A double-enzyme cascade system for the efficient conversion of ethylene glycol to glycolic acid in a waste pet depolymerization system and application thereof

Abstract

The application discloses a kind of for waste PET depolymerization system glycol directional efficient conversion of glycolic acid two enzyme level connection system and its application, belong to biological catalysis and resource recycling technical field.The system includes a kind of mutant alcohol dehydrogenase with high selectivity to glycol and a mutant aldehyde dehydrogenase ALDH-V7.Said mutant alcohol dehydrogenase is obtained in at least one combination mutation in 97 and 101 and / or 145 and 148 in the basis of ADH5 enzyme derived from acetic acid bacillus ( Gluconobacter oxydans ).The system can efficiently and directionally oxidize low-value glycol in waste PET chemical or biological depolymerization product to high-value-added product glycolic acid under mild conditions, effectively avoiding peroxidation and C-C bond rupture.The application provides an efficient and green biological catalysis solution for upgrading and recycling of waste PET, and realizes a circular economy path of "waste plastics-degradable plastic monomers".

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis and resource recovery technology, specifically relating to a method for utilizing *Acetobacter* spp. (… Gluconobacter oxydans This study describes a method for efficiently and selectively oxidizing ethylene glycol (EG) in the depolymerization products of polyethylene terephthalate (PET) waste into glycolic acid (GA), a high-value-added chemical, by constructing a dual-enzyme cascade catalytic system using specific dehydrogenases discovered in PET. It also describes the application of this system to achieve a circular economy in plastics. Background Technology

[0002] Currently, ethylene glycol (EG), produced from the chemical or biological depolymerization of waste PET, is often oxidized into CO2 as a sacrificial agent due to its low market value and high separation costs, resulting in a waste of carbon resources. Selectively oxidizing EG to glycolic acid (GA)—a key monomer of the biodegradable plastic polyglycolic acid (PGA)—is an ideal pathway to upgrade and recycle PET.

[0003] However, existing chemical catalysis methods (such as noble metal Pd-Ni catalysts) are costly, and the selective oxidation of the two identical hydroxymethyl groups in the EG molecule is extremely challenging, easily leading to overoxidation or C / C bond breakage. A research team from Shandong University published a study that constructed a Pd@Ni(OH)2-NiO catalyst with metal-support interaction (MSI) and tip-enhanced electric field effect through a three-step hydrothermal-annealing-electrodeposition method. Theoretical calculations show that this bifunctional mechanism can reduce the Gibbs free energy of the EG oxidation reaction (EGOR) while inhibiting C / C bond breakage in the *OC-CH2OH intermediate (Li, Wenbo, et al. Metal-support interaction and tip-enhanced electric field effect co-enhancing oxygen species adsorption / enrichment for efficient electrooxidation of plastic waste derived alcohol into glycolic acid). Applied Catalysis B: Environmental , 374.000(2025). Although the bioenzymatic method has the advantages of high site selectivity and mild conditions, it lacks enzyme elements that can efficiently and specifically catalyze the incomplete oxidation of EG to GA, and the existing enzymes have insufficient activity and stability in complex depolymerization systems, which limits its industrial application.

[0004] In the prior art, such as the invention patent with publication number CN113337001A, a method for the combined degradation of polyethylene terephthalate by bacteria and enzymes is disclosed, using Klebsiella variegata SY1 ( Klebsiella variicola Using a cell suspension obtained through expanded culture (SY1) and Candida antarctica cutinase HiC as a catalyst, polyethylene terephthalate (PET) as a substrate, and a buffer solution with pH 7.0-9.0 as the reaction medium, a degradation reaction was carried out at 30-60℃ to degrade the polymer PET into monomeric compounds. The combined application of bacteria and enzymes relieved the product inhibition of the PET degradation enzymes, improving the degradation efficiency. However, this invention still has shortcomings in terms of degradation effect. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a highly efficient, low-cost, and selective biocatalytic method and dedicated enzyme system for converting EG from waste PET into GA.

[0006] The core of this invention lies in the fact that, for the first time, a species of *Acetobacter* possessing the ability to incompletely oxidize polyols (… Gluconobacter oxydans In the genome, a series of incomplete alcohol dehydrogenases (ADHs) with high activity and selectivity for EG, along with their matching aldehyde dehydrogenases (AldDHs), were discovered and obtained through a sequence-structure combined intelligent screening strategy. Furthermore, protein engineering techniques were used to rationally modify the enzymes, enhancing their activity and stability under industrial conditions, ultimately constructing a highly efficient dual-enzyme cascade catalytic system.

[0007] The technical solution of this invention is summarized as follows: 1. Discovery and identification of enzyme elements: From Gluconobacter oxydans Dehydrogenase sequences were retrieved from genomic databases. Candidate genes were narrowed down using methods such as primary structure stability prediction, CD-HIT clustering for redundancy removal, and phylogenetic tree analysis. Based on protein three-dimensional structure prediction and molecular docking simulation, candidate genes for alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) that may have catalytic activity against EG were screened. After heterologous expression, purification, and enzyme activity testing, active enzymes that specifically catalyze the oxidation of EG to hydroxyacetaldehyde (or directly to GA) were obtained.

[0008] Based on this, the present invention provides a mutant alcohol dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO. 1 derived from... Gluconobacter oxydans Based on the amino acid sequence of the wild-type alcohol dehydrogenase ADH5, it contains amino acid mutations selected from any one or more of the following groups: (a) Serine at position 97 is mutated to tyrosine, and isoleucine at position 101 is mutated to leucine; (b) The glutamic acid at position 145 is mutated to lysine, and the aspartic acid at position 148 is mutated to glutamic acid.

[0009] This invention also provides an aldehyde dehydrogenase (ALDH-V7, derived from an inhibitory modification) that has been modified to resist inhibition. Pseudomonas putida In previous work, similar computational strategies have been used to improve its specificity to hydroxyacetaldehyde and its tolerance to aromatic acids. The amino acid sequence is shown in SEQ ID NO. 2.

[0010] 2. Construction of a two-enzyme cascade system: Highly active ADH obtained through screening was combined with AldDH to construct a two-enzyme cascade catalytic reaction system of "EG → hydroxyacetaldehyde → GA". This system can operate under mild conditions (e.g., pH 7.5-8.5, temperature 30-37°C) with the assistance of NAD(P). + The / NAD(P)H cofactor cycle enables efficient and targeted conversion of EG to GA, with GA selectivity reaching over 90%.

[0011] Specifically, the present invention provides a two-enzyme cascade system for converting ethylene glycol to glycolic acid, comprising: (i) the mutant alcohol dehydrogenase of claim 1; and (ii) The mutant aldehyde dehydrogenase ALDH-V7 with the amino acid sequence shown in SEQ ID NO. 2.

[0012] First enzyme (ethylene glycol incomplete dehydrogenase): from Gluconobacter oxydans Selected from the genome, site mutations guided by computational simulation (such as substrate pocket hydrophobic residue substitution and flexible region rigidification) enhance the selective oxidation ability of EG, avoiding peroxidation or CC breakage.

[0013] The second enzyme (aldehyde dehydrogenase) matches the hydroxyacetaldehyde intermediate produced by the first enzyme and efficiently converts it into glycolic acid; it also undergoes anti-inhibition mutation (such as reducing affinity for MHET / TPA) and thermal stability enhancement (introduction of salt bridges / disulfide bonds).

[0014] Preferably, the mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 exist in the form of a mixture of free enzymes; Alternatively, the mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 may exist in the form of a gene fusion protein; Alternatively, the mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 may be co-immobilized on the same vector.

[0015] Furthermore, the carrier is a magnetic nanoparticle or a metal-organic framework material.

[0016] Furthermore, when present in the form of a gene fusion protein, the mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 are linked by a linker peptide.

[0017] 3. Rational Design and Modification of Enzymes: Addressing the issue of insufficient activity or stability of initially screened enzymes in complex systems (such as PET depolymerization solutions containing TPA, MHET, BHET, etc.), a computational simulation-guided site-saturation mutagenesis and iterative combined mutagenesis strategy is employed to modify the substrate-binding pocket and flexible regions of the enzyme protein. Interactions such as disulfide bonds, salt bridges, and hydrogen bonds are introduced or optimized to improve the enzyme's thermal stability, acid and alkali resistance, and resistance to substrate / product inhibition.

[0018] 4. Integrated Application Method: The optimized dual-enzyme system described above is directly applied to simulated or real waste PET depolymerization reaction solutions to achieve in-situ conversion of EG. Subsequently, high-purity GA products are separated and extracted from the reaction solution by calcium salt precipitation or ion exchange methods, and can be further used as monomers to prepare biodegradable plastic polyglycolic acid (PGA), forming a complete technical closed loop of "waste PET → depolymerization → EG → enzyme catalysis → GA → PGA".

[0019] Specifically, the present invention provides a method for converting ethylene glycol into glycolic acid, wherein ethylene glycol is contacted with the aforementioned dual-enzyme cascade system in a reaction system to convert ethylene glycol into glycolic acid.

[0020] Preferably, the pH of the reaction system is 7.5-8.5 and the temperature is 30-60℃.

[0021] Furthermore, the reaction system also contains NAD(P). + Cofactor or cofactor regeneration system.

[0022] The present invention also provides the application of the aforementioned dual-enzyme cascade system in the catalytic conversion of ethylene glycol in the depolymerization products of waste polyethylene terephthalate into glycolic acid.

[0023] This invention also provides a method for upgrading and recycling waste polyethylene terephthalate, comprising the following steps: (1) Waste polyethylene terephthalate is enzymatically hydrolyzed under the action of hydrolytic enzymes to generate a depolymerization solution containing terephthalic acid and ethylene glycol. (2) Add the dual-enzyme cascade system to the depolymerization solution to convert the ethylene glycol in the depolymerization solution into glycolic acid.

[0024] The hydrolytic enzyme can be FAST-PETase / MHETase. Together with a two-enzyme cascade system, it forms a three-enzyme total depolymerization-upgrading recovery system.

[0025] The beneficial effects of this invention are: 1. High value and green: Converting low-value component EG in waste PET into high-value chemical GA extends the PET recycling industry chain and provides a green and low-cost monomer source for bio-based biodegradable plastic PGA, which has both environmental benefits and economic value.

[0026] 2. High efficiency and specificity: Based on the specific dehydrogenase obtained by genome mining, it has excellent selective oxidation ability of EG hydroxyl groups, effectively avoiding side reactions such as peroxidation and C / C bond breakage, and the target product GA has high yield and selectivity.

[0027] 3. Mild conditions and stable system: Biocatalytic reactions operate under mild conditions with low energy consumption. Through rational modification of the enzyme, its catalytic activity and operational stability in real, complex depolymerization systems have been significantly improved, demonstrating its potential for industrial application.

[0028] 4. Closed-loop technology: This invention provides a complete technical path from waste plastics to high-value chemicals, providing an innovative "upgraded recycling" paradigm for the plastic circular economy. Attached Figure Description

[0029] Figure 1 Phylogenetic tree of candidate sequences for alcohol dehydrogenase (ADH). Detailed Implementation

[0030] Example 1 This embodiment is intended to be from Gluconobacter oxydans Dehydrogenases with ethylene glycol (EG) oxidation activity were systematically screened and validated in strain 621H, providing candidate enzyme elements for the subsequent construction of an efficient two-enzyme cascade system.

[0031] First, based on the publicly available whole genome sequence of this strain, combined with functional annotation and conserved domain analysis (such as Rossmann fold NAD(P)), + Based on motif, transmembrane region prediction, and transcriptome response data, 18 potential dehydrogenase encoding genes were initially screened; further, based on their expression upregulation under ethylene glycol culture conditions and phylogenetic relationships (…),… Figure 1 Ultimately, 10 high-potential candidate genes were selected and named ADH1 to ADH10.

[0032] Table 1: Sequence nouns in the developmental tree corresponding to ADH1-10 Subsequently, these genes were cloned into the pET-28a(+) vector (provided by Qingke Biotechnology Co., Ltd.), and recombinant plasmids with an N-terminal 6×His tag were constructed. E. coliExpression was induced at low temperature in BL21(DE3). After purification by Ni-NTA affinity chromatography, the NADH production rate was monitored spectrophotometrically at 340 nm to evaluate the catalytic activity of each recombinase for ethylene glycol.

[0033] As shown in Table 2, ADH3, ADH5, ADH7, and ADH9 exhibited significant activity, with ADH5 showing the highest activity at 18.3 ± 1.2 U / mg, and this activity was strictly dependent on NAD+. + The cofactor maintains good stability within the pH range of 7.5-8.5.

[0034] Table 2: ADH1-10 Activity Test Results To further confirm its reaction specificity, DNPH derivatization combined with GC-MS was used to analyze the ADH5 catalytic products. The results showed that it mainly selectively oxidizes ethylene glycol to hydroxyacetaldehyde, and no over-oxidation products (such as glyoxylic acid or oxalic acid) were detected, confirming that it has the ideal characteristics as a first-step catalyst for cascade reactions.

[0035] This embodiment successfully established a complete process from genome mining to functional verification, laying a key foundation for subsequent enzyme engineering and the construction of a directional conversion system for ethylene glycol in waste PET depolymerization solution.

[0036] Example 2 Based on the optimal candidate enzyme ADH5 (amino acid sequence shown in SEQ ID NO.1) obtained in Example 1, this study adopted a rational design strategy combining "PRODA (Protein Design Automation) + molecular dynamics (MD) simulation" to deeply optimize its structure and function, so as to improve its catalytic efficiency (kcat / Km) and thermal stability (Tm), thereby adapting to the high temperature and complex component reaction environment in the waste PET enzymatic hydrolysis system.

[0037] First, the three-dimensional structure of ADH5 was predicted using AlphaFold2, and high-throughput virtual mutation scanning of the active pocket and flexible region was performed using the PRODA platform, focusing on the following three types of sites: (1) residues in the substrate-binding pocket that may affect ethylene glycol orientation and affinity; (2) cofactor NAD +(2) Polar / aromatic residues near the interface can enhance electron transfer efficiency; (3) Sites in the flexible loop region of the surface can be rigidified by introducing salt bridges or proline to improve thermal stability. Based on energy scores, sequence conservation analysis and spatial accessibility assessment, five groups of high-potential single-point or combined mutation schemes were finally selected and named ADH5-M1 to ADH5-M5 respectively (where M1: F130W / L172K; M2: V89P; M3: E145K / D148E; M4: A201F; M5: S97Y / I101L).

[0038] Subsequently, the mutant gene was synthesized and cloned into the pET-28a(+) vector. E. coli The recombinant protein was expressed and purified in BL21(DE3) to obtain high-purity recombinant protein.

[0039] As shown in Table 3, the enzymatic performance tests revealed that all mutants maintained soluble expression, with ADH5-M3 and ADH5-M5 exhibiting significantly enhanced catalytic efficiency: ADH5-M5 achieved a kcat / Km of 12.6 s. -1 •mM -1 Compared to the wild type (5.8 s), -1 •mM -1 The affinity of ethylene glycol increased by about 2.2 times, mainly due to the decrease in Km value from 8.7 mM to 3.9 mM, indicating a significant enhancement in its affinity for ethylene glycol. ADH5-M3, on the other hand, showed outstanding performance in thermal stability. Its melting temperature (Tm) was determined to be 64.3°C by differential scanning fluorometry (DSF), which is more than 12°C higher than that of the wild type (52.1°C). This is attributed to the effective stabilization of the local secondary structure by the newly formed E145K-D148E salt bridge.

[0040] Table 3: Results of Enzyme Performance Tests Furthermore, MD simulations (100 ns) further validated the conformational stability of the mutants at 60°C: the wild type exhibited active pocket collapse in the later stages of the simulation, while M3 and M5 maintained a stable substrate channel and cofactor binding conformation throughout the simulation.

[0041] In summary, through computation-driven precise design, an ADH5 mutant with both high catalytic efficiency and high thermal stability was successfully obtained, providing a key enzyme element for constructing an efficient dual-enzyme cascade system suitable for actual PET depolymerization solutions.

[0042] Example 3 To verify the functional performance of the designed enzyme system in a scenario closely resembling actual waste PET recycling, this study constructed an enzyme system consisting of an optimized ethylene glycol dehydrogenase (ADH5-M5) and an anti-inhibition modified aldehyde dehydrogenase (ALDH-V7, derived from...). Pseudomonas putida The amino acid sequence is shown in SEQ ID NO.2. In previous work, a similar computational strategy was used to improve its specificity to hydroxyacetaldehyde and its tolerance to aromatic acids. The two-enzyme cascade system was used to evaluate its ability to efficiently convert ethylene glycol (EG) into the high-value-added product glycolic acid (GA) in a simulated PET enzymatic hydrolysate environment.

[0043] The simulated depolymerization solution was prepared according to the final system of a typical PET biodepolymerization reaction, containing 50 mM terephthalic acid (TPA), 20 mM mono(2-hydroxyethyl) terephthalate (MHET), 10 mM bis(2-hydroxyethyl) terephthalate (BHET), and 50 mM ethylene glycol, in 50 mM Tris-HCl buffer (pH 8.0) to realistically reproduce the potential inhibitory effect of complex components on enzyme activity after enzymatic hydrolysis of waste PET. The dual-enzyme system was added in free enzyme form at final concentrations of 0.5 mg / mL for ADH5-M5 and 0.4 mg / mL for ALDH-V7, and the reaction was carried out at 60°C and 200 rpm for 12 hours. Samples were taken periodically during the reaction, filtered through a 0.22 μm filter, and analyzed quantitatively for substrate consumption and product formation using high-performance liquid chromatography (HPLC) combined with differential refractive index and ultraviolet dual detectors.

[0044] As shown in Table 4, the two-enzyme system still efficiently drives the cascade reaction in this complex system: ethylene glycol is almost completely converted within 8 hours, and the final glycolic acid yield reaches 92.3 ± 2.1% (based on the initial molar amount of EG), which is significantly higher than the yield of the wild-type two-enzyme system alone (only 58.7%). Notably, even in the presence of high concentrations of MHET and TPA (known to non-competitively inhibit multiple dehydrogenases), the optimized ADH5-M5 and ALDH-V7 maintain stable activity, indicating that their anti-inhibition ability effectively improves the robustness of the system.

[0045] Table 4: Reaction efficiency and yield tests of wild-type and mutant two-enzyme systems Control experiments further confirmed that no significant GA accumulation could be detected if either enzyme component was omitted, indicating that the two-step reaction strictly depends on the synergistic effect of the two enzymes. Furthermore, no accumulation of hydroxyacetaldehyde was detected in the reaction solution (<0.1 mM), indicating that the oxidation rate of the second step was sufficient to match the formation rate of the first step, avoiding intermediate toxicity or side reactions.

[0046] This embodiment successfully demonstrates that the constructed dual-enzyme cascade system can achieve efficient and directional conversion of ethylene glycol to glycolic acid in a highly simulated PET depolymerization solution, providing key technical support for the integrated "depolymerization-upgrading" process of waste PET plastics.

[0047] Example 4 To achieve a one-step bio-upgrade recycling of waste PET plastics from solid-phase raw materials to high-value chemicals, this study integrates the aforementioned optimized dual-enzyme cascade system (ADH5-M5+ALDH-V7) with the commercially available high-performance PET hydrolase FAST-PETase (developed by Carbios or purified based on its publicly available sequence) to construct an integrated whole-enzyme catalytic system. Using untreated commercially available PET powder (intrinsic viscosity of approximately 0.6 dL / g and crystallinity of approximately 35%) as substrate, the system simultaneously completes PET depolymerization and directional conversion of ethylene glycol in a single reactor, with the final products being terephthalic acid (TPA) and glycolic acid (GA).

[0048] Specifically, the reaction system contained 50 mM Tris-HCl buffer (pH 8.0), 2% (w / v) PET powder, 0.8 mg / mL FAST-PETase, 0.5 mg / mL ADH5-M5 and 0.4 mg / mL ALDH-V7, supplemented with 1 mM NAD. + As a cofactor for dehydrogenase, the reaction was continued for 48 hours at 60°C and 250 rpm. To maintain cofactor regeneration, 0.2 U / mL of thermophilic formate dehydrogenase (FDH) and 20 mM formate were additionally added to the system to achieve NADH→NAD. + The reaction is cyclically regenerated to avoid cost limitations of cofactors. Samples are taken periodically during the reaction, and after centrifugation to remove insoluble residues, the supernatant is quantitatively analyzed using an HPLC-UV / RI system to determine changes in the concentrations of TPA, MHET, BHET, EG, and GA.

[0049] The results showed that FAST-PETase rapidly hydrolyzed PET within the first 12 hours, releasing a large amount of MHET and small amounts of TPA and EG. As the reaction proceeded, MHET was further hydrolyzed by FAST-PETase into TPA and EG, while the newly generated EG was immediately captured by the dual-enzyme cascade system and efficiently converted into GA. By the end of the 48-hour reaction, the weight loss rate of PET powder reached 89.5%, the TPA concentration in the liquid phase reached 42.3 mM (theoretical yield of approximately 85%), and the GA concentration reached 38.7 mM (yield of 82.1% based on the theoretically releaseable EG amount). No significant EG or hydroxyacetaldehyde residues were detected.

[0050] Control experiments showed that if any dehydrogenase component was omitted, EG accumulated in large quantities while GA was hardly generated; if only wild-type dehydrogenase was used, the GA yield dropped to less than 50%, and the enzyme activity was significantly reduced in the later stages of the reaction.

[0051] The results fully demonstrate that the three-enzyme integrated system constructed in this invention can achieve seamless coupling of "depolymerization-conversion" in a real PET solid-liquid two-phase system, producing two high-purity platform chemicals in one step (TPA can be used for recycled PET, and GA can be used to synthesize biodegradable polyglycolic acid PGA). This not only enhances the resource utilization value of waste PET, but also significantly simplifies the process flow, providing an innovative solution for the plastic circular economy that combines technical feasibility and economic potential.

Claims

1. A mutant alcohol dehydrogenase, characterized in that, Its amino acid sequence is derived from the sequence shown in SEQ ID NO.

1. Gluconobacteroxydans Based on the amino acid sequence of the wild-type alcohol dehydrogenase ADH5, it contains amino acid mutations selected from any one or more of the following groups: (a) Serine at position 97 is mutated to tyrosine, and isoleucine at position 101 is mutated to leucine; (b) The glutamic acid at position 145 is mutated to lysine, and the aspartic acid at position 148 is mutated to glutamic acid.

2. A two-enzyme cascade system for converting ethylene glycol to glycolic acid, characterized in that, Include: (i) the mutant alcohol dehydrogenase of claim 1; and (ii) The mutant aldehyde dehydrogenase ALDH-V7 with the amino acid sequence shown in SEQ ID NO.

2.

3. The dual-enzyme cascade system according to claim 2, characterized in that, The mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 exist in the form of a mixture of free enzymes; Alternatively, the mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 may exist in the form of a gene fusion protein; Alternatively, the mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 may be co-immobilized on the same vector.

4. The dual-enzyme cascade system according to claim 3, characterized in that, The carrier is a magnetic nanoparticle or a metal-organic framework material.

5. The dual-enzyme cascade system according to claim 3, characterized in that, When present as a gene fusion protein, the mutant alcohol dehydrogenase and the mutant aldehyde dehydrogenase ALDH-V7 are linked by a linker peptide.

6. A method for converting ethylene glycol into glycolic acid, characterized in that, In the reaction system, ethylene glycol is contacted with the dual-enzyme cascade system according to any one of claims 2-5, thereby converting ethylene glycol into glycolic acid.

7. The method according to claim 6, characterized in that, The reaction system has a pH of 7.5-8.5 and a temperature of 30-60℃.

8. The method according to claim 6 or 7, characterized in that, The reaction system also contains NAD(P). + Cofactor or cofactor regeneration system.

9. The application of the dual-enzyme cascade system according to any one of claims 2-5 in the catalytic conversion of ethylene glycol in the depolymerization product of waste polyethylene terephthalate to glycolic acid.

10. A method for upgrading and recycling waste polyethylene terephthalate, characterized in that, Includes the following steps: (1) Waste polyethylene terephthalate is enzymatically hydrolyzed under the action of hydrolytic enzymes to generate a depolymerization solution containing terephthalic acid and ethylene glycol. (2) Add the dual-enzyme cascade system according to any one of claims 2-5 to the depolymerization solution to convert the ethylene glycol in the depolymerization solution into glycolic acid.

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