BhrPETase mutants and their applications

Abstract

This invention belongs to the field of enzyme engineering technology and discloses a BhrPETase mutant and its applications. The BhrPETase mutants provided by this invention include the IYL-S27L mutant and the IYL-S29V mutant, whose amino acid sequences are shown in SEQ ID No. 2 or 3. At 70°C, compared with wild-type BhrPETase, the PET degradation activity of the BhrPETase mutants provided by this invention is increased by 13.9%–16.4%. Furthermore, the IYL-S27L mutant exhibits good degradation efficiency for various types of PET products, including amorphous PET, untreated commercial PET (PET powder and cake boxes), and PET polyester yarn, meeting the needs of industrial applications. This invention can be applied to the degradation of PET, MHET, or BHET, and the preparation of degradation agents for PET and its degradation products.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to an MHET degrading enzyme, specifically a BhrPETase mutant and its applications. Background Technology

[0002] With the continuous increase in global plastic production, the treatment and recycling of waste polyethylene terephthalate (PET) has become an urgent environmental and economic issue. Traditional mechanical recycling methods are difficult to efficiently process contaminated or mixed plastics and can lead to a decline in the performance of recycled materials, resulting in downgraded recycling. Chemical recycling methods generally suffer from high energy consumption, complex processes, and potential secondary pollution. In contrast, bio-enzymatic degradation, due to its mild conditions, relatively simple products, and environmentally friendly characteristics, is widely recognized as a key technological path for promoting the "upgraded recycling" of PET and building a closed-loop circular economy.

[0003] However, the high-efficiency degradation performance of existing PET hydrolases and their mutants has mostly been verified on pretreated, clean, low-crystallinity PET flakes or powders. In contrast, real-world industrial PET waste (such as waste textiles, labeled or oil-contaminated containers) has a complex composition, containing various additives, pigments, and other impurities. These impurities can severely inhibit enzyme activity, resulting in degradation efficiency in actual waste far lower than ideal laboratory data. This hinders the full effectiveness of existing PET hydrolases and impedes the application of enzymatic recycling technology in the degradation of actual PET substrates.

[0004] As a thermophilic PET-degrading enzyme, wild-type BhrPETase possesses advantages such as good thermal stability and high PET catalytic activity. However, there is still room for improvement in its PET degradation activity, and the range of applicable PET substrates needs to be further expanded to better match the needs of industrial applications. Summary of the Invention

[0005] In view of the above-mentioned problems in the prior art, the present invention provides two BhrPETase mutants with significantly improved degradation activity by modifying the BhrPETase-IYL mutant, and expands the range of PET substrate types, providing technical support for enzymatic degradation of PET.

[0006] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions:

[0007] In a first aspect, the present invention provides a BhrPETase mutant, which is based on the amino acid sequence of the BhrPETase-IYL mutant shown in SEQ ID No.1, by site-directed mutation of serine at position 27 to leucine or serine at position 29 to valine, while the amino acid residues at other positions remain unchanged. The two mutants are named IYL-S27L mutant and IYL-S29V mutant, respectively.

[0008] While ensuring that the Tm value was essentially equivalent to that of the BhrPETase-IYL mutant, further mutations were performed on the BhrPETase-IYL mutant to obtain the IYL-S27L mutant and the IYL-S29V mutant. Compared to the BhrPETase-IYL mutant, the PET degradation activity of these two mutants at 70℃ was increased by 13.9%-16.4%.

[0009] Preferably, the BhrPETase mutant is the IYL-S27L mutant, which is based on the amino acid sequence of the BhrPETase mutant shown in SEQ ID No. 1, with the serine at position 27 being mutated to leucine, while the amino acid residues at other positions remain unchanged.

[0010] The Tm value of the IYL-S27L mutant was 93.93℃ (0.94℃ higher than that of the BhrPETase-IYL mutant), and its PET degradation activity at 70℃ was 16.4% higher than that of the BhrPETase mutant.

[0011] Secondly, the present invention provides a recombinant plasmid that can express the above-mentioned BhrPETase mutant.

[0012] Thirdly, the present invention provides a BhrPETase mutant recombinant strain comprising the recombinant plasmid described in the second aspect.

[0013] Fourthly, the application of the BhrPETase mutant provided in the first aspect above in the degradation of PET, MHET, or BHET.

[0014] Fifthly, the application of the BhrPETase mutant provided in the first aspect above in the preparation of PET degrading agents, MHET degrading agents or BHET degrading agents.

[0015] In a sixth aspect, the present invention provides a method for degrading PET products, specifically comprising: contacting the PET products with the BhrPETase mutant provided in the first aspect of the invention to perform an enzymatic hydrolysis reaction.

[0016] Preferably, the enzymatic hydrolysis reaction is carried out at a temperature of 70℃-74℃, a pH of 8-10, and a time of 4h-6h.

[0017] Preferably, the concentration of the BhrPETase mutant in the enzymatic hydrolysis reaction is 400 nM-600 nM.

[0018] More preferably, the PET products include PET textiles, PET containers, PET films, or PET engineering parts with a crystallinity of 8%-41%.

[0019] BhrPETase, a thermophilic PET-degrading enzyme, possesses both high thermal stability and excellent PET catalytic activity. This invention further mutates the BhrPETase-IYL mutant to obtain the IYL-S27L and IYL-S29V mutants. While maintaining a Tm value essentially equivalent to the BhrPETase-IYL mutant, the PET degradation activity of these two mutants is increased by 13.9%-16.4%. Among them, the IYL-S27L mutant exhibits good degradation efficiency for various types of PET products, including amorphous PET, untreated commercial PET (PET powder and cake boxes), and PET yarn, meeting the needs of industrial applications. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results of PET degradation activity determination of wild-type BhrPETase and its related mutants in Example 1 of the present invention;

[0022] Figure 2 The results of Tm value determination for wild-type BhrPETase and its related mutants in Example 1 of the present invention;

[0023] Figure 3 The results of the concentration determination of degradation products of wild-type BhrPETase and IYLL mutant on different types of PET substrates in Example 3 of the present invention are shown. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. The naming of mutants described in this invention follows the conventional naming methods used by those skilled in the art.

[0025] Example 1

[0026] This embodiment provides a method for preparing a BhrPETase mutant, as detailed below:

[0027] This invention obtains the mutated target gene using polymerase chain reaction (PCR), prepares recombinant plasmids using molecular biology methods such as DMT enzyme (TransGen, GD111) and seamless cloning, and transforms them into E. coli BL21(DE3) (TransGen, CD601) competent cells. After culturing, the target protein is obtained through heterologous expression. The specific process is as follows:

[0028] I. Construction of Recombinant Plasmids

[0029] 1. Construction of recombinant plasmid pET-22b-BhrPETase

[0030] This invention commissioned Genewiz to synthesize recombinant plasmids. The amino acid sequence of wild-type BhrPETase is shown in SEQ ID No. 4. Its coding gene was obtained through codon optimization, and the coding gene is shown in SEQ ID No. 5. The synthesized E. coli pET-22b plasmid carrying the wild-type BhrPETase coding gene is designated as recombinant plasmid pET-22b-BhrPETase. The construction method of this recombinant plasmid is described in Chinese Patent CN202311211045.9.

[0031] 2. Construction of recombinant plasmid pET-22b-BhrPETase-IYL and related recombinant plasmids

[0032] The first step involved PCR using the recombinant plasmid pET-22b-BhrPETase as a template, with F208I-F as the forward primer and F208I-R as the reverse primer, to obtain a linearized plasmid fragment containing the F208I mutation. The obtained fragment was then digested with DMT enzyme (TransGen, GD111), and further validated using molecular biology methods such as seamless cloning and Sanger sequencing, successfully constructing the recombinant plasmid pET-22b-F208I.

[0033] The second step involved using the constructed recombinant plasmid pET-22b-F208I as a template, H183Y-F as the forward primer, and H183Y-R as the reverse primer, to perform PCR again, obtaining a linearized plasmid fragment containing the F208I-H183Y mutation. Following the same template digestion, seamless cloning, and Sanger sequencing steps described above, the recombinant plasmid pET-22b-F208I-H183Y was successfully constructed.

[0034] The third step involves using the constructed recombinant plasmid pET-22b-F208I-H183Y as a template, Q142L-F as the forward primer, and Q142L-R as the reverse primer, and performing PCR again to obtain a linearized plasmid fragment containing the F208I-H183Y-Q142L mutation. After the same template digestion, seamless cloning, and Sanger sequencing steps described above, the recombinant plasmid pET-22b-F208I-H183Y-Q142L (also referred to as recombinant plasmid pET-22b-IYL) was successfully constructed.

[0035] Finally, using the constructed recombinant plasmid pET-22b-IYL as a template, PCR was performed with a total of 12 primer pairs, namely S27L-F / S27L-R, S27V-F / S27V-R, S29L-F / S29L-R, S29V-F / S29V-R, S32L-F / S32L-R, S95P-F / S95P-R, S95V-F / S95V-R, S101L-F / S101L-R, Q189L-F / Q189L-R, N190L-F / N190L-R, N214T-F / N214T-R, and Q258R-F / Q258R-R. The following compounds were obtained: F208I-H183Y-Q142L-S27L, F208I-H183Y-Q142L-S27V, F208I-H183Y-Q142L-S29L, F208I-H183Y-Q142L-S29V, F208I-H183Y-Q142L-S32L, F208I-H183Y-Q142L-S95P, and F208I-H Twelve different linearized plasmid fragments were obtained: 183Y-Q142L-S95V, F208I-H183Y-Q142L-S101L, F208I-H183Y-Q142L-Q189L, F208I-H183Y-Q142L-N190L, F208I-H183Y-Q142L-N214T, and F208I-H183Y-Q142L-Q258R. The templates for these 12 different linearized plasmid fragments were then digested using DMT enzyme (TransGen, GD111). Further verification was performed using molecular biology methods such as seamless cloning and Sanger sequencing, successfully obtaining 12 recombinant plasmids containing four-point mutations, including pET-22b-F208I-H183Y-Q142L-S27L (IYLL).

[0036] The PCR reaction system consisted of 20 μL of PCR reagent, including 1 μL of template (plasmid), 1 μL of forward primer (F), 1 μL of reverse primer (R), 10 μL of high-fidelity amplification reagent, and the remainder being enzyme-free water. The PCR reaction conditions were as follows: pre-denaturation at 98℃ for 3 min; followed by 30 cycles, each cycle consisting of: denaturation at 98℃ for 15 s, annealing at 66℃ for 15 s, extension at 72℃ for 3 min; and final extension at 72℃ for 5 min.

[0037] The statistical table of site-directed mutagenesis primers used in the construction of the above mutant recombinant plasmids is shown in Table 1 below.

[0038] Table 1

[0039]

[0040] 3. Expression and purification of recombinant proteins

[0041] The recombinant plasmid was introduced into BL21 competent cells (TransGen, CD601), gently mixed, and incubated on ice for 30 min. Then, a heat shock was performed at 42°C for 45 s, followed by rapid transfer to ice for 2 min. 500 μL of sterile LB medium (antibiotic-free) was added, mixed, and incubated at 37°C and 200 rpm for 1 hour to allow bacterial recovery. After recovery, the cells were centrifuged at 6000 rpm for 90 s, and 450 μL of supernatant was discarded. The remainder was added to LB agar, and the cells were spread evenly until the liquid was absorbed. The plates were inverted and incubated at 37°C for 12 h. Then, 1 mL of medium was drawn from a 5 mL tube of LB medium (containing 100 mg / L ampicillin) and slowly poured onto a plate confluent with monoclonal colonies. A 1 mL pipette tip was used to gently scrape the medium, ensuring half of the monoclonal colonies were distributed in the liquid medium. The resulting liquid was then transferred back to the original small tube of medium using a pipette. The small test tubes were placed in a shaker at 37℃ and 220 rpm for 3 h; then transferred to a shaker flask containing 80 mL of ZYM self-inducing medium for fermentation, and induced to express for 20 h at 21℃ and 160 rpm to obtain a bacterial culture rich in PET degrading enzyme.

[0042] Bacterial cells were collected using a high-speed refrigerated centrifuge (8000xg, 5 min), and resuspended in 10 mL of lysis buffer. The collected cells were then lysed using a high-pressure homogenizer. After lysis, the cells were centrifuged at 10000 rpm for 1 h to remove cell debris. The supernatant was the total protein solution containing wild-type BhrPETase and mutants such as BhrPETase-IYL, IYL-S27L, and IYL-S29V. The total protein solution was filtered through a 0.45 μm filter to remove impurities, followed by purification using a Ni-NTA packed column with gradient elution to obtain the target protein. The specific steps were as follows: first, equilibration was performed with lysis buffer for 2 min; then, the filtered total protein solution was re-coated onto the column three times, washed three times with washing buffer to remove contaminating proteins, and finally eluted with elution buffer to further concentrate the protein and remove high concentrations of imidazole, thus obtaining the concentrated protein solution. The amino acid sequences of the obtained BhrPETase-IYL mutant are shown in SEQ ID No. 1, the amino acid sequences of the IYL-S27L mutant are shown in SEQ ID No. 2, and the amino acid sequences of the IYL-S29V mutant are shown in SEQ ID No. 3.

[0043] The raw materials for ZYM self-induction medium are as follows: per 1L, 10g tryptone, 5g yeast extract, 25mM Na2HPO4·12H2O, 25mM KH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4·7H2O, 5g glycerol, 0.5g anhydrous glucose and 2g lactose monohydrate, with the remainder being water;

[0044] Each 1L of lysis buffer contains: 50mM Tris-HCl, 150mM NaCl and 10mM imidazole, with the remainder being water, pH=7.5;

[0045] Each 1L of washing buffer contains: 50mM Tris-HCl, 150mM NaCl and 40mM imidazole, with the remainder being water, pH=7.5;

[0046] Each 1L of elution buffer contains: 50 mM Tris-HCl, 300 mM NaCl and 300 mM imidazole, with the remainder being water, pH=7.5.

[0047] Example 1

[0048] This invention measures the PET degradation activity and Tm value of wild-type BhrPETase, BhrPETase-IYL mutant, IYL-S27L mutant, IYL-S27V mutant, IYL-S29L mutant, IYL-S29V mutant, IYL-S32L mutant, IYL-S95P mutant, IYL-S95V mutant, IYL-S101L mutant, IYL-Q189L mutant, IYL-N190L mutant, IYL-N214T mutant, and IYL-Q258R mutant, as detailed below:

[0049] (1) Method for determining PET degradation activity

[0050] The PET substrate selected in this invention is an amorphous model substrate (crystallinity of about 8%) purchased from GoodFellow. The model substrate was washed sequentially with 1% SDS, anhydrous ethanol and double-distilled water, and then punched into discs with a diameter of 6 mm (weighing about 8 mg, accurately recorded). One disc was taken for each enzymatic hydrolysis reaction.

[0051] The concentrated protein solution was added to 300 μL of reaction solution (containing 100 mM potassium phosphate buffer, pH=8) at a concentration of 500 nM. A PET disc was added to the reaction system, and the mixture was reacted in a 70°C water bath for 5 h. After the reaction was completed, acetonitrile was added to terminate the reaction, and the TPA, MHET, and BHET produced in the reaction were analyzed by high performance liquid chromatography (HPLC). The sum of the concentrations of TPA, MHET, and BHET was used to characterize the PET degradation activity.

[0052] The HPLC determination conditions are as follows:

[0053] Column: ZORBAX Eclipse Plus C18 reversed-phase column; Column temperature: 30℃;

[0054] Mobile phase: Mobile phase A is 0.1% formic acid-distilled water, and mobile phase B is acetonitrile;

[0055] Flow rate: 0.8 mL / min;

[0056] Gradient elution: The mobile phase changes from 95% mobile phase A to 30% mobile phase A within 20 min;

[0057] Detection wavelength: 240nm.

[0058] (2) Method for determining Tm value

[0059] Differential scanning fluorescence (DSF) was used to determine the melting temperature of proteins. Protein samples were loaded into 96-well plates at a volume of 25 μL per well, consisting of 15 μL of enzyme stock solution (containing 20 mM Tris-HCl and 300 mM NaCl per liter), 9 μL of protein solution (protein concentration of 0.4 mg / mL), and 1 μL of SYPRO Orange dilution solution. DSF experiments were performed using a real-time quantitative PCR system with 465 nm excitation and a 580 nm emission filter. Samples were heated from 25 °C to 100 °C at a rate of 0.3 °C / s, and fluorescence was measured every 0.03 s. The melting temperature (Tm) was determined using a first derivative curve.

[0060] The PET degradation activity and Tm values ​​of wild-type BhrPETase and its related mutants are shown in Table 2. The PET degradation activity of wild-type BhrPETase and its related mutants are as follows: Figure 1 As shown; the Tm value determination results of wild-type BhrPETase and its related mutants are as follows. Figure 2 As shown. Figure 1-2 In the text, Bhr represents wild-type BhrPETase, and IYL represents the BhrPETase-IYL mutant.

[0061] Table 2

[0062]

[0063] The results showed that, compared to the BhrPETase-IYL mutant, the 12 further modified BhrPETase mutants exhibited varying degrees of change in PET degradation activity and thermal stability. Except for the IYL-S27V, IYL-Q189L, and IYL-N214T mutants, whose PET degradation activities were lower than those of the BhrPETase-IYL mutant, the PET degradation activities of the other nine mutants were increased by 3.8%–16.4% compared to BhrPETase-IYL, and by 4.3–5.1 times compared to wild-type BhrPETase. Among them, the IYL-S27L mutant showed the best degradation effect on the PET model substrate, with a 5.1-fold increase in PET degradation activity compared to wild-type BhrPETase and a 16.4% increase compared to the BhrPETase-IYL mutant. The IYL-S29V mutant also showed a significant improvement in PET degradation activity, increasing by 4.97 times compared to wild-type BhrPETase and by 13.9% compared to the BhrPETase-IYL mutant. Regarding thermal stability, the Tm value of the IYL-S27V mutant was 1.85℃ higher than that of the BhrPETase-IYL mutant, and the Tm value of the IYL-Q189L mutant was 1.43℃ higher. The thermal stability of the IYL-S27L and IYL-S29V mutants, which exhibited superior PET degradation activity, was essentially comparable to that of the BhrPETase-IYL mutant. Among them, the Tm value of the IYL-S27L mutant (93.94℃) was 0.95℃ higher than that of the BhrPETase-IYL mutant (92.99℃), while the Tm value of the IYL-S29V mutant (92.08℃) was slightly lower than that of the BhrPETase-IYL mutant (92.99℃).

[0064] Example 2

[0065] Based on Example 1, this invention optimized the reaction conditions of the IYL-S27L mutant (named IYLL mutant), which showed significantly enhanced PET degradation activity. Specifically, it used potassium phosphate buffer (100 mM) at different temperatures (68℃, 70℃, 72℃, 74℃) and different pH values ​​(7, 8, 9, 10). The substrate was the model substrate used in the screening process described above. The reaction time was 5 h, and the enzyme concentration was 500 nM. The concentration data of degradation products under different conditions are shown in Tables 3-4 below. Specifically, the pH of the potassium phosphate buffer was 8 during the temperature-only experiment, and the temperature was set to 72℃ during the pH-only experiment.

[0066] Table 3

[0067]

[0068] Table 4

[0069]

[0070] The results show that the optimal temperature for both wild-type BhrPETase and the IYLL mutant is 72℃. The PET degradation activity of BhrPETase at 72℃ is 3.7% higher than that at 70℃, while the PET degradation activity of the IYLL mutant at 72℃ is 7.1% higher than that at 70℃. After temperature optimization, the PET degradation activity of the IYLL mutant at 72℃ is 6.3 times that of the wild-type enzyme. Furthermore, the optimal pH for wild-type BhrPETase is found to be 8, and for the IYLL mutant, it is 9. At pH 9, the PET degradation activity of the IYLL mutant is 8.9% higher than that at pH 8. After pH optimization, the PET degradation activity of the IYLL mutant at pH 9 is 6.9 times that of wild-type BhrPETase.

[0071] Example 3

[0072] This invention further investigated the degradation ability of the IYLL mutant on PET substrates with different crystallinity or different forms, as detailed below:

[0073] Based on Example 2, the optimal reaction conditions for the IYLL mutant—72℃, pH=9, and 100mM potassium phosphate buffer—were used to degrade four different types of PET substrates using wild-type BhrPETase and the IYLL mutant, both at a concentration of 500nM. The substrates used included a PET model substrate (LcPET, 8% crystallinity), untreated commercial PET powder (HcPET, 41% crystallinity, purchased from Far Eastern Textiles (Shanghai) Co., Ltd., 30-mesh PET powder), commercial cake boxes (PcPET, 11% crystallinity, purchased from Hengshui Rongfa Packaging Products Co., Ltd.), and PET polyester yarn (TfPET, 36% crystallinity, yarn diameter approximately 0.15mm). The degradation product formation after 3 hours was investigated at a substrate concentration of 30 g / L. The concentrations of degradation products from wild-type BhrPETase and the IYLL mutant for different types of PET substrates are shown below. Figure 3 As shown in Table 5.

[0074] Table 5

[0075]

[0076] The above results show that the IYLL mutant exhibits 68.4%–538.7% higher degradation activity for four different types of PET substrates compared to the wild-type BhrPETase. For the low-crystallinity substrates LcPET and PcPET, the IYLL mutant demonstrates a significant improvement in degradation efficiency, with the total release of TPA and MHET products being 6.44 and 4.87 times that of the wild-type BhrPETase, respectively. For LcPET and PcPET, the TPA yield after degradation by the IYLL mutant is 4.02 and 3.17 times higher than that of the wild-type, respectively. Furthermore, the IYLL mutant also shows improved degradation efficiency for the high-crystallinity substrates HcPET and TfPET, with the total release of TPA and MHET products being 1.58 and 2.63 times higher than that of the wild-type, respectively. TPA yield is increased by 69.1% and 148.6% compared to the wild-type. These results indicate that the IYLL mutant possesses high monomer conversion capacity and initial reaction rate for different types of PET substrates, demonstrating excellent potential for industrial applications.

[0077] Given that the IYL-S27L mutant and IYL-S29V mutant provided by the present invention can significantly improve PET degradation activity while maintaining or significantly increasing the Tm value, especially the IYL-S27L mutant which has high monomer conversion ability and initial reaction rate for different types of PET substrates, the BhrPETase mutant provided by the present invention can be used to prepare PET degrading agents, MHET degrading agents or BHET degrading agents with water.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A BhrPETase mutant, characterized in that: It is based on the amino acid sequence of the BhrPETase-IYL mutant shown in SEQ ID No.1, by site-directed mutation of serine at position 27 to leucine or serine at position 29 to valine, while the amino acid residues at other positions remain unchanged.

2. The BhrPETase mutant as described in claim 1, characterized in that: It is based on the amino acid sequence of the BhrPETase mutant shown in SEQ ID No.1, with the serine at position 27 being mutated to leucine, while the amino acid residues at other positions remain unchanged.

3. A recombinant plasmid, characterized in that: The recombinant plasmid can express the BhrPETase mutant as described in claim 1 or 2.

4. A BhrPETase mutant recombinant strain, characterized in that: It contains the recombinant plasmid as described in claim 3.

5. The application of the BhrPETase mutant as described in claim 1 or 2 in the degradation of PET, MHET or BHET.

6. The use of the BhrPETase mutant as described in claim 1 or 2 in the preparation of PET degrading agents, MHET degrading agents or BHET degrading agents.

7. A method for degrading PET products, characterized in that: The PET product is brought into contact with the BhrPETase mutant according to claim 1 or 2 to carry out an enzymatic hydrolysis reaction.

8. The method for degrading PET products as described in claim 7, characterized in that: The enzymatic hydrolysis reaction was carried out at a temperature of 70℃-74℃, a pH of 8-10, and a time of 4-6 hours.

9. The method for degrading PET products as described in claim 7, characterized in that: The concentration of the BhrPETase mutant in the enzymatic hydrolysis reaction is 400 nM-600 nM.

10. The method for degrading PET articles according to any one of claims 7 to 9, characterized in that: The PET products include PET textiles, PET containers, PET films, or PET engineering parts with a crystallinity of 8%-41%.

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