A heat-resistant plastic hydrolase mutant and its application

Abstract

The present application discloses a heat-resistant plastic hydrolase mutant and its application. In particular, the present invention provides a heat-resistant plastic hydrolase ThPETase mutant and its application. Specifically, the present invention is to synthesize a heat-resistant plastic hydrolase mutant derived from a thermophilic microorganism. Thermobifida halotolerant The mutant Th_DCTPC (amino acid sequence shown in SEQ ID NO: 1) of the protein ThPETase (GeneBank ID: WP_243597587.1) was further mutated to produce a mutant with significantly improved enzyme activity and thermal stability. The present invention also includes methods for producing the aforementioned heat-resistant plastic hydrolase mutant and its use in degrading PET or PBAT plastics.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a heat-resistant plastic hydrolase mutant and application thereof. Background Art

[0002] Plastic pollution has become a pressing global environmental challenge, garnering widespread attention and significant public concern. Among the many environmentally friendly materials currently available, polybutylene adipate / terephthalate (PBAT) is emerging as a key breakthrough in combating white pollution due to its biodegradable properties. PBAT, a copolymer of 1,4-butanediol, adipic acid, and terephthalic acid, boasts excellent ductility and biocompatibility due to its unique molecular structure. Its excellent processing properties make it particularly advantageous in the packaging industry, particularly in food-grade packaging films, agricultural mulch films, and biodegradable tableware.

[0003] PET (polyethylene terephthalate) is a very common plastic. PET plastic boasts high strength, excellent transparency, and excellent chemical stability, making it widely used in beverage bottles, electronic product casings, packaging materials, and many other fields. However, PET plastic is difficult to degrade naturally and can persist in the environment for decades or even centuries. The large amount of PET plastic waste has become a major source of plastic pollution.

[0004] Studies have found that the number of microbial populations in nature that have the ability to hydrolyze PET and PBAT is limited. This lack of biodegradation efficiency has made the development of efficient PET and PBAT hydrolase systems a key area that needs to be tackled in the field of environmental biotechnology. Chinese patent ZL202411540951.8 (authorization announcement number CN119040295B) discloses ThPETase (derived from thermophilic microorganisms) Thermobifida halotolerans , GeneBank ID: WP_243597587.1) and Th_DCTPC (ThPETase mutant, amino acid sequence shown in SEQ ID NO: 1) have certain PET and PBAT degradation capabilities, but their hydrolysis efficiency at high temperatures (50-70°C) is limited, restricting their potential for application under harsh industrial conditions. Therefore, there is still a need for high-temperature-resistant plastic hydrolases. Summary of the Invention

[0005] In order to solve the deficiencies in the prior art, the present invention provides a heat-resistant plastic hydrolase mutant and its application, in particular a heat-resistant plastic hydrolase ThPETase mutant and its application. Specifically, the present invention is to Thermobifida halotoleransA mutant of the protein ThPETase (GeneBank ID: WP_243597587.1), Th_DCTPC (amino acid sequence shown in SEQ ID NO: 1), was further mutated to produce a mutant with significantly improved enzyme activity and thermal stability. The present invention also includes methods for producing the aforementioned heat-resistant plastic hydrolase mutant and its use in degrading PET or PBAT plastics.

[0006] Specifically, the present invention includes but is not limited to the following technical solutions:

[0007] In one aspect, the present invention provides a mutant of a plastic hydrolase ThPETase, wherein the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1 at a position selected from positions 68, 105, 194 and 232:

[0008] (1) S68K (i.e., replacing serine at position 68 with lysine);

[0009] (2) D105R (i.e., aspartic acid at position 105 is replaced by arginine);

[0010] (3) G194Q (i.e., glycine at position 194 is replaced by glutamine);

[0011] (4) D232K (i.e., substitution of aspartic acid at position 232 with lysine); or

[0012] (5) S68K+D105R+G194Q+D232K (i.e., replacing serine at position 68 with lysine, aspartic acid at position 105 with arginine, glycine at position 194 with glutamine, and aspartic acid at position 232 with lysine).

[0013] In one aspect, the plastic hydrolase ThPETase mutant of the present invention is obtained by performing the following mutation on position 68 of the amino acid sequence shown in SEQ ID NO: 1: S68K (ie, replacing the serine at position 68 with lysine).

[0014] In one aspect, the plastic hydrolase ThPETase mutant of the present invention is obtained by performing the following mutation on position 105 of the amino acid sequence shown in SEQ ID NO: 1: D105R (ie, replacing aspartic acid at position 105 with arginine).

[0015] In one aspect, the plastic hydrolase ThPETase mutant of the present invention is obtained by performing the following mutation on position 194 of the amino acid sequence shown in SEQ ID NO: 1: G194Q (ie, replacing glycine at position 194 with glutamine).

[0016] In one aspect, the plastic hydrolase ThPETase mutant of the present invention is obtained by performing the following mutation on position 232 of the amino acid sequence shown in SEQ ID NO: 1: D232K (ie, replacing aspartic acid at position 232 with lysine).

[0017] In one aspect, the plastic hydrolase ThPETase mutant described in the present invention is obtained by performing the following mutations on positions 68, 105, 194 and 232 of the amino acid sequence shown in SEQ ID NO: 1: S68K+D105R+G194Q+D232K (i.e., replacing the serine at position 68 with lysine, the aspartic acid at position 105 with arginine, the glycine at position 194 with glutamine and the aspartic acid at position 232 with lysine).

[0018] On the one hand, the present invention also provides a plastic hydrolase ThPETase mutant, which is obtained by performing the following mutations on positions 68, 105, 194 and 232 of the amino acid sequence shown in SEQ ID NO: 1: S68K+D105R+G194Q+D232K (i.e., replacing the serine at position 68 with lysine, the aspartic acid at position 105 with arginine, the glycine at position 194 with glutamine and the aspartic acid at position 232 with lysine).

[0019] In another aspect, the present invention provides a polynucleotide encoding the plastic hydrolase ThPETase mutant of the present invention.

[0020] In another aspect, the present invention also provides a recombinant vector comprising the polynucleotide of the present invention.

[0021] In one aspect, the present invention provides a host cell comprising the polynucleotide of the present invention or the recombinant vector of the present invention.

[0022] In another aspect, the present application provides the use of the plastic hydrolase ThPETase mutant described in the present invention or the host cell described in the present invention in degrading PET or PBAT plastics.

[0023] In one aspect, the application of the present invention comprises contacting an object containing PET and / or PBAT plastic with the plastic hydrolase ThPETase mutant to perform a degradation reaction. Preferably, the object containing PET and / or PBAT plastic according to the present invention is PET or PBAT plastic powder or PET or PBAT plastic film. More preferably, the object containing PET and / or PBAT plastic according to the present invention is PET or PBAT plastic powder. Most preferably, the PBAT plastic powder according to the present invention is PBAT powder with a particle size of 100 mesh. Most preferably, the PET plastic powder according to the present invention is PET powder with a particle size of 1000 mesh.

[0024] In one aspect, in the application of the present invention, based on PBAT plastic, the addition amount of the plastic hydrolase ThPETase mutant is: 10 μg / mL-200 μg / mL, calculated as a mass percentage; the reaction temperature is: 37-70°C; the reaction time is: 1 h-7 d; and the solution pH is: 7.0-10.0.

[0025] In one aspect, the contacting of the object containing PET and / or PBAT plastic with the plastic hydrolase ThPETase mutant according to the present invention occurs in the presence of Tris-HCl buffer or potassium dihydrogen phosphate-NaOH buffer solution.

[0026] In another aspect, the present application provides a method for producing the plastic hydrolase ThPETase mutant of the present invention, the method comprising:

[0027] (a) culturing the host cell of the present invention under conditions suitable for the expression of the plastic hydrolase ThPETase mutant;

[0028] (b) Recovering the plastic hydrolase ThPETase mutant.

[0029] In one aspect, the host cell of the present invention is a fungal cell, a bacterial cell or a plant cell.

[0030] In another aspect, the host cell of the present invention is a bacterial cell, preferably, the bacterial cell is an Escherichia coli cell. In one aspect, the Escherichia coli cell is preferably an Escherichia coli BL21 (DE3) cell.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention molecularly modifies a mutant of ThPETase, Th_DCTPC, resulting in a mutant that not only exhibits improved enzymatic activity but also significantly enhanced heat resistance. Specifically, while Th_DCTPC exhibits low hydrolysis activity against PET and PBAT at high temperatures, the mutant (specifically, DCTPC_4M) obtained in this invention exhibits significantly enhanced hydrolysis of PET and PBAT at high temperatures (e.g., 70°C), increasing these activities by 2.0-fold and 2.3-fold, respectively, suggesting significant potential for industrial application.

[0033] In this application, the amino acid sequence of Th_DCTPC is shown in SEQ ID NO.1, which is derived from thermophilic microorganisms. Thermobifida halotolerans The protein amino acid sequence (ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTNETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGH SMGGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELDGASHFAPNLSNTTIAKYSISWLKRFVDDDTRYTQFLCPGPSTGLFGEVEEYRSTCPF, that is, SEQ ID NO: 3) by performing N48D+D204C+L213T+S214P+E253C mutations (i.e., asparagine at position 48 is mutated to aspartic acid, aspartic acid at position 204 is mutated to cysteine, leucine at position 213 is mutated to threonine, serine at position 214 is mutated to proline, and glutamic acid at position 253 is mutated to cysteine).

[0034] SEQ ID NO: 1:

[0035] ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTDETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGHSMGGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELCGASHFAPNTPNTTIAKYSISWLKRFVDDDTRYTQFLCPGPSTGLFGEVCEYRSTCPF

[0036] The nucleotide sequence of the gene encoding SEQ ID NO: 1 SEQ ID NO: 2:

[0037] GCTAATCCTTATGAAAGGGGACCCAACCCAACGAACAGCTCTATCGAAGCGCTGCGTGGTCCGTATTCTGTTTCCGAAGATTCCGTGAGCTCCCTGGTTAGCGGTTTCGGTGGCGGCACGATCTACTACCCGACTGGCACGGATGAAACCTTCGGCGCGGTCGCAATTTCGCCGGGCTACACCGGCACCCAAAGCTCGATCAGCTGGTTAGGTCCACGTTTGGCGTCACAGGGTTTTGTCGTGATGACCATCGACACCAACACCACATTGGACCAGCCGGATTCCAGAGCAAGCCAACTGGATGCTGCGCTGGACTACATGGTTAACCGTTCTTCCTCTACTGTACGTAATCGTATTGATAGCAGTCGCTTGGCGGCTATGGGTCATAGCATGGGTGGTGGCGGAACCCTCCGTCTTGCGGAGCGCCGTCCGGACCTGCAGGCAGCGATTCCGCTGACGCCTTGGCATACCGATAAGACCTGGGGTTCCGTTCGCGTGCCGACCCTGATTATTGGTGCCGAGAACGATACTATCGCCAGCGTGCGCAGCCACAGCGAGCCGTTCTATAACAGCTTGCCGGGTAGCCTGGACAAAGCATATCTGGAGCTGTGCGGCGCGAGCCACTTTGCTCCGAATACCCCGAATACCACCATCGCCAAATACAGCATCAGCTGGCTCAAGCGCTTCGTGGATGACGACACCCGTTATACCCAATTTCTGTGTCCGGGCCCAAGCACCGGTCTGTTTGGCGAAGTTTGCGAGTACCGTTCGACGTGCCCGTTC。 Description of the Drawings

[0038] Figure 1 The stability screening of mutants of Th_DCTPC is shown, where S68K, D85K, T89R, D105R, V108R, D145K, Q147K, D197K, Y221K, D232K, T237R, G194Q, and E251A on the abscissa are all mutations made on the basis of Th_DCTPC.

[0039] Figure 2 The Tm value analysis graph of different mutants is shown; among them, DCTPC represents Th_DCTPC, DCTPC-68 represents S68K mutation further made on the basis of Th_DCTPC; DCTPC-105 represents D105R mutation further made on the basis of Th_DCTPC; DCTPC-194 represents G194Q mutation further made on the basis of Th_DCTPC; DCTPC-232 represents D232K mutation further made on the basis of Th_DCTPC; DCTPC-4M (or Th_DCTPC-4M) represents S68K+D105R+G194Q+D232K mutation further made on the basis of Th_DCTPC.

[0040] Figure 3 A comparative graph showing the activity of Th_DCTPC and its mutant Th_DCTPC-4M (abbreviated as DCTPC_4M) in degrading PET powder to produce TPA is shown, where DCTPC represents the mutant Th_DCTPC, DCTPC_4M represents the mutant Th_DCTPC-4M, and the vertical axis represents the amount of TPA produced by degrading PET.

[0041] Figure 4 A graph comparing the activities of Th_DCTPC and its mutant Th_DCTPC-4M (abbreviated as DCTPC-4M) in degrading PBAT powder to produce TPA is shown, wherein WT represents the wild-type ThPETase enzyme, DCTPC represents the mutant Th_DCTPC, DCTPC_4M represents the mutant Th_DCTPC-4M, and the vertical axis represents the amount of TPA produced by degrading PBAT.

[0042] Figure 5A comparative graph showing the degradation activity of PBAT film by ThPETase and its mutant Th_DCTPC-4M (abbreviated as DCTPC-4M) is shown, wherein WT represents the wild-type ThPETase enzyme, ThCC represents the mutant ThCC of ThPETase (disclosed in Chinese patent ZL202411540951.8 (authorization announcement number CN119040295B)), DCTPC_4M represents the mutant Th_DCTPC-4M, FastPETase is a plastic hydrolase reported in the journal Nature (the relevant article is titled: Machine learning-aided engineering of hydrolases for PET depolymerization). The amino acid sequence of FastPETase is shown in SEQ ID NO: 5. The gene of the enzyme (SEQ ID NO: 32) was synthesized by Nanjing GenScript Co., Ltd., and enzyme cleavage sites were added upstream and downstream of the target gene. Nde I (CATATG) and Xho I (CTCGAG), vector and target gene were double-digested at 37℃ ( Nde I and Xho I), recovered by gel ligation, ligated with T4 ligase at 4°C overnight, loaded into the pET21a vector, and then introduced into BL21 (DE3) and fermented.

[0043] ThCC:ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTNETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGHS MGGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELCGASHFAPNLSNTTIAKYSISWLKRFVDDDTRYTQFLCPGSTGLFGEVCEYRSTCPF (SEQ ID NO: 4)

[0044] FastPETase:QTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS(SEQ ID NO: 5)

[0045] FastPETase gene sequence: (SEQ ID NO: 32).

[0046] Figure 6 The flow chart shows the degradation of PBAT film to produce TPA powder by the mutant Th_DCTPC-4M (abbreviated as DCTPC-4M). After 3 days of reaction at 37°C, 32 mg of PBAT agricultural mulch film finally produced 8.2 mg of TPA with a purity of 99%. TPA is the raw material for the production of plastic PET and PBAT. DETAILED DESCRIPTION

[0047] Example 1 Construction of mutants

[0048] 1. Construction of mutants

[0049] (1) PCR amplification was performed using the circular plasmid containing pET-21a-ThPETase as a template.

[0050] Circular plasmid PCR system: 1 μL template; 1 μL each of 10 μM forward and reverse primers (primers see Table 1); 4 μL of 2.5 mM dNTPs; 5 μL of 10× high-fidelity enzyme buffer; 1 μL of high-fidelity enzyme; add water to a 50 μL reaction system.

[0051] PCR thermal cycling conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at (Tm-5)°C for 20 s; extension at 72°C for 4 min 20 s; insulation at 4°C; PCR amplification was performed for 30 cycles under the following reaction conditions.

[0052] After digesting the amplified product with Dpn I, 5 μL was added to 50 μL of DH5α competent cells for transformation. The cells were incubated on ice for 20 min, heat-shocked at 42°C for 45 s, and placed on ice for 3 min. The cells were then activated with 800 μL of LB medium and incubated at 37°C, 220 rpm, for 1 h. The cells were then spread on plates and cultured for 14–16 h until mature single colonies formed. The cells were then picked and verified by sequencing.

[0053] Table 1 Mutant primer sequences

[0054]

[0055] 2. Construction of engineered bacteria expressing Th_DCTPC and its mutants

[0056] Th_DCTPC and its mutant genes were synthesized by GenScript and cloned into pET21a with restriction enzymes NdeI and XhoI. The resulting strains were BL21(DE3) / pET21a-DCTPC, BL21(DE3) / pET21a-DCTPC-68, BL21(DE3) / pET21a-DCTPC-105, BL21(DE3) / pET21a-DCTPC-194, BL21(DE3) / pET21a-DCTPC-232, and DCTPC_4M. The strains were plated on LB agar plates containing ampicillin and cultured for 14–16 hours until single mature colonies formed, yielding engineered strains expressing the wild-type enzyme and mutants.

[0057] Example 2 Expression and purification of DCTPC and its mutants

[0058] The constructed mutant strains BL21(DE3) / pET21a-DCTPC, BL21(DE3) / pET21a-DCTPC-68, BL21(DE3) / pET21a-DCTPC-105, BL21(DE3) / pET21a-DCTPC-194, BL21(DE3) / pET21a-DCTPC-232, and DCTPC_4M were inoculated into 5 mL of LB medium (containing ampicillin) and seeded for 13 hours. Then, 2 mL of the overnight culture was inoculated into 100 mL of fresh LB medium (containing antibiotics, in a 500 mL Erlenmeyer flask) and incubated at 37°C at 250 rpm for approximately 2 hours until the logarithmic growth phase (OD600 = 0.8-1). IPTG was added for induction at a final concentration of 0.1 mM. After induction for 24 hours at 16°C, the cells were harvested at 7000 rpm for 10 minutes. The cells were suspended in 10 mL of lysis buffer solution, disrupted by ultrasonication (2 s, 3 s, 30 min, 70%), and centrifuged at 8000 rpm for 15 min to obtain the supernatant.

[0059] Pre-wash the equilibrated column with two column volumes of lysis buffer containing 10 mM imidazole to allow the buffer to slowly drain from the resin; then slowly load the supernatant after lysis centrifugation (two column volumes of sample solution); wash with lysis buffer for 20 column volumes, and then wash with wash buffer containing 20 mM imidazole for 10 column volumes; finally, elute the sample with elution buffer containing 250 mM imidazole in a volume of 3 mL.

[0060] Ni-NTA purification was performed, and the purified protein was concentrated by centrifugation using a 10 kD ultrafiltration tube, and the elution buffer containing a high concentration of imidazole was replaced with a reaction buffer solution.

[0061] The formula of LB liquid medium is as follows: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L;

[0062] The formula of lysis buffer is: 50 mM Tris-HCl, 150 mM sodium chloride, 10 mM imidazole, pH = 7.5;

[0063] The formula of the washing buffer is: 50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH = 7.5;

[0064] The formula of elution buffer is: 50 mM Tris-HCl, 300 mM sodium chloride, 300 mM imidazole, pH = 7.5;

[0065] The formula of the reaction buffer is: 0.1 M potassium dihydrogen phosphate-sodium hydroxide buffer solution, pH = 8.0.

[0066] Finally, the soluble proteins DCTPC, DCTPC_68, DCTPC_105, DCTPC_194, DCTPC_232 and DCTPC_4M were purified and used for the next step of detection.

[0067] Example 3 Screening and thermal stability experiments of Th_DCTPC and its mutants

[0068] p-Nitrophenol acetate (pNPA, which, like PBAT, is an ester containing a benzene ring) was used to evaluate and screen the stability of mutants: the total reaction volume was 200 μL, containing 197 μL of 0.1 M potassium dihydrogen phosphate-sodium hydroxide buffer (pH 8), 2 μL of 0.1 M pNPA, and 1 μL of 1.0 mg / mL enzyme solution (incubated at 50°C or 60°C for 30 minutes). Kinetic measurements were performed, with the maximum reaction rate representing the enzyme activity. For each batch, the highest activity within the group was normalized to 100% to facilitate comparison of relative activities. Figure 1 As shown, the results showed that the following mutants including DCTPC_68, DCTPC_105, DCTPC_194, and DCTPC_232 all had improved performance relative to DCTPC, indicating that these mutants are beneficial mutants.

[0069] The thermal stability of the enzyme was analyzed using a protein stability analyzer (nanoDSF, NanoTemper Technologies Prometheus NT.48). 10 μL of sample was loaded into a capillary tube. The concentration of the loaded protein (ThPETase protein and its mutants) was uniformly quantified at 1.0 mg / mL. The temperature was raised from 30°C to 95°C at a rate of 1°C / min. The protein fluorescence ratio at 330 nm and 350 nm was recorded. Each protein sample was measured in triplicate. The test results are shown in Figure 2. Figure 2 shown.

[0070] Depend on Figure 2As can be seen, the Tm value of DCTPC is 69°C. The Tm of mutant DCTPC-68 increased to 69.1°C, approximately 0.1°C higher than DCTPC. The Tm of mutant DCTPC-105 increased to 70.1°C, approximately 1.1°C higher than DCTPC. The Tm of mutant DCTPC-194 increased to 69.7°C, approximately 0.7°C higher than DCTPC. The Tm of mutant DCTPC-232 increased to 72.3°C, approximately 3.3°C higher than DCTPC. Combining the above four single-point mutations, we found that the Tm of mutant DCTPC-4M increased to 74.2°C, approximately 5.2°C higher than DCTPC.

[0071] Example 4 Hydrolysis of PET / PBAT using ThPETase and DCTPC_4M

[0072] Degradation of PET / PBAT plastic powder for activity testing

[0073] The enzyme prepared in Example 2 was used to degrade PET / PBAT plastic powder, and ultra-performance liquid chromatography (UPLC) was used to analyze the TPA (terephthalic acid) released during the depolymerization of PBAT. MHET was measured using a C18 column (4.6 × 250 mm, 5 µm) with a detection wavelength of 240 nm. The column temperature was maintained at 40°C, and the mobile phase consisted of 0.1% formic acid in water and acetonitrile at a constant flow rate of 0.1 mL / min. The injection volume was 2 µL, and the acetonitrile content of the mobile phase was reduced from 50% to 20% over 4 minutes. The TPA peak elutes at approximately 2.8 minutes.

[0074] Reaction system: Add 5 mg / mL of 100-mesh PBAT powder or 1000-mesh PET powder to a 0.1 M potassium dihydrogen phosphate-NaOH buffer solution at pH 8.0, then add purified DCTPC and its mutant proteins to a final concentration of 0.02 mg / mL, and react at 60-70°C for 2 h. After the reaction, centrifuge the supernatant, add an equal volume of acetonitrile, inject the sample by UPLC, and calculate the product (TPA / MHET) yield based on the peak area. Figure 3 and Figure 4 The results showed that DCTPC_4M showed enhanced hydrolysis of PET and PBAT powders compared to DCTPC at both 60°C and 70°C. In particular, at 70°C, product yields increased by 2.0 and 2.3 times, respectively. Clearly, the modified DCTPC_4M is more effective in degrading PET and PBAT materials under harsh industrial conditions.

[0075] Example 5 Hydrolysis of PET / PBAT powder using DCTPC_4M

[0076] To evaluate the degradation efficiency of the mutant DCTPC_4M on PBAT agricultural film (Zhejiang Jialemi Horticultural Technology Co., Ltd. (tobacco-specific mulch film) and the purity and yield of the resulting TPA, approximately 2 cm × 2 cm or 4 cm × 5 cm black agricultural film was incubated in 0.1 mmol / L Tris-HCl buffer (pH 8.0) or KH2PO4-NaOH buffer (0.1 mol / L, pH 8.0). Purified FastPETase, ThPETase, its mutants ThCC, and DCTPC_4M were added to a final concentration of 0.02 mg / mL, and the enzyme reactions were tested and compared at different temperatures.

[0077] Depend on Figure 5 As can be seen, after 10 hours of reaction at 50°C, ThCC and DCTPC_4M had already degraded the PBAT film into powder, while wild-type ThPETase and the reported enzyme FastPETase showed no significant degradation. DCTPC_4M also significantly outperformed ThCC in terms of degradation efficiency. After 5 hours of reaction at 60°C, only DCTPC_4M showed degradation, while the other three groups (ThPETase, ThCC, and FastPETase) showed no degradation.

[0078] Depend on Figure 6 It can be seen that after reacting at 37°C for 3 days, DCTPC_4M degraded the PBAT film into powder and generated TPA powder with a purity of up to 99%. TPA is a key raw material for making plastics, completing the recycling of plastic monomers.

[0079] Figure 5 and Figure 6 It shows that the effect of DCTPC_4M in degrading PBAT film is better than that of ThPETase and its variants disclosed in Chinese patent ZL202411540951.8 (authorization announcement number CN119040295B).

Claims

1. A plastic hydrolase ThPETase mutant, characterized in that: The mutant is obtained by performing the following mutation on a position selected from the group consisting of position 68, position 105, position 194 and position 232 of the amino acid sequence shown in SEQ ID NO: 1: (1) D232K; or (2)S68K+D105R+G194Q+D232K.

2. The plastic hydrolase ThPETase mutant according to claim 1, characterized in that The mutant is obtained by performing the following mutations on positions 68, 105, 194 and 232 of the amino acid sequence shown in SEQ ID NO: 1: S68K+D105R+G194Q+D232K.

3. A polynucleotide, characterized in that Encoding the plastic hydrolase ThPETase mutant according to claim 1 or 2.

4. A recombinant vector, characterized in that Comprising the polynucleotide according to claim 3.

5. A host cell, characterized in that Comprising the polynucleotide according to claim 3 or the recombinant vector according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell is a fungal cell or a bacterial cell.

7. The host cell according to claim 6, characterized in that The host cell is a bacterial cell.

8. The host cell according to claim 7, characterized in that The bacterial cells are Escherichia coli cells.

9. Use of the plastic hydrolase ThPETase mutant according to claim 1 or 2 or the host cell according to any one of claims 5-8 in degrading PET or PBAT plastics.

10. The use according to claim 9, characterized in that The application pathways are: An object containing PET and / or PBAT plastic is brought into contact with a mutant of the plastic hydrolase ThPETase to carry out a degradation reaction.

11. A method for producing the mutant of plastic hydrolase ThPETase according to claim 1 or 2, characterized in that: The method includes: (a) culturing the host cell according to any one of claims 5 to 8 under conditions suitable for expression of the plastic hydrolase ThPETase mutant; and (b) Recovering the plastic hydrolase ThPETase mutant.

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