PET hydrolase mutant as well as preparation method and application thereof

Abstract

The invention discloses a PET hydrolase mutant as well as a preparation method and application thereof, and belongs to the field of enzyme engineering. On the basis of FAST-PETase, in combination with deep learning prediction and enzyme engineering optimization strategies, a plurality of mutants with specific performance advantages are systematically constructed and screened, the method is suitable for application scenes with different temperatures, substrate loads and degradation efficiency requirements, and experimental basis and technical support are provided for constructing a diversified and combinable PET degrading enzyme system. The mutants show diversified advantages in different performance dimensions, for example, the enzyme activity of the K233R mutant is 1.62 times that of FAST-PETase, and the K233R mutant still keeps good thermal stability (1.03 times) after being subjected to heat treatment for 2 hours at the temperature of 50 DEG C; q224K is more remarkably improved in the aspect of thermal stability, and the residual activity after heat treatment is 1.64 times that of FAST-PETase.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to a PET hydrolase mutant and a preparation method and application thereof. Background Art

[0002] With the mass production and consumption of plastic products, plastic pollution is becoming increasingly serious and has become a prominent challenge to global ecological security and sustainable development. Polyethylene terephthalate (PET) has become a key target in plastic pollution control due to its widespread use, strong chemical inertness, and long environmental residual life. Biocatalysis, as a green and efficient PET recycling method, is becoming an important research direction to replace traditional mechanical and chemical recycling methods due to its mild reaction conditions and easy product handling.

[0003] In recent years, PET hydrolase (PETase) derived from Ideonella sakaiensis has attracted widespread attention because of its ability to efficiently break the ester bond in the PET backbone under neutral and room temperature conditions. FAST-PETase obtained by enzyme engineering has significantly improved enzyme activity and thermal stability compared to natural enzymes (Lu H., Diaz DJ, Cuisinier M., et al. Machine learning-aided engineering of hydrolases for PET depolymerization [J]. Nature, 2022, 604 (7907): 662-667.), laying the foundation for the application of enzymatic degradation of PET. However, in actual industrial applications, different scenarios (such as different substrate types, reaction temperatures, degradation cycles, etc.) have significant differences in the specific requirements for enzyme performance, and a single mutant is difficult to meet all conditions at the same time. For example, some application scenarios may prioritize the high temperature stability of the enzyme, while other scenarios pay more attention to substrate binding ability or initial reaction rate.

[0004] Therefore, developing a series of PETase mutants with differentiated functional advantages and establishing a library of enzyme variants that can be flexibly selected have become important directions in current PET enzymatic degradation research and are of great significance for adapting to diverse application conditions.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a PET hydrolase mutant, its preparation method, and its application. Based on FAST-PETase, the present invention uses deep learning-assisted screening and high-throughput experimental verification to obtain a variety of mutants with advantages in enzyme activity or thermal stability, such as K233R and Q224K, all of which exhibit properties superior to the original enzyme in different dimensions. The present invention places greater emphasis on the balance and selectivity of multidimensional performance indicators, providing a more adaptable enzyme variant reserve for subsequent applications in complex or changing environments.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A PET hydrolase mutant, wherein the amino acid sequence of SEQ ID NO. 1 is obtained by any one of the following mutations:

[0009] K95E, T140D, I168V, S169A, N212E, Q224K, K233R or T140D\I168V\Q224K\K233R; T140D means the 140th amino acid is mutated from T to D, and the same applies to the others;

[0010] A gene encoding the mutant.

[0011] The mutant-related biological materials are any one or more combinations of the following biological materials:

[0012] (1) an expression cassette containing the above-mentioned encoding gene;

[0013] (2) a recombinant expression vector containing the above-mentioned encoding gene;

[0014] (3) a recombinant expression vector containing the expression cassette described in (1);

[0015] (4) Recombinant microorganisms containing the above-mentioned encoding genes;

[0016] (5) A recombinant microorganism containing the expression cassette described in (1);

[0017] (6) A recombinant microorganism containing the recombinant expression vector described in (2) or (3).

[0018] Furthermore, the starting vector of the recombinant expression vector in (2) and (3) is a pET series vector or a pPICZα vector, etc.; preferably, it is a pET-22a(+) vector or a pPICZαA vector.

[0019] Furthermore, the host microorganism corresponding to the recombinant microorganism in (4), (5) and (6) is selected from prokaryotes, yeasts or higher eukaryotic cells; the prokaryotes include bacteria such as Escherichia, Bacillus, Salmonella, Pseudomonas or Streptomyces; the yeast includes yeasts such as Pichia pastoris. More specifically, the prokaryote is Escherichia, preferably Escherichia coli (E. coli), specifically Escherichia coli BL21 (DE3) or Escherichia coli Origami 2 (DE3); the yeast is Pichia pastoris X33 or GS115.

[0020] Application of the mutant, encoding gene, and mutant-related biological materials in the preparation of PET hydrolase mutants.

[0021] Furthermore, the application of the mutant, encoding gene, and mutant-related biomaterials is one of the following applications:

[0022] (a) Application in PET degradation;

[0023] (b) Application in plastic degradation.

[0024] Preferably, the crystallinity of the PET is less than 20%, further 5-20%, further 6-8%, and further 6.7%.

[0025] A method for obtaining the above mutant comprises the following steps: performing site-directed mutagenesis on a gene encoding a PET hydrolase having an amino acid sequence as shown in SEQ ID NO. 1 and then expressing the site-directed mutagenesis to obtain a PET hydrolase mutant.

[0026] Furthermore, a mutation was introduced into the gene encoding the PET hydrolase having an amino acid sequence as shown in SEQ ID NO. 1 by site-directed mutagenesis. After correct sequencing, the gene was transformed into Escherichia coli for expression to obtain a PET hydrolase mutant.

[0027] The present invention has the following advantages and effects compared to the prior art:

[0028] (1) Based on FAST-PETase, the present invention combines deep learning prediction with enzyme engineering optimization strategies to systematically construct and screen multiple mutants with specific performance advantages. Using the CPD-ResNet50 model prediction and combined with manual evaluation, 11 single-point mutants to be verified that may improve thermal stability or enzyme activity were selected. Combined with the high-throughput p-NPA screening platform and the PET substrate evaluation system, 8 advantageous mutants including K233R, Q224K, N212E, etc. were screened. Further, a library of 247 combination mutants was constructed through a 2-8 site combination strategy, and amorphous PET film (amoPET, crystallinity 6.7%) was used as a substrate for screening. Multiple mutants with outstanding performance in enzyme activity or thermal stability were obtained, which are suitable for application scenarios with different temperatures, substrate loads and degradation efficiency requirements, providing experimental basis and technical support for the construction of a diversified and combinatorial PET degradation enzyme system.

[0029] (2) The mutants of the present invention exhibit diverse advantages in different performance dimensions. For example, the K233R mutant has an enzymatic activity 1.62 times that of FAST-PETase and maintains good thermal stability (1.03 times) after heat treatment at 50°C for 2 hours. The Q224K mutant has an even more significant improvement in thermal stability, with a residual activity of 1.64 times that of FAST-PETase after heat treatment. The relevant mutants have been successfully expressed and purified efficiently using the cSAT 2.0 expression system, providing a good foundation for application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result graph of TPA+MHET+BHET production of each mutant before heat treatment at a PET enzyme loading of 0.3 mg / g.

[0031] Figure 2 The graph shows the results of TPA+MHET+BHET production of each mutant after heat treatment at 50°C for 2 hours at a PET enzyme loading of 0.3 mg / g. DETAILED DESCRIPTION

[0032] The present invention is described in further detail below with reference to the examples and accompanying drawings, but the present invention is not limited thereto. Experimental methods in the following examples, where specific experimental conditions are not specified, generally followed conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0033] The culture medium in the embodiment:

[0034] Preparation of LSG culture medium: take 20 mL of 50×L salt solution (containing 25 mM Na2HPO4, 25 mM KH2PO4, 50 mMNH4Cl, 5 mM Na2SO4), 12.5 mL of 40% glucose solution (final 0.5%), 20 mL of 1 M succinic acid solution (final 20 mM), 2 mL of 1 M MgSO4 solution (final 2 mM) and 2 mL of 1000× metal ion mixed solution (final 1×), add ultrapure water to make up to 1 L, mix well and autoclave at 121°C for 20 min, cool and store at 4°C for later use.

[0035] Preparation of LS-5052 autoinduction medium: Prepare 20 mL of 50×L salt solution (containing 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, and 5 mM Na₂SO₄), 20 mL of 50×L 5052 carbon source mixture (containing 0.5% glycerol, 0.05% glucose, and 0.2% α-lactose), 20 mL of 1M succinic acid solution (final volume: 20 mM), 2 mL of 1M MgSO₄ solution (final volume: 2 mM), and 2 mL of 1000×L metal ion mixture (final volume: 1×), and add ultrapure water to bring the volume to 1 L. Mix all components except the 5052 carbon source mixture, then autoclave at 121°C for 20 min. Cool to room temperature, then aseptically add the 5052 carbon source mixture. Mix thoroughly and store at 4°C until ready for use.

[0036] Example 1 Construction of single-point mutants of PET hydrolase (PETase)

[0037] The PET hydrolase (PETase) used in the examples is FAST-PETase, whose amino acid sequence is shown in SEQ ID NO. 1, and the nucleotide sequence of the encoding gene after codon optimization is shown in SEQ ID NO. 2, which is synthesized by a commercial company.

[0038] 1. Obtaining single-point mutants based on the CPD-ResNet50 deep learning model

[0039] First, the CPD-ResNet50 deep learning model (software name: protein mutation site prediction software, registration number: 2023SR1616838) predicts the possible mutation sites and amino acids and sorts them from large to small according to probability.

[0040] Secondly, based on the above prediction results and combined with manual evaluation, the following mutants (11 in total) were selected as additional single-point mutants to be verified: S92E, K95E, Q127L, T140D, I168V, S169A, N212E, S213T, Q224K, K233R, and A240C.

[0041] 2. Construction and Verification of Single-point Mutant Recombinant Plasmids

[0042] (1) Recombinant plasmid cloning

[0043] Using the recombinant plasmid pET-22a(+)-FAST nucleotide sequence as a template, a full-plasmid polymerase chain reaction (PCR) was performed using primers containing the mutation site. The PCR reaction system consisted of 25 μL of 2× KOD buffer, 10 μL of 2 mM dNTPs, 1.5 μL of each upstream and downstream primer, 20 ng of plasmid template, 1 μL of KOD enzyme, and ddH2O added to a final volume of 50 μL. Reaction conditions were 94°C for 3 min, followed by 30 cycles of 98°C for 30 s, 58°C for 30 s, and 68°C for 3 min 30 s, followed by 68°C for 2 min, and then storage at 4°C.

[0044] The recombinant plasmid pET-22a(+)-FAST was constructed by inserting the nucleotide sequence of the gene encoding FAST-PETase (bp 2-290 in SEQ ID NO.1) (bp 4-870 in SEQ ID NO.2) into the region between BstB I and Xho I of the pET-22a(+) vector.

[0045] (2) Product assembly

[0046] The above product was diluted with 15 μL of Gibson Mix (fragment 1: 25 fmol, fragment 2: 25 fmol), and ddH2O to make up to 20 μL. The mixture was reacted at 50°C for 1 h.

[0047] (3) Recombinant plasmid transformation and verification

[0048] Transformation of recombinant plasmid: Transform 10 μL of the above linked product into 100 μL of E. coli DH5α competent cells, spread on LB solid plates containing a final concentration of 100 μg / mL carbenicillin, and culture inverted at 37°C overnight for about 12-16 hours.

[0049] Verification of transformed clones: Randomly pick 2-4 monoclonal colonies and perform sequencing verification.

[0050] 3. Fermentation and induced expression of single-point mutants

[0051] Take 5 μL of the plasmid after successful sequencing verification and transform it into 100 μL of Escherichia coli BL21 (DE3) competent cells, spread it on an LB solid plate containing a final concentration of 100 μg / mL carbenicillin, and culture it in a 37°C constant temperature incubator overnight for 12-16 hours.

[0052] Pick the colonies, and after verification by colony PCR, streak the strain containing the target plasmid on an LB plate containing the corresponding antibiotic resistance and culture it at 37°C overnight.

[0053] Pick a single colony into a 96-well plate (flat bottom, with lid) containing 150 μL of the corresponding antibiotic resistance LSG medium and culture it overnight in a shaking incubator at 37°C and 220 rpm.

[0054] Transfer 2 μL of the overnight cultured bacterial solution to a 96-well plate (flat-bottom, with lid) containing 198 μL of LS-5052 autoinduction medium of the corresponding resistance at a ratio of 1:100, and culture at 20°C and 220 rpm for 48 hours.

[0055] The expression product was centrifuged at 3,320 g for 15 min, and the supernatant was collected for the measurement of initial enzyme activity in the next step.

[0056] Note: All mutants were activated, transferred and induced under the same conditions.

[0057] Example 2 Screening of single-point mutants of PET hydrolase

[0058] For the 11 single-point mutants, the residual enzyme activity of their fermentation supernatants after heat treatment at 45°C for 30 minutes was measured, as well as the enzyme activity before heat treatment. The activity was then compared with that of FAST-PETase, allowing for the identification of single-point mutants with improved thermostability and relative enzyme activities greater than 0.5. Ultimately, eight single-point mutants with improved thermostability and relative enzyme activities greater than 0.5 were obtained (Table 1): K95E, T140D, I168V, S169A, N212E, S213T, Q224K, and K233R.

[0059] PET hydrolase activity assay: Enzyme activity was determined using 405 nm absorbance, using p-NPA as the substrate. One unit of activity is defined as the yield of 1 μmol of p-NA (p-nitroaniline) in 1 minute at room temperature at pH 8.0. A 5 μL aliquot of the enzyme solution was mixed with 145 μL of phosphate buffer (pH 8.0, 100 mM) and 50 μL of the model substrate, p-NPA (dissolved in ethanol to a 4 mM concentration). The absorbance at 405 nm was immediately measured at room temperature. The relative activity of each mutant was determined by comparing the activity of the mutant with that of FAST-PETase (see Table 1).

[0060] PET hydrolase thermal stability assay: 5 μL of enzyme solution was mixed with 145 μL of phosphate buffer (pH 8.0, 100 mM), sealed with film, and heat-treated at 45°C for 30 minutes. After heat treatment, the solution was returned to room temperature and then mixed with 50 μL of the model substrate p-NPA (dissolved in ethanol at a concentration of 4 mM). The absorbance at a wavelength of 405 nm was immediately measured at room temperature. The enzyme activity was determined by comparing the post-heat-treatment activity with the pre-heat-treatment activity to determine the residual activity of each mutant. The residual activity of the mutants was compared with the residual activity of FAST-PETase to determine the relative thermal stability of each mutant (see Table 1).

[0061] Table 1 Relative enzyme activity and relative thermal stability of the fermentation supernatant of single-point mutants after heat treatment at 45°C for 30 min

[0062] Location Original amino acids Mutated amino acid Relative enzyme activity Relative thermal stability FAST-PETase - - 1.00 1.00 S92E S(agc) E(gag) 0.52 1.04 K95E K(aag) E(gag) 1.35 1.27 Q127L Q(cag) L(ctg) 0.2 - T140D T(acc) D(gac) 0.75 1.17 I168V I(att) V(gtt) 1.12 1.30 S169A S(agc) A(gcg) 1.35 1.26 N212E N(aac) E(gag) 0.64 1.60 S213T S(agc) T(acg) 0.84 1.32 Q224K Q(cag) K(aag) 0.77 1.06 K233R K(aag) R(cgg) 0.71 1.32 A240C A(gcc) C(tgt) 0.34 -

[0063] Example 3 Construction of a PET Hydrolase Combinatorial Mutation Library

[0064] The eight beneficial single-point mutants in Example 2 were randomly combined and a combinatorial mutant library was constructed using the DNAwork method. A total of 247 combinatorial mutants were constructed using the random combination of 2 to 8 sites.

[0065] Through the mutant construction and induced expression technology described in Example 1, and the mutant enzyme activity and thermal stability determination method described in Example 2, a beneficial combination mutant was screened out: T140D\I168V\Q224K\K233R, with a relative enzyme activity of 0.6 and a relative thermal stability of 1.3.

[0066] Example 4 PET degradation assay of some PET hydrolase single-site and combined mutants

[0067] 1. Recombinant construction and purification of beneficial mutants

[0068] (1) Based on the cSAT 2.0 method, some single-site and combined mutants of PET hydrolase were recombinantly constructed. During the construction process, the signal peptide of the target gene (some single-site and combined mutants of PET hydrolase) was removed. The amino acid sequence of the signal peptide is shown in SEQ ID NO.1, 1-27aa, and the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO.2, 1-81bp.

[0069] (2) Expression of single-site and combined mutants of some PET hydrolases based on the cSAT 2.0 method

[0070] The dominant mutants based on the cSAT 2.0 method were plasmidized and transformed into Escherichia coli Origami 2 (DE3) competent cells, and grown overnight at 37°C for 12-16 h on LB plates containing 100 μL / mL carbenicillin.

[0071] A well-growing single colony was transferred to 5 mL of liquid LB medium containing 100 μL / mL carbenicillin and cultured at 37°C and 200 rpm for 18 h.

[0072] Transfer 2 mL of seed solution to 200 mL of fresh liquid 2YT medium containing 100 μL / mL carbenicillin and culture at 37 °C and 200 rpm until OD 600 Reach 0.6-0.8.

[0073] Protein expression was induced with 0.2 mM IPTG and cultured at 18°C and 200 rpm for 20 hours. The cSAT 2.0 bacterial pellet was then collected by centrifugation at 4,000 rpm for 20 minutes.

[0074] (3) Purification of some PET hydrolase single-site and combined mutants was performed based on the cSAT 2.0 method. Detection was performed by SDS-PAGE electrophoresis to obtain the purified PET hydrolase single-site and combined mutants, which were recorded as pure enzymes.

[0075] Among them, the cSAT 2.0 method is disclosed in the document “Huang Y, Zhang Y, Yang X, et al. A high-performance protein preparation approach in a single column-free step [J]. Trends in biotechnology: S0167-7799 (24) 00290-7. DOI: 10.1016 / j.tibtech.2024.10.008.”.

[0076] 2. Enzyme activity and thermal stability testing of beneficial mutants for PET degradation

[0077] For single-point beneficial mutants and combined beneficial mutants, the residual enzyme activity of the pure enzyme after heat treatment at 50°C for 2 hours and then reaction for 3 hours and the enzyme activity after reaction at 50°C for 3 hours were measured. Before heat treatment, the enzyme activity of the mutants was compared with the enzyme activity of FAST-PETase to obtain the relative enzyme activity of each mutant. The residual activity was calculated by comparing the enzyme activity after heat treatment with the enzyme activity before heat treatment; the relative thermal stability of the mutants was obtained by comparing the residual activity of the mutants with the residual activity of FAST-PETase, as shown in Table 2 and Figure 1-2 From Table 2 and Figure 1-2It can be seen that compared with FAST-PETase, the enzyme activity of the single-point mutant K233R was improved, which was 1.62 times that of FAST-PETase. The thermal stability of the single-point mutant Q224K was improved after heat treatment at 50°C for 2 hours, and the residual activity of Q224K was 1.64 times that of FAST-PETase.

[0078] PET hydrolase activity assay: Amorphous PET membrane (amoPET, 6.7% crystallinity) (Guttfu Co., Ltd., ES301445) was used as a substrate. 6 mm diameter discs weighing approximately 8.5 mg were prepared and then soaked in 1% SDS, 20% ethanol, and deionized water for 30 min each, air-dried, and used as the actual substrate for HPLC analysis. 200 nM pure enzyme was added to a 96-well plate in 436 μL of 50 mM glycine buffer (pH 9.2) at 50°C for an enzyme loading of 0.3 mg / g PET. The 6 mm diameter amorphous PET membrane was added directly to the plate and reacted for 3 h. After the reaction, an equal volume (436 μL) of cold methanol and trifluoroacetic acid was added to each reaction system as a stop solution to precipitate unreacted macromolecules and enzyme protein, thereby removing potential analytical interferences. The yield of each product was analyzed by HPLC. HPLC analysis was performed using a 1260 Infinity II system equipped with XB-C18 chromatographic column. Mobile phase A is an aqueous solution containing 0.1% formic acid, mobile phase B is 100% acetonitrile, and the flow rate is set to 1.5 mL / min. The initial elution condition is 13% B to separate TPA and MHET, then the gradient is increased to 95% B, and finally adjusted back to 13% B to rebalance the chromatographic column. By comparing the corresponding relationship between the product peak area and the standard, the three products of terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET) are calculated. The results are as follows Figure 1 shown.

[0079] Method for determining the thermal stability of PET degradation by PET hydrolase: a pretreated 6 mm amorphous PET film (crystallinity 6.7%) was used as the substrate. The total volume in a 96-deep-well plate was 436 μL of glycine buffer (50 mM, pH 9.2), which contained 200 nM pure enzyme and an enzyme loading of 0.3 mg / g PET. After heat treatment at 50°C for 2 h, an amorphous PET film with a diameter of 6 mm was added and the reaction was continued for 3 h. After the reaction was completed, an equal volume (436 μL) of a mixture of cold methanol and trifluoroacetic acid was added to each reaction system as a reaction termination solution to precipitate unreacted macromolecules and enzyme proteins, thereby removing potential analytical interferences. HPLC analysis was performed according to the above method. The results are shown in FIG. Figure 2 shown.

[0080] Table 2 Relative enzyme activity and relative thermal stability of beneficial mutants at a uniform enzyme loading of 0.3 mg / g PET at 50°C

[0081] mutation site Relative enzyme activity Residual activity Relative thermal stability K233R 1.62 0.84 1.03 T140D 1.56 0.94 1.16 K95E 1.15 1.02 1.26 N212E 1.05 1.16 1.43 T140D\I168V\Q224K\K233R 1.05 1.14 1.40 I168V 0.99 1.04 1.28 S169A 0.93 1.02 1.25 Q224K 0.60 1.33 1.64 FAST-PETase 1.00 0.81 1.00

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A PET hydrolase mutant, characterized in that The amino acid sequence of the mutant is obtained by subjecting SEQ ID NO.1 to any of the following mutations: K95E, T140D, I168V, S169A, N212E, Q224K, K233R or T140D\I168V\Q224K\K233R.

2. A gene encoding the PET hydrolase mutant according to claim 1.

3. The biomaterial related to the PET hydrolase mutant according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (1) An expression cassette containing the gene according to claim 2; (2) a recombinant expression vector containing the gene according to claim 2; (3) a recombinant expression vector containing the expression cassette described in (1); (4) A recombinant microorganism containing the gene according to claim 2; (5) A recombinant microorganism containing the expression cassette described in (1); (6) A recombinant microorganism containing the recombinant expression vector described in (2) or (3).

4. The biomaterial according to claim 3, characterized in that: The host microorganism corresponding to the recombinant microorganisms described in (4), (5) and (6) is selected from prokaryotes, yeasts or higher eukaryotic cells.

5. The biomaterial according to claim 4, characterized in that: The prokaryotic organism is Escherichia coli; the yeast is Pichia pastoris.

6. Use of the PET hydrolase mutant according to claim 1, the gene according to claim 2, or the biomaterial according to any one of claims 3 to 5 in the preparation of a PET hydrolase mutant.

7. Use of the PET hydrolase mutant according to claim 1, the gene according to claim 2, or the biomaterial according to any one of claims 3 to 5, characterized in that: One of the following applications: (a) Application in PET degradation; (b) Application in plastic degradation.

8. The use according to claim 7, characterized in that: The crystallinity of the PET is less than 20%.

9. A method for obtaining the PET hydrolase mutant according to claim 1, characterized in that: The method comprises the following steps: performing site-directed mutagenesis on a gene encoding a PET hydrolase having an amino acid sequence as shown in SEQ ID NO. 1 by using a site-directed mutagenesis technique and then expressing the gene to obtain the PET hydrolase mutant according to claim 1.

10. The method according to claim 9, characterized in that A mutation is introduced into the gene encoding the PET hydrolase having an amino acid sequence as shown in SEQ ID NO. 1 by site-directed mutagenesis technology, and after correct sequencing, the gene is transformed into Escherichia coli for expression to obtain the PET hydrolase mutant according to claim 1.

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