/S269V/N114I, named QM-PETase-1 and QM-PETase-2.

3.2. Enzymatic performance evaluation of PETase mutants

To verify the hydrolytic activity of the obtained excellent PETase mutants, the PET degradation products were quantitatively detected by chromatography based on a 72 h degradation reaction using the purified enzymes (Fig. S3). Here, the concentrations of MHET and TPA, representing the total degradation products, were quantified. And the series of the screened mutants exhibited higher activity at 40 °C and 45 °C (Fig. 2a and b). As shown in Fig. 2a, the hydrolytic activity steadily increased with the number of mutations, in agreement with results from high-throughput preliminary screening, confirming the effectiveness of the screening strategy. Among the mutants, the quadruple mutants QM-PETase-1 and QM-PETase-2 produced 5347 μM and 4944 μM of total degradation products at 40 °C, with 5.4-fold and 4.9-fold improvements over IsPETase, respectively, which represent the more efficient degradation capability. Noteworthy, although the overall degradation activity of the selected PETase mutants at 45 °C was lower than that at 40 °C, the activity increase of PETase mutants was more significant compared with that of the wild-type IsPETase, such as the activity of QM-PETase-2 even increased to more than 17.9-fold. This result implies that the thermal stability of the obtained mutants, especially QM-PETase-1 and QM-PETase-2, has also been significantly improved.

Fig. 2.

Enzyme activity verification and specific structure analysis of the screened mutants. PET-hydrolytic activity of IsPETase and mutants was indicated by the sum of TPA and MHET released from PETase mutants after 72 h of incubation at temperatures of 40 °C (a) and 45 °C (b). All measurements were conducted in triplicate (n = 3); (c) Specific structure analysis of QM-PETase-2 (N233K/N205K/S269V/N114I). Local structure regions characteristics of mutation sites (K205, K233, I114 and V269 showed by blue, yellow, cyan and magenta spheres) are shown as amplified panels and highlighted as green sticks.

The overall structural analysis of the mutation site showed the four mutation sites, I114, K205, K233 and V269, were located in the loop region at the distal end of the enzyme active center (Fig. 2c). Such mutant structural characteristics should have an effect on the improvement of enzyme performance, especially thermal stability. What's more, differential scanning calorimetry (DSC) analysis results revealed that QM-PETase-1 and QM-PETase-2 had melting temperatures (T_m) of 57.0 °C and 60.2 °C, respectively, which were both markedly higher than that of IsPETase (47.8 °C; Fig. S4a–c). This further validated and explained the experimental results of the corresponding mutants under relatively high temperatures. In comparison, given the relatively superior degradation performance, QM-PETase-2 was selected for subsequent studies.

3.3. Molecular dynamics (MD) simulations reveal structural mechanisms underlying enhanced catalytic performance

To gain mechanistic insights into the improved catalytic efficiency of the QM-PETase-2 mutant, we constructed a 3PET-bound complex model based on the crystal structure of IsPETase (PDB: 5XJH). And the complex model was optimized by 200 ns MD simulations. Based on this, top and side views of the surface structure of complex model were analyzed, focusing on the substrate binding grooves (Fig. 3). From the top view (Fig. 3a), the width of the substrate binding cell increases from 7.8 Å of IsPETase to 10.0 Å of QM-PETase-2; on the other hand, the side view (Fig. 3b) shows that the depth of the substrate binding cell is reduced from 7.0 Å of IsPETase to 5.6 Å of QM-PETase-2. This widening and shallower groove would favor the enzymatic substrate accessibility and reaction process. Here, we proposed the structural change trend of the expansion of the substrate binding region of PETase mutant. Interestingly, this structural change trend is very similar to the structural characteristics triggered by small-sized double mutants (Ser/Ile DM) revealed by Guo et al. [43]. Accordingly, such common structural characteristics play an important role in improving the plastics degradation ability of PETase, which will guide the engineering modification of PETase.

Fig. 3.

Surface representation of substrate binding groove of IsPETase (left) and QM-PETase-2 (right). The substrate model of 3PET was docked into IsPETase and QM-PETase-2, and 3PET is shown in magenta sticks. The protein surface of IsPETase and QM-PETase-2 is colored by green and cyan. The width (a) and depth (b) of the substrate binding groove is measured separately.

Next, the change characteristics of QM-PETase-2 were analyzed on the two aspects of hydrogen bond interactions and solvent accessible surface area (SASA) parameters (Fig. 4). After the mutation, the original two hydrogen bonds at residue sites N205 and N233 disappeared, and the removal of this constraint loosened the loop region (denoted in orange), which resulted in a conformational rearrangement and expanded the substrate binding area (Fig. 4a). SASA analysis revealed that mutations N114I and S269V enhanced the local hydrophobicity and significantly expanded solvent-accessible surface area for QM-PETase-2, compared with the corresponding characteristic parameters for IsPETase (Fig. 4b). In particular, the mutations of N114I and S269V, with the introduction of hydrophobic residues, disrupted the original polar hydrogen bond network, thereby enhancing the interactions of enzyme with hydrophobic substrate and surrounding solvent. Meanwhile, these findings suggest that the QM-PETase-2 enhances catalytic performance by increasing the scale of the substrate binding region and the accessibility of the hydrophobic substrate.

Fig. 4.

Structural analysis of four mutation sites in PETase and surface morphology analysis of GF-PET films. (a) A close up of the IsPETase (left) and QM-PETase-2 (right) mutation sites in conjunction with the docked 3PET structure. Mutation residues are shown in stick representations. N205, K205 and N233, K233 are shown in light blue, and G234 and T279, which form hydrogen bonds (highlighted as black dashed lines and are marked with concrete distances (Å)) with N205 and N233, are shown in gray. The loop region where 205 is located is colored by orange. The docked 3PET is shown in yellow stick representation; (b) Solvent Accessible Surface Area (SASA) distribution of IsPETase and QM-PETase-2. 114 (left) and 269 (right) of the hydrogen bonds disappeared after mutation were analyzed separately; (c) Scanning electron microscopy (SEM) analysis of Gf-PET film at 5000× magnification. The PET films were treated with enzymes for 72 h in 100 mM Glycine-NaOH (pH 9.0) buffer at different temperature. The SEM images from left to right respectively represent the blank control group of the Gf-PET film without enzyme treatment, and the two experiment groups of the Gf-PET film treated by the wild-type IsPETase and QM-PETase-2.

While the experiments to investigating the morphological effect of Gf-PET film treated by PETase were conducted and analyzed. As shown in SEM images of Fig. 4c, the surface of the blank control Gf-PET film without enzyme treatment remained smooth and intact; the surface of the Gf-PET film treated by the wild-type IsPETase only exhibited slight erosion; remarkably, the surface of the same Gf-PET film treated by QM-PETase-2 exposed obvious erosion and cracks. These observations further demonstrate the superior hydrolytic activity of QM-PETase-2 toward Gf-PET films.

3.4. Generalizability of the mutation combination across high-performance PETase

Furthermore, the mutation set of QM-PETase-2 was introduced into several high-performance PETase mutants, including FAST-PETase, PA-PETase, and DepoPETase, yielding new recombinant mutants of QMFAST-PETase, QMPA-PETase, and QM-DepoPETase. And the new recombinant mutants all exhibited better plastic degradation performance, in terms of both activity and thermal stability. QMFAST-PETase produced 6818 μM of hydrolysis products at the high temperature of 55 °C, with the 17 % enzymatic activity increase over FAST-PETase (Fig. 5a); more significantly, QMPA-PETase produced 6103 μM of hydrolysis products at the high temperature of 50 °C, with the 314 % enzymatic activity increase over PA-PETase (Fig. 5b); QM-DepoPETase produced 6838 μM of hydrolysis products at the high temperature of 55 °C with the 68 % enzymatic activity increase over DepoPETase (Fig. 5c). Simultaneously, the results of differential scanning calorimetry (DSC) confirmed the increases on thermal stability of the recombinant mutant mentioned above, and the T_m values were raised by 4.0 °C, 3.1 °C, and 3.7 °C for QMFAST-PETase, QMPA-PETase, and QM-DepoPETase, respectively (Fig. S4d–f). These data demonstrate that the new quadruple mutation set we discovered can significantly enhance the activity and thermal stability of the enzyme. Even the mutation set is not only effective against IsPETase, but also applicable to the reported high-performance IsPETase mutants. This indicates that the mutation strategy we proposed has good universality and guiding significance, for the development of PET-degrading enzymes with high activity and high thermal stability.

Fig. 5.

The activity and thermal stability of the combined mutants of IsPETase. (a)–(c) Three histograms show the PET-hydrolytic activity of the combined mutants compare to that of the control PETases at 72 h. All measurements were conducted in triplicate (n = 3). (a) QM-PETase-2 binds across FAST-PETase, named QMFAST-PETase. The reaction temperatures of enzymes is 55 °C; (b) QM-PETase-2 binds across PA-PETase, named QMPA-PETase. The reaction temperatures of enzymes is 45 °C; (c) QM-PETase-2 binds across DepoPETase, named QM-DepoPETase. The reaction temperature of enzymes is 55 °C. (d) The RMSD (Å) and (e) RMSF (Å) values of the combined mutants of IsPETase. The RMSD (Å) and RMSF (Å) were calculated by simulation; (f) Thermal stability of the wild-type and mutants of IsPETase. The thermal stability is characterized by half-life measurements of the enzyme at a certain heat treatment temperature. IsPETase and QM-PETase-2 were subjected to heat treatment at 40 °C; PA-PETase and QMPA-PETase were subjected to heat treatment at 50 °C; FAST-PETase, QMFAST-PETase, DepoPETase, and QM-DepoPETase were subjected to heat treatment at 55 °C. And the enzymes with heat treatment for different durations were taken for relative reaction activity measurement, displaying their percentage of the corresponding initial activity.

Structural stability and flexibility were assessed by analyzing RMSD and RMSF. RMSD simulation results showed that the multiple mutants (QM-PETase-2, QMFAST-PETase, QMPA-PETase, and QM-DepoPETase) obtained in this paper exhibited the reduced overall structural fluctuations relative to the that of IsPETase (Fig. 5d & Fig. S5), that indicated the enhanced conformational stability. Also, RMSF analysis results showed the lower fluctuation peak at the mutation sites of the quadruple mutations set, which implied the better structural stability in corresponding loop regions, particularly in the regions of N205K and N233K (Fig. 5e).

In addition, the thermal stability of IsPETase and its mutants were evaluated by measuring the half-life of the enzyme at a certain heat treatment temperature. In Fig. 5f, the residual activities of IsPETase and its mutants were measured at different time periods and displayed as a relative percentage of their corresponding initial activities. In detail, QM-PETase-2 were able to retain over 90 % of initial activities after 20 h of preincubation at 40 °C, whereas IsPETase were completely inactivated under the same conditions. After calculation, the half-life of QM-PETase-2 at 40 °C reached approximately 40 h, which was much longer than the half-life (about 4 h) of IsPETase. Similarly, based on the percentage curve of residual activity at 55 °C, the half-life of FAST-PETase was approximately 48 h. Surprisingly, the half-life of QMFAST-PETase was over 60 h. Even after being subjected to heat treatment for 60 h, the activity of QMFAST-PETase can still reach over 60 % of the initial activity. Other, at a heat treatment temperature of 50 °C, the calculated half-life of QMPA-PETase was approximately 20 h, which was much longer than the 5-h half-life of PA-PETase; and the calculated half-life of QM-DepoPETase was 33 h, while that of DepoPETase was 25 h, at a heat treatment temperature of 55 °C. From the above results, by introducing the discovered quadruple mutations in this study, the half-life of all the combined mutants was significantly increased, meaning that the thermal stability was also enhanced. This has significant practical implications for the development of PETase with high activity and high thermal stability.

3.5. Morphological characterization of PET film degradation by PETase mutants

To further evaluate the degradation capabilities of the engineered PETase mutants on actual PET substrates, scanning electron microscopy (SEM) was used to examine surface morphological changes of Gf-PET films before and after enzymatic treatment (Fig. 6). The surface of the control samples treated without enzymes was smooth and intact, indicating no intrinsic physical damage (Fig. 6a–c). In contrast, Gf-PET films treated with enzymes for 72 h showed obvious signs of surface erosion, including visible cracks, porosity formation, and localized spalling. And Gf-PET films treated with FAST-PETase (Fig. 6d), PA-PETase (Fig. 6e), and DepoPETase (Fig. 6f) showed pronounced cracks and extensive delamination. Notably, Gf-PET films exposed to QMFAST-PETase (Fig. 6g), QMPA-PETase (Fig. 6h) and QM-DepoPETase (Fig. 6i) highlights more intense surface degradation phenomenon, which declared the excellent degradation performance by introducing the quadruple mutations set. These results demonstrate that the engineered PETase mutants not only possess enhanced catalytic performance to PET materials in the suspension, but also exhibit robust activity against large-sized Gf-PET films, underscoring their potential for real-world plastic biodegradation applications.

Fig. 6.

Scanning electron microscopy (SEM) analysis of Gf-PET film at 5000× magnification. The Gf-PET films were treated with enzymes for 72 h in 100 mM Glycine-NaOH (pH 9.0) buffer at different temperature. (a)–(c) were the blank control groups, which indicated the SEM of three different areas substrate samples from the same Gf-PET film without enzyme treatment. The rest were surface morphological changes after 72 h of enzymatic treatment: (d) FAST-PETase; (e) PA-PETase; (f) DepoPETase; (g) QMFAST-PETase; (h) QMPA-PETase; (i) QM-DepoPETase.

4. Conclusions

In this study, we employed a strategy combining semi-rational design and directed evolution to engineer the PET hydrolase IsPETase, yielding a quadruple mutant QM-PETase-2 with a 4.9-fold increase in catalytic activity and a T_m of 12.4 °C improvement in thermal stability. Molecular dynamics simulations revealed that QM-PETase-2 possesses a more stable structure, a more open substrate-binding groove, and optimized loop rigidity, providing molecular-level insights into its enhanced catalytic efficiency. Furthermore, when the quadruple mutation sites of QM-PETase-2 was introduced into three reported high-performance PETase mutants (FAST-PETase, PA-PETase, and DepoPETase), the obtained combinatorial mutants exhibited higher activity and thermal stability. These findings highlight the newly discovered quadruple mutations set is a promising and broadly applicable engineering strategy, which is expected to promote the development and application of highly efficient PETase.

CRediT authorship contribution statement

Yuxin Han: Writing – review & editing, Writing – original draft, Validation, Investigation, Formal analysis, Data curation. Shilong Xing: Writing – review & editing, Writing – original draft, Validation, Investigation, Formal analysis, Data curation. Mingzhu Ding: Funding acquisition, Conceptualization. Ying Wang: Supervision, Methodology, Funding acquisition. Wenhai Xiao: Supervision, Conceptualization. Yixun Jiang: Writing – review & editing, Formal analysis, Data curation. Mingdong Yao: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Plasmids constructed in this study (Table S1); Primers used in this study (Table S2); The sequences of genes involved in this study (Table S3); Error-prone PCR system (Table S4); Error-prone PCR procedure (Table S5); The standard curve of A254nm and the PET monomers released. It has a good linear relationship (Fig. S1); Phylogenetic tree and sequence conservation analysis of IsPETase (Fig. S2); SDS-PAGE after protein purification (Fig. S3); The enthalpy curves of IsPETase and its mutants were determined by DSC (Fig. S4).