Name: IsPETase, BBa_25RH3V8U

Base Pairs: 879bp

Origin: Ideonella sakaiensis

Properties:

A bacterial strain Ideonella sakaiensis capable of efficiently degrading PET has been discovered. At the same time, a hydrolase secreted by this bacterium with the ability to degrade PET, named PETase, has also been reported. This enzyme can hydrolyze PET into bis-2-hydroxyethyl-terephthalate (BHET), mono-2-hydroxyethyl-terephthalate (MHET), and TPA [1].

Usage and Biology:

In the existing literature, Ideonella sakaiensis is known to thrive under mild environmental conditions, and accordingly, its enzyme IsPETase exhibits optimal activity at ambient temperature. Although IsPETase demonstrates generally higher PET degradation activity compared to other known enzymes, it suffers from relatively poor stability [2]. After incubation at 37 °C for 24 hours, the enzyme nearly loses all activity [3], and operates only at temperatures well below the glass transition temperature of PET. These limitations restrict its degradation efficacy and hinder its practical application in industrial settings. For instance, under conditions of 30 °C and pH 7.0, when 50 nmol/L of IsPETase was reacted with low-crystallinity PET film for 18 hours, only approximately 300 μmol/L of degradation products were detected [4]. Furthermore, IsPETase shows efficient degradation mainly toward low-crystallinity PET substrates, while exhibiting minimal activity against high-crystallinity PET materials.

Experimental data:

In order to construct the plasmid pET28a-IsPETase, the target gene IsPETase was amplified by PCR with primer IsPETase-F(5’-catatgaattttcccagggcttcaagactaatg-3’) and IsPETase-R(5’-ctcgaggctgcagttcgcggtgcg-3’). In Fig 2, it is clear to see that our sample are mainly in the range between 500 bp and 1000bp, which accurately matched with theoretical length, indicating a successful polymerase chain reaction.

Fig 2. The gel electrophoresis of IsPETase nucleic acids. The length nucleic acids of 879bp

Name：dsPETase, BBa_25S16DKP

Base Pairs: 816bp

Origin: Hydrothermal vent metagenome

Properties: A novel halophilic PET plastic-degrading enzyme with high activity derived from the hydrothermal vent metagenome (of deep-sea origin) was reported. Meanwhile, a hydrolase with PET-degrading activity, namely PETase, secreted by this bacterium was also reported. This enzyme can hydrolyze PET into bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET), and terephthalic acid (TPA) [5]

Usage and Biology: This highly active new salt-loving PET plastic degradation enzyme originating from deep-sea sources’s degradation rate of PET film reached 83 percent in three day, which is 44 times the activity of the reported IsPETase plastic degradation enzyme. But the content about research on dsPETase enzyme activity is little[5].

Experimental data:

To construct the expression plasmid dsPETase, firstly, the gene encoding dsPETase was amplified using polymerase chain reaction (PCR) with primer. dsPETase-F(5’-gaattcatgacaaatcccggggggggaggcgga-3’) and dsPETase-R(5’-ctcgagttagttaatgcctttacgacgcacgtcccagtca--3’). As shown in Fig 3, compared with the DNA marker, a significant band was seenin the range between 500 bp and 1000bp, indicating a successful polymerase chain reaction.

Fig 3. The gel electrophoresis of dsPETase nucleic acids

dsPETase Mutant：

Name: Gly8-GIn(BBa_2542VYML), Gly8-Ser(BBa_25N2AZ4A), Gly8-Thr(BBa_25655FKG), Gly8-Cys(BBa_25EL7FQM), Gly8-Asn(BBa_25NSRU73), Gly8-Tyr(BBa_2578647W), Arg47-Lys(BBa_25VTOT4S), and Arg47-His(BBa_2543N0QT)

Base Pairs: 816 bp

Origin: Hydrothermal vent metagenome

Properties: Derived from the hydrothermal vent metagenome, a highly active novel halophilic PET plastic-degrading enzyme from the deep sea has been reported, along with the hydrolytic enzyme PETase secreted by this bacterium, which can hydrolyze PET into bis-2-hydroxyethyl) terephthalate (BHET), mono-2-hydroxyethyl terephthalate (MHET), and TPA. The Gly8-GIn, Gly8-Ser, Gly8-Thr, Gly8-Cys, Gly8-Asn, Gly8-Tyr, Arg47-Lys, and Arg47-His are all mutants of dsPETase, exhibiting the same functionality.

Usage and Biology: The highly active novel halophilic PET plastic-degrading enzyme from the deep sea achieves a degradation rate of 83% for PET films within 3 days, which is 44 times the activity of the previously reported IsPETase plastic-degrading enzyme. There is relatively little research on the enzyme activity of dsPETase. Gly8-GIn, Gly8-Ser, Gly8-Thr, Gly8-Cys, Gly8-Asn, and Gly8-Tyr are mutations of the eighth amino acid of dsPETase to similar amino acids (Fig. 4), while Arg47-Lys and Arg47-His are mutants of dsPETase (Fig. 5), where Arg at position 47 is mutated to Lys and His, showing high homology[5].

Fig 4. The Mutant of GLY8-GIn，GLY8-Ser，GLY8-Thr，GLY8-Cys，GLY8-Asn，GLY8-Tyr

Fig 5. The Mutant of Arg47-Lys and Arg47-His

Name：MHETase,BBa_25ZYO9Z7

Base Pairs: 1818bp

Origin: Ideonella sakaiensis

Properties:

Mono-2-hydroxyethyl terephthalate (MHET) is an intermediate with a competitive inhibitory effect in the degradation process of PET, which can be hydrolyzed from terephthalic acid (TPA) and ethylene glycol (EG) catalyzed by MHET degradation enzymes.

Usage and Biology:

MHET hydrolase is mainly used for degrading plastics. Studies on its enzymatic properties indicate that the optimal pH and temperature for this enzyme are 7.5 and 40 °C, respectively, and it is relatively stable under conditions of pH 7.5−10.0 and 30−45 °C; the kinetic parameters measured with MHET as the substrate show kcat of (24.2±0.5) s–1 and Km of (1.8±0.2) μmol/L.[8]

Experimental data:

DNA encoding MHETase was optimised according to the codon preference of E. coli and synthesised. To construct the plasmid for MHETase, the gene encoding MHETase was amplified using polymerase chain eaction (PCR) with primer MHETase-F (5’-catatgcaaacgactgtaacaacaatgttactag-3’) and MHETase-R (5’-ctcgagcggaggggctgcg--3’).Fig. 6shows representative photos of the DNA encoding MHETase .And the gel electrophoresis results showed that our sample are in the range between 500 bp and 1000bp, suggesting that MHETase was successful polymerase chain reaction.

Fig. 6 The gel electrophoresis of MHETase nucleic acids

Engineering Principle:

PETase is a hydrolase enzyme that can hydrolyze PET into bis(2-hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET). PETase from different sources exhibits varying enzyme activity characteristics, with proper protein folding having a significant impact on enzyme activity. Therefore, firstly, PETase from Ideonella sakaiensis and hydrothermal vent metagenome was screened using data blocks, and molecular docking was employed to assist in verifying key active sites of PETase and mutating those key sites. Furthermore, HPLC was used to determine the content of MHET and high-activity mutant sites were screened. Finally, the selected mutant dsPETase and MHETase were used in conjunction, and scanning electron microscopy (SEM) was employed to observe the changes in surface morphology of PET films before and after degradation, along with HPLC measurements of the degradation products which are terephthalic acid (TPA) and ethylene glycol (EG), calculating the degradation rate. Ultimately, this demonstrates a high efficiency in degrading specific plastics (such as PET), which could be applied to plastic pollution management or industrial degradation processes (Fig 7).

Fig. 7.The engineering schematic diagram of the project design

1. pET28a- IsPETase(BBa_25BUBMDI)

Construction Design:

Selection of PETase from Ideonella sakaiensis was conducted in NCBI database, named IsPETase. The genes encoding IsPETase fused with 6×His-tag at its C-terminus were optimized and chemically synthesized. The plasmid map was constructed by snapgene software(Fig. 8), and inserted into the expression vector pET28a by homologous recombination method, the recombinant plasmids pET28a-IsPETase were further transformed into E.Coli DH5α.



Fig 8. The plasmid map of pET28a-IsPETase

Experimental Approach:



Fig. 9.The electrophoresis, plate Assay, and sequencing of IsPETase. Note: IsPETase：879bp; pET28a：5323bp

The pET-28a plasmid vector was linearized for the construction of the expression plasmid. The plasmid containing the IsPETase gene was constructed using homologous recombination, followed by confirmation of successful construction via agarose gel electrophoresis. The Fig 9A showed that the IsPETase gene is between 500bp and 1000bp markers. The results are showed in Fig 9B, the linearized plasmid pET28a is highlighted 5000bp. The expansion length of gene is consistent with the DNA encoding and indicate the succeed of amplification. The Fig 9C showed that the recombinant plasmids was transformed into processed E.coli DH5α to copy the plasmids. As shown in Fig 9D, the sequencing results revealed a perfect match to the target gene sequence without any mutations, demonstrating the successful construction of the plasmid pET28a-IsPETase.

2. pET28a-dsPETase(BBa_25GLIJTL)

Construction Design:

The genes encoding dsPETase fused with 6×His-tag at its C-terminus were optimized and chemically synthesized. The plasmid map was constructed by snapgene software(Fig 10), and inserted into the expression vector pET28a by homologous recombination method, the recombinant plasmids pET28a-dsPETase were further transformed into E.Coli DH5α.



Fig 10. The plasmid map of pET28a-dsPETase

Experimental Approach:



Fig. 11.The electrophoresis, plate Assay, and sequencing of dsPETase

The construction of the dsPETase plasmid employed an identical strategy to that of the IsPETase plasmid, with both based on the method of homologous recombination. As shown in Fig 11A, the dsPETase gene migrated between 500 and 1000 bp. This is consistent with its actual length of 816 bp, which was determined by sequencing. The linearized pET28a line is around 5000bp and its original length is 5329bp. We was transformed the pET28a-dsPETase plasmid into E.coli in the Fig 11C. In Fig 11D, sequencing of the plasmid confirmed that the inserted DNA fragment matched the target gene sequence, demonstrating successful ligation into the vector.

3. dsPETase mutant: pET28a-GLY8-GIn, ET28a-GLY8-Ser, pET28a-GLY8-Thr, pET28a-GLY8-Cys, pET28a-GLY8Asn, pET28a-GLY8Tyr, pET28a-Arg47Lys and pET28a-Arg47His

Construction Design:

To identify key active sites in dsPETase for mutagenesis, we constructed a three-dimensional protein structure of dsPETase and performed molecular docking with the chemical structure of PET(Fig 12). The docking score (binding energy) serves as an indicator to evaluate the affinity between PET and protein dsPETase. A stronger affinity results in tighter binding and more stable molecular interactions. A more negative docking score corresponds to a stronger affinity between the molecules.



Fig 12. Docking results of dsPETase with the PET molecule; Red represents the PET molecule.



Fig 13. Schematic diagram of the active site of dsPETase with the PET molecule.

The optimal binding mode between the PET molecule and dsPETase is shown in Figs 12 and 13. The molecular docking binding energy was calculated to be −3.249 kcal/mol, with stabilization primarily achieved through hydrogen bonding (Table 1). Key amino acid residues forming hydrogen bonds with the PET molecule were glycine at position 8 (Gly8) and arginine at position 47 (Arg47), which enhanced the stability of the PET molecule within the binding pocket.The molecular docking results validated the feasibility of selecting Gly8 and Arg47 for mutagenesis, a decision initially made based on expert opinions during the experimental design phase. However, experimental validation is required to confirm the degradation efficiency of the mutants and ensure the reliability of the result.

Based on the advice of biology Professor Yao, we performed site-directed mutagenesis on the active site residues glycine (Gly8) and arginine (Arg47). Specifically, Gly8 was mutated to amino acids with similar properties—namely, polar neutral R-group residues such as glutamine (Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), and tyrosine (Tyr). This strategy helps minimize structural perturbations that could lead to loss of protein activity. The arginine at position 47 was mutated to amino acids with similar properties, specifically basic R-group residues such as lysine (Lys) and histidine (His). The resulting mutants were designated as Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr. As shown in Table 1, the docking scores of these mutants were significantly improved compared to that of wild-type dsPETase, indicating stronger binding affinity with PET.

Table 1. Docking scores (binding energy, kcal/mol) of PET with dsPETase and mutants.

The plasmid map was constructed by snapgene software(Fig 14), and inserted into the expression vector pET28a by homologous recombination method, the recombinant plasmids were further transformed into E.Coli DH5α.

Fig 14. The plasmid map of pET28a-IsPETase

Cultivation, Purification and SDS-PAGE:

1. Mutant Arg47-Lys and Arg47-His



Fig 15. Molecular interactions between the protein and the ligand within the binding pocket. Note: The protein structures of the Arg47-Lys and Arg47-His mutants are depicted in blue. The PET molecule is shown in orange.

The binding energies between the PET molecule and the mutant proteins Arg47-Lys and Arg47-His were −5.076 kcal/mol and −5.012 kcal/mol, respectively, indicating strong molecular interactions. As shown in Fig 15, the PET molecule interacts with the Arg47-Lys mutant primarily through hydrogen bonds and hydrophobic interactions. Hydrogen bonds were formed with LEU-70 and ALA-71, while hydrophobic interactions occurred with VAL-43, LEU-70, ALA-71, and ARG-68. In the Arg47-His mutant, the PET molecule was involved in hydrophobic interactions with the specific residues PHE-44, TYR-118, and THR-117 (Fig 15).

2. Mutant (Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr)



Fig 16. Molecular interactions between the protein and ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted in orange.

The binding energy between the PET molecule and the Gly8-Asn mutant protein was calculated to be −5.002 kcal/mol, indicating strong binding affinity. As shown in Fig 16, the PET molecule interacts with the Gly8-Asn mutant primarily through hydrogen bonding and hydrophobic interactions. Specifically, a hydrogen bond was formed with TYR-106, while hydrophobic interactions were observed with PHE-44, THR-117, and TYR-118.

The binding energy for the PET molecule with the Gly8-Cys mutant was −4.945 kcal/mol, demonstrating favorable binding interactions. Fig 16 shows that the PET molecule engages in hydrophobic interactions with LEU-103 and LYS-102, as well as electrostatic interactions with LYS-102 and ARG-99 in the Gly8-Cys mutant.



Fig 17. Molecular interactions between the protein and ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted in orange.

The binding energies between the PET molecule and the Gly8-Gln and Gly8-Ser mutant proteins were calculated to be −5.103 kcal/mol and −4.932 kcal/mol, respectively, indicating strong binding affinity. As shown in Fig 17, the PET molecule interacts with the Gly8-Gln mutant primarily through hydrogen bonding and hydrophobic interactions. Specifically, a hydrogen bond was formed with TYR-106, while hydrophobic interactions were observed with PHE-44, THR-117, and TYR-118. Fig 17 illustrates that the PET molecule engages in hydrophobic interactions with LEU-103 and LYS-102, as well as electrostatic interactions with LYS-102 and ARG-99 in the Gly8-Ser mutant.



Fig 18. Molecular interactions between the protein and ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted in orange.

The binding energies between the PET molecule and the Gly8-Thr and Gly8-Tyr mutant proteins were calculated to be −5.151 kcal/mol and −5.204 kcal/mol, respectively, indicating strong binding interactions. In Fig 18, the PET molecule engages in hydrophobic interactions with PHE-44, TYR-118, and THR-117 in the Gly8-Thr mutant. Fig 18 illustrates that the PET molecule interacts with the Gly8-Tyr mutant primarily through hydrogen bonding and hydrophobic interactions. Specifically, a hydrogen bond was formed with TYR-106, while hydrophobic interactions were observed with PHE-44 and TYR-118.



Fig 19. Agarose gel electrophoresis analysis of the mutant

We performed site-directed mutagenesis using PCR. As shown in Fig 19, the DNA fragments migrated between 5000 bp and 7500 bp, with all mutants (Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr) exhibiting a consistent length of 6190 bp. The plasmids were subsequently sent for commercial sequencing(Fig 20), and the results confirmed that the introduced mutations matched the expected designs.



Fig 20. The sequencing of the mutant

As shown in Fig 21, no significant difference was observed between the growth curves of the control group and the dsPETase and mutant strain, indicating robust bacterial growth.



Fig 21.The growth curve of dsPETase and mutant

As shown in Fig 22A, lanes 1–2 exhibited diffuse bands in the crude protein samples, suggesting the presence of a substantial amount of non-specific proteins. Lanes 3–5, which served as blank controls, showed no detectable target band corresponding to dsPETase. In lanes 6–9, clear protein bands were observed within the molecular weight range of 25–35 kDa. These samples were purified by nickel-affinity chromatography, wherein Ni-NTA resin selectively captured His-tagged proteins, wash buffer removed non-specifically bound impurities, and the target protein was subsequently eluted under appropriate conditions. Fig 22B displays the protein expression profiles of various mutants. Band intensity and thickness reflect relative expression levels, with the Gly8-Ser mutant showing the darkest and thickest band, indicating its highest expression among all variants.



Fig 22. The SDS-PAGE of dsPETase and mutant. M:marker 1,2:rough protein; 3-5:Wash buffer 6-9:purified protein. DsPETase:31.5kDa

Characterization/Measurement:

The chromatographic conditions were optimized using a Thermo Scientific C18 column (250 × 4.6 mm, 5 μm) with a mobile phase consisting of phosphate buffer (containing 5 mmol/L potassium dihydrogen phosphate and 0.04% phosphoric acid) and methanol in a ratio of 70:30 (v/v). According to the experimental protocol, standard solutions were serially diluted to appropriate concentrations to establish a calibration curve. The peak area was plotted on the Y-axis against the concentration of the reference standard on the X-axis, and regression analysis was performed to obtain the equation for each standard. A good linear relationship was observed within the concentration range of 1–50 mg/L (Table 2).

Table 2. The standard curve of MHET

Concentrations (mg/L)	Peak area
1	58.41312
2	130.59349
5	357.34796
10	697.24371
20	1393.50171

We incubated 500 mg of PET plastic substrates with IsPETase, dsPETase, and their variants at a final concentration of 1 mg/mL, then collected samples after 48 and 72 hours of degradation at room temperature. The peak area of MHET was measured by HPLC, and its concentration in the experimental samples was calculated based on the standard curve.

As shown in Fig 23A, MHET was detected in the experimental groups of IsPETase, dsPETase, and their mutants at 48-hour, whereas MHET was undetectable in the CK control group. Comparative analysis revealed no significant difference in MHET levels between IsPETase and dsPETase at the 48-hour. In contrast, all tested mutants—namely Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr—exhibited significantly higher MHET accumulation compared to dsPETase. Notably, the Gly8-Ser mutant generated markedly elevated MHET levels relative to the other mutants. Furthermore, there was no statistically significant difference in MHET production between the Arg47-Lys and Arg47-His variants.

In Fig 23B, MHET was detected in the experimental groups of IsPETase, dsPETase, and their mutants after 72 hours, while no MHET was observed in the CK control group. The MHET level produced by dsPETase was significantly higher than that of IsPETase at 72h. The MHET concentration of Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr mutants exhibited markedly higher than dsPETase. In contrast, no significant difference in MHET levels was observed among Arg47-Lys, Arg47-His, and dsPETase. These results suggest that Gly8 may represent a critical active site in dsPETase. Notably, the substitution of Gly8 with Ser significantly enhanced the degradation efficiency of PET.

Fig 23. MHET content of IsPETase, dsPETase, and mutantsArg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr) at 48 and 72 hours.

Construction Design:

Selection of PETase from Ideonella sakaiensis was conducted in NCBI database, and abbreviated as MHETase. The genes encoding MHETase. fused with 6×His-tag at its C-terminus were optimized and chemically synthesized. The plasmid map was constructed by snapgene software(Fig 24), and inserted into the expression vector pET28a by homologous recombination method, the recombinant plasmids pET28a- MHETase. were further transformed into E.Coli DH5α.

Fig 24. The plasmid map of pET28a- MHETase.

Cultivation, Purification and SDS-PAGE:

Fig 25.The electrophoresis, plate Assay, and sequencing of MHETase.

Note:the length of MHETase is 1818bp. the length of pET28a is 5324b

The agarose gel electrophoresis result in Fig 25 A and B showed a band of the MHETase gene at approximately 1500 bp and a linearized plasmid band near the 3000 bp marker. After ligation, the recombinant plasmids were transformed into E. coli DH5α competent cells for amplification in the Fig25 C. The subsequent sequencing analysis confirmed the successful insertion of the target gene MHETase into the vector(Fig 25D).

Fig 26.The growth curve and SDS-PAGE of IsPETase. Note:The size of MHETase is 64kDa.

To express the target protein, the plasmid was transformed into E. coli BL21(DE3) cells and induced with 1 mM IPTG, followed by protein extraction and purification. In Fig 26A, no significant difference was observed between the growth curves of the control group and the dsPETase and mutant strain, indicating robust bacterial growth. The SDS-PAGE analysis showed that the purified MHETase protein was approximately 63 kDa, which is consistent with its actual molecular size(Fig 26 B).

Characterization/Measurement:

1. Scanning electron microscopy (SEM)



Fig. 27. Results of scanning electron microscopy (SEM) of PET. A: control group; B:IsPETase+MHETase; C:DsPETase+MHETase; D:Gly8-Ser+MHETase

Fig 27A shows the PET film from the control group without enzyme addition, exhibiting a smooth surface without cracks or pores. Fig 27B presents the experimental group treated with IsPETase and MHETase, where faint fissures observed on the film surface indicate initial enzymatic activity, though the degradation effect remains suboptimal. In Fig 27C, the sample co-treated with dsPETase and MHETase demonstrates more distinct fissure structures, suggesting enhanced degradation efficacy compared to the IsPETase group. Fig 27D displays the Gly8-Ser variant + MHETase group. Numerous pronounced fissures can be observed on the film surface, demonstrating the superior degradation performance of the Gly8-Ser variant.

2. HPLC determination of concentration TPA and EG

The experimental instrument parameters were adjusted as follows: the column was a Thermo Fisher C18 (250 mm × 4.6 mm, 5 μm), with a column temperature of 30°C, flow rate of 1 mL/min, and a mobile phase consisting of phosphate buffer solution (containing 5 mmol/L potassium dihydrogen phosphate and 0.04% phosphoric acid) and methanol at a ratio of 70:30.

According to the experimental protocol, the standard was diluted to appropriate concentrations to establish a calibration curve. Using the peak area as the ordinate (Y) and the mass concentration of the reference standard as the abscissa (X), regression analysis was performed to derive the equation for each standard. A good linear relationship was demonstrated within the concentration range of 1–50 mg/L. The tested concentrations and corresponding calibration curve are presented in Table 3

Table 3. The standard curve of TPA

Concentrations (mg/L)	Peak area
1	85.65078
2	204.93451
5	584.46924
20	2220.17896
50	6006.84082

We incubated 1 g PET plastic with 1 mg/mL IsPETase, dsPETase, and the Gly8-Ser variant, along with MHETase at a final concentration of 1 mg/mL. Under the catalytic action of PETase and MHETase, PET can be degraded to TPA, which was quantified using HPLC. As shown in Fig 28, the control group (pET28a vector) showed no peak at 10–12 minutes, whereas distinct peaks were observed for the IsPETase + MHETase, dsPETase + MHETase, and Gly8-Ser variant + MHETase groups.



Fig. 28. Chromatographic peak of TPA

As illustrated in Fig 29, no TPA was detected in the control (CK) group. The TPA concentrations produced in dsPETase + MHETase and Gly8-Ser + MHETase were significantly higher than IsPETase + MHETase. Furthermore, the TPA concentrations from the Gly8-Ser+ MHETase combination was markedly higher than that of dsPETase + MHETase. These results indicate that the degradation efficiency of dsPETase + MHETase exceeds that of IsPETase + MHETase, and the Gly8-Ser variant in combination with MHETase demonstrated superior performance compared to all other experimental groups. This evidence confirms that site-directed mutagenesis enhanced the enzymatic activity of PETase, thereby significantly improving the overall degradation efficiency.



Fig. 29. The concentration of TPA of CK, IsPETase + MHETase, dsPETase + MHETase, and Gly8-Ser+ MHETase

EG (Ethylene Glycol) was qualitatively and quantitatively analyzed in complex mixtures using Gas Chromatography-Mass Spectrometry (GC-MS). The instrument parameters were set as follows:

Gas Chromatograph: Agilent GC 8860 equipped with CTC PAL3 autosampler

Mass Spectrometer: Agilent 5977B MSD

Column: Agilent DB-WAX (30 m × 0.25 μm)

Carrier Gas: High-purity helium (≥99.99999%)

Chromatographic conditions:

Detector temperature: 240°C

Injector temperature: 220°C

Flow rate: 1.0 mL/min

Temperature gradient: Initial temperature 80°C (hold 5 min), then ramp at 5°C/min to 240°C (hold 1 min).



Fig. 30. The chromatographic peak, ratio, mass scharge ratio of EG.

As shown in Fig 30, the detection results of ethylene glycol (EG) in the samples were obtained using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The control group exhibited no detectable peaks, indicating the absence of EG in the sample. Distinct chromatographic peaks were observed in the experimental groups containing IsPETase + MHETase, dsPETase + MHETase, and the Gly8-Ser + MHETase.The concentration of EG in the samples was calculated based on the chromatographic peak areas using the standard curve equation y = 6.306x + 363.8. As shown in Fig 31A, the calibration curve exhibited an excellent fit with a coefficient of determination (R²) greater than 0.99.

In Fig 31B, the EG concentration obtained with dsPETase + MHETase was significantly higher than that with IsPETase + MHETase. Notably, the Gly8-Ser variant + MHETase combination yielded a substantially greater EG concentration compared to both the dsPETase and IsPETase. These results indicate that the Gly8-to-Ser mutation at position 8 in dsPETase enhances enzymatic activity, significantly improving the conversion efficiency of EG.



Fig 31. The concentration of EG of CK, IsPETase + MHETase, dsPETase + MHETase, and Gly8-Ser+MHETase