Improve:pET28a- IsPETase, BBa_25BUBMDI

Old part: IsPETase, BBa_K2010999

PETase serves as a plastic-degrading enzyme capable of hydrolyzing polyethylene terephthalate (PET). In this study, we engineered a new plasmid, pET28a-IsPETase (BBa_25BUBMDI), based on the old part IsPETase (BBa_K2010999). This construct serves as a positive control, enabling comparative analysis of PET degradation efficiency between the IsPETase and our deep-sea PET hydrolase (dsPETase) under identical experimental conditions. Meanwhile, we employed IsPETase (or DspPETase) and MHETase in a synergistic two-enzyme system, enabling the complete degradation of PET into TPA and EG[1-3].

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(Figure. 1), and inserted into the expression vector pET28a by homologous recombination method, the recombinant plasmids pET28a-IsPETase were further transformed into E.Coli DH5α.

Figure 1. The plasmid map of pET28a-IsPETase

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’). 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 Figure 2A showed that the IsPETase gene is between 500bp and 1000bp markers. The results are showed in Figure 2B, 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 Figure 2C showed that the recombinant plasmids was transformed into processed E.coli DH5α to copy the plasmids. As shown in Figure 2D, the sequencing results revealed a perfect match to the target gene sequence without any mutations, demonstrating the successful construction of the plasmid pET28a-IsPETase.

Figure.2.The electrophoresis, plate Assay, and sequencing of IsPETase. Note: IsPETase：879bp; pET28a：5323bp

1. Protein Expression



Figure 3.The growth curve and SDS-PAGE of IsPETase

We measured the OD₆₀₀ at various time points. As shown in Figure 3A, no significant difference was observed between the growth curves of the control group and the IsPETase-expressing strain, indicating robust bacterial growth.

We engineered the expression construct to incorporate a specific gene sequence encoding a hexahistidine (His-tag) affinity tag, enabling ribosomal synthesis of the recombinant protein with a C-terminal His-tag. The His-tagged protein was subsequently purified using Ni-NTA affinity chromatography, whereby the immobilized nickel ions selectively bind the His-tag. Non-specifically bound proteins were removed by washing with buffer, and the target protein was eluted under native conditions using imidazole-containing elution buffer.

In SDS-PAGE analysis, the crude protein dsPETase exhibited diffuse banding patterns, indicative of the presence of non-target cellular proteins. Following purification, the Figure 3B result demonstrated that the molecular weight of the recombinant protein was between 25 kDa and 35 kDa, which is consistent with its theoretical molecular mass of 33.5 kDa.

2. Enzyme activity

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 1).

Table 1. 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
50	3568.67041

We incubated 500 mg of PET plastic substrates with IsPETas and dsPETase at a final concentration of 1 mg/mL, then collected samples after 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.

In Figure 4, MHET was detected in the experimental groups of dsPETase and IspETase after 72 hours, while no MHET was observed in the CK control group. The enzyme activity of dsPETase was significantly higher than that of IsPETase.



Figure 4. Enzymatic Activity of PETase

3. Scanning electron microscopy (SEM)



Figure. 5. Results of scanning electron microscopy (SEM) of PET. A: control group; B: IsPETase+MHETase; C:DsPETase+MHETase; D:Gly8-Ser+MHETase

Figure 5A shows the PET film from the control group without enzyme addition, exhibiting a smooth surface without cracks or pores. Figure 5B 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 Figure 5 C, the sample co-treated with dsPETase and MHETase demonstrates more distinct fissure structures, suggesting enhanced degradation efficacy compared to the IsPETase group. Figure5 D displays the Gly8-Ser variant + MHETase group, which showed the optimal HPLC results. Numerous pronounced fissures can be observed on the film surface, demonstrating the superior degradation performance of the Gly8-Ser variant.

4 . 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 2.

Table 2. 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 Figure 6, 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.



Figure 6. Chromatographic peak of TPA

As illustrated in Figure 7, 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[4-5].



Figure. 7. The concentration of TPA of CK, IsPETase + MHETase, dsPETase + MHETase, and Gly8-Ser+ MHETase

As shown in Figure 8, 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 Figure 22A, the calibration curve exhibited an excellent fit with a coefficient of determination (R²) greater than 0.99.



Figure. 8. The chromatographic peak, ratio, mass scharge ratio of EG.

In Figure 9B, 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.



Figure 9. The concentration of EG of CK, IsPETase + MHETase, dsPETase + MHETase, and Gly8-Ser+MHETase

1. An engineered PET-depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216-219.
2. Arnal, G., et al. (2023). The diversity of PET degrading enzymes: A systematic review of sequence, structure, and function. Protein Science, 34(10), e70282. https://doi.org/10.1002/pro.70282
3. Guo, Z., Li, Y., Wang, M., & Ma, D. (2025). Catalytic upcycling of PET: From waste to chemicals and degradable polymers. Accounts of Chemical Research.
4. Guangyi Fan, Global marine microbial diversity and its potential in bioprospecting, Nature (2024). DOI: 10.1038/s41586-024-07891-2. www.nature.com/articles/s41586-024-07891-2
5. La Mantia, F. P., Titone, V., Botta, L., & Glerean, P. (2025). Mechanical recycling processes of PET in bottle-to-bottle circular manufacturing. Journal of Applied Polymer Science.