Heat-resistant plastic hydrolase mutant and application thereof

Abstract

The invention discloses a heat-resistant plastic hydrolase mutant and application thereof. In particular, the invention provides a heat-resistant plastic hydrolase ThPETase mutant and an application of the heat-resistant plastic hydrolase ThPETase mutant. Specifically, a mutant ThDCTPC (an amino acid sequence is shown as SEQ ID NO: 1) of a protein ThPETase (GeneBank ID: WP243597587.1) derived from a thermophilic microorganism Thermofida halotolans is further mutated, so that the mutant of which the enzyme activity and the thermal stability are greatly improved is obtained, and the mutant of which the enzyme activity and the thermal stability are greatly improved is obtained by using the mutant of the protein ThPETase (GeneBank ID: WP243597587.1) and the mutant of the protein ThPETase (GeneBank ID: WP243597587.1). The invention also comprises a method for producing the heat-resistant plastic hydrolase mutant, and an application of the heat-resistant plastic hydrolase mutant in degradation of PET (polyethylene terephthalate) or PBAT (poly (butyleneadipate-co-terephthalate)) plastics.

Description

Technical Field

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

[0002] Plastic pollution has evolved into a global urgent environmental problem, which has attracted extensive attention and high emphasis from all sectors of society. Among numerous current environmental protection materials, poly(butylene adipate-co-terephthalate) (PBAT) is gradually becoming a key breakthrough in controlling white pollution due to its biodegradable characteristics. PBAT is copolymerized from 1,4-butanediol, adipic acid, and terephthalic acid. Its unique molecular structure endows it with good ductility and biocompatibility. Its excellent processing performance gives it great advantages in the packaging field, especially showing huge market potential in the manufacture of food-grade packaging films, agricultural mulch films, and degradable tableware.

[0003] In the plastic family, polyethylene terephthalate (PET) plastic is also a very common one. PET plastic has characteristics such as high strength, good transparency, and excellent chemical stability, and is widely used in many fields such as beverage bottles, electronic product casings, and packaging materials. However, PET plastic is difficult to degrade naturally and can exist in the environment for decades or even hundreds of years. A large number of PET plastic product wastes have become one of the important sources of plastic pollution.

[0004] Research has found that the number of microbial populations in nature with the ability to hydrolyze PET and PBAT is limited. Due to this insufficient biodegradation efficiency, developing an efficient PET and PBAT hydrolase system has become a key direction that urgently needs to be overcome in the field of environmental biotechnology. Chinese Patent ZL202411540951.8 (authorized announcement number CN119040295B) discloses that ThPETase (derived from a thermophilic microorganism Thermobifida halotolerans , GeneBank ID: WP_243597587.1) and Th_DCTPC (a ThPETase mutant with an amino acid sequence shown in SEQ ID NO: 1) have certain PET and PBAT degradation abilities, but their hydrolysis effects at high temperatures (50 - 70 °C) are limited, restricting the potential for application under harsh industrial conditions. Therefore, there is still a need for heat-resistant plastic hydrolases in the existing technology. Summary of the Invention

[0005] To solve the deficiencies in the existing technology, the present invention provides a heat-resistant plastic hydrolase mutant and its application, especially a heat-resistant plastic hydrolase ThPETase mutant and its application. Specifically, the present invention modifies a thermophilic microorganism-derived Thermobifida halotoleransThe 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 obtain mutants with significantly improved enzyme activity and thermal stability. The present invention also includes a method for producing the aforementioned heat-resistant plastic hydrolase mutants, and the application of the heat-resistant plastic hydrolase mutants in degrading PET or PBAT plastics.

[0006] Specifically, the present invention includes but is not limited to the following technical solutions: In one aspect, the present invention provides a mutant of the plastic hydrolase ThPETase, which is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1 at positions selected from the 68th, 105th, 194th, and 232nd positions as follows: (1) S68K (i.e., replacing serine at the 68th position with lysine); (2) D105R (i.e., replacing aspartic acid at the 105th position with arginine); (3) G194Q (i.e., replacing glycine at the 194th position with glutamine); (4) D232K (i.e., replacing aspartic acid at the 232nd position with lysine); or (5) S68K+D105R+G194Q+D232K (i.e., replacing serine at the 68th position with lysine, replacing aspartic acid at the 105th position with arginine, replacing glycine at the 194th position with glutamine, and replacing aspartic acid at the 232nd position with lysine).

[0007] In one aspect, the mutant of the plastic hydrolase ThPETase of the present invention is obtained by mutating the 68th position of the amino acid sequence shown in SEQ ID NO: 1 as follows: S68K (i.e., replacing serine at the 68th position with lysine).

[0008] In one aspect, the mutant of the plastic hydrolase ThPETase of the present invention is obtained by mutating the 105th position of the amino acid sequence shown in SEQ ID NO: 1 as follows: D105R (i.e., replacing aspartic acid at the 105th position with arginine).

[0009] In one aspect, the mutant of the plastic hydrolase ThPETase of the present invention is obtained by mutating the 194th position of the amino acid sequence shown in SEQ ID NO: 1 as follows: G194Q (i.e., replacing glycine at the 194th position with glutamine).

[0010] In one aspect, the mutant of the plastic hydrolase ThPETase of the present invention is obtained by mutating the 232nd position of the amino acid sequence shown in SEQ ID NO: 1 as follows: D232K (i.e., replacing the aspartic acid at the 232nd position with lysine).

[0011] In one aspect, the mutant of the plastic hydrolase ThPETase of the present invention is obtained by mutating the 68th, 105th, 194th, and 232nd positions of the amino acid sequence shown in SEQ ID NO: 1 as follows: S68K+D105R+G194Q+D232K (i.e., replacing the serine at the 68th position with lysine, replacing the aspartic acid at the 105th position with arginine, replacing the glycine at the 194th position with glutamine, and replacing the aspartic acid at the 232nd position with lysine).

[0012] In one aspect, the present invention also provides a mutant of the plastic hydrolase ThPETase, which is obtained by mutating the 68th, 105th, 194th, and 232nd positions of the amino acid sequence shown in SEQ ID NO: 1 as follows: S68K+D105R+G194Q+D232K (i.e., replacing the serine at the 68th position with lysine, replacing the aspartic acid at the 105th position with arginine, replacing the glycine at the 194th position with glutamine, and replacing the aspartic acid at the 232nd position with lysine).

[0013] In another aspect, the present invention provides a polynucleotide encoding the mutant of the plastic hydrolase ThPETase of the present invention.

[0014] In another aspect, the present invention also provides a recombinant vector comprising the polynucleotide of the present invention.

[0015] 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.

[0016] In yet another aspect, the present application provides the use of the mutant of the plastic hydrolase ThPETase of the present invention or the host cell of the present invention in degrading PET or PBAT plastics.

[0017] In one aspect, the application path of the present invention is as follows: an object containing PET and / or PBAT plastic is contacted with the plastic hydrolase ThPETase mutant for a degradation reaction. Preferably, the object containing PET and / or PBAT plastic in 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 in the present invention is PET or PBAT plastic powder. Most preferably, the PBAT plastic powder in the present invention is PBAT powder with a particle size of 100 mesh. Most preferably, the PET plastic powder in the present invention is PET powder with a particle size of 1000 mesh.

[0018] In one aspect, in the application of the present invention, based on PBAT plastic, by mass percentage, the addition amount of the plastic hydrolase ThPETase mutant is: 10 μg / mL - 200 μg / mL; the temperature of the reaction is: 37 - 70 °C; the reaction time is: 1 h - 7 d; the solution pH is: 7.0 - 10.0.

[0019] In one aspect, the contact of the object containing PET and / or PBAT plastic with the plastic hydrolase ThPETase mutant in the present invention occurs in the presence of Tris-HCl buffer or potassium dihydrogen phosphate-NaOH buffer solution.

[0020] In yet another aspect, the present application provides a production method of the plastic hydrolase ThPETase mutant of the present invention, and the method includes: (a) Culturing the host cell of the present invention under conditions suitable for the expression of the plastic hydrolase ThPETase mutant; (b) Recovering the plastic hydrolase ThPETase mutant.

[0021] In one aspect, the host cell of the present invention is a fungal cell, a bacterial cell or a plant cell.

[0022] In yet 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 Escherichia coli BL21(DE3) cell.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention performs molecular modification on the mutant Th_DCTPC of ThPETase. The obtained mutant not only has improved enzyme activity but also significantly enhanced heat resistance. Specifically, when the hydrolysis activities of Th_DCTPC towards PET and PBAT are relatively low at high temperatures, the mutant obtained in the present invention (especially, DCTPC_4M) has a significantly improved hydrolysis ability towards PET and PBAT at high temperatures (e.g., 70 °C). The hydrolysis abilities towards these two plastics are enhanced by 2.0 times and 2.3 times respectively, showing great potential for industrial applications.

[0024] In the present application, the amino acid sequence of Th_DCTPC is shown in SEQ ID NO.1, which is obtained 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) on the amino acid sequence of the protein (ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTNETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGHSMGGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELDGASHFAPNLSNTTIAKYSISWLKRFVDDDTRYTQFLCPGPSTGLFGEVEEYRSTCPF, i.e., SEQ ID NO: 3) derived from a thermophilic microorganism. Thermobifida halotolerans

[0025] SEQ ID NO: 1: ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTDETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGHSMGGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELCGASHFAPNTPNTTIAKYSISWLKRFVDDDTRYTQFLCPGPSTGLFGEVCEYRSTCPF The nucleotide sequence of the gene encoding SEQ ID NO: 1 SEQ ID NO: 2: GCTAATCCTTATGAAAGGGGACCCAACCCAACGAACAGCTCTATCGAAGCGCTGCGTGGTCCGTATTCTGTTTCCGAAGATTCCGTGAGCTCCCTGGTTAGCGGTTTCGGTGGCGGCACGATCTACTACCCGACTGGCACGGATGAAACCTTCGGCGCGGTCGCAATTTCGCCGGGCTACACCGGCACCCAAAGCTCGATCAGCTGGTTAGGTCCACGTTTGGCGTCACAGGGTTTTGTCGTGATGACCATCGACACCAACACCACATTGGACCAGCCGGATTCCAGAGCAAGCCAACTGGATGCTGCGCTGGACTACATGGTTAACCGTTCTTCCTCTACTGTACGTAATCGTATTGATAGCAGTCGCTTGGCGGCTATGGGTCATAGCATGGGTGGTGGCGGAACCCTCCGTCTTGCGGAGCGCCGTCCGGACCTGCAGGCAGCGATTCCGCTGACGCCTTGGCATACCGATAAGACCTGGGGTTCCGTTCGCGTGCCGACCCTGATTATTGGTGCCGAGAACGATACTATCGCCAGCGTGCGCAGCCACAGCGAGCCGTTCTATAACAGCTTGCCGGGTAGCCTGGACAAAGCATATCTGGAGCTGTGCGGCGCGAGCCACTTTGCTCCGAATACCCCGAATACCACCATCGCCAAATACAGCATCAGCTGGCTCAAGCGCTTCGTGGATGACGACACCCGTTATACCCAATTTCTGTGTCCGGGCCCAAGCACCGGTCTGTTTGGCGAAGTTTGCGAGTACCGTTCGACGTGCCCGTTC。 Description of the Drawings

[0026] Figure 1 Shows the stability screening of mutants of Th_DCTPC, where S68K, D85K, T89R, D105R, V108R, D145K, Q147K, D197K, Y221K, D232K, T237R, G194Q, E251A on the abscissa are all mutations made on the basis of Th_DCTPC.

[0027] Figure 2 The figure shows the Tm value analysis diagrams of different mutants; among them, DCTPC represents Th_DCTPC, DCTPC-68 represents the S68K mutation further made on the basis of Th_DCTPC; DCTPC-105 represents the D105R mutation further made on the basis of Th_DCTPC; DCTPC-194 represents the G194Q mutation further made on the basis of Th_DCTPC; DCTPC-232 represents the D232K mutation further made on the basis of Th_DCTPC; DCTPC-4M (or Th_DCTPC-4M) represents the S68K+D105R+G194Q+D232K mutation further made on the basis of Th_DCTPC.

[0028] Figure 3 The figure shows the activity comparison diagram of Th_DCTPC and its mutant Th_DCTPC-4M (abbreviated as DCTPC_4M) for degrading PET powder to generate TPA. Among them, DCTPC represents the mutant Th_DCTPC, DCTPC_4M represents the mutant Th_DCTPC-4M, and the ordinate represents the amount of TPA generated by degrading PET.

[0029] Figure 4 The figure shows the activity comparison diagram of Th_DCTPC and its mutant Th_DCTPC-4M (abbreviated as DCTPC-4M) for degrading PBAT powder to generate TPA. Among them, WT represents the wild-type enzyme of ThPETase, DCTPC represents the mutant Th_DCTPC, DCTPC_4M represents the mutant Th_DCTPC-4M, and the ordinate represents the amount of TPA generated by degrading PBAT.

[0030] Figure 5Shows a comparison chart of the activities of ThPETase and its mutant Th_DCTPC-4M (abbreviated as DCTPC-4M) in degrading PBAT films. Among them, WT represents the wild-type enzyme of ThPETase, ThCC represents the mutant ThCC of ThPETase (disclosed in Chinese Patent ZL202411540951.8 (authorized announcement number CN119040295B)), DCTPC_4M represents the mutant Th_DCTPC-4M, FastPETase is a plastic hydrolase reported in the journal "Nature" (the relevant article name is: Machine learning-aided engineering of hydrolases for PET depolymerization), the amino acid sequence of FastPETase is shown in SEQ ID NO: 5, and the gene (SEQ ID NO: 32) of this enzyme was synthesized by Nanjing Genscript Corporation, with restriction enzyme sites added to the upstream and downstream of the target gene respectively. Nde I (CATATG) and Xho I (CTCGAG), the vector and the target gene were double digested at 37°C ( Nde I and Xho I), then recovered by gel extraction, and ligated overnight at 4°C using T4 ligase. After loading onto the vector pET21a, it was then introduced into BL21(DE3) and fermented to obtain.

[0031] ThCC: ANPYERGPNPTNSSIEALRGPYSVSEDSVSSLVSGFGGGTIYYPTGTNETFGAVAISPGYTGTQSSISWLGPRLASQGFVVMTIDTNTTLDQPDSRASQLDAALDYMVNRSSSTVRNRIDSSRLAAMGHSMGGGGTLRLAERRPDLQAAIPLTPWHTDKTWGSVRVPTLIIGAENDTIASVRSHSEPFYNSLPGSLDKAYLELCGASHFAPNLSNTTIAKYSISWLKRFVDDDTRYTQFLCPGPSTGLFGEVCEYRSTCPF (SEQ ID NO: 4) FastPETase: QTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS (SEQ ID NO: 5) Gene sequence of FastPETase: CAAACAAATCCCTATGCTAGGGGACCCAACCCGACGGCTGCGAGCTTGGAGGCATCAGCCGGTCCGTTTACCGTGCGCTCGTTCACCGTCAGCCGTCCGAGCGGCTATGGTGCAGGCACCGTGTATTACCCGACGAACGCGGGTGGCACCGTGGGTGCGATCGCGATCGTGCCGGGTTATACCGCGCGTCAAAGCTCTATCAAGTGGTGGGGACCACGTCTGGCATCCCACGGCTTTGTTGTTATTACCATTGATACCAACTCCACCCTGGATCAGCCGGAAAGCCGTAGCTCCCAACAAATGGCAGCGTTAAGACAGGTTGCAAGCTTGAATGGTACTTCATCTTCCCCGATTTACGGCAAAGTTGATACGGCGCGTATGGGTGTGATGGGCTGGTCAATGGGTGGCGGCGGCAGCCTGATCTCCGCTGCTAACAACCCTAGCCTGAAAGCAGCGGCGCCACAGGCGCCGTGGCACAGCAGCACGAACTTTAGCTCTGTTACCGTCCCGACCCTGATTTTTGCGTGCGAAAACGACTCCATTGCGCCGGTGAATAGCTCTGCTCTGCCGATCTACGACAGCATGTCGCAAAACGCCAAACAGTTCCTGGAGATCAAGGGCGGTAGCCATTCTTGTGCTAACAGTGGTAATTCCAATCAGGCTCTCATCGGTAAGAAGGGTGTTGCCTGGATGAAACGTTTCATGGATAATGACACTCGCTACAGCACCTTCGCGTGCGAGAACCCGAATAGCACCGCCGTAAGCGACTTCCGCACCGCGAACTGCAGCCTCGAG (SEQ ID NO:32).

[0032] Figure 6 The flow chart showing the degradation of PBAT film by mutant Th_DCTPC-4M (abbreviated as DCTPC-4M) to produce TPA powder is presented. After a 3-day reaction at 37°C, 32 mg of agricultural film made of PBAT finally produced 8.2 mg of TPA with a purity of 99%. TPA is a raw material for the production of plastics PET and PBAT. Detailed implementation mode

[0033] Example 1 Construction of mutants 1. Construction of mutants (1)Using the circular plasmid containing pET-21a-ThPETase as a template, PCR amplification was carried out.

[0034] Circular plasmid PCR system: 1 μL of template; 1 μL each of forward and reverse primers (primers are shown in Table 1) at 10 μM; 4 μL of dNTP (2.5 mM); 5 μL of 10× high-fidelity enzyme buffer; 1 μL of high-fidelity enzyme; make up the reaction system to 50 μL with water.

[0035] 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; incubation at 4°C; perform 30 cycles of PCR amplification under the reaction conditions.

[0036] After the amplification product was digested with Dpn I, 5 μL was taken and added to 50 μL of DH5α competent cells for transformation. Incubate on ice for 20 min; heat shock at 42°C for 45 s; place on ice for 3 min; add 800 μL of LB medium, activate at 37°C and 220 rpm for 1 h, then spread on plates and culture for 14 h - 16 h until mature single colonies grew out, and pick colonies for sequencing verification.

[0037] Table 1 Mutant primer sequences

[0038] 2. Construction of engineering bacteria expressing Th_DCTPC and its mutants The genes of Th_DCTPC and its mutants were synthesized by GenScript, cloned into pET21a respectively, with the restriction enzyme sites NdeI and XhoI, and finally introduced into BL21(DE3) to obtain 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. Spread on LB agar plates containing ampicillin and culture for 14 h - 16 h until mature single colonies grew out, and finally obtain wild-type enzyme expression engineering bacteria and mutant expression engineering bacteria.

[0039] Example 2 Expression and purification of DCTPC and its mutants 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 culture medium (containing ampicillin) and cultured as seeds for 13 h. Then, 2 mL of the overnight culture was inoculated into 100 mL of fresh LB medium (containing antibiotics, in a 500 mL conical flask) and cultured at 37°C and 250 rpm for about 2 h until the logarithmic growth phase (OD600 = 0.8 - 1). IPTG was added at a final concentration of 0.1 mM for induction. After continued induction culture at 16°C for 24 h, the cells were harvested by centrifugation at 7000 rpm for 10 min. The cells were suspended in 10 mL of lysis buffer, sonicated (2 s, 3 s, 30 min, 70%), and centrifuged at 8000 rpm for 15 min to obtain the supernatant.

[0040] Previously, the column was rinsed and equilibrated with two column volumes of lysis buffer containing 10 mM imidazole to slowly drain the buffer from the resin; then the supernatant after lysis and centrifugation was slowly loaded (two column volumes of sample solution); the column was washed with 20 column volumes of lysis buffer and then with 10 column volumes of washing buffer containing 20 mM imidazole; finally, the sample was eluted with elution buffer containing 250 mM imidazole, and the elution volume was 3 mL.

[0041] Ni-NTA purification was performed. 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 reaction buffer.

[0042] Among them, the formula of LB liquid medium is: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L; The formula of lysis buffer is: 50 mM Tris-HCl, 150 mM sodium chloride, 10 mM imidazole, pH = 7.5; The formula of washing buffer is: 50 mM Tris-HC, 150 mM sodium chloride, 20 mM imidazole, pH = 7.5; The formula of elution buffer is: 50 mM Tris-HCl, 300 mM sodium chloride, 300 mM imidazole, pH = 7.5; The formula of reaction buffer is: 0.1 M potassium dihydrogen phosphate-sodium hydroxide buffer solution, pH = 8.0.

[0043] Finally, soluble proteins DCTPC, DCTPC_68, DCTPC_105, DCTPC_194, DCTPC_232 and DCTPC_4M were purified for the next step of detection.

[0044] Example 3 Screening and Thermal Stability Experiment of Th_DCTPC and Its Mutants The stability of mutants was evaluated and screened using p-nitrophenyl acetate (pNPA, an ester containing a benzene ring like PBAT): The total volume of the reaction system 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, and the maximum reaction rate was used to represent enzyme activity. For each batch, the highest activity within the group was normalized to 100% for comparison of relative activities. As Figure 1 shown, the results indicate that the following mutants, including DCTPC_68, DCTPC_105, DCTPC_194, DCTPC_232, have improved performance relative to DCTPC, indicating that these mutants are all beneficial mutants.

[0045] The thermal stability of the enzyme was analyzed using a protein stability analyzer (nanoDSF, NanoTemper Technologies Prometheus NT.48). 10 μL was loaded onto the capillary, and the concentration of the loaded protein (ThPETase protein and its mutants) was uniformly quantified as 1.0 mg / mL. The temperature was raised from 30 °C to 95 °C at a rate of 1 °C / min, and the protein fluorescence ratio at 330 nm and 350 nm was recorded. Each protein sample was measured in three parallel replicates. The test results are as Figure 2 shown.

[0046] It can be seen from Figure 2 that the Tm value of DCTPC is 69 °C. The Tm of the mutant DCTPC-68 was increased to 69.1 °C, about 0.1 °C higher than DCTPC. The Tm of the mutant DCTPC-105 was increased to 70.1 °C, about 1.1 °C higher than DCTPC. The Tm of the mutant DCTPC-194 was increased to 69.7 °C, about 0.7 °C higher than DCTPC. The Tm of the mutant DCTPC-232 was increased to 72.3 °C, about 3.3 °C higher than DCTPC. By combining the above 4 single-point mutations, we found that the Tm of the mutant DCTPC-4M was increased to 74.2 °C, about 5.2 °C higher than DCTPC.

[0047] Example 4 Hydrolysis of PET / PBAT by ThPETase and DCTPC_4M Conducting activity tests on the degraded PET / PBAT plastic powder Using the enzyme prepared in Example 2 to degrade the PET / PBAT plastic powder, and analyzing the TPA (terephthalic acid) released during the depolymerization of PBAT by ultra-high performance liquid chromatography (UPLC). MHET was measured using a C18 column (4.6×250 mm, 5 µm), and the detection wavelength was 240 nm. The column temperature was maintained at 40 °C, an aqueous solution of 0.1% formic acid and acetonitrile were used as the mobile phase, the flow rate was fixed at 0.1 mL / min, the injection volume was 2 μL, and the proportion of acetonitrile in the mobile phase decreased from 50% to 20% within 4 min. The peak time of TPA was approximately 2.8 min.

[0048] Reaction system: Add 5 mg / mL of PBAT powder with a particle size of 100 mesh or 1000 mesh of PET powder to a 0.1 M potassium dihydrogen phosphate-NaOH buffer solution with a pH value of 8.0, and then add the purified DCTPC and its mutant protein with a final concentration of 0.02 mg / mL. React at 60 - 70 °C for 2 h. After the reaction, centrifuge and take the supernatant, add an equal volume of acetonitrile, inject into UPLC, and calculate the product (TPA / MHET) yield according to the peak area. The degradation effect is shown in Figure 3 and Figure 4 . The results show that at 60 °C and 70 °C, the hydrolysis effect of DCTPC_4M on PET and PBAT powders is enhanced compared with DCTPC. Especially at 70 °C, the product yields are increased by 2.0 and 2.3 times respectively. Obviously, the modified DCTPC_4M will be more helpful for degrading PET and PBAT materials under harsh industrial conditions.

[0049] Example 5 Hydrolyzing PET / PBAT powder using DCTPC_4M To evaluate the degradation efficiency of the mutant DCTPC_4M on PBAT agricultural films (tobacco special plastic films from Zhejiang Jialemi Horticultural Technology Co., Ltd.), as well as the purity and yield of TPA generated after degradation, we incubated black agricultural films of about 2 cm × 2 cm or 4 cm × 5 cm in 0.1 mmol / L Tris-HCl buffer (pH 8.0) or KH2PO4-NaOH buffer (0.1 mol / L, pH 8.0). Add the purified FastPETase, ThPETase and their mutants ThCC and DCTPC_4M with a final concentration of 0.02 mg / mL, and test and compare the enzyme reaction effects at different temperatures.

[0050] By Figure 5It can be seen that after reacting at 50 °C for 10 h, ThCC and DCTPC_4M have degraded the PBAT film into powder, while the wild-type ThPETase and the enzyme FastPETase reported in the literature have not shown obvious degradation reactions. In terms of the degradation effect, DCTPC_4M is also significantly better than ThCC. After reacting at 60 °C for 5 h, only DCTPC_4M degrades, and the other three groups (ThPETase, ThCC, and FastPETase) do not show degradation reactions.

[0051] It can be seen from Figure 6 that after reacting at 37 °C for 3 days, DCTPC_4M degraded the PBAT film into powder and produced TPA powder with a purity of up to 99%. TPA is a key raw material for making plastics, completing the recycling of plastic monomers.

[0052] Figure 5 and Figure 6 show that the degradation effect of DCTPC_4M on the PBAT film is better than that of ThPETase and its variants disclosed in Chinese Patent ZL202411540951.8 (authorized announcement number CN119040295B).

Claims

1. A mutant of plastic hydrolase ThPETase, characterized in that, The mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1 at positions selected from the 68th, 105th, 194th, and 232nd positions as follows: (1) S68K; (2) D105R; (3) G194Q; (4) D232K; or (5) S68K+D105R+G194Q+D232K.

2. A mutant of the plastic hydrolase ThPETase, characterized in that, The mutant is obtained by mutating the 68th, 105th, 194th, and 232nd positions of the amino acid sequence shown in SEQ ID NO: 1 as follows: S68K+D105R+G194Q+D232K.

3. A polynucleotide, characterized in that, Encoding the mutant of the plastic hydrolase ThPETase 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, a bacterial cell, or a plant cell.

7. The host cell according to claim 6, wherein The host cell is a bacterial cell. Preferably, the bacterial cell is an Escherichia coli cell.

8. Use of the mutant of the plastic hydrolase ThPETase according to claim 1 or 2 or the host cell according to any one of claims 5-7 in degrading PET or PBAT plastics.

9. The application according to claim 8, wherein The way of the said use is: Contacting an object containing PET and / or PBAT plastics with the mutant of the plastic hydrolase ThPETase for a degradation reaction.

10. A method for producing the plastic hydrolase ThPETase mutant as described in claim 1 or 2, characterized in that, This method includes: (a) Culturing the host cell according to any one of claims 5-7 under conditions suitable for the expression of the mutant of the plastic hydrolase ThPETase; and (b) Recovering the mutant of the plastic hydrolase ThPETase.

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