PET hydrolase mutant with improved enzyme activity and application thereof

Abstract

The invention discloses a PET hydrolase mutant with improved enzyme activity and application thereof. Starting from FAST-PETase, research is carried out on the basis of deep learning design, enzyme engineering optimization and application. 11 to-be-verified single-point mutants capable of improving the enzyme performance are selected through model prediction and manual evaluation, and then experimental verification and enzyme engineering modification are carried out. The method comprises the following steps: comprehensively selecting better single-point mutants and combined mutants, and evaluating by taking an amorphous PET (Polyethylene Terephthalate) film (with the crystallinity of 6.7%) as a substrate to obtain dominant mutants. The dominant mutant is efficiently expressed and purified based on cSAT 2.0, the enzyme activity of the optimal mutant T140DS213T is two times that of FAST-PETase, and the thermal stability of the optimal mutant T140DS213T is equivalent to that of the FAST-PETase after the optimal mutant is subjected to heat treatment at 50 DEG C for 2 hours. A protein Tm test on the optimal mutant T140DS213T shows that the Tm of the optimal mutant T140DS213T is increased by 1 DEG C.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering, and particularly relates to a PET hydrolase mutant with improved enzyme activity and its application. Background Art

[0002] Polyethylene terephthalate (PET) is a thermoplastic polyester widely used in industrial fields such as packaging materials, synthetic fibers, and plastic containers. It has good thermal stability and chemical inertness and is difficult to degrade in the natural environment. With the continuous growth of PET product consumption, its waste accumulates in the environment for a long time, becoming an important source of waste plastic pollution. Although PET has the potential for recycling, the existing recycling methods still have obvious limitations. Currently, the recycling methods of PET mainly include mechanical recycling and chemical recycling. Mechanical recycling realizes the reuse of PET through physical methods, with simple operation, but the performance of the material is prone to decline after multiple recycling, restricting its application in high-performance products. Chemical recycling degrades PET into its monomers or oligomers through reactions such as hydrolysis and alcoholysis, which can realize the regeneration and synthesis of materials, but this type of method generally has problems such as harsh process conditions, high energy consumption, and difficulty in treating by-products.

[0003] In comparison, enzymatic degradation of PET has been proposed as an alternative in related fields due to its advantages such as mild reaction conditions, clear products, and environmental friendliness. It is known that PET hydrolase (PETase) can break the ester bonds in PET molecules under suitable temperature and pH conditions to generate degradation products such as terephthalic acid (TPA) and ethylene glycol (EG). Existing studies have shown that Ideonella sakaiensis PETase and MHETase from can synergistically achieve the decomposition of PET and are representative in biocatalytic degradation technology. However, natural PETase still has deficiencies in aspects such as enzyme catalytic efficiency, thermal stability, and substrate adaptability, and it is difficult to adapt to industrial conditions such as high substrate concentration, long reaction time, or relatively high temperature. To improve its performance, existing studies have used enzyme engineering methods such as directed evolution and rational design to develop improved enzyme variants. Among them, mutants represented by FAST-PETase have improved both in activity and stability compared to natural enzymes and have become an important tool in current enzymatic PET degradation technology (Lu H., Diaz D. J., Cuisinier M., et al. Machine learning-aided engineering of hydrolases for PET depolymerization [J]. Nature, 2022, 604(7907): 662-667.). However, FAST-PETase still has problems such as insufficient reaction efficiency and limited optimization space for enzyme stability in specific application environments, and its performance still needs to be further optimized to meet the actual application requirements.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies of the prior art, the object of the present invention is to provide a PET hydrolase mutant with improved enzyme activity and its application. The present invention combines deep learning model design and enzyme engineering optimization strategies to develop a high-performance PET hydrolase mutant that better meets the requirements of industrial applications.

[0006] The object of the present invention is achieved by the following technical solutions: A PET hydrolase mutant with improved enzyme activity, the amino acid sequence of which is obtained by any one of the following mutations of SEQ ID NO.1: S213T, T140D\S213T, S169A\S213T or N212E\S213T; wherein, S213T means that the 213th amino acid is mutated from S to T, and the same applies to others; A coding gene of the above mutant.

[0007] The biological material related to the above mutant is any one or a combination of the following biological materials: (1) An expression cassette containing the above coding gene; (2) A recombinant expression vector containing the above coding gene; (3) A recombinant expression vector containing the expression cassette described in (1); (4) A recombinant bacterium containing the above coding gene; (5) A recombinant bacterium containing the expression cassette described in (1); (6) A recombinant bacterium containing the recombinant expression vector described in (2) or (3).

[0008] Furthermore, the starting vector of the recombinant expression vectors described in (2) and (3) is a vector of the pET series or the pPICZα vector, etc.; preferably the pET-22a(+) vector or the pPICZαA vector.

[0009] Furthermore, the host bacteria corresponding to the recombinant bacteria described in (4), (5), and (6) are selected from prokaryotes, yeasts, or higher eukaryotic cells, etc.; the prokaryotes include Escherichia ( Escherichia ), Bacillus ( Bacillus ), Salmonella ( Salmonella ), Pseudomonas ( Pseudomonas ), or Streptomyces ( Streptomyces ) and other bacteria; the yeasts include yeasts such as Pichia pastoris. More specifically, the prokaryote is Escherichia, preferably Escherichia coli ( Escherichia coli , E. coli), specifically, it can be Escherichia coli BL21(DE3) or Escherichia coli Origami 2(DE3); the yeast is Pichia pastoris X33 or GS115.

[0010] Use of the above-mentioned mutant, coding gene, and biological material related to the mutant in preparing a PET hydrolase mutant with improved enzyme activity.

[0011] Furthermore, use of the above-mentioned mutant, coding gene, and biological material related to the mutant in degrading PET.

[0012] Preferably, the crystallinity of the PET is below 20%, further 5 - 20%, still further 6 - 8%; even further 6.7%.

[0013] A method for obtaining the above-mentioned mutant, comprising the following steps: performing site-directed mutagenesis on the gene encoding the PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 by site-directed mutagenesis technology and then expressing it to obtain a PET hydrolase mutant.

[0014] Furthermore, introduce mutations into the gene encoding the PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 by site-directed mutagenesis technology. After correct sequencing, transform it into Escherichia coli for expression to obtain a PET hydrolase mutant.

[0015] The present invention has the following advantages and effects compared with the prior art: The present invention starts from FAST-PETase and conducts research based on deep learning design, enzyme engineering optimization, and application. First, use the CPD-ResNet50 deep learning model to predict and combine with manual evaluation to select 11 single-site mutants to be verified that may improve enzyme performance, and then conduct experimental verification and enzyme engineering transformation. Construct a high-throughput PETase enzyme activity screening method based on a secretion expression system and the model substrate 4-nitrophenyl acetate ( p -NPA), and use a relative enzyme activity greater than 0.5 (mutant / FAST-PETase) and a relative thermal stability greater than 1.0 (mutant / FAST-PETase) as the criteria to screen out 8 dominant single-site mutants; then construct and evaluate a library of 247 combinatorial mutants containing 2 - 8 mutation sites, comprehensively select better single-site mutants and combinatorial mutants, and evaluate them using an amorphous PET film (amoPET, crystallinity 6.7%) as the substrate to obtain dominant mutants. The dominant mutant was efficiently expressed and purified based on the cleavable self-aggregation tag method (cSAT 2.0). The enzyme activity of the optimal mutant T140D\S213T is 2 times that of FAST-PETase, and its thermal stability after heat treatment at 50°C for 2 hours is equivalent to that of FAST-PETase. Perform protein melting temperature (Tm ) The test shows that its T m increases by 1 °C. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a result graph of the yields of TPA + MHET + BHET before heat treatment of each mutant at a PET enzyme loading of 0.3 mg / g PET.

[0017] Figure 2 It is a result graph of the yields of TPA + MHET + BHET after heat treatment of each mutant at 50 °C for 2 hours at a PET enzyme loading of 0.3 mg / g PET. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto. The test methods without specific experimental conditions noted in the following embodiments are generally in accordance with conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial sources.

[0019] Example 1 Construction of Single-Point Mutants of PET Hydrolase (PETase) The PET hydrolase (PETase) used in the example is FAST-PETase, whose amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene after codon optimization is shown in SEQ ID NO.2, which is synthesized by a commercial company.

[0020] 1. Obtaining single-point mutants based on the CPD-ResNet50 deep learning model First, the sites and amino acids that may be mutated predicted by the CPD-ResNet50 deep learning model (software name: Protein Mutation Site Prediction Software, registration number: 2023SR1616838) are sorted according to probability from large to small.

[0021] Second, based on the above prediction results and combined with manual evaluation, the following mutants (a total of 11) are selected as additional single-point mutants to be verified: S92E, K95E, Q127L, T140D, I168V, S169A, N212E, S213T, Q224K, K233R, A240C.

[0022] 2. Construction and verification of single-point mutant recombinant plasmids (1) Cloning of recombinant plasmids Using the recombinant plasmid pET-22a(+)-FAST nucleotide sequence as a template, perform whole plasmid PCR polymerase chain reaction with primers containing mutation sites. The PCR reaction system is 25 μL of 2×KOD buffer, 10 μL of 2 mM dNTP, 1.5 μL each of upstream and downstream primers, 20 ng of plasmid template, 1 μL of KOD enzyme, and ddH 2 O is added to make the final volume of the system 50 μL. The reaction conditions are 3 min at 94°C; 30 s at 98°C, 30 s at 58°C, 3 min 30 s at 68°C, for 30 cycles; 2 min at 68°C; and stored at 4°C.

[0023] Among them, the recombinant plasmid pET-22a(+)-FAST is constructed by inserting the nucleotide sequence of the gene encoding FAST-PETase (2-290aa in SEQ ID NO.1) (4-870bp in SEQ ID NO.2) into the BstB I to Xho I of the pET-22a(+) vector.

[0024] (2)Product assembly Mix the above products in the ratio of fragment 1: 25 fmol, fragment 2: 25 fmol, 15 μL of Gibson Mix, and ddH 2 O is added to make the final volume 20 μL. React at 50°C for 1 h.

[0025] (3)Transformation and verification of recombinant plasmid Recombinant plasmid transformation: Transform 10 μL of the above ligation product into 100 μL of Escherichia coli DH5α competent cells, coat on an LB solid plate containing 100 μg / mL carbenicillin at a final concentration, and incubate overnight at 37°C for about 12-16 hours.

[0026] Verification of transformed clones: Randomly pick 2-4 monoclonal colonies for sequencing verification.

[0027] 3. Fermentation and induced expression of single point mutants Take 5 μL of the plasmid after successful sequencing verification and transform it into 100 μL of Escherichia coli BL21(DE3) competent cells, coat on an LB solid plate containing 100 μg / mL carbenicillin at a final concentration, and incubate overnight in a 37°C constant temperature incubator for 12-16 h.

[0028] Pick colonies, after verifying correctly by colony PCR, streak the strain containing the target plasmid on an LB plate with the corresponding antibiotic resistance and incubate overnight at 37°C.

[0029] Pick a single colony into a 96-well plate (flat bottom, with lid) containing 150 μL of the corresponding antibiotic-resistant LSG medium, and culture overnight at 37 °C and 220 rpm in a shaking incubator.

[0030] Transfer 2 μL of the overnight-cultured bacterial solution according to a 1:100 ratio into a 96-well plate (flat bottom, with lid) containing 198 μL of the LS-5052 autoinduction medium with the corresponding resistance, and culture at 20 °C and 220 rpm for 48 hours.

[0031] Centrifuge the expressed product at 3,320 g for 15 min, collect the supernatant, and use it for the measurement of the initial enzyme activity in the next step.

[0032] Note: All mutants were activated, transferred, and induced under the same conditions.

[0033] Among them, the preparation of the LSG medium: Take 20 mL of 50×L salt solution (containing 25 mM Na 2 HPO 4 、25 mM KH 2 PO 4 、50 mM NH 4 Cl、5 mM Na 2 SO 4 ), 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 MgSO 4 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, autoclave at 121 °C for 20 min, and store at 4 °C for later use after cooling.

[0034] The preparation of the LS-5052 autoinduction medium: Take 20 mL of 50×L salt solution (containing 25 mM Na 2 HPO 4 、25 mM KH 2 PO 4 、50 mM NH 4 Cl、5 mM Na 2 SO 4 ), 20 mL of 50×5052 carbon source mixed solution (containing 0.5% glycerol, 0.05% glucose, 0.2% α-lactose), 20 mL of 1 M succinic acid solution (final 20 mM), 1 M MgSO 42 mL of the solution (final concentration 2 mM) and 2 mL of the 1000× metal ion mixed solution (final concentration 1×) were added, and ultrapure water was added to make up to 1 L. Except for the 5052 carbon source mixture, other components were mixed well and autoclaved at 121 °C for 20 min. After cooling to room temperature, the 5052 carbon source mixture was added under sterile conditions. After mixing well, it was stored at 4 °C for later use.

[0035] Example 2 Screening of single-point mutants of PET hydrolase For the above 11 single-point mutants, by measuring the enzyme activity after heat treatment for 30 min at 45 °C and the enzyme activity before heat treatment of their fermentation supernatants, and comparing with FAST-PETase, single-point mutants with improved thermal stability and relative enzyme activity greater than 0.5 were screened out. Finally, 8 single-point mutants with improved thermal stability and relative enzyme activity greater than 0.5 were obtained (Table 1): K95E, T140D, I168V, S169A, N212E, S213T, Q224K, K233R.

[0036] Method for determining the enzyme activity of PET hydrolase: The enzyme activity was determined by the light absorption method at 405 nm, using p -NPA as the substrate to determine the enzyme activity of PET hydrolase. One enzyme activity unit refers to the reaction efficiency of producing 1 μmol p -NA (p-nitroaniline) within 1 min under the conditions of pH 8.0 and room temperature; Take a 5 μL enzyme solution and mix it with 145 μL of phosphate buffer (pH 8.0, 100 mM) respectively, and mix it with 50 μL of the model substrate p -NPA (dissolved in ethanol, concentration 4 mM), and immediately measure the absorbance at a wavelength of 405 nm at room temperature. Determine its enzyme activity, and obtain the relative enzyme activity of each mutant by comparing the mutant enzyme activity with the FAST-PETase enzyme activity, as shown in Table 1.

[0037] Method for determining the thermal stability of PET hydrolase: Take a 5 μL enzyme solution and mix it with 145 μL of phosphate buffer (pH 8.0, 100 mM) respectively, stick on the sealing film, and heat-treat at 45 °C for 30 minutes. After the heat treatment is completed and cooled to room temperature, mix it with 50 μL of the model substrate p -NPA (dissolved in ethanol, concentration 4 mM), and immediately measure the absorbance at a wavelength of 405 nm at room temperature. Determine its enzyme activity, and obtain the residual activity of each mutant by comparing the enzyme activity after heat treatment with the enzyme activity before heat treatment; The residual activity of the mutant was compared with the residual activity of FAST-PETase to obtain the relative thermal stability of each mutant, as shown in Table 1.

[0038] Table 1 Relative enzyme activity and relative thermal stability of the fermentation supernatants of single-point mutants after heat treatment at 45 °C for 30 min

[0039] Example 3 Construction of a combinatorial mutant library of PET hydrolase The 8 beneficial single mutants in Example 2 were randomly combined, and the DNAwork method was used to construct a combinatorial mutant library. A total of 247 combinatorial mutants were constructed in the form of random combinations at positions 2-8.

[0040] Through the mutant construction and induced expression techniques described in Example 1, and through the mutant enzyme activity and thermal stability determination methods described in Example 2, 2 beneficial combinatorial mutants were screened out: N212E\S213T, S169A\S213T, with relative enzyme activities of 1.35 and 1.27 respectively, and relative thermal stabilities of 0.92 and 1.16 respectively. At the same time, based on the dominant single mutant T140D, the combinatorial mutant T140D\S213T was also used for subsequent PET degradation assays.

[0041] Example 4 PET degradation assay of some single and combinatorial mutants of PET hydrolase 1. Recombinant construction and purification of beneficial mutants (1) Based on the cSAT 2.0 method, some single and combinatorial mutants of PET hydrolase were recombinantly constructed. During the construction process, the signal peptide of the target gene (some single and combinatorial mutants of PET hydrolase) needed to be removed. The amino acid sequence of this signal peptide is shown as the 1-27aa in SEQ ID NO.1, and the nucleotide sequence encoding the signal peptide is shown as the 1-81bp in SEQ ID NO.2.

[0042] (2) Expression of some single and combinatorial mutants of PET hydrolase based on the cSAT 2.0 method The plasmid of the dominant mutant based on the cSAT 2.0 method was transferred into Escherichia coli Origami 2(DE3) competent cells, and grown overnight at 37°C for 12-16 h on an LB plate containing 100 μL / mL carbenicillin.

[0043] The well-grown single colonies were transferred to 5 mL of liquid LB medium containing 100 μL / mL carbenicillin and cultured at 37°C and 200 rpm for 18 h.

[0044] 2 mL of the seed solution was transferred to 200 mL of fresh liquid 2YT medium containing 100 μL / mL carbenicillin and cultured at 37°C and 200 rpm until the OD 600 reached 0.6-0.8.

[0045] Protein expression was induced with 0.2 mM IPTG and further cultured at 18 °C and 200 rpm for 20 hours. Then, the cells were centrifuged at 4,000 rpm for 20 min to collect the cSAT 2.0 cell pellet.

[0046] (3)Purify some single-point and combinatorial mutants of PET hydrolase based on the cSAT 2.0 method. Detection was carried out using SDS-PAGE electrophoresis to obtain the purified single-point and combinatorial mutants of PET hydrolase, denoted as pure enzymes.

[0047] Among them, the cSAT 2.0 method was disclosed in the literature "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."

[0048] 2. Enzyme activity and thermal stability tests of beneficial mutants for degrading PET For single-point beneficial mutants and combinatorial beneficial mutants, the enzyme activity after heat treatment and the enzyme activity before heat treatment were measured by measuring the pure enzyme after heat treatment at 50 °C for 2 hours and then reacting for 3 hours and reacting at 50 °C for 3 hours before heat treatment. Before heat treatment, the enzyme activity of the mutants was compared with that of FAST-PETase to obtain the relative enzyme activity of each mutant. By comparing the enzyme activity after heat treatment with the enzyme activity before heat treatment, its residual activity was calculated; the residual activity of the mutants was compared with the residual activity of FAST-PETase to obtain its relative thermal stability, as shown in Table 2 and Figure 1 、 Figure 2 。From Table 2 and Figure 1 、 Figure 2 It can be seen that compared with FAST-PETase, the enzyme activities of the single-point mutant S213T and the combinatorial mutants T140D\S213T, S169A\S213T, and N212E\S213T were further improved, and their thermal stabilities were comparable or even improved.

[0049] Method for determining the enzymatic activity of PET hydrolase in degrading PET: Using an amorphous PET film (crystallinity 6.7%) (Goodfellow Corporation, ES301445) as the substrate, first cut it into discs with a diameter of 6 mm and a mass of approximately 8.5 mg, then soak them in 1% SDS, 20% ethanol, and deionized water for 30 min each, and air-dry them to serve as the actual substrate for HPLC testing. In a 96-well deep-well plate, the total volume is 436 μL of glycine buffer (50 mM, pH 9.2), which contains 200 nM of pure enzyme, and the enzyme loading is 0.3 mg / g PET. At 50 °C, directly add the amorphous PET film with a diameter of 6 mm and carry out a 3-h reaction. After the reaction, add an equal volume (436 μL) of a cold methanol and trifluoroacetic acid mixture to each reaction system as the reaction terminating solution to precipitate unreacted macromolecules and enzyme proteins, thereby removing potential analytical interferents. Finally, analyze the production amount of each product by HPLC. The HPLC analysis uses a 1260 Infinity II system equipped with a Kinetex® XB-C18 chromatographic column. Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is 100% acetonitrile. The flow rate is set at 1.5 mL / min. The initial elution condition is 13% B to separate TPA and MHET, then gradually increase to 95% B, and finally return to 13% B to re-equilibrate the chromatographic column. Calculate the three products of terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET) by comparing the corresponding relationship between the product peak area and the standard. The results are as Figure 1 shown.

[0050] Method for determining the thermal stability of PET hydrolase in degrading PET: Using a pretreated 6-mm amorphous PET film (crystallinity 6.7%) as the substrate. In a 96-well deep-well plate, the total volume is 436 μL of glycine buffer (50 mM, pH 9.2), which contains 200 nM of pure enzyme, and the enzyme loading is 0.3 mg / g PET. After heat treatment at 50 °C for 2 h, then add the amorphous PET film with a diameter of 6 mm and continue the 3-h reaction. After the reaction, add an equal volume (436 μL) of a cold methanol and trifluoroacetic acid mixture to each reaction system as the reaction terminating solution to precipitate unreacted macromolecules and enzyme proteins, thereby removing potential analytical interferents. Perform HPLC analysis according to the above method. The results are as Figure 2 shown.

[0051] Table 2 Relative enzymatic activity and relative thermal stability of beneficial mutants with a unified enzyme loading of 0.3 mg / g PET at 50 °C

[0052] Example 5 Determination of the protein melting temperature (T m ) of the optimal mutant T140D\S213T of PET hydrolase The protein melting temperature was evaluated by differential scanning calorimetry. The experiment was carried out using a MicroCal PEAQ-DSC microcalorimetric differential scanning calorimeter (Malvern Panalytical). The specific method was as follows: The purified protein sample was first concentrated to 1 mg / mL by a Microcon® 10 kDa ultrafiltration centrifugal device (Merck Millipore) and replaced with an intramolecular peptide cleavage buffer (Buffer B2, 50 mM Bis-Tris, pH 6.2) to maintain consistent buffer conditions. After injecting 325 μL of the concentrated protein sample and an equal volume of blank Buffer B2 into the sample cell and the reference cell respectively, the temperature program was set as follows: linearly heating from 15°C to 85°C at a rate of 1.0°C·min⁻¹, maintaining at 85°C for 2 minutes to eliminate the thermal history effect, and then cooling to 15°C at the same rate. High-purity nitrogen gas (99.999%) was continuously introduced during the experiment to prevent solution evaporation. After baseline correction and asymmetric peak fitting of the heat flow data by the MicroCal PEAQ-DSC analysis software (v1.41), the melting temperature (T m ) was defined as the temperature corresponding to the peak of the first derivative of the heat capacity change curve (ΔCp), as shown in Table 3. As can be seen from Table 3, the T m value of the mutant T140D\S213T increased by 1.06°C.

[0053] Table 3 Protein melting temperatures of PET hydrolase

[0054] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A PET hydrolase mutant with improved enzyme activity, characterized in that: The amino acid sequence of the mutant is obtained by subjecting SEQ ID NO.1 to any of the following mutations: S213T, T140D\S213T, S169A\S213T, or N212E\S213T.

2. A gene encoding the PET hydrolase mutant with improved enzyme activity according to claim 1.

3. The biomaterial related to the PET hydrolase mutant with improved enzyme activity 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 bacterium containing the gene of claim 2; (5) A recombinant bacterium containing the expression cassette described in (1); (6) A recombinant bacterium containing the recombinant expression vector described in (2) or (3).

4. The biomaterial according to claim 3, characterized in that: The host bacteria corresponding to the recombinant bacteria described in (4), (5) and (6) are selected from prokaryotes, yeast 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 with improved enzyme activity 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 the PET hydrolase mutant with improved enzyme activity.

7. Use of the PET hydrolase mutant with improved enzyme activity according to claim 1, the gene according to claim 2 or the biomaterial according to any one of claims 3 to 5 in degrading PET.

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 with improved enzyme activity 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 and then expressing the site-directed mutagenesis technique to obtain the PET hydrolase mutant with improved enzyme activity as claimed in claim 1.

10. The method according to claim 9, characterized in that A mutation is introduced into the gene encoding the PET hydrolase whose amino acid sequence is 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 with improved enzyme activity as claimed in claim 1.

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