PET degrading enzyme recombinant protein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a PET (Polyethylene Terephthalate) degrading enzyme recombinant protein which is a 12-polymer nanoparticle formed by fusing a DPS protein and a PET degrading enzyme. In order to promote the PETase to better exert the degradation activity, in combination with a DPS application thought, DPS is utilized to provide a degradation reaction microenvironment for the PETase, DPS-PETase nanoparticles are constructed, and under the assistance of a DPS nanocage, the effective contact area of the PETase nanoparticles and a PET polymer is increased, and the stability of the PETase is enhanced. The invention provides a new thought for the application of PETase and lays a foundation for the degradation research of PET materials.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a recombinant protein of a PET-degrading enzyme, a preparation method thereof, and an application thereof. Background Art

[0002] PET is a thermoplastic plastic with high crystallinity synthesized by polycondensation of terephthalic acid and ethylene glycol. Due to its high rigidity, high impact strength, resistance to acids, dilute alkalis, oils, lipids, good barrier properties, high transparency, good gloss, ability to block ultraviolet rays, odorless, non-toxic, good biosecurity, and low cost, the PET material is widely used in various industries. Due to the chemical inertness of the PET material, PET is resistant to natural degradation. Therefore, the accumulation of PET materials causes serious environmental problems.

[0003] Plastic recycling through chemical, mechanical, and biological processes is the main conventional method used so far. Eliminating plastics in landfills is the simplest and most common method in the world, but due to the anaerobic conditions around landfills, the degradation rate of plastic waste is slow, so it has many hazards and disadvantages. Plastic waste accumulates in landfills, rivers, oceans, and terrestrial environments and pollutes groundwater. This pollution has a fatal impact on marine animals through ingestion or being trapped in plastic debris. Incineration is seriously harmful to the atmospheric environment due to the release of toxic substances such as furans and dioxins. Mechanical recycling, which converts plastic waste into reusable materials, is an alternative method to overcome plastic waste pollution, but this method is limited to single-layer plastics rather than multi-layer plastics, and plastics sensitive to temperature cannot be mechanically processed. Chemical recycling is usually carried out through alkaline, acidic, and neutral methods to depolymerize plastics using hydrolysis, glycolysis, and methanolysis to obtain their monomers or other useful products. This chemical process requires selective and active catalysts, which are both expensive and energy-consuming, and high temperatures are required to produce a product mixture that is difficult to separate, which limits its industrial application. Using microorganisms to biodegrade plastic waste is a cost-effective environmental protection strategy, which can be applied through the cultivation of microorganisms or the extraction of enzymes. The enzymatic activity of these enzymes catalyzes the cleavage of polymer bonds into monomers.

[0004] DPS protein (DNA protection during starvation protein) is a dodecameric bacterial ferritin that protects DNA from oxidative stress and is related to bacterial survival and virulence. The DPS protein is composed of 12 identical monomers that assemble together to form a roughly spherical dodecagon with 23-fold symmetry. This structure is assembled from 12 identical subunits and consists of a conserved four-helix bundle. The iron-binding site (ferroxidase center), where iron ions are oxidized by hydrogen peroxide or molecular oxygen, is located at each dimer interface and is formed by amino acid residues of adjacent subunits; on both sides of the four-helix bundle are N-terminal and C-terminal extensions of variable length and amino acid composition, and these extensions are usually unstructured and flexible. The DPS protein is often used as a cis-replacement protein complex for Ferritin nanoparticle research. The smaller and simpler DPS than Ferritin may perform better in the research of expression on the outer side of the nanocage, but there is no corresponding research report yet. Summary of the Invention

[0005] The object of the present invention is to provide a PET-degrading enzyme recombinant protein DPS-5XJH in view of the problem that PET plastics cannot be effectively degraded.

[0006] To achieve the above object, the present invention provides a PET-degrading enzyme recombinant protein, which is a dodecameric nanoparticle formed by fusing DPS protein and PET-degrading enzyme.

[0007] Furthermore, the amino acid sequence of the nanoparticle is shown in SEQ ID NO.1.

[0008] The present invention also provides a gene fragment that encodes the aforementioned PET-degrading enzyme recombinant protein.

[0009] Furthermore, its nucleotide sequence is shown in SEQ ID NO.2.

[0010] The present invention also provides a recombinant vector, which is a plasmid containing the aforementioned gene fragment.

[0011] Furthermore, the plasmid is a prokaryotic expression plasmid.

[0012] Even further, the prokaryotic expression plasmid includes pET-20b(+).

[0013] The present invention also provides a recombinant cell, which is a prokaryotic cell including the aforementioned recombinant vector.

[0014] Furthermore, the prokaryotic cell includes Escherichia coli cells.

[0015] Even further, the Escherichia coli cells include E.coli C43 / DE3.

[0016] The present invention also provides a method for preparing the aforementioned recombinant protein of PET degrading enzyme, comprising the following steps:

[0017] Take the gene fragment with the nucleotide sequence shown in SEQ ID NO.2, introduce it into a prokaryotic expression plasmid to obtain a recombinant plasmid, and then introduce it into prokaryotic cells for recombinant protein expression, and collect and purify to obtain.

[0018] Furthermore, the prokaryotic expression plasmid includes pET-20b(+); the prokaryotic cells include E.coli C43 / DE3, preferably E.coli BL21(DE3); the purification is first with a nickel column and then with a Superose 6 Increase 10 / 300GL size exclusion chromatography column.

[0019] Finally, the present invention provides an application of the aforementioned recombinant protein of PET degrading enzyme in PET degradation.

[0020] Furthermore, the method of the application includes the following steps: take the recombinant protein of PET degrading enzyme and degrade PET under the condition of 20-40°C.

[0021] In order to promote the better degradation activity of PETase, the present invention combines the idea of DPS application, uses DPS to provide a microenvironment for the degradation reaction of PETase, constructs nanoparticles of DPS-PETase, and with the assistance of DPS nanocages, increases the effective contact area between PETase nanoparticles and PET polymers, and enhances the stability of PETase. The present invention provides new ideas for the application of PETase and lays a foundation for the degradation research of PET materials.

[0022] Advantages of the present invention:

[0023] Compared with the prior art, the nano-cage type recombinant protein of PET degrading enzyme DPS-5XJH provided by the present invention optimizes the codons of the gene sequence according to the codon preference of Escherichia coli, and uses the method of total gene synthesis to fuse the PET degrading enzyme genes 5xjh and dps genes, and clone them into the expression vector pET-20b(+), transform the Escherichia coli competent cell E.coli BL21(DE3), and the conditions for protein induction expression obtained by optimization are: the final concentration of IPTG is 1 mM, and induce culture at 16°C for 18 h. Through protein purification and verification by SDS-PAGE and size exclusion chromatography column purification, a recombinant protein of PET degrading enzyme with a nano-cage structure formed by 12 identical subunits and high purity is obtained. With the assistance of the DPS nanocage, this recombinant protein increases the effective contact area between PETase nanoparticles and PET polymers, enhances the stability of PETase, and lays a foundation for the functional research and development application of PET degradation.

[0024] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0025] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a map of the recombinant protein DPS-5XJH expression vector.

[0027] Figure 2 It is a size exclusion chromatogram of the recombinant protein DPS-5XJH expressed at different temperatures.

[0028] Figure 3 It is a negative staining image of the recombinant protein DPS-5XJH.

[0029] Figure 4 PET thin film degradation reaction result diagram. A: blank group; B: 5XJH; C: DPS-5XJH.

[0030] Figure 5 Scanning electron microscope image. a, b, c: PET thin films after reaction in the blank group; d, e, f: PET thin films in the 5XJH group; g, h, i: PET thin films in the DPS-5XJH group.

[0031] Figure 6 TPA standard curve.

[0032] Figure 7 HPLC detection curve. a: HPLC detection full process curve data diagram; b: HPLC detection peak comparison and fusion diagram; green: methanol solution; orange: blank control group; red: TPA standard; blue: 5XJH sample; pink: DPS-5XJH sample. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0034] Example 1 Construction of Expression Vector

[0035] After optimizing the gene sequence using codon optimization software, the PET-degrading enzyme genes 5xjh and dps gene were synthesized. Using the synthesized gene sequence as a template, PCR amplification was carried out, and the dps gene and 5xjh gene were fused and ligated through the GGGS linker to the NcoI and HindIII double digestion sites of the prokaryotic expression vector pET-20b(+) by homologous recombination to construct the prokaryotic expression recombinant plasmid pET20b-8his-dps-linker-5xjh carrying an 8×His tag at the N-terminus, as Figure 1 shown. The amino acid sequence of the recombinant protein DPS-5XJH is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1:

[0037] MKYLLPTAAAGLLLLAAQPAMAMHHHHHHHHPMAETQTLLRNFGNVY

[0038] DNPVLLDRSVTAPVTEGFNVVLASFQALYLQYQKHHFVVEGSEFYSLHEF

[0039] FNESYNQVQDHIHEIGERLDGLGGVPVATFSKLAELTCFEQESEGVYSSRQ

[0040] MVENDLAAEQAIIGVIRRQAAQAESLGDRGTRYLYEKILLKTEERAYHLS

[0041] HFLAKDSLTLGFVQAAGGGSMRGPNPTAASLEASAGPFTVRSFTVSRPSG

[0042] YGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITID

[0043] TNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSM

[0044] GGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVN

[0045] SSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRF

[0046] MDNDTRYSTFACENPNSTRVSDFRTANCSLEDPAANKARKEAELAAATAEQ*

[0047] SEQ ID NO.2:

[0048] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGCATCATCATCATCACCATCATCATccgatggccGAAACACAAACCTTACTGCGTAACTTCGGTAATGTGTATGATAATCCGGTTCTGCTGGATCGCAGCGTGACCGCACCGGTTACCGAAGGCTTCAATGTGGTTCTGGCATCATTCCAGGCACTGTATCTGCAGTATCAGAAACATCACTTCGTGGTGGAAGGCAGCGAGTTCTATAGCCTGCATGAGTTCTTCAATGAAAGCTATAATCAGGTTCAGGATCATATTCATGAAATTGGCGAACGTCTGGATGGTCTGGGCGGTGTGCCGGTGGCCACCTTCAGTAAACTGGCCGAACTGACCTGCTTCGAACAGGAAAGCGAAGGCGTGTATAGCAGTCGTCAGATGGTGGAAAATGATCTGGCAGCAGAACAGGCAATTATTGGCGTGATTCGCCGTCAGGCCGCACAGGCCGAAAGTCTGGGTGATCGTGGCACCCGTTATCTGTATGAAAAAATTCTGCTGAAAACCGAAGAACGTGCATATCATCTGAGTCACTTCCTGGCCAAAGATAGCCTGACCCTGGGCTTCGTTCAGGCAgccGGAGGAGGAAGCATGAGAGGTCCCAACCCTACTGCTGCTAGCCTCGAAGCCTCTGCTGGCCCCTTCACCGTCAGATCTTTCACTGTGTCTAGGCCTTCTGGCTACGGCGCCGGTACTGTCTACTACCCTACCAACGCTGGTGGCACTGTCGGTGCCATCGCTATCGTGCCTGGTTACACCGCCAGACAGAGCAGCATCAAGTGGTGGGGCCCCAGACTGGCCTCTCACGGTTTCGTCGTGATCACCATCGACACTAACTCTACTCTGGACCAGCCTTCTAGCCGTTCTAGCCAGCAGATGGCTGCTCTCCGTCAGGTGGCTAGCCTCAACGGTACCTCTAGCTCTCCCATCTACGGTAAAGTCGACACCGCCCGTATGGGTGTGATGGGCTGGTCTATGGGTGGCGGCGGTTCTCTCATCTCTGCTGCTAACAACCCCAGCCTGAAGGCCGCCGCCCCCCAAGCTCCTTGGGACTCTTCTACCAACTTCTCTAGCGTCACTGTCCCCACCCTGATCTTCGCCTGCGAAAACGACAGCATCGCTCCCGTCAACAGCTCTGCCCTCCCTATCTACGACTCTATGTCTCGTAACGCTAAGCAGTTCCTGGAAATCAACGGCGGCAGCCACTCTTGCGCTAACAGCGGTAACTCTAACCAGGCCCTCATCGGTAAAAAGGGCGTGGCCTGGATGAAGAGATTCATGGACAACGACACCAGATACAGCACCTTCGCCTGCGAGAACCCTAACTCTACCCGTGTGTCTGACTTCCGTACCGCCAACTGCTCTCTGGAAGACCCCGCTGCCAACAAGGCTAGAAAGGAAGCTGAACTCGCTGCTGCCACTGCCGAACAAtga

[0049] The constructed recombinant plasmid was transformed into competent Escherichia coli DH5α cells, and positive clones were screened. The plasmid was extracted and verified by PCR, showing specific bands of the expected size. The sequencing sequence of the correctly verified recombinant plasmid was aligned and confirmed to be correct, indicating that the recombinant plasmid was successfully constructed and could be used for the expression of the target protein.

[0050] Example 2 Expression and purification of recombinant protein DPS-5XJH of PET degrading enzyme

[0051] The recombinant plasmid pET20b-8his-dps-linker-5xjh constructed in Example 1 was separately transformed into competent BL21(DE3) cells to prepare the genetically engineered bacterium pET20b-8his-dps-linker-5xjh-BL21(DE3). A single colony of this engineered bacterium was picked and inoculated into 100 mL of LB liquid medium containing 100 μg / ml ampicillin, and cultured at 37 °C and 220 rpm for 12 h to prepare a seed solution. Then, the seed solution was inoculated into 1 L of LB liquid medium containing 100 μg / ml ampicillin at a volume ratio of 2%, and cultured at 37 °C and 220 rpm until the OD600 value reached 0.8. IPTG was added to a final concentration of 0.5 mM, and then induced at 37 °C for 3 h and at 16 °C for 18 h, respectively. The induced bacterial solution was centrifuged at 4 °C and 3500 rpm for 15 min to collect the cell precipitate. The cell precipitate was resuspended in lysis buffer (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 2% glycerol) at a ratio of 1 g / 10 ml. The resuspended solution was ultrasonically disrupted (250 W, 10 min, repeated 3 times), and then centrifuged at 4 °C and 17000 rpm for 60 min to collect the supernatant containing the target protein, which was separated and purified by passing through a nickel column and size exclusion chromatography column.

[0052] The specific method for nickel column separation and purification is as follows:

[0053] 1) Equilibration: The nickel column was equilibrated with 20 column volumes of Balance Buffer (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 2% glycerol).

[0054] 2) Column passing: The collected supernatant containing the protein was passed through the nickel column at a flow rate of 1 ml / min.

[0055] 3) Elute the miscellaneous proteins: Elute the miscellaneous proteins successively with 20 column volumes of Wash Buffer 1 (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 2% glycerol, 20 mM Imidazole) and 20 column volumes of Wash Buffer 2 (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 2% glycerol, 50 mM Imidazole) at a flow rate of 1 ml / min;

[0056] 4) Elute the target protein: Elute the target protein with 20 column volumes of Elute Buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 2% glycerol, 350 mM Imidazole).

[0057] 5) Concentration and SDS-PAGE analysis: Use a 100 KDa concentrator tube for the eluted target protein.

[0058] The specific method for purification by size exclusion chromatography column is as follows: Purify the concentrated target protein solution with a Superose 6 Increase 10 / 300 GL size exclusion chromatography column. The buffer components used are: 25 mM Tris-HCl pH 8.0, 200 mM NaCl, 2% glycerol. The buffer is filtered through a 0.22 μm filter membrane before use. Equilibrate the chromatography column with 25 mL of buffer at a flow rate of 0.3 mL / min. Before loading the protein solution, wash the sample loop successively with UP water and buffer. After injecting the protein solution into the sample loop, set the flow rate to 0.3 mL / min, collect the peak volume, and conduct experiments with the pressure value. After the experiment, analyze the protein collected in each tube by SDS-PAGE electrophoresis.

[0059] The pET20b-8his-dps-linker-5xjh-BL21(DE3) genetically engineered bacteria are induced and cultured at 16 °C for 18 h and at 37 °C for 3 h, and DPS-5XJH nanoparticles are expressed under both conditions. The proteins expressed under these two conditions are separated and purified by nickel column, and the protein solution eluted with 350 mM Imidazole is purified by molecular sieve chromatography with a Superose 6 Increase 10 / 300 GL column. From Figure 2As can be seen from the results, the molecular sieve peak shapes of the protein solutions eluted after nickel column separation and purification under the two induction conditions are similar. However, there is a peak at the 11.13 mL (peak1) position in the molecular sieve results under the condition of induction culture at 16 °C for 18 h, while there is no peak at this position after induction culture at 37 °C for 3 h. The molecular weight of the protein solution eluted at the 11.13 mL (peak1) position is close to the molecular weight of the DPS-5XJH nanoparticles. Therefore, it is speculated that the protein eluted at this position may be the DPS-5XJH nanoparticles. Take the protein solution eluted at the 11.13 mL (peak1) position for negative staining to further confirm whether the DPS-5XJH nanoparticle molecules are formed.

[0060] Take the protein eluted at the 11.13 mL elution position for negative staining, and the negative staining results Figure 3 are shown as follows. As can be seen from the negative staining result diagram, the DPS-5XJH recombinant protein forms nanoparticle molecules (indicated by the arrows in the figure). The negative staining results show that the DPS-5XJH PETase nanoparticles have been successfully expressed in the E. coli BL21(DE3) receptor strain that highly expresses toxic proteins, laying a foundation for the large-scale expression of DPS-5XJH nanoparticles and the analysis of protein structure.

[0061] Example 3 Structure confirmation of the PET-degrading enzyme recombinant protein DPS-5XJH

[0062] To determine whether the recombinant protein DPS-5XJH expressed by the prokaryotic cell E. coli BL21(DE3) in Example 2 forms 12-mer nanoparticle molecules, take the protein solution eluted at the 11.13 mL (peak1) position in the size exclusion chromatography result for negative staining.

[0063] As can be seen from the negative staining result ( Figure 3 ), the DPS-5XJH recombinant protein forms nanoparticle molecules (indicated by the arrows in the figure). Among them, the nanoparticles are evenly distributed, the morphology is relatively clear, and the size is about 20 nm. The negative staining results show that the DPS-5XJH PETase nanoparticles have been successfully expressed in the E. coli BL21(DE3) receptor strain that highly expresses toxic proteins.

[0064] Example 4 Efficacy verification of the PET-degrading enzyme recombinant protein DPS-5XJH in degrading PET

[0065] 1. Method

[0066] PET degradation reaction system: Add 5 mg of pretreated PET flakes (rectangles of 5 mm × 5 mm) to the reaction Buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl), then add 0.03 mg of PET-degrading enzyme and incubate with shaking at 200 rpm at 40 °C for 24 h, and then detect the degradation products. The blank group is 0.03 mg of the DPS-5XJH PETase enzyme solution inactivated at 85 °C for 15 min and confirmed by the structure in Example 3; positive control group: 0.03 mg of 5XJH PETase enzyme solution (enzyme expressed by the PET-degrading enzyme gene 5xjh, reaction temperature 37 °C); the experimental group is 0.03 mg of the DPS-5XJH PETase enzyme solution confirmed by the structure in Example 3.

[0067] PET flake pretreatment method: Cut the PET film into rectangles of 5 mm × 5 mm, ultrasonically wash with ultrapure water for 30 min, and then dry at 37 °C for 12 h; the PET films dried at 37 °C for 12 h are ultrasonically washed with 0.1% SDS, absolute ethanol, and ultrapure water for 30 min respectively, and then dried at 37 °C for 48 h.

[0068] Detect the degradation ability of DPS-5XJH PETase on PET by surveying the surface changes of the PET flakes after the reaction through a scanning electron microscope.

[0069] The amount of the product (TPA) generated after the reaction was quantitatively detected by HPLC. A standard curve of TPA was plotted to convert the detection peak shape and peak area of the product into the corresponding generated amount, and then the degradation ability of the corresponding enzyme was compared and analyzed. For the TPA standard curve, methanol was used as the blank control vial. Weigh 1.51 mg, 2.63 mg, 3.88 mg, 4.77 mg, and 5.72 mg of TPA standard powder respectively, use the reaction buffer as the liquid environment, add a small amount of base to completely dissolve the standard, and finally dilute them to concentrations of 12.58 μg / mL, 21.92 μg / mL, 32.33 μg / mL, 39.75 μg / mL, and 47.67 μg / mL respectively. HPLC detection method: Use a C18 chromatographic column, and liquid A (0.1% formic acid aqueous solution) and liquid B (methanol) as the mobile phase during the detection process. Detection procedure: Set the system flow rate to 0.8 mL / min, the temperature in the column oven to 30 °C, the detection wavelength to 240 nm, and the single injection volume to 10 μL. The program operation parameters are as follows: Run with 25% liquid B for 5 min, then increase the gradient to 100% liquid B within 10 min, then continuously wash with 100% liquid B for 5 min, and finally adjust back to the initial mobile phase gradient of 25% liquid B to balance for 5 min, with a total of 25 min for one process. Record their corresponding retention times, peak heights, and peak areas respectively. Taking the concentration of the standard product TPA as the abscissa and the peak area of the HPLC detection peak as the ordinate, the standard curve of TPA was plotted as Figure 6 shown.

[0070] 2. Results

[0071] Figure 4 The results showed that: Compared with the blank group, it could be seen that white flocculent precipitate-like substances were generated by the degradation of PET thin slices under the action of PET degrading enzyme within 24 h.

[0072] From Figure 5 the scanning electron microscope results, it could be seen that the surface morphology characteristics of the PET thin slices were significantly different from those of the blank control group under the action of PET degrading enzyme. In the 5XJH and DPS-5XJH groups, there were obvious cracks and cracking of the reaction layer on the surface of the PET thin slices, and the surface was rough, showing an obvious degradation trend.

[0073] From the HPLC detection results ( Figure 7 ), it could be seen that the degradation product TPA was generated at the position corresponding to the standard product, indicating that DPS-5XJH degraded PET to generate TPA. By comparing the TPA standard curve and taking the average value, the generated TPA concentrations were obtained as 5XJH: 24.42 μg / mL, DPS-5XJH: 21.99 μg / mL.

[0074] Most evidence indicates that there are significant differences in the activity of enzymes between 40 °C and 37 °C. Enzymes usually exhibit optimal activity at 37 °C, and when the temperature rises to 40 °C and above, the activity of the enzymes will decrease significantly. However, with the assistance of DPS nanocages, when the temperature for degrading PET is increased to 40 °C, DPS-5XJH still maintains an enzyme activity comparable to that at 37 °C for PET degradation, enabling DPS-5XJH to be applied to PET degradation under variable environmental temperatures.

[0075] In summary, the high-purity recombinant protein of the PET-degrading enzyme with a nanocage structure formed by 12 subunits obtained in the present invention can effectively contact with PET polymers and has strong stability of PETase, laying a foundation for the functional research and development application of PET degradation.

Claims

1. A PET degrading enzyme recombinant protein, characterized in that: It is a 12-mer nanoparticle formed by the fusion of DPS protein and PET degrading enzyme.

2. The PET degrading enzyme recombinant protein according to claim 1, characterized in that: The amino acid sequence of the nanoparticle is shown in SEQ ID NO.

1.

3. A gene fragment, characterized in that: It encodes the PET degrading enzyme recombinant protein according to claim 1 or 2.

4. The gene fragment according to claim 3, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

5. A recombinant vector, characterized in that: The plasmid comprises the gene fragment according to claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that: The plasmid is a prokaryotic expression plasmid; The prokaryotic expression plasmid includes pET-20b(+).

7. A recombinant cell, characterized in that: The prokaryotic cell comprises the recombinant vector according to any one of claims 5 to 6.

8. The recombinant cell according to claim 7, characterized in that: The prokaryotic cells include Escherichia coli cells; The E. coli cells include E. coli C43 / DE3, preferably E. coli BL21 (DE3).

9. A method for preparing the PET degrading enzyme recombinant protein according to claim 1 or 2, characterized in that: The steps include: Take a gene fragment with a nucleotide sequence as shown in SEQ ID NO.2, introduce it into a prokaryotic expression plasmid to obtain a recombinant plasmid, then introduce it into a prokaryotic cell to express the recombinant protein, collect and purify it; The prokaryotic expression plasmid includes pET-20b(+); the prokaryotic cell includes E.coli C43 / DE3, preferably E.coliBL21(DE3); the purification is first performed using a nickel column and then using a Superose 6Increase10 / 300GL size exclusion chromatography column.

10. Use of the PET degrading enzyme recombinant protein according to claim 1 or 2 in PET degradation.

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