Chimeric enzyme for degrading pet, and related biological material and use thereof

Abstract

Provided are a chimeric enzyme for degrading PET, and a related biological material and the use thereof. The provided chimeric enzyme for degrading PET is obtained by replacing at least one fragment in an amino acid sequence shown as SEQ ID No. 1-12 on the basis of IsPETase. Compared with IsPETase, the provided chimeric enzyme for degrading PET exhibits a 4.8-31.7°C increase in melting temperature, and a 1.5-44.5-fold increase in the concentration of PET degradation product TPA. The present invention expands the application conditions for PET degradation, and further greatly improves PET degradation efficiency and improves the recycling rate of the high value-added degradation product TPA, thereby achieving important economic value and environmental benefits. The present invention can be used in the fields of PET degradation, preparation of a PET degradation agent and preparation of PET degradation products, etc.

Description

Chimeric enzymes for degrading PET and their related biomaterials and applications

[0001] This patent application claims priority to Chinese Patent Application No. CN 202510174110.8, filed on February 18, 2025. The disclosure of the earlier application is incorporated herein by reference in its entirety. Technical Field

[0002] This application relates to the field of bioengineering technology, specifically to a chimeric enzyme for degrading PET and its related biomaterials and applications. Background Technology

[0003] Polyethylene terephthalate (PET) has been widely used in various fields due to its superior physicochemical properties. Especially since the outbreak of COVID-19 in 2020, the use of disposable plastic protective equipment such as masks, gloves, face shields, and protective clothing has surged, with global PET production capacity exceeding 100 million tons in 2020. However, improper disposal of used PET has posed a serious threat to the ecological environment, making the management of plastic waste a key issue.

[0004] Currently, biodegradation, especially enzymatic degradation, has been applied to the treatment of waste PET due to its advantages such as low cost, mild reaction conditions, and green and pollution-free operation. Various enzymes capable of hydrolyzing PET have been discovered, such as esterases, keratinases, lipases, PETase, and MHETase. Among them, IsPETase is a PETase derived from the bacterium *Ideonella sakaiensis* (i.e., an enzyme that degrades PET), belonging to the α / β hydrolase superfamily. Compared with other PET hydrolases, IsPETase has advantages such as high degradation efficiency, suitability for medium-temperature degradation, specific degradation of PET, and the ability to degrade highly crystalline PET films. However, the hydrolysis of PET by IsPETase mainly occurs in the amorphous portion, while the glass transition temperature (Tg) of PET is between 65℃ and 71℃. This places higher demands on the thermal stability of PET hydrolases. However, the enzyme's thermal stability and degradation activity at high temperatures are poor, thus limiting its practical application in PET degradation. Technical issues

[0005] To address the above-mentioned technical problems, this application designs and improves IsPETase, providing a chimeric enzyme for degrading PET, along with related biomaterials and applications. Compared to the wild-type IsPETase, this chimeric enzyme for degrading PET not only exhibits higher thermal stability but also significantly improved PET degradation activity. Technical solutions

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution:

[0007] In a first aspect, this application provides a chimeric enzyme for degrading PET. This chimeric enzyme is obtained by dividing the IsPETase amino acid sequence (as shown in SEQ ID No. 37) into 12 amino acid sequences as shown in SEQ ID No. 1 to SEQ ID No. 12, and replacing at least one of the 12 amino acid sequences, wherein:

[0008] Replace the amino acid sequence shown in SEQ ID No. 1 with the amino acid sequence shown in SEQ ID No. 13 or SEQ ID No. 25;

[0009] Replace the amino acid sequence shown in SEQ ID No. 2 with the amino acid sequence shown in SEQ ID No. 14 or SEQ ID No. 26;

[0010] Replace the amino acid sequence shown in SEQ ID No. 3 with the amino acid sequence shown in SEQ ID No. 15 or SEQ ID No. 27;

[0011] Replace the amino acid sequence shown in SEQ ID No. 4 with the amino acid sequence shown in SEQ ID No. 16 or SEQ ID No. 28;

[0012] Replace the amino acid sequence shown in SEQ ID No. 5 with the amino acid sequence shown in SEQ ID No. 17 or SEQ ID No. 29;

[0013] Replace the amino acid sequence shown in SEQ ID No. 6 with the amino acid sequence shown in SEQ ID No. 18 or SEQ ID No. 30;

[0014] Replace the amino acid sequence shown in SEQ ID No. 7 with the amino acid sequence shown in SEQ ID No. 19 or SEQ ID No. 31;

[0015] Replace the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 20 or SEQ ID No. 32;

[0016] Replace the amino acid sequence shown in SEQ ID No. 9 with the amino acid sequence shown in SEQ ID No. 21 or SEQ ID No. 33;

[0017] Replace the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 22 or SEQ ID No. 34;

[0018] Replace the amino acid sequence shown in SEQ ID No. 11 with the amino acid sequence shown in SEQ ID No. 23 or SEQ ID No. 35;

[0019] Replace the amino acid sequence shown in SEQ ID No. 12 with the amino acid sequence shown in SEQ ID No. 24 or SEQ ID No. 36.

[0020] Compared to the wild-type IsPETase enzyme, the thermal melting temperature (T0) of the chimeric enzyme for degrading PET provided in this application is higher. m The temperature was increased by 4.8℃ to 31.7℃, and the concentration of TPA, the degradation product after PET degradation, increased by 1.5 to 44.5 times. Given that the chimeric enzyme for PET degradation constructed in this application has higher PET degradation activity and thermal stability, it can not only broaden the application conditions for PET degradation but also significantly improve PET degradation efficiency, thus contributing to increasing the recycling rate of the high-value-added degradation product TPA.

[0021] Among them, the amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 12 are derived from IsPETase, and their specific amino acid sequences are detailed in Table 1.

[0022] Table 1

[0023] The amino acid sequences shown in SEQ ID No. 13 to SEQ ID No. 24 are derived from TfCut2, and their specific amino acid sequences are detailed in Table 2.

[0024] Table 2

[0025] The amino acid sequences shown in SEQ ID No. 25 to SEQ ID No. 36 are derived from LCC, and their specific amino acid sequences are detailed in Table 3.

[0026] Table 3

[0027] The amino acid sequence of IsPETase is shown in SEQ ID No. 37, specifically:

[0028] The amino acid sequence of TfCut2 is shown in SEQ ID No. 38, specifically:

[0029] The amino acid sequence of LCC is shown in SEQ ID No. 39, specifically:

[0030] For example, as a preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0031] For example, as a preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23.

[0032] Exemplarily, as a preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0033] Exemplarily, as a preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0034] By way of example, as a preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0035] Compared to the wild-type IsPETase enzyme, the preferred PET-degrading chimeric enzyme provided in this application has a thermal melting temperature that is 19.8℃ to 31.7℃ higher, and the concentration of the degradation product TPA after PET degradation is increased by 29.4 to 36.6 times.

[0036] Exemplarily, as a further preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 33, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23, while the amino acid sequence shown in SEQ ID No. 14 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 14 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 15 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, the The amino acid sequence shown in No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 36.

[0037] Exemplarily, as a further preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, and the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, while the amino acid sequence shown in SEQ ID No. 13 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 22, and the amino acid sequence shown in SEQ ID No. 13 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30, the amino acid The amino acid sequence shown in No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0038] For example, as a more preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0039] By way of example, as a further preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 26, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, and the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22.

[0040] By way of example, as a further preferred technical solution, the amino acid sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0041] Compared to the wild-type IsPETase enzyme, the further preferred chimeric enzyme for PET degradation provided in this application has a thermal melting temperature that is increased by 4.8℃ to 31.5℃, and the concentration of the degradation product TPA after PET degradation is increased by 36.9 to 44.5 times.

[0042] Secondly, this application provides biomaterials related to the chimeric enzymes that degrade PET described above, wherein the biomaterials are any one of (I) to (IV) below:

[0043] (I) The nucleic acid molecule (gene) encoding the chimeric enzyme that degrades PET as described above;

[0044] (II) An expression cassette containing the nucleic acid molecule described in (I);

[0045] (III) A recombinant vector containing the nucleic acid molecule described in (I), or a recombinant vector containing the expression cassette described in (II);

[0046] (IV) A recombinant strain containing the nucleic acid molecule described in (I), or a recombinant strain containing the expression cassette described in (II), or a recombinant strain containing the recombinant vector described in (III).

[0047] Thirdly, this application also provides a method for constructing a chimeric enzyme recombinant vector for degrading PET, the method comprising: transforming a gene encoding the chimeric enzyme for degrading PET into T1 competent cells, and obtaining the recombinant vector by screening with ampicillin plate culture medium, plasmid extraction and sequencing verification.

[0048] Fourthly, this application also provides a method for constructing a chimeric recombinant strain of PET-degrading enzyme, the method comprising: transforming the recombinant vector constructed in the third aspect into competent Escherichia coli cells, plating it on ampicillin agar plates, and culturing to obtain positive recombinants, thereby obtaining the chimeric recombinant strain of PET-degrading enzyme.

[0049] Fifthly, this application also provides the application of the PET-degrading chimeric enzyme provided in the first aspect in the degradation of PET, the preparation of PET degradation agents or the preparation of PET degradation products (especially the recovery of high-value-added TPA), which has broad application value.

[0050] Sixthly, this application also provides the application of the biomaterials related to the chimeric enzymes for degrading PET provided in the second aspect in the degradation of PET, the preparation of PET degradation agents, or the preparation of PET degradation products. Beneficial effects

[0051] The research approach of this application is to use IsPETase, TfCut2, and LCC as parents under the guidance of the SCHEMA recombination method. Based on the residue contacts between the parents, the structural domains of the parent proteins are divided. Each domain is then randomly replaced with a corresponding domain from a different parent source to obtain progeny recombinant proteins. In this application, the crystal structures of the three parent proteins, IsPETase, TfCut2, and LCC, are superimposed. Crossover sites are located using the RASPP algorithm, and the structures of the three enzymes are divided into 12 fragments to generate progeny chimeric sequences. After expression and measurement of their fitness (activity, thermostability) data, high-fitness chimeras are selected for subsequent synthesis and expression assays, and further screening and optimization are performed.

[0052] After optimization, the chimeric enzyme for degrading PET provided in this application exhibits high PET degradation activity and thermal stability, with a thermal melting temperature T0. m The maximum temperature can be increased to 78.86℃. In a degradation system containing PET film and glycine-NaOH buffer at pH 9.0, after adding a chimeric enzyme or wild-type PET degrading enzyme to a final concentration of 500 nM and incubating for 1 day at 40℃–60℃ and 250 rpm, the TPA concentrations in the wild-type IsPETase, TfCut2, or LCC degradation systems were 216.78 μM, 207.81 μM, and 1085.35 μM, respectively, while the TPA yield in the degradation system of the chimeric enzyme for PET degradation provided in this application can reach 535.97 μM–9955.22 μM. Given the excellent thermal stability and PET degradation activity of the chimeric enzyme for PET degradation provided in this application, it can be applied to the degradation of PET, the preparation of PET degrading agents, or the preparation of TPA, and has significant economic value and environmental benefits. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 is a schematic diagram of the recombinant plasmid pET-22b-IsPETase-12×Chimera6 in Example 34 of this application, wherein P-1 represents the coding gene for the amino acid shown in SEQ ID No. 1, T-2 represents the coding gene for the amino acid shown in SEQ ID No. 14, T-3 represents the coding gene for the amino acid shown in SEQ ID No. 15, P-4 represents the coding gene for the amino acid shown in SEQ ID No. 4, T-5 represents the coding gene for the amino acid shown in SEQ ID No. 17, T-6 represents the coding gene for the amino acid shown in SEQ ID No. 18, T-7 represents the coding gene for the amino acid shown in SEQ ID No. 19, T-8 represents the coding gene for the amino acid shown in SEQ ID No. 20, L-9 represents the coding gene for the amino acid shown in SEQ ID No. 33, T-10 represents the coding gene for the amino acid shown in SEQ ID No. 22, T-11 represents the coding gene for the amino acid shown in SEQ ID No. 23, and L-12 represents the coding gene for the amino acid shown in SEQ ID No. 36.

[0055] Figure 2 is a liquid chromatogram of the TPA concentration in the degradation system after the addition of chimeric enzyme 6 in this application. Embodiments of the present invention

[0056] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0057] The solution of this application will be described below through specific embodiments.

[0058] Unless otherwise specified, the reagents used in the following examples are all commercially available or obtained using methods known in the art.

[0059] Example 1

[0060] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), by replacing the amino acid sequence shown in SEQ ID No. 5 with the amino acid sequence shown in SEQ ID No. 29, replacing the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 34, and replacing the amino acid sequence shown in SEQ ID No. 12 with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 1.

[0061] Example 2

[0062] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 2.

[0063] Example 3

[0064] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is denoted as chimeric enzyme 3.

[0065] Example 4

[0066] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 4.

[0067] Example 5

[0068] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 5.

[0069] Example 6

[0070] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 33; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 33. The amino acid sequence shown in No. 23 is replaced with the amino acid sequence shown in SEQ ID No. 12, which is replaced with the amino acid sequence shown in SEQ ID No. 36. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 6.

[0071] Example 7

[0072] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), by replacing the amino acid sequence shown in SEQ ID No. 1 with the amino acid sequence shown in SEQ ID No. 13, replacing the amino acid sequence shown in SEQ ID No. 2 with the amino acid sequence shown in SEQ ID No. 14, replacing the amino acid sequence shown in SEQ ID No. 3 with the amino acid sequence shown in SEQ ID No. 15, replacing the amino acid sequence shown in SEQ ID No. 4 with the amino acid sequence shown in SEQ ID No. 16, replacing the amino acid sequence shown in SEQ ID No. 6 with the amino acid sequence shown in SEQ ID No. 30, replacing the amino acid sequence shown in SEQ ID No. 7 with the amino acid sequence shown in SEQ ID No. 19, replacing the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 32, replacing the amino acid sequence shown in SEQ ID No. 9 with the amino acid sequence shown in SEQ ID No. 21, and replacing the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 22. Simultaneously, the amino acid sequence shown in SEQ ID No. 37 is replaced with the amino acid sequence shown in SEQ ID No. 22. The amino acid sequence shown in ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 7.

[0073] Example 8

[0074] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 8.

[0075] Example 9

[0076] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 26; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; and the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 9.

[0077] Example 10

[0078] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 10.

[0079] Example 11

[0080] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 26; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34; the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 11.

[0081] Example 12

[0082] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 34. The amino acid sequence shown in No. 35 is replaced with the amino acid sequence shown in SEQ ID No. 12, which is replaced with the amino acid sequence shown in SEQ ID No. 36. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 12.

[0083] Example 13

[0084] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 26; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 13.

[0085] Example 14

[0086] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 14.

[0087] Example 15

[0088] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 15.

[0089] Example 16

[0090] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 33; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 16.

[0091] Example 17

[0092] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; and the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 17.

[0093] Example 18

[0094] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 33; the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 18.

[0095] Example 19

[0096] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; and the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 19.

[0097] Example 20

[0098] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: replacing the amino acid sequence shown in SEQ ID No. 1 with the amino acid sequence shown in SEQ ID No. 13; replacing the amino acid sequence shown in SEQ ID No. 2 with the amino acid sequence shown in SEQ ID No. 14; replacing the amino acid sequence shown in SEQ ID No. 3 with the amino acid sequence shown in SEQ ID No. 15; replacing the amino acid sequence shown in SEQ ID No. 4 with the amino acid sequence shown in SEQ ID No. 28; replacing the amino acid sequence shown in SEQ ID No. 5 with the amino acid sequence shown in SEQ ID No. 29; replacing the amino acid sequence shown in SEQ ID No. 7 with the amino acid sequence shown in SEQ ID No. 19; replacing the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 32; replacing the amino acid sequence shown in SEQ ID No. 9 with the amino acid sequence shown in SEQ ID No. 33; replacing the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 22; and replacing the amino acid sequence shown in SEQ ID No. 37 with the amino acid sequence shown in SEQ ID No. 32. The amino acid sequence shown in No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 20.

[0099] Example 21

[0100] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 21.

[0101] Example 22

[0102] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; and the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22. The chimeric enzyme for degrading PET provided in this embodiment is denoted as chimeric enzyme 22.

[0103] Example 23

[0104] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 36. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 23.

[0105] Example 24

[0106] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 36. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 24.

[0107] Example 25

[0108] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 25.

[0109] Example 26

[0110] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 33; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 26.

[0111] Example 27

[0112] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 26; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 27.

[0113] Example 28

[0114] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30; the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34; and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 28.

[0115] Example 29

[0116] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 26; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 29.

[0117] Example 30

[0118] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 26; the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; and the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 30.

[0119] Example 31

[0120] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25; the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14; the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15; the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29; the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18; the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20; the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 33; and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 31.

[0121] Example 32

[0122] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: replacing the amino acid sequence shown in SEQ ID No. 1 with the amino acid sequence shown in SEQ ID No. 25; replacing the amino acid sequence shown in SEQ ID No. 2 with the amino acid sequence shown in SEQ ID No. 14; replacing the amino acid sequence shown in SEQ ID No. 3 with the amino acid sequence shown in SEQ ID No. 15; replacing the amino acid sequence shown in SEQ ID No. 4 with the amino acid sequence shown in SEQ ID No. 28; replacing the amino acid sequence shown in SEQ ID No. 5 with the amino acid sequence shown in SEQ ID No. 17; replacing the amino acid sequence shown in SEQ ID No. 6 with the amino acid sequence shown in SEQ ID No. 18; replacing the amino acid sequence shown in SEQ ID No. 7 with the amino acid sequence shown in SEQ ID No. 31; replacing the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 32; replacing the amino acid sequence shown in SEQ ID No. 9 with the amino acid sequence shown in SEQ ID No. 21; and replacing the amino acid sequence shown in SEQ ID No. 37 with the amino acid sequence shown in SEQ ID No. 31. The amino acid sequence shown in No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 32.

[0123] Example 33

[0124] This application provides a chimeric enzyme for degrading PET. This chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No. 37), with the following modifications: replacing the amino acid sequence shown in SEQ ID No. 1 with the amino acid sequence shown in SEQ ID No. 13; replacing the amino acid sequence shown in SEQ ID No. 2 with the amino acid sequence shown in SEQ ID No. 26; replacing the amino acid sequence shown in SEQ ID No. 3 with the amino acid sequence shown in SEQ ID No. 27; replacing the amino acid sequence shown in SEQ ID No. 4 with the amino acid sequence shown in SEQ ID No. 16; replacing the amino acid sequence shown in SEQ ID No. 6 with the amino acid sequence shown in SEQ ID No. 18; replacing the amino acid sequence shown in SEQ ID No. 7 with the amino acid sequence shown in SEQ ID No. 31; replacing the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 20; replacing the amino acid sequence shown in SEQ ID No. 9 with the amino acid sequence shown in SEQ ID No. 21; replacing the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 22; and replacing the amino acid sequence shown in SEQ ID No. 37 with the amino acid sequence shown in SEQ ID No. 22. The amino acid sequence shown in No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this embodiment is designated as chimeric enzyme 33.

[0125] Example 34

[0126] This application provides recombinant vectors containing genes encoding chimeric enzymes that degrade PET as described in Examples 1 to 33, recombinant bacteria, and methods for constructing them. In this embodiment, the recombinant vector is illustrated using a recombinant plasmid as an example.

[0127] The method for constructing a recombinant plasmid containing the encoding gene of the chimeric enzyme for degrading PET as described in Examples 1-33 specifically includes the following steps:

[0128] 1. Construction of recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP, and pET-22b-LCC-cSP

[0129] (1) Construction of recombinant plasmids pET-22b-IsPETase, pET-22b-TfCut2 and pET-22b-LCC

[0130] Using molecular biology methods such as DMT enzyme (commercially available product, TransGen Biotech, GD111) and seamless cloning, the wild-type IsPETase gene (derived from Ideonella sakaiensis, NCBI database GI number 1028065175, denoted as gene IsPETase) was ligated into the pET-22b plasmid to obtain the recombinant plasmid pET-22b-IsPETase;

[0131] Using molecular biology methods such as DMT enzyme (TransGen Biotech, GD111) and seamless cloning, the wild-type TfCut2 gene (derived from Thermobifida fusca, accession number JN129500.1 in GenBank database, denoted as gene TfCut2) was ligated into the pET-22b plasmid to obtain the recombinant plasmid pET-22b-TfCut2;

[0132] Using molecular biology methods such as DMT enzyme (TransGen Biotech, GD111) and seamless cloning, the wild-type LCC gene (derived from uncultured bacterium, accession number HQ704839.1 in GenBank database, denoted as gene LCC) was ligated into the pET-22b plasmid to obtain the recombinant plasmid pET-22b-LCC.

[0133] (2) Construction of recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP and pET-22b-LCC-cSP

[0134] Using recombinant plasmids pET-22b-IsPETase, pET-22b-TfCut2, and pET-22b-LCC as templates, and IsPETase-cSP-F / R, TfCut2-cSP-F / R, and LCC-cSP-F / R as upstream / downstream primers, linear vector fragments of 6.2kb, 6.2kb, and 6.2kb corresponding to each template were amplified by PCR. The linear vector fragments obtained from each template were mixed with ligase to form three circular recombinant plasmids. These three circular recombinant plasmids were transformed into T1 competent cells, and the recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP, and pET-22b-LCC-cSP were obtained through screening on ampicillin agar plates, plasmid extraction, and sequencing verification.

[0135] The nucleotide sequence of the IsPETase-cSP gene (where cSP stands for cut off signal peptide) in the recombinant plasmid pET-22b-IsPETase-cSP is shown in SEQ ID No. 40; the nucleotide sequence of the TfCut2-cSP gene in the recombinant plasmid pET-22b-TfCut2-cSP is shown in SEQ ID No. 41; and the nucleotide sequence of the LCC-cSP gene in the recombinant plasmid pET-22b-LCC-cSP is shown in SEQ ID No. 42.

[0136] The formulation of the ampicillin plate culture medium is as follows: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L, agar 15g / L, ampicillin 50mg / L;

[0137] The nucleotide sequences of primers IsPETase-cSP-F / R, TfCut2-cSP-F / R, and LCC-cSP-F / R are detailed in Table 4.

[0138] Table 4

[0139] The PCR reaction system and PCR procedure were based on those of Beijing TransGen Biotech Co., Ltd. The requirements for the FastPfu Fly DNA Polymerase kit are as shown in Table 5:

[0140] Table 5

[0141] The nucleotide sequence of the IsPETase-cSP gene is shown in SEQ ID No. 40, specifically:

[0142] The nucleotide sequence of the TfCut2-cSP gene is shown in SEQ ID No. 41, specifically:

[0143] The nucleotide sequence of the LCC-cSP gene is shown in SEQ ID No. 42, specifically as follows:

[0144] 2. Constructing a recombinant plasmid containing a chimeric enzyme gene encoding PET degradation and a recombinant bacterial strain (Example 6 is used as an example in this application).

[0145] (1) Design and synthesis of required primers

[0146] The primers designed and synthesized in this step are shown in Table 6.

[0147] Table 6

[0148] (2) Construction of chimeric recombinant plasmids for PET degradation

[0149] Following the requirements of the seamless recombination kit, a recombinant plasmid containing the chimeric enzyme gene encoding PET degradation in Example 6 was constructed using seamless ligation and PCR technology. The main steps included: (1) Using the recombinant plasmid pET-22b-IsPETase-cSP as a template, PCR amplification was performed with upstream primer 6-1 and downstream primer 6-1 as primers. The PCR product was then digested with DMT enzyme, subjected to nucleic acid electrophoresis, and gel extraction to obtain the purified linear gene fragment 6-1; (2) Using the recombinant plasmid pET-22b-TfCut2-cSP as a template, PCR amplification was performed with upstream primer 6-2 and downstream primer 6-2 as primers. The PCR product was digested with DMT enzyme, subjected to nucleic acid electrophoresis and gel extraction to obtain purified linear gene fragment 6-2; purified linear gene fragments 6-3 to 6-12 were obtained in the same manner; (3) after ligating gene fragments 6-1 to 6-12 under the action of seamless recombinant ligase, the seamless recombinant product was transformed into T1 competent cells, and the recombinant plasmid pET-22b-IsPETase-12×Chimera6 containing the chimeric enzyme gene encoding the PET degradation in Example 6 was obtained by screening with ampicillin plate medium, plasmid extraction and sequencing verification. The plasmid map is shown in Figure 1, where P-1 represents SEQ The gene encoding the amino acid shown in SEQ ID No. 1, T-2 represents the gene encoding the amino acid shown in SEQ ID No. 14, T-3 represents the gene encoding the amino acid shown in SEQ ID No. 15, P-4 represents the gene encoding the amino acid shown in SEQ ID No. 4, T-5 represents the gene encoding the amino acid shown in SEQ ID No. 17, T-6 represents the gene encoding the amino acid shown in SEQ ID No. 18, T-7 represents the gene encoding the amino acid shown in SEQ ID No. 19, T-8 represents the gene encoding the amino acid shown in SEQ ID No. 20, L-9 represents the gene encoding the amino acid shown in SEQ ID No. 33, T-10 represents the gene encoding the amino acid shown in SEQ ID No. 22, T-11 represents the gene encoding the amino acid shown in SEQ ID No. 23, and L-12 represents the gene encoding the amino acid shown in SEQ ID No. 36.

[0150] When constructing recombinant plasmids corresponding to the chimeric enzymes that degrade PET in Examples 1-5 and Examples 7-33, primers were designed based on the coding genes corresponding to the amino acid sequences of each chimeric enzyme. The primer pair design strategy was as follows: the upstream primer consisted of a 5'-15-25 bp homologous sequence from the end of the upstream vector + a gene-specific primer - 3', and the downstream primer consisted of a 5'-15-25 bp homologous sequence from the end of the downstream vector + a gene-specific primer - 3'. After designing and synthesizing the primer pairs required for recombination, recombinant plasmids containing the chimeric enzymes encoding each PET degradation enzyme were constructed using seamless ligation and PCR technology, following the requirements of the seamless recombination kit.

[0151] (3) Construction of chimeric recombinant strains for PET degradation

[0152] The PET-degrading chimeric recombinant plasmid pET-22b-IsPETase-12×Chimera6 obtained in step (2) was transformed into BL21(DE3) competent cells, plated on ampicillin agar plates, and cultured at 37°C for 12 h. Positive recombinants were screened to obtain the PET-degrading chimeric recombinant strain, which was denoted as recombinant strain-chimeric enzyme 6.

[0153] The construction process of the recombinant strains of PET-degrading chimeric enzymes in Examples 1-5 and Examples 7-33 is basically the same as the construction process of recombinant strain-chimeric enzyme 6, the difference being the selection of different recombinant plasmids.

[0154] Example 35

[0155] This application uses recombinant strain-chimeric enzyme 6 as an example to illustrate the method for inducing expression of the PET-degrading recombinant strain and purifying the target protein:

[0156] The recombinant strain-chimeric enzyme 6 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm. The overnight culture was then inoculated into fresh LB liquid medium at a 5% inoculation rate and cultured at 37°C and 220 rpm until OD500 was reached. 600 The pH was approximately 0.8. 0.1% (v / v) IPTG was added, and the mixture was cooled to 16℃ to induce expression for 20 h. Wet cells of the recombinant strain were collected by centrifugation at 4000 rpm for 15 min.

[0157] The recombinant strain of wet cells was resuspended in lysis buffer, and the resuspended wet cells were lysed using a high-pressure cell disruptor. After lysis, the bacterial suspension was centrifuged at 10,000 rpm for 60 min to remove cell debris. The supernatant was then passed through a Ni-NTA packed column to adsorb the target protein. Non-specifically adsorbed proteins were washed away using a washing buffer. The target protein was eluted using an elution buffer, and the eluent was concentrated using a protein concentration tube to obtain chimeric enzyme 6.

[0158] The LB liquid culture medium formula is as follows: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L;

[0159] The formulation of the lysis buffer is: 50mM Tris-HCl, 150mM NaCl, 10mM Imidazole, pH=7.5;

[0160] The washing buffer solution is formulated as follows: 50 mM Tris-HCl, 150 mM NaCl, 20 mM Imidazole, pH = 7.5;

[0161] The elution buffer formulation is: 50mM Tris-HCl, 300mM NaCl, 300mM Imidazole, pH=7.5.

[0162] Example 36

[0163] This application provides the application of the chimeric enzymes for degrading PET described in Examples 1-33 in the hydrolysis of PET, and the specific methods are as follows:

[0164] Add a PET film (purchased from Good Fellow, with a crystallinity of approximately 8% and a diameter of approximately [missing information]) to a 50 mM glycine-NaOH buffer solution with a pH of 9.0. Add a PET-degrading chimeric enzyme to a final concentration of 500 nM, and incubate at 40℃~60℃ (select the optimal temperature for different PET-degrading chimeric enzymes) and 250 rpm for 1~3 days. Then, determine the concentration of TPA in the degradation system using HPLC.

[0165] During extensive testing, this application discovered that, based on IsPETase, arbitrarily substituting the amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 12 alters the thermal melting temperature (T) of many chimeric enzymes. m (and / or PET enzyme degradation activity is inferior to that of wild-type enzymes.)

[0166] Test Example

[0167] To examine whether the thermostability and PET degradation activity of the chimeric enzymes in this application are improved compared to the wild-type enzymes, this test example examines the PET-degrading chimeric enzymes provided in Examples 1-33, as well as the TT of the wild-type enzymes IsPETase, TfCut2, and LCC. m The values ​​and the concentration of TPA in the degradation system under the conditions provided in Example 36 (this test example uses an incubation period of 1 day as an example) were measured, and the results are shown in Table 7.

[0168] In this application, the Tm value was measured using a Q-PCR instrument;

[0169] The concentration of TPA in the degradation system was determined by HPLC under the following conditions: detection wavelength: 240 nm; column: ZORBAX Eclipse Plus C18 reversed-phase column (5 μm, 250 mm × 4.6 mm); mobile phase: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile, with mobile phase B increasing from 5% to 70% within 20 min; column temperature: 30℃; injection volume: 10 μL; flow rate: 0.8 mL / min. The HPLC chromatogram of the TPA concentration in the degradation system after the addition of chimeric enzyme 6 is shown in Figure 2.

[0170] The specific measurement results are shown in Table 7.

[0171] Table 7

[0172] As shown in Table 7, the chimeric enzyme for PET degradation provided in this application exhibits excellent PET degradation activity and thermal stability. Compared to the wild-type IsPETase, the thermal melting temperatures (T1-33) of the chimeric enzymes are significantly higher. m The temperature was increased by 4.8℃ to 31.7℃, and the concentration of TPA, the degradation product after PET degradation, increased by 1.5 to 44.5 times. Given that the chimeric enzyme for PET degradation constructed in this application has higher PET degradation activity and thermal stability, it can be used in fields such as PET degradation, preparation of PET degradation agents, or preparation of PET degradation products. While broadening the application conditions for PET degradation, it can also significantly improve PET degradation efficiency and increase the recycling rate of the high-value-added degradation product TPA, thus possessing significant economic value and environmental benefits.

[0173] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A chimeric enzyme for degrading PET, characterized in that, The amino acid sequence of IsPETase is sequentially divided into 12 amino acid sequences as shown in SEQ ID No. 1 to SEQ ID No.

12. The PET-degrading chimeric enzyme is obtained by replacing at least one of the 12 amino acid sequences, wherein: Replace the amino acid sequence shown in SEQ ID No. 1 with the amino acid sequence shown in SEQ ID No. 13 or SEQ ID No. 25; Replace the amino acid sequence shown in SEQ ID No. 2 with the amino acid sequence shown in SEQ ID No. 14 or SEQ ID No. 26; Replace the amino acid sequence shown in SEQ ID No. 3 with the amino acid sequence shown in SEQ ID No. 15 or SEQ ID No. 27; Replace the amino acid sequence shown in SEQ ID No. 4 with the amino acid sequence shown in SEQ ID No. 16 or SEQ ID No. 28; Replace the amino acid sequence shown in SEQ ID No. 5 with the amino acid sequence shown in SEQ ID No. 17 or SEQ ID No. 29; Replace the amino acid sequence shown in SEQ ID No. 6 with the amino acid sequence shown in SEQ ID No. 18 or SEQ ID No. 30; Replace the amino acid sequence shown in SEQ ID No. 7 with the amino acid sequence shown in SEQ ID No. 19 or SEQ ID No. 31; Replace the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 20 or SEQ ID No. 32; Replace the amino acid sequence shown in SEQ ID No. 9 with the amino acid sequence shown in SEQ ID No. 21 or SEQ ID No. 33; Replace the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 22 or SEQ ID No. 34; Replace the amino acid sequence shown in SEQ ID No. 11 with the amino acid sequence shown in SEQ ID No. 23 or SEQ ID No. 35; Replace the amino acid sequence shown in SEQ ID No. 12 with the amino acid sequence shown in SEQ ID No. 24 or SEQ ID No.

36.

2. The chimeric enzyme for degrading PET according to claim 1, characterized in that, The amino sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24; or Based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23; or Based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24; or Based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24; or Based on IsPETase, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No.

24.

3. The chimeric enzyme for degrading PET according to claim 1, characterized in that, The amino sequence of the PET-degrading chimeric enzyme is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 33, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No.

14. The amino acid sequence shown in No. 36; or Based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 30, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24; or Based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 32, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24; or Based on IsPETase, replace the amino acid sequence shown in SEQ ID No. 2 with the amino acid sequence shown in SEQ ID No. 26, replace the amino acid sequence shown in SEQ ID No. 3 with the amino acid sequence shown in SEQ ID No. 15, replace the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 20, replace the amino acid sequence shown in SEQ ID No. 9 with the amino acid sequence shown in SEQ ID No. 21, and simultaneously replace the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 22; or Based on IsPETase, the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 25, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 27, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 28, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 31, the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20, the amino acid sequence shown in SEQ ID No. 9 is replaced with the amino acid sequence shown in SEQ ID No. 21, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 22, the amino acid sequence shown in SEQ ID No. 11 is replaced with the amino acid sequence shown in SEQ ID No. 35, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No.

24.

4. A biomaterial associated with a chimeric enzyme for degrading PET according to any one of claims 1 to 3, characterized in that: The biomaterial is any one of (I) to (IV) below: (I) A nucleic acid molecule encoding the chimeric enzyme that degrades PET as described in any one of claims 1 to 3; (II) An expression cassette containing the nucleic acid molecule described in (I); (III) A recombinant vector containing the nucleic acid molecule described in (I), or a recombinant vector containing the expression cassette described in (II); (IV) A recombinant strain containing the nucleic acid molecule described in (I), or a recombinant strain containing the expression cassette described in (II), or a recombinant strain containing the recombinant vector described in (III).

5. A method for constructing a chimeric recombinant vector for degrading PET, characterized in that: The gene encoding the chimeric enzyme that degrades PET as described in any one of claims 1 to 3 was transformed into T1 competent cells, and the recombinant vector was obtained through screening, plasmid extraction and sequencing verification.

6. A method for constructing a chimeric recombinant strain of PET-degrading enzyme, characterized in that: The recombinant vector constructed in claim 5 was transformed into competent Escherichia coli cells, plated on ampicillin agar plates, and cultured to obtain positive recombinants, thus obtaining the PET-degrading chimeric recombinant strain.

7. The use of the PET-degrading chimeric enzyme according to any one of claims 1 to 3 in the degradation of PET, the preparation of PET degradation agents, or the preparation of PET degradation products.

8. The application of the biomaterial associated with the chimeric enzyme for degrading PET as described in claim 4 in the degradation of PET, the preparation of PET degradation agents, or the preparation of PET degradation products.

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