A polyethylene terephthalate hydrolase pmPETase and application thereof

Abstract

This invention discloses a polyethylene terephthalate (PET) hydrolase, PmPETase, and its applications. The hydrolase PmPETase is a protein with the amino acid sequence shown in SEQ ID NO:1. This invention constructs an expression system suitable for PmPETase, obtains a recombinant enzyme protein with catalytic activity, and comprehensively analyzes its substrate specificity, catalytic properties, and potential plastic degradation ability. This provides a new theoretical basis and application reference for the development and utilization of PET degrading enzymes and green treatment technology for polymer materials.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis and plastic degradation technology, specifically relating to a polyethylene terephthalate hydrolase PmPETase and its application. Background Technology

[0002] Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester materials globally, extensively used in beverage bottles, synthetic fibers, food and daily chemical packaging, and its production and consumption continue to grow. Due to its high crystallinity and excellent chemical stability, PET is difficult to rapidly degrade by physical, chemical, or biological processes in the natural environment. Waste PET accumulates over time and gradually breaks down into smaller particles (less than 5 mm) of PET microplastics (PET-MPs). These microplastics are widely distributed in oceans, water bodies, soil, and sediments, and can migrate and persist in the environment for extended periods, posing a potential threat to ecosystem structure and function, as well as the health of individual organisms. Existing research indicates that PET microplastics can accumulate across multiple trophic levels through the food chain and may induce adverse effects such as cytotoxicity, endocrine disruption, chronic inflammation, and oxidative stress, gradually evolving into one of the important global environmental safety and human health issues.

[0003] Traditional treatment methods for PET pollution include landfill, incineration, and mechanical recycling. However, landfilling can lead to long-term environmental burdens, incineration releases greenhouse gases and harmful gases, and mechanical recycling is limited by the degree of pollution and material quality, making it difficult to achieve efficient, low-carbon, and high-value utilization. In contrast, enzyme- or microbial biodegradation strategies offer advantages such as mild conditions, low energy consumption, and environmental friendliness, and are considered a potential green approach to addressing PET, especially microplastic pollution. However, currently reported microbial-derived PET hydrolases still have significant shortcomings in terms of substrate adaptability, catalytic efficiency, and environmental stability, particularly in their limited ability to degrade highly crystalline PET.

[0004] Currently identified typical PET hydrolases include IsPETase from Ideonella sakaiensis, TfCut2 from Thermobifida fusca, and LCC (leaf and branch compost cutinase) from composting environments. These enzymes exhibit some activity in degrading low-crystallinity PET films or pretreated PET substrates, but their catalytic efficiency often decreases significantly with substrate crystallinity, resulting in insufficient degradation effects on high-crystallinity PET waste and aged PET microplastics in real-world environments. Furthermore, many enzymes exhibit decreased activity and stability in neutral aquatic environments or near-natural temperature, pH, and salinity conditions, making them difficult to apply directly to complex environmental systems or long-cycle reaction processes. These issues severely limit the widespread application of existing PET hydrolases in practical engineering remediation and industrial settings. Therefore, developing novel PET hydrolases from diverse sources, possessing higher hydrolytic capacity for high-crystallinity PET, and exhibiting good stability under mesophilic conditions, along with establishing heterologous expression systems capable of obtaining sufficient quantities of high-purity enzyme proteins, are crucial scientific and technological steps in promoting the application of PET biodegradation technology.

[0005] Marine environments are characterized by high salinity, intense sunlight, nutrient depletion, and the coexistence of multiple pollutants. Marine microbial communities exposed to plastic surfaces for extended periods may evolve unique polyester recognition and degradation capabilities during stress adaptation. Seawater-plastic interface biofilms and coastal sediments are considered important ecological niches for enriching microorganisms capable of plastic degradation. During the genome analysis of marine-derived *Priscilla auriculata* Jou-S14 (strain accession CGMCC No. 33815), this invention identified a gene, PmPETase, capable of degrading highly crystalline PET microplastics. Its amino acid sequence contains a typical α / β-hydrolase fold structure and a conserved catalytic triplet, Ser. 151 -Asp 222 -His 253 This suggests that it may possess interfacial ester hydrolysis activity. However, the amount of naturally secreted enzymes from environmental strains is limited, and purification is difficult, making it challenging to support systematic studies of their enzymatic properties and structural functions, and also hindering subsequent process scale-up and application evaluation. Therefore, it is necessary to construct an efficient heterologous expression system for PmPETase to obtain sufficient, soluble, and catalytically active recombinant enzyme protein, and to systematically characterize its optimal reaction conditions, pH and temperature adaptation range, structural characteristics, and degradation ability against PET-related substrates, particularly its hydrolytic potential for highly crystalline PET microplastics. Based on these needs, this invention constructed a PmPETase heterologous expression system derived from *Priscilla auriculi* Jou-S14, and systematically studied its enzymatic properties and catalytic characteristics, providing a new enzyme resource and technological foundation for the biodegradation of highly crystalline PET microplastics. Summary of the Invention

[0006] The background section mentions that existing PET microplastics, especially highly crystalline PET microplastics (hcPET-MPs), are difficult to degrade effectively in the natural environment due to their dense crystalline regions and limited effective exposed surface area, leading to persistent pollution over long periods. Although PET hydrolases such as IsPETase, LCC, and TfCut2 have been reported, they often suffer from low hydrolysis efficiency on highly crystalline PET substrates, decreased activity and stability under moderate temperature and neutral / weakly alkaline conditions, and insufficient production of naturally sourced enzymes, making it difficult to obtain large quantities of high-purity enzymes for degradation system construction and scale-up applications, thus failing to meet environmental remediation and engineering requirements. Therefore, this paper proposes a polyethylene terephthalate hydrolase, PmPETase, and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first objective of this invention is to provide a polyethylene terephthalate hydrolase, PmPETase, characterized in that: the hydrolase PmPETase is a protein having the amino acid sequence shown in SEQ ID NO:1.

[0009] Furthermore, the hydrolase PmPETase comprises Ser 151 -Asp 222 -His 253 The catalytic triplet is formed.

[0010] A second objective of this invention is to propose a nucleic acid molecule encoding polyethylene terephthalate hydrolase PmPETase, said nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:2.

[0011] A third objective of this invention is to provide a recombinant expression vector containing nucleic acid molecules.

[0012] The fourth objective of this invention is to provide a recombinant engineered bacterium, characterized in that the recombinant engineered bacterium contains a recombinant expression vector.

[0013] A fifth objective of this invention is to provide a degradation agent for polyethylene terephthalate, comprising the hydrolytic enzyme PmPETase.

[0014] The sixth object of the present invention is to propose the application of hydrolytic enzyme PmPETase or degrading agent in the degradation of polyethylene terephthalate products.

[0015] Furthermore, the hydrolase PmPETase reached its highest relative enzyme activity of 22.75 U / mL at pH 8.0 and 35 °C.

[0016] The seventh object of the present invention is to provide a method for degrading polyethylene terephthalate by contacting polyethylene terephthalate with a hydrolytic enzyme PmPETase or a degrading agent.

[0017] The above technical solution can achieve the following beneficial effects:

[0018] This invention relates to a novel PET hydrolase, PmPETase (GenBank: PX379572.1), and its heterologous expression system derived from *Priscilla auriculi* Jou-S14 (strain accession number CGMCC No. 33815). This enzyme contains Ser... 151 -Asp 222 -His 253 The enzyme catalyzes the triplet and binds stably to BHET. Its pNPB activity is 22.75 U / mL at pH 8.0 and 35 °C, and it maintains high activity within the pH 5.0-10.0 and 25-50 °C range. After treating hcPET-MPs with purified PmPETase for 24 hours at pH 8.0 and 35 °C, TPA 8.49 μM and BHET 1.75 μM were detected. Simultaneously, the enzyme exhibits dual BHETase / MHETase activity, hydrolyzing 60% of BHET within 24 hours to generate MHET and TPA. Therefore, this invention specifically addresses the following issues: (1) the problem that existing enzymes are "insufficiently active and have poor stability" under mild conditions, which prevents them from continuously hydrolyzing PET model substrates in a medium-temperature neutral / weakly alkaline environment; (2) the problem that natural bacteria produce insufficient enzymes directly and it is difficult to prepare sufficient high-purity enzymes for system evaluation and process scale-up; (3) the lack of core enzymes with dual BHETase / MHETase activity in existing systems, which makes intermediate products such as BHET and MHET easy to accumulate and incompletely convert, resulting in insufficient TPA generation and low yield; and (4) the lack of core enzyme tools that can be used to construct a degradation system for highly crystalline PET microplastics, providing a directly applicable enzymatic basis and technical path for the subsequent green treatment of highly crystalline PET microplastics. Attached Figure Description

[0019] Figure 1 This is a phylogenetic tree of the PmPETase gene, where family I: IsPETase-type PET hydrolase; family II: keratinase-type PET hydrolase; * indicates the gene sequence of PmPETase.

[0020] Figure 2This is the result of PmPETase multiple sequence alignment.

[0021] Figure 3 (A) Secondary structure of PmPETase; (B) Tertiary structure of PmPETase.

[0022] Figure 4 It is a molecular docking of PmPETase with BHET.

[0023] Figure 5 (A) Schematic diagram of the structure of PmPETase-pET-28a (+); (B) Agarose gel electrophoresis of PmPETase gene digested with EcoRI / XhoI (M: DL5000 DNA marker; Lane 1: PmPETase gene digested with EcoRI / XhoI); (C) pET-28a (+) vector digested with EcoRI / XhoI (M: D2000 plus DNA marker; Lane 1: pET-28a (+) vector digested with EcoRI / XhoI); (D) PCR verification diagram of PmPETase-pET-28a (+) transformed into E. coli Rosetta (DE3).

[0024] Figure 6 This is the SDS-PAGE result of PmPETase-pET28a (+) - E. coli Rosetta (DE3) (electrophoresis performed in 12% Tirs-glycine buffer). Lane M: molecular weight marker protein (12-250 kDa); Lane 1: supernatant of uninduced cell lysate; Lane 2: supernatant of induced cell lysate; Lanes 3 and 4: purified PmPETase.

[0025] Figure 7 This is the protein curve of PmPETase purified by AKTA.

[0026] Figure 8 This is the standard curve of pNP under absorbance conditions of 405nm.

[0027] Figure 9 It represents the relative enzyme activity of PmPETase at pH 5.0-10.0.

[0028] Figure 10 It represents the relative enzyme activity of PmPETase at temperatures of 25-50 °C.

[0029] Figure 11(A) The state of PmPETase after 24 hours of degradation of hcPET-MPs (left: control; right: PmPETase); (B) The state of PmPETase after 24 hours of degradation of BHET (left: control; right: PmPETase).

[0030] Figure 12 (A) Content of BHET and TPA released after PmPETase degrades hcPET-MPs for 24 hours; (B) HPLC chromatogram of the degradation products after PmPETase degrades hcPET-MPs for 24 hours; (C) Content and conversion rate of BHET after PmPETase degrades for 24 hours; (D) HPLC chromatogram of BHET after PmPETase degrades for 24 hours.

[0031] Figure 13 (A) HPLC standard curve of BHET (0-400 μM); (B) HPLC chromatogram of BHET (50 μM).

[0032] Figure 14 (A) HPLC standard curve of TPA (0-400 μM); (B) HPLC chromatogram of TPA (50 μM). Detailed Implementation

[0033] The following examples and appendices Figure 1-14 Further explanation of the present invention:

[0034] This embodiment discloses a novel PET hydrolase, PmPETase (GenBank: PX379572.1), derived from *Priscilla argentea* Jou-S14. The hydrolase PmPETase is a protein with the amino acid sequence shown in SEQ ID NO:1, which is as follows: VQIKKDERQSFYAGCQFVDIEDQENEHVFPLLIMYPTYKEETVEKIGPYT

[0035] ISVAQDAPLSDGLFPLVIIISHGDGSTPFAYRTIAQFLARHGFIVGIPQHPFNNRENNTLSGTIGNLKNRPTHIRTVIDWFLKESSFSPSIKSNNISLIGHSMGGYTALAVAGGVPTSFPSESPDQKPYCLSVDHDKRVQSLILLAPATGWFRERGALEDVNIPILMITGEKDTITPSFHGEFVLNGVSDAERVQHIVIENGGHFSFLSPFPDFMKSPTFLPSQDPEGFNRKEFHEDLQNTILDFLLRSF.

[0036] The nucleotide sequence of its coding gene is shown in SEQ ID NO: 2, and the SEQ ID NO: 2 sequence is as follows: GTGCAAATAAAAAAAGATGAAAGACAGTCATTTTATGCCGGATGTCAATTCGTAGATATAGAAGATCAAGAAAATGAACACGTCTTTCCTCTGCTTATTATGTATCCTACATATAAAGAAGAAACAGTTGAAAAGATCGGACCTTACACGATAAGCGTTGCGCAAGATGCTCCGCTTTCAGATGGATTATTTCCTTTGGTCATTATTTCTCATGGAGATGGAAGCACACCTTTTGCTTATCGAACAATTGCTCAGTTTTTAGCACGTCATGGTTTTATTGTAGGAATACCTCAGCATCCGTTTAATAATCGAGAGAATAATACGCTATCTGGAACAATAGGTAATCTAAAAAATCGGCCTACCCATATACGTACTGTAATTGATTGGTTTTTGAAAGAAAGTTCCTTTTCTCCATCCATTAAATCAAATAATATTTCTCTTATTGGCCATTCAATGGGAGGATATACGGCTCTTGCTGTAGCAGGTGGAGTTCCCACTTCTTTTCCTTCTGAATCTCCTGATCAAAAGCCTTATTGTTTATCTGTTGATCATGATAAGCGGGTTCAATCACTTATTCTTTTAGCGCCAGCCACCGGATGGTTCAGAGAAAGAGGAGCTCTTGAAGACGTGAATATACCTATTTTAATGATTACAGGCGAAAAGGATACGATTACTCCTTCTTTTCACGGGGAATTTGTATTAAATGGGGTTTCTGACGCAGAAAGAGTTCAGCATATAGTTATCGAAAATGGAGGGCATTTTTCATTTCTCAGCCCATTTCCCGACTTTATGAAATCTCCTACCTTCCTCCCTTCTCAGGATCCGGAAGGCTTTAATCGCAAAGAGTTTCATGAGGACCTGCAAAACACTATATTGGACTTTTTATTACGTTCTTTTTAA。

[0037] In existing technologies, enzymes related to PET degradation mainly include: PETases (Family I) represented by IsPETase from Ideonella sakaiensis, cutinases (such as TfCut2) from Thermobifida, and leaf-branch compost cutinases (Family II), as well as highly active variants obtained through rational design or directed evolution based on the above frameworks (such as engineered PETase / LCC variants commonly seen in the literature). In general, the efficient degradation of existing PET hydrolysis systems often relies on two preconditions: firstly, increasing the reaction temperature to enhance the mobility of PET chain segments (especially crucial for highly crystalline PET); and secondly, reducing crystallinity and increasing the proportion of amorphous regions through pretreatment with alkali / heat / solvents, thereby improving the enzyme's accessibility to the substrate. For highly crystalline PET microplastics (hcPET-MPs), due to the dense crystalline regions, low chain segment accessibility, and limited enzyme adsorption and entry interfaces, even various reported PETases or keratinases usually face the problem of limited degradation efficiency under mild temperatures (such as 30-40 °C) and without significant pretreatment. However, increasing the temperature or using strong pretreatment will lead to problems such as increased energy consumption, process complexity, increased equipment tolerance requirements, or potential secondary environmental burden.

[0038] Compared with the existing technologies, the advantages of PmPETase in this invention lie in its ability to effectively act on highly crystalline substrates and promote the formation of the final product even under mild conditions. At pH 8.0 and 35 °C, PmPETase can produce a detectable hydrolytic effect on hcPET-MPs and release TPA (8.49 μM) and BHET (1.75 μM), indicating that it still possesses interfacial hydrolytic ability for highly crystalline PET without relying on high temperatures or significant reduction in crystallinity pretreatment. More importantly, many existing PET hydrolases mainly exhibit initial cleavage ability of the PET backbone, and the accumulation of soluble intermediates such as BHET and MHET during the reaction process easily leads to a conversion bottleneck between "releasing intermediates" and "obtaining the final product TPA," thereby limiting the yield of the final product and the sustainability of the process. This invention, PmPETase, possesses dual activity as both BHETase and MHETase, enabling continuous cleavage and transformation of oligomers / intermediates generated from PET hydrolysis. Within 24 hours, it can degrade approximately 60% of BHET, generating MHET and TPA. This demonstrates that it not only initiates surface hydrolysis of highly crystalline PET but also simultaneously promotes the transformation of intermediates towards the final product, reducing intermediate accumulation and promoting the continuous generation of TPA. Therefore, this invention overcomes the common limitations of existing PET hydrolases—insufficient activity / stability under mild conditions, poor accessibility to highly crystalline PET, and low final product yield due to incomplete intermediate transformation—through its three differentiating characteristics: usability under mild conditions, degradability under high crystallinity, and sustainable intermediate transformation. It offers comprehensive advantages for practical environmental remediation and mild process scale-up.

[0039] Example: (1) Materials and methods:

[0040] Highly crystalline PET microplastics (hcPET-MPs) (crystallinity 52 ± 3%, melting point 250 ± 5 ℃, average molecular weight 50,000 Da, density in the range of 1.401-1.446 g / cm³) were purchased from China Huachuang Plastics Co., Ltd.; terephthalic acid (TPA) and bis(2-hydroxyethyl) terephthalate (BHET) were purchased from Beijing Puxitang Biotechnology Co., Ltd.; p-nitrophenol (pNP) and p-nitrophenyl butyrate (pNPB) were purchased from Shanghai Maclean Biochemical Co., Ltd. All other reagents and solvents (purity >98%) were provided by Sangon Biotech (Shanghai) Co., Ltd.

[0041] (2) Three-dimensional structure and molecular docking analysis of PmPETase

[0042] Based on gene annotation results, the focus was on screening for carboxylesterase (CES) families related to ester bond hydrolysis. GC content statistics and open reading frame (ORF) prediction were performed on the target DNA sequence to determine start and stop codons and translate the protein. Molecular weight, isoelectric point (pI), and total average hydrophobicity index (GRAVY) were calculated, and transmembrane helix, signal peptide, and subcellular localization tendency were predicted. Further multiple sequence alignment analysis was performed using Clustal X 2.1 and ESPript 3.0, and a phylogenetic tree was constructed using MEGA 6.0 to analyze the homology of this gene with previously reported lipid hydrolases. To verify its catalytic properties, the three-dimensional structure of PmPETase predicted by AlphaFold3 was used, and molecular docking with the substrate BHET (PubChem CID: 13739) was performed using AutoDock Vina.

[0043] (3) Construction and protein expression of PmPETase-pET28a (+)

[0044] Using the genomic DNA of marine-derived Jou-S14 strain as a template, primers for amplifying the PmPETase gene were designed:

[0045] PmS14-F (5'-GATCCGAATTCAAATAAAAAAAGATGAAAGACAGTC-3'),

[0046] PmS14-R (5'-TGGTGCTCGAGAAAAGAACGTAATAAAAAGTCCAA-3').

[0047] Subsequently, the PmPETase fragment and the vector pET-28a(+) were ligated after double digestion with Xho I and EcoRI restriction endonucleases to construct the recombinant plasmid PmPETase-pET28a(+). The recombinant plasmid was transformed into E. coli Rosetta(DE3) and cultured at OD500. 600 After reaching the optimal value, 0.1 mM IPTG was added, and expression was induced at 16 ℃ for 12 hours. After induction, the bacterial cells were collected, sonicated, and centrifuged to remove cell debris, yielding a supernatant containing soluble protein. The supernatant was purified by affinity chromatography using the AKTA system to obtain purified PmPETase protein.

[0048] (4) pNPB colorimetric method for detecting universal esterase activity

[0049] Using p-nitrophenylbutyrate (pNPB) as a substrate, the absorbance of its hydrolysis product, p-nitrophenol (pNP), was measured at 405 nm. Esterase activity was determined using the pNPB colorimetric method, and the optimal pH and temperature for the enzyme were determined accordingly. Standard solutions of different concentrations of pNP were prepared and standard curves were plotted to calculate the linear relationship between absorbance and enzyme activity, ensuring the accuracy and comparability of the results. Enzyme activity (U) was defined as the amount of enzyme released per minute (1 μmol pNP) per milliliter of reaction system at 35 °C and pH 8.0, which is 1 U (U / mL).

[0050] (5) HPLC quantitative method for the detection of hcPET-MPs degradation products

[0051] The purified PmPETase enzyme solution was added to potassium phosphate buffer containing hcPET-MPs (the reaction system consisted of 90 mM inorganic salt solution, 10 g / L hcPET-MPs (or 0.3 mM BHET), 10 mM CaCl2, 0.1% Tween-80, and 5% glycerol), and reacted at 35 ℃ for 24 hours. After the reaction, the sample was centrifuged at high speed to remove solid particles and filtered through a 0.22 μm filter membrane to obtain a clear supernatant. The degradation products were then quantitatively analyzed using HPLC (Zorbax SB-C18, 4.6 × 250 mm, 5 μm, Agilent). The mobile phase was a mixture of acetonitrile and 0.1% formic acid, the column temperature was set at 40 ℃, the flow rate was 1 mL / min, and the detection wavelength was 254 nm. By preparing TPA and BHET standard solutions and plotting standard curves, the HPLC peak area was converted into product concentration, enabling quantitative analysis of the degradation efficiency of hcPET-MPs. This method directly reflects the catalytic ability of PmPETase in the actual degradation of highly crystalline PET microplastics and is corroborated with the results of the pNPB colorimetric method, thus comprehensively revealing its enzymatic characteristics.

[0052] Experimental results:

[0053] (1) Results of PETase mining

[0054] A novel carboxylesterase (CE) has been identified in the carboxylesterase (CE) family derived from *Priestia aryabhattai* Jou-S14 (this strain was deposited on March 14, 2025, at the China General Microbiological Culture Collection Center, accession number CGMCC No. 33815) (GenBank: PX379572.1). VQIKKDERQSFYAGCQFVDIEDQENEHVFPLLIMYPTYKEETVEKIGPYTISVAQDAPLSDGLFPLVIISHGDGSTPFAYRTIAQFLARHGFIVGIPQHPFNNRENNTLSGTIGNLKNRPTHIRTVIDWFLKESSFSSPIKSNNISLIGH SMGGYTALAVAGGVPTSFPSESPDQKPYCLSVDHDKRVQSLILLAPATGWFRERGALEDVNIPILMITGEKDTITPSFHGEFVLNGVSDAERVQHIVIENGGHFSFLSPFPDFMKSPTFLPSQDPEGFNRKEFHEDLQNTILDFLLRSF. ), named PmPETase. The length is 900 bp, and the GC content of its full sequence is approximately 38.0%. Open reading frame (ORF) prediction analysis revealed a major coding region on the +1 reading frame of the positive chain: its start codon is ATG (located at position 100, 1-based), and its stop codon is TAA (located at positions 898-900), forming an ORF length of 801 bp (including the stop codon), which can be translated into a protein of 266 amino acids (aa). Further prediction of the protein's physicochemical properties and subcellular characteristics showed that its theoretical molecular weight is approximately 29.52 kDa, and its theoretical isoelectric point (pI) is approximately 5.65; its overall average hydrophobicity (GRAVY) is approximately -0.20, indicating that the protein is generally hydrophilic and has a tendency to form soluble proteins; no typical continuous long hydrophobic fragments were detected at its N-terminus, nor were obvious transmembrane helical features observed, suggesting that this protein is not a membrane-integrated protein; at the same time, its N-terminus does not exhibit the typical hydrophobic core structure of a signal peptide, suggesting that it is less likely to be a classic secretory protein.

[0055] Based on the above predictions, the protein is more likely to exist as a non-membrane-bound, intracellularly soluble protein, with its subcellular localization predominantly in the cytoplasm. Furthermore, sequence feature identification based on the translated protein sequence revealed a GXSXG motif near amino acid position 151, associated with α / β-hydrolase folding, with a GHSMGG fragment visible at positions 149-154. Simultaneously, the protein contains serine, aspartic acid, and histidine residues at positions 151, 222, and 253, respectively, constituting a Ser... 151 -Asp 222 -His 253 Catalytic triplet.

[0056] Furthermore, phylogenetic analysis using known PET hydrolases (such as IsPETase, LCC, and TfCut2) revealed that PmPETase belongs to family I and is an IsPETase-type PET hydrolase (see...). Figure 1 ).

[0057] (2) Three-dimensional structure and molecular docking analysis results of PmPETase

[0058] Multiple sequence alignment results indicate that PmPETase contains a conserved GXSXG motif, with the GHSMGG fragment visible at amino acid positions 149-154, and possesses Ser... 151 Asp 222 and His 253 The typical catalytic triplet (see) Figure 2 ).

[0059] Structural predictions based on AlphaFold3 show that PmPETase has a typical α / β-hydrolase fold structure, consisting of 10 α-helices and 10 β-sheets (see [link to AlphaFold3]). Figure 3 ).

[0060] Molecular docking analysis showed that the substrate BHET could stably bind near the catalytic center of PmPETase and interact with Ser. 151 His 253 Asp 222 Key amino acid residues form hydrogen bonds and hydrophobic interactions, thereby stabilizing a substrate conformation favorable for ester bond cleavage. Further structural analysis shows that the distances between the residues at the three sites are all less than 5.0 Å, synergistically forming a stable catalytic core, among which Ser... 151 His acts as a nucleophilic group to attack the carbonyl carbon atom of the ester bond. 253 Responsible for mediating proton transfer, Asp 222 Then used to stabilize His253 The protonated state provides the structural basis for the ester bond hydrolysis reaction of PmPETase (see...). Figure 4 ).

[0061] (3) Results of heterologous expression of PmPETase

[0062] As shown in the schematic diagram of the structure of PmPETase-pET-28a (+) (see...) Figure 5 A), after Xho I / EcoRI digestion (see...) Figure 5 The PmPETase gene was successfully ligated into the pET-28a (+) vector. After transforming the ligation product into E. coli Rosetta (DE3), a specific band of approximately 900 bp was obtained by colony PCR, consistent with the theoretical fragment size, indicating that the PmPETase gene had been correctly inserted and successfully transformed into the host bacterium (see BC). Figure 5 D).

[0063] Subsequently, SDS-PAGE analysis of the transformed bacterial expression product revealed a soluble protein band of approximately 37 kDa, whose molecular weight was consistent with the predicted value of the PmPETase amino acid sequence (see [link to analysis]). Figure 6 ).

[0064] Further affinity chromatography purification using the AKTA system yielded a single elution peak (see...). Figure 7 The study successfully obtained high-purity PmPETase protein, indicating that the enzyme was solublely expressed and efficiently purified in E. coli Rosetta (DE3).

[0065] (4) Validation results of PmPETase esterase activity (pNP)

[0066] The accuracy of activity calculations is ensured by using a pNP standard curve (see [link]). Figure 8 );

[0067] The purified PmPETase exhibited significant hydrolytic activity against the substrate pNPB, verifying its stable catalytic function. In the optimal condition screening experiment, PmPETase achieved its highest relative enzyme activity (22.75 U / mL) at pH 8.0 and 35 °C, demonstrating excellent catalytic efficiency. Further evaluation of pH adaptability showed that PmPETase maintained significant activity within the pH range of 5.0–10.0, exhibiting the most stable and efficient performance under weakly alkaline conditions (pH 7.0–9.0) (see [link to relevant documentation]). Figure 9 ).

[0068] Temperature optimization showed that PmPETase maintained its activity within the range of 25-50 °C, with the highest activity and best stability observed in the 30-40 °C range (see [link to product description]). Figure 10 The activity characteristics of this enzyme under medium temperature and weakly alkaline conditions indicate that PmPETase is not only widely adaptable, but also maintains high catalytic activity in mild environments.

[0069] (5) HPLC analysis results of PmPETase-catalyzed hcPET-MPs products

[0070] After the purified PmPETase enzyme solution reacted with hcPET-MPs for 24 hours, the reaction solution became cloudy and produced a yellow suspension (see...). Figure 11 A).

[0071] HPLC results showed significant accumulation of BHET and TPA products (see...). Figure 12 A), HPLC chromatograms showed characteristic peaks of BHET and TPA in the reaction solution, confirming the formation of hcPET-MPs degradation products (see A). Figure 12 B). Based on the standard curves of BHET and TPA (see...). Figure 13-14 The results showed that the contents of TPA and BHET were 8.49 μM and 1.75 μM, respectively, while the Jou-S14 strain produced only 9.94 μM and 2.30 μM after 30 days.

[0072] Furthermore, when the intermediate product BHET is used as a substrate, the phenomenon is similar to that when hcPET-MPs is used as a substrate, the reaction system gradually changes from clear to turbid (see...). Figure 12 B) indicates that insoluble / microscopic products were generated or product precipitation occurred during enzymatic hydrolysis. After 24 hours of reaction, the BHET content decreased by 60% compared to the initial value, while the TPA characteristic peak simultaneously increased (see [link to reaction]). Figure 12 The CD indicates that PmPETase can continuously transform intermediates in the PET degradation process and promote the accumulation of final products, confirming that it has the catalytic activity of both BHETase and MHETase, thereby reducing the accumulation of intermediate products, improving the efficiency of subsequent hydrolysis and promoting TPA generation.

[0073] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

Claims

1. The application of the hydrolase PmPETase in the degradation of polyethylene terephthalate products, characterized in that, hydrolytic enzymes PmPETase Application in degradable polyethylene terephthalate products ， hydrolytic enzymes Pm PETase is the protein shown in SEQ ID NO:1, a hydrolytic enzyme. Pm PETase reaches its highest relative enzyme activity of 22.75 U / mL at pH 8.0 and 35 °C.

2. The application of the hydrolytic enzyme PmPETase according to claim 1 in the degradation of polyethylene terephthalate products, characterized in that: The method of use is to use the hydrolytic enzyme. PmPETase Contact with polyethylene terephthalate.

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