Pet hydrolytic enzyme mutant with improved enzyme activity and application thereof

Abstract

The application discloses a PET hydrolytic enzyme mutant with improved enzyme activity and application thereof. The application is based on the research on design based on deep learning, enzyme engineering optimization and application of FAST-PETase. Model prediction and artificial evaluation are combined, 11 single-point mutants which may improve enzyme performance are selected, and then experiment verification and enzyme engineering modification are carried out. The better single-point mutants and combined mutants are comprehensively selected, and the non-crystalline PET film (crystallinity 6.7%) is used as a substrate to evaluate the dominant mutant. The dominant mutant is purified based on cSAT 2.0 high-efficiency expression, and the enzyme activity of the optimal mutant T140D\S213T is twice that of FAST-PETase. The thermal stability of the optimal mutant T140D\S213T after 2 hours of heat treatment at 50 DEG C is equivalent to that of FAST-PETase. The protein T m The test shows that the T m is improved by 1 DEG C.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, and specifically relates to a PET hydrolase mutant with enhanced enzyme activity and its application. Background Technology

[0002] Polyethylene terephthalate (PET) is a thermoplastic polyester widely used in industrial applications such as packaging materials, synthetic fibers, and plastic containers. It possesses good thermal stability and chemical inertness, making it difficult to degrade in the natural environment. With the continuous growth in PET product consumption, its waste accumulates in the environment over a long period, becoming a significant source of waste plastic pollution. Although PET has recycling potential, existing recycling methods still have significant limitations. Currently, PET recycling methods mainly include mechanical recycling and chemical recycling. Mechanical recycling achieves PET reuse through physical methods, which is simple to operate, but the material's performance tends to decline after multiple recyclings, limiting its application in high-performance products. Chemical recycling degrades PET into its monomers or oligomers through reactions such as hydrolysis and alcoholysis, enabling the regeneration and synthesis of materials. However, these methods generally suffer from harsh process conditions, high energy consumption, and difficulties in handling byproducts.

[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, well-defined products, and environmental friendliness. It is known that PET hydrolases (PETases) can break the ester bonds in PET molecules under suitable temperature and pH conditions, generating degradation products such as terephthalic acid (TPA) and ethylene glycol (EG). Existing research indicates that... Ideonella sakaiensis PETase and MHETase can synergistically decompose PET, representing a significant advancement in biocatalytic degradation technology. However, natural PETase still suffers from limitations in enzyme catalytic efficiency, thermal stability, and substrate adaptability, making it difficult to adapt to industrial conditions such as high substrate concentrations, long reaction times, or high temperatures. To improve its performance, studies have employed enzyme engineering methods such as directed evolution and rational design to develop modified enzyme variants. Among these, mutants, represented by FAST-PETase, have shown improvements in both activity and stability compared to natural enzymes, becoming an important tool in current enzymatic PET degradation technology (Lu H., Diaz DJ, Cuisinier M., et al. Machine learning-aided engineeringofhydrolases for PET depolymerization [J]. Nature, 2022, 604(7907): 662-667.). However, FAST-PETase still faces challenges such as insufficient reaction efficiency and limited room for optimization in specific application environments, requiring further performance optimization to meet practical application needs.

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

[0005] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a PET hydrolase mutant with enhanced enzyme activity and its applications. This invention combines deep learning model design with enzyme engineering optimization strategies to develop a high-performance PET hydrolase mutant better suited to industrial application needs.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A mutant PET hydrolase with enhanced enzyme activity, the amino acid sequence of which is SEQ ID NO.1, is obtained by any of the following mutations:

[0008] S213T, T140D\S213T, S169A\S213T or N212E\S213T; among them, S213T means that the 213th amino acid is mutated from S to T, and the others are similar;

[0009] The coding gene of one of the mutants.

[0010] The aforementioned mutant-related biological materials are any one or more combinations of the following biological materials:

[0011] (1) Expression cassettes containing the above-mentioned coding genes;

[0012] (2) Recombinant expression vectors containing the above-mentioned coding genes;

[0013] (3) A recombinant expression vector containing the expression cassette described in (1);

[0014] (4) Recombinant bacteria containing the above-mentioned encoding genes;

[0015] (5) Recombinant bacteria containing the expression cassette described in (1);

[0016] (6) Recombinant bacteria containing the recombinant expression vector described in (2) or (3).

[0017] Furthermore, the starting vector for the recombinant expression vector mentioned in (2) and (3) is a pET series vector or a pPICZα vector, etc.; preferably a pET-22a(+) vector or a pPICZαA vector.

[0018] Furthermore, the host bacteria corresponding to the recombinant bacteria mentioned in (4), (5), and (6) are selected from prokaryotes, yeast, or higher eukaryotic cells, etc.; the prokaryotes include Escherichia coli (Escherichia coli genus). Escherichia ), Bacillus spp. ( Bacillus Salmonella ( Salmonella), Pseudomonas spp. Pseudomonas ) or Streptomyces ( Streptomyces Bacteria such as *Pichia pastoris*; the yeast includes *Pichia pastoris* and other yeasts. More specifically, the prokaryotes are *Escherichia coli*, 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.

[0019] The above-mentioned mutants, encoding genes, and mutant-related biomaterials are used in the preparation of PET hydrolase mutants with enhanced enzyme activity.

[0020] Furthermore, the above-mentioned mutants, encoding genes, and mutant-related biomaterials are used in the degradation of PET.

[0021] Preferably, the crystallinity of the PET is below 20%, further to 5-20%, even further to 6-8%, and even further to 6.7%.

[0022] A method for obtaining the above-mentioned mutant includes the following steps: expressing the gene encoding the amino acid sequence of PET hydrolase as shown in SEQ ID NO.1 by site-directed mutagenesis to obtain the PET hydrolase mutant.

[0023] Furthermore, a mutation was introduced into the gene encoding the PET hydrolase, as shown in SEQ ID NO.1, using site-directed mutagenesis. After the sequence was confirmed to be correct, the mutant was transformed into E. coli for expression, thus obtaining the PET hydrolase mutant.

[0024] The present invention has the following advantages and effects compared with the prior art:

[0025] This invention starts with FAST-PETase and conducts research on enzyme design, engineering optimization, and application based on deep learning. First, using the CPD-ResNet50 deep learning model for prediction combined with manual evaluation, 11 single-point mutants that could potentially improve enzyme performance were selected for validation. Subsequently, experimental verification and enzyme engineering modification were performed. A secretory expression system and the model substrate 4-nitrophenylacetate (FAST-PETase) were constructed. pA high-throughput PETase activity screening method (NPA) was used, with relative enzyme activity greater than 0.5 (mutant / FAST-PETase) and relative thermostability greater than 1.0 (mutant / FAST-PETase) as criteria, to screen out 8 dominant single-site mutants. A library of 247 combined mutants containing 2–8 mutation sites was then constructed and evaluated. Superior single-site mutants and combined mutants were selected, and dominant mutants were evaluated using amorphous PET membrane (amoPET, 6.7% crystallinity) as a substrate. The dominant mutant was efficiently expressed and purified using the cSAT 2.0 method. The optimal mutant T140D\S213T showed twice the enzyme activity of FAST-PETase, and its thermostability after heat treatment at 50℃ for 2 hours was comparable to that of FAST-PETase. The optimal mutant T140D\S213T was further tested by measuring its protein melting temperature (T). m Tests show that its T m Increase by 1℃. Attached Figure Description

[0026] Figure 1 This is a graph showing the TPA+MHET+BHET yields of each mutant before heat treatment at a PET enzyme loading of 0.3 mg / g.

[0027] Figure 2 This is a graph showing the TPA+MHET+BHET yields of each mutant after heat treatment at 50°C for 2 hours with a PET enzyme loading of 0.3 mg / g. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0029] Example 1: Construction of a single-point mutant of PETase hydrolase

[0030] The PET hydrolase used in the examples 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 was synthesized by a commercial company.

[0031] 1. Obtaining single-point mutants based on the CPD-ResNet50 deep learning model

[0032] First, the possible mutation sites and amino acids predicted by the CPD-ResNet50 deep learning model (software name: protein mutation site prediction software, registration number: 2023SR1616838) were sorted in descending order of probability.

[0033] Secondly, based on the above prediction results and combined with manual evaluation, the following mutants (a total of 11) were selected as additional single-point mutants to be verified: S92E, K95E, Q127L, T140D, I168V, S169A, N212E, S213T, Q224K, K233R, and A240C.

[0034] 2. Construction and validation of single-point mutant recombinant plasmids

[0035] (1) Cloning of recombinant plasmids

[0036] Using the recombinant plasmid pET-22a(+)-FAST nucleotide sequence as a template, whole-plasmid PCR polymerase chain reaction 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 each of forward and reverse primers, 20 ng of plasmid template, 1 μL of KOD enzyme, and ddH2O added to a final volume of 50 μL. The reaction conditions were: 94℃ for 3 min; 98℃ for 30 s, 58℃ for 30 s, 68℃ for 3 min 30 s, 30 cycles; 68℃ for 2 min; and storage at 4℃.

[0037] The recombinant plasmid pET-22a(+)-FAST is formed by inserting the nucleotide sequence (4-870 bp in SEQ ID NO. 2) of the gene encoding FAST-PETase (SEQ ID NO. 1, nucleotides 2-290aa) into the pET-22a(+) vector. BstB I to Xho It is constructed from I.

[0038] (2) Product assembly

[0039] The above products were mixed at a ratio of fragment 1:25 fmol, fragment 2:25 fmol, and 15 μL of Gibson Mix was added, with the final volume of ddH2O brought to 20 μL. The mixture was reacted at 50 °C for 1 h.

[0040] (3) Transformation and validation of recombinant plasmids

[0041] Recombinant plasmid transformation: 10 μL of the above-linked product was transformed into 100 μL of E. coli DH5α competent cells, plated on LB agar plates containing a final concentration of 100 μg / mL carbenicillin, and incubated upside down at 37°C overnight for about 12-16 hours.

[0042] Transformed clone validation: Randomly select 2-4 single-clone colonies for sequencing validation.

[0043] 3. Fermentation and induced expression of single-point mutants

[0044] 5 μL of the plasmid, after successful sequencing verification, was transformed into 100 μL of E. coli BL21(DE3) competent cells, plated on LB agar plates containing a final concentration of 100 μg / mL carbenicillin, and incubated overnight at 37°C for 12–16 h.

[0045] After selecting colonies and verifying their correctness through colony PCR, streak the strains containing the target plasmid onto LB plates containing the corresponding antibiotic resistance and incubate overnight at 37°C.

[0046] Pick a single colony into a 96-well plate (flat bottom, with cap) containing 150 μL of LSG medium with the corresponding antibiotic resistance, and incubate overnight in a shaking incubator at 37°C and 220 rpm.

[0047] The overnight culture was transferred at a ratio of 1:100, 2 μL to a 96-well plate (flat bottom, with lid) containing 198 μL of LS-5052 self-induction medium with the corresponding resistance, and cultured at 20℃ and 220 rpm for 48 hours.

[0048] The expression product was expressed at 3,320. g Centrifuge for 15 min, collect the supernatant, and use it for the next step of measuring initial enzyme activity.

[0049] Note: All mutants were activated, transferred, and induced under the same conditions.

[0050] The LSG medium was prepared as follows: 20 mL of 50×L salt solution (containing 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, and 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×) were added to make up to 1 L with ultrapure water. After mixing, the mixture was autoclaved at 121℃ for 20 min, cooled, and stored at 4℃ for later use.

[0051] Preparation of LS-5052 self-induction medium: Take 20 mL of 50×L salt solution (containing 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, and 5 mM Na2SO4), 20 mL of 50×5052 carbon source mixture (containing 0.5% glycerol, 0.05% glucose, and 0.2% α-lactose), 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 mixture (final 1×), and add ultrapure water to a final volume of 1 L. Except for the 5052 carbon source mixture, after mixing all other components, autoclave at 121℃ for 20 min. After cooling to room temperature, add the 5052 carbon source mixture under aseptic conditions, mix well, and store at 4℃ for later use.

[0052] Example 2 Screening of single-point mutants of PET hydrolase

[0053] For the above 11 single-point mutants, the enzyme activity of their fermentation supernatant after heat treatment at 45℃ for 30 min and before heat treatment were measured and compared with FAST-PETase. This allowed for the screening of single-point mutants with improved thermostability and relative enzyme activity greater than 0.5. Ultimately, eight single-point mutants with improved thermostability and relative enzyme activity greater than 0.5 were obtained (Table 1): K95E, T140D, I168V, S169A, N212E, S213T, Q224K, and K233R.

[0054] PET hydrolase activity assay method: Enzyme activity was measured using the 405 nm light absorption method. p -NPA is a substrate used to determine the enzyme activity of PET hydrolase. One unit of enzyme activity refers to the production of 1 μmol of enzyme per minute at pH 8.0 and room temperature. p The reactivity of -NA (p-nitroaniline); take 5 μL of enzyme solution and mix it with 145 μL of phosphate buffer (pH 8.0, 100 mM), and add 50 μL of model substrate. p Mix -NPA (dissolved in ethanol, concentration 4 mM) and immediately measure absorbance at 405 nm at room temperature. Determine enzyme activity by comparing mutant enzyme activity with FAST-PETase enzyme activity to obtain the relative enzyme activity of each mutant, as shown in Table 1.

[0055] Method for determining the thermostability of PET hydrolase: Mix 5 μL of enzyme solution with 145 μL of phosphate buffer (pH 8.0, 100 mM), cover with a sealing film, and heat-treat at 45°C for 30 minutes. After heat treatment, allow to return to room temperature, then mix with 50 μL of model substrate. pMix -NPA (dissolved in ethanol, concentration 4 mM) and immediately measure absorbance at 405 nm at room temperature. Determine enzyme activity by comparing the activity after heat treatment with the activity before heat treatment to obtain the residual activity of each mutant; compare the residual activity of the mutant with the residual activity of FAST-PETase to obtain the relative thermal stability of each mutant, as shown in Table 1.

[0056] Table 1. Relative enzyme activity and relative thermal stability of fermentation supernatant from single-point mutants after heat treatment at 45℃ for 30 min.

[0057]

[0058] Example 3 Construction of a PET hydrolase combinatorial mutant library

[0059] The eight beneficial single-point mutants from Example 2 were randomly combined, and a combined mutant library was constructed using the DNAwork method. A total of 247 combined mutants were constructed using random combinations of 2 to 8 sites.

[0060] Using the mutant construction and induced expression techniques described in Example 1, and the mutant enzyme activity and thermostability determination methods described in Example 2, two beneficial mutant combinations were screened: N212E\S213T and S169A\S213T, with relative enzyme activities of 1.35 and 1.27, and relative thermostabilities of 0.92 and 1.16, respectively. Simultaneously, based on the advantageous single-point mutant T140D, the mutant combination T140D\S213T was also used for subsequent PET degradation determination.

[0061] Example 4: Determination of PET degradation by single-site and combined mutants of some PET hydrolases

[0062] 1. Construction and purification of beneficial mutant recombination

[0063] (1) Based on the cSAT 2.0 method, some PET hydrolase single-point and combined mutants were recombinantly constructed. During the construction process, the target gene (some PET hydrolase single-point and combined mutants) needs to have its signal peptide removed. The amino acid sequence of the signal peptide is shown as 1-27aa in SEQ ID NO.1, and the nucleotide sequence encoding the signal peptide is shown as 1-81bp in SEQ ID NO.2.

[0064] (2) Expression of some PET hydrolases by single-site and combined mutants based on cSAT 2.0 method

[0065] The dominant mutant based on the cSAT 2.0 method was plasmidized and transformed into Escherichia coli Origami 2(DE3) competent cells and grown overnight at 37°C for 12–16 h in LB plates containing 100 μL / mL carbenicillin.

[0066] A single colony with good growth was transferred to 5 mL of liquid LB medium containing 100 μL / mL carbenicillin and incubated at 37°C and 200 rpm for 18 h.

[0067] Transfer 2 mL of seed culture to 200 mL of fresh liquid 2YT medium containing 100 μL / mL carbenicillin, and incubate at 37°C and 200 rpm until OD500 is reached. 600 It reaches 0.6-0.8.

[0068] Protein expression was induced with 0.2 mM IPTG, and after further culturing at 18°C ​​and 200 rpm for 20 hours, cSAT 2.0 cell pellets were collected by centrifugation at 4,000 rpm for 20 min.

[0069] (3) Single-site and combined mutants of some PET hydrolases were purified using the cSAT 2.0 method. The purified single-site and combined mutants of PET hydrolases were detected by SDS-PAGE electrophoresis and recorded as pure enzymes.

[0070] The cSAT 2.0 method is disclosed in the literature “Huang Y, Zhang Y, Yang X, et al. A high-performance protein preparation approach in a singlecolumn-free step[J].Trends inbiotechnology:S0167-7799(24)00290-7.DOI:10.1016 / j.tibtech.2024.10.008.”

[0071] 2. Enzymatic activity and thermal stability tests of beneficial mutants in degrading PET

[0072] For single-point beneficial mutants and combined beneficial mutants, the enzyme activity of the pure enzyme after heat treatment at 50℃ for 2 hours followed by a 3-hour reaction was measured, as well as the enzyme activity before heat treatment at 50℃ for 3 hours. 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. The residual activity was calculated by comparing the enzyme activity after heat treatment with that before heat treatment; the relative thermostability was obtained by comparing the residual activity of the mutants with that of FAST-PETase, as shown in Table 2. Figure 1 , Figure 2 From Table 2 and Figure 1 , Figure 2 It can be seen that, compared with FAST-PETase, the enzyme activity of the single-point mutant S213T, the combined mutants T140D\S213T, S169A\S213T, and N212E\S213T was further improved, and the thermal stability was comparable or even improved.

[0073] Method for determining the activity of PET hydrolase in PET degradation: Amorphous PET membrane (6.7% crystallinity) (Gutefu Co., ES301445) was used as the substrate. Discs with a diameter of 6 mm and a coarse weight of approximately 8.5 mg were prepared and then soaked in 1% SDS, 20% ethanol, and deionized water for 30 min each, followed by air drying. These discs were then used as the actual substrate for HPLC analysis. A total volume of 436 μL of glycine buffer (50 mM, pH 9.2) containing 200 nM purified enzyme (enzyme loading of 0.3 mg / g PET) was added directly to a 6 mm diameter amorphous PET membrane in a 96-well plate at 50 °C and allowed to react for 3 h. After the reaction, an equal volume (436 μL) of a mixture of cold methanol and trifluoroacetic acid was added to each reaction system as a stop solution to precipitate unreacted macromolecules and enzyme proteins, thereby removing potential analytical interferences. The amount of each product generated was finally analyzed by HPLC. HPLC analysis was performed using a 1260 Infinity II system equipped with a Kinetex® XB-C18 column. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was 100% acetonitrile, with a flow rate set at 1.5 mL / min. Initial elution conditions were 13% B to separate TPA and MHET, followed by a gradient increase to 95% B, and finally back to 13% B to reequilibrate the column. The peak areas of the products were compared with those of the standards to calculate the concentrations of terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). Results are as follows: Figure 1 As shown.

[0074] Method for determining the thermal stability of PET degraded by PET hydrolase: A pretreated 6 mm amorphous PET membrane (6.7% crystallinity) was used as the substrate. A total volume of 436 μL of glycine buffer (50 mM, pH 9.2) containing 200 nM pure enzyme (enzyme loading 0.3 mg / g PET) was added to a 96-well plate. After heat treatment at 50 °C for 2 h, the 6 mm diameter amorphous PET membrane was added, and the reaction continued for another 3 h. After the reaction, an equal volume (436 μL) of a mixture of cold methanol and trifluoroacetic acid was added to each reaction system as a stop solution to precipitate unreacted macromolecules and enzyme proteins, thereby removing potential analytical interferences. HPLC analysis was performed according to the above method. Results are as follows: Figure 2 As shown.

[0075] Table 2. Relative enzyme activity and relative thermal stability of beneficial mutants with a uniform enzyme load of 0.3 mg / g PET at 50℃.

[0076]

[0077] Example 5: Protein melting temperature (T) of the optimal mutant T140D / S213T of PET hydrolase m Determination of )

[0078] Protein melting temperatures were assessed using differential scanning calorimetry (DSC). The experiment employed a MicroCal PEAQ-DSC differential scanning calorimeter (Malvin Panaco), and the specific method was as follows: Purified protein samples were first concentrated to 1 mg / mL using a Microcon® 10 kDa ultrafiltration centrifuge (Merck Millipore), and then replaced with peptide cleavage buffer (Buffer B2, 50 mM Bis-Tris, pH 6.2) to maintain consistent buffer conditions. 325 μL of concentrated protein sample and an equal volume of blank Buffer B2 were injected into the sample and reference cells, respectively. The temperature program was set as follows: linearly increasing from 15 °C to 85 °C at a rate of 1.0 °C·min⁻¹, holding at 85 °C for 2 minutes to eliminate thermal history effects, and then cooling to 15 °C at the same rate. High-purity nitrogen (99.999%) was continuously purged throughout the experiment to prevent solution evaporation. After baseline correction and asymmetric peak fitting of the heat flow data using MicroCalPEAQ-DSC analysis software (v1.41), the melting temperature (T) was determined. m The value of ΔCp is defined as the temperature corresponding to the peak value of the first derivative of the heat capacity change curve (ΔCp), as shown in Table 3. Table 3 shows that the temperature of ΔCp for the mutant T140D\S213T is... m The value increased by 1.06℃.

[0079] Table 3 Melting temperature of PET hydrolysate protein

[0080]

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A PET hydrolytic enzyme mutant with improved enzyme activity, characterized in that, The amino acid sequence of the mutant is obtained by mutating SEQ ID NO. 1 at T140D\S213T.

2. A PET hydrolytic enzyme mutant having improved enzyme activity and thermal stability, characterized in that, The amino acid sequence of the mutant is obtained by mutating SEQ ID NO. 1 at any of the following: S213T, S169A\S213T or N212E\S213T.

3. A gene encoding the PET hydrolytic enzyme mutant of claim 1 or 2.

4. The PET hydrolytic enzyme mutant related biomaterial according to claim 1 or 2, characterized in that, Any one or combination of the following biological materials: (1) an expression cassette containing the gene of claim 3; (2) a recombinant expression vector containing the gene of claim 3; (3) a recombinant expression vector containing the expression cassette of (1); (4) a recombinant bacterium containing the gene of claim 3; (5) a recombinant bacterium containing the expression cassette of (1); (6) a recombinant bacterium containing the recombinant expression vector of (2) or (3).

5. The biological material of claim 4, wherein: The host bacterium corresponding to the recombinant bacterium in (4), (5) and (6) is selected from prokaryotes, yeast or higher eukaryotic cells.

6. The biological material of claim 5, wherein: The prokaryote is Escherichia coli and the yeast is Pichia pastoris.

7. Use of the gene of claim 3 or the biological material of any one of claims 4-6 in the preparation of a PET hydrolytic enzyme mutant with improved enzyme activity.

8. Use of the PET hydrolytic enzyme mutant of any one of claims 1-2, the gene of claim 3 or the biological material of any one of claims 4-6 in the degradation of PET.

9. Use according to claim 8, characterized in that: The crystallinity of the PET is 20% or less.

10. A method of obtaining the PET hydrolytic enzyme mutant of claim 1 or 2, characterized in that, The method comprises the following steps: mutating a gene encoding a PET hydrolytic enzyme with an amino acid sequence as shown in SEQ ID NO. 1 by site-directed mutation technology, and then expressing the gene to obtain the PET hydrolytic enzyme mutant of claim 1 or 2.

11. The method of claim 10, wherein, The method comprises the following steps: mutating a gene encoding a PET hydrolytic enzyme with an amino acid sequence as shown in SEQ ID NO. 1 by site-directed mutation technology, and then expressing the gene to obtain the PET hydrolytic enzyme mutant of claim 1 or 2.

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