Chimeric enzyme for degrading PET (Polyethylene Terephthalate) as well as related biological material and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses a chimeric enzyme for degrading PET (Polyethylene Terephthalate) as well as a related biological material and application of the chimeric enzyme. The chimeric enzyme for degrading PET provided by the invention is obtained by replacing at least one fragment in an amino acid sequence as shown in SEQ ID No.1-12 on the basis of IsPETase. Compared with IsPETase, the chimeric enzyme for degrading PET provided by the invention has the advantages that the thermal melting temperature is increased by 4.8-31.7 DEG C, the concentration of a degradation product TPA after PET degradation is increased by 1.5-44.5 times, while the application conditions of PET degradation are widened, the PET degradation efficiency can be greatly improved, the recycling rate of the degradation product TPA with high added value is increased, and the chimeric enzyme has important economic value and environmental benefit. The method can be used in the fields of PET degradation, PET degradation agent preparation or PET degradation product preparation and the like.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a chimeric enzyme for degrading PET and related biomaterials and applications thereof. Background Art

[0002] Polyethylene terephthalate (PET) has been widely used in various fields due to its superior physical and chemical properties. However, due to improper disposal of PET after use, it poses a serious threat to the ecological environment, and how to deal with plastic waste has become a focal issue.

[0003] At present, biodegradation, especially enzymatic degradation, has been applied to the treatment of waste PET due to its advantages of low cost, mild reaction conditions, and green and pollution-free. At present, many enzymes that can hydrolyze PET have been discovered, such as esterase, cutinase, lipase, PETase and MHETase. Among them, IsPETase is a bacterial Ideonella sakaiensis PETase (i.e., an enzyme that degrades PET, called IsPETase), belongs to the α / β hydrolase superfamily. Compared with other PET hydrolases, IsPETase has the advantages of high degradation efficiency, suitable for degradation at medium temperature, specific degradation of PET, and degradation of highly crystalline PET films. However, the hydrolysis of PET by IsPETase mainly occurs in the amorphous part, and the glass transition temperature (Tg) of PET is between 65°C and 71°C, which puts higher requirements on the thermal stability of PET hydrolase. However, the thermal stability and degradation activity of the enzyme at high temperature are poor, so it is subject to certain limitations in the actual application of PET degradation. Summary of the invention

[0004] In view of the above technical problems, the present invention designs and improves IsPETase, and provides a chimeric enzyme for degrading PET and its related biomaterials and applications. Compared with wild IsPETase enzyme, the chimeric enzyme for degrading PET not only has higher thermal stability, but also significantly improves PET degradation activity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a chimeric enzyme for degrading PET, wherein the chimeric enzyme is obtained by sequentially 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 sequence in the 12 amino acid sequences, wherein: The amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.13 or SEQ ID No.25; The amino acid sequence shown in SEQ ID No.2 is replaced by the amino acid sequence shown in SEQ ID No.14 or 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 or SEQ ID No.27; The amino acid sequence shown in SEQ ID No.4 is replaced by the amino acid sequence shown in SEQ ID No.16 or SEQ ID No.28; The amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.17 or SEQ ID No.29; The amino acid sequence shown in SEQ ID No.6 is replaced by the amino acid sequence shown in SEQ ID No.18 or SEQ ID No.30; The amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19 or 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 or SEQ ID No.32; The amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.21 or SEQ ID No.33; The amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.22 or 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.23 or SEQ ID No.35; The amino acid sequence shown in SEQ ID No.12 is replaced with the amino acid sequence shown in SEQ ID No.24 or SEQ ID No.36.

[0006] Compared with the wild-type IsPETase enzyme, the thermal melting temperature (T m) increased by 4.8°C to 31.7°C, and the concentration of the degradation product TPA after PET degradation increased by 1.5 times to 44.5 times. In view of the fact that the chimeric enzyme for degrading PET constructed by the present invention has higher PET degradation activity and thermal stability, it can not only broaden the application conditions of PET degradation, but also greatly improve the PET degradation efficiency, which is helpful to improve the recovery rate of the high value-added degradation product TPA.

[0007] Among them, the amino acid sequences shown in SEQ ID No.1 to SEQ ID No.12 are derived from IsPETase, and the amino acid sequences thereof are shown in Table 1 for details.

[0008] Table 1

[0009] The amino acid sequences shown in SEQ ID No. 13 to SEQ ID No. 24 are derived from TfCUT2, and their amino acid sequences are shown in Table 2 for details.

[0010] Table 2

[0011] The amino acid sequences shown in SEQ ID No.25 to SEQ ID No.36 are derived from LCC, and their amino acid sequences are shown in Table 3 for details.

[0012] Table 3

[0013] The amino acid sequence of IsPETase is shown in SEQ ID No. 37, specifically: QTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGW SMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS.

[0014] The amino acid sequence of TfCUT2 is shown in SEQ ID No. 38, specifically: ANPYERGPNPTDALLEARSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSM GGGGSLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPF.

[0015] The amino acid sequence of LCC is shown in SEQ ID No. 39, specifically: SNPYQRGPNPTRSALTADGPFSVATYTVSRLSVSGFGGGVIYYPTGTSLTFGGIAMSPGYTADASSLAWLGRRLASHGFVVLVINTNSRFDYPDSRASQLSAALNYLRTSSPSAVRARLDANRLAVAGH SMGGGGTLRIAEQNPSLKAAVPLTPWHTDKTFNTSVPVLIVGAEADTVAPVSQHAIPFYQNLPSTTPKVYVELDNASHFAPNSNNAAISVYTISWMKLWVDNDTRYRQFLCNVNDPALSDFRTNNRHCQ.

[0016] Exemplarily, as a preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: based on IsPETase, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.29, the amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.34, and the amino acid sequence shown in SEQ ID No.12 is replaced by the amino acid sequence shown in SEQ ID No.24.

[0017] Exemplarily, as a preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: 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.

[0018] Exemplarily, as a preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: 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.

[0019] Exemplarily, as a preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: 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.

[0020] Exemplarily, as a preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: 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.

[0021] Compared with the wild-type IsPETase enzyme, the thermal melting temperature of the preferred chimeric enzyme for degrading PET provided by the present invention is increased by 19.8°C to 31.7°C, and the concentration of the degradation product TPA after PET degradation is increased by 29.4 to 36.6 times.

[0022] Exemplarily, as a further preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.2 is replaced by the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced by the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19, the amino acid sequence shown in SEQ ID No.8 is replaced by the amino acid sequence shown in SEQ ID No.20, the amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.33, the amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.22, the amino acid sequence shown in SEQ ID No.11 is replaced by the amino acid sequence shown in SEQ ID No.23, and the amino acid sequence shown in SEQ ID No. The amino acid sequence shown in No.12 is replaced by the amino acid sequence shown in SEQ ID No.36.

[0023] Exemplarily, as a further preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: on the basis of 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. The amino acid sequence shown in No.12 is replaced by the amino acid sequence shown in SEQ ID No.24.

[0024] Exemplarily, as a more preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: on the basis of 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.

[0025] Illustratively, as a further preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: on the basis of 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.

[0026] Exemplarily, as a further preferred technical solution, the amino acid sequence of the chimeric enzyme for degrading PET is: on the basis of 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.

[0027] Compared with the wild-type IsPETase enzyme, the thermal melting temperature of the further preferred chimeric enzyme for degrading PET provided by the present invention is increased by 4.8°C to 31.5°C, and the concentration of the degradation product TPA after degrading PET is increased by 36.9 times to 44.5 times.

[0028] In a second aspect, the present invention provides a biomaterial related to the chimeric enzyme for degrading PET, wherein the biomaterial is any one of the following (I) to (IV): (I) a gene encoding the chimeric enzyme for degrading PET as claimed in claim 1; (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).

[0029] In a third aspect, the present invention also provides a method for constructing a recombinant vector of a chimeric enzyme that degrades PET, the construction method comprising: transforming the gene encoding the above-mentioned chimeric enzyme that degrades PET into T1 competent cells, and obtaining the recombinant vector through ampicillin plate culture medium screening, plasmid extraction and sequencing verification.

[0030] In a fourth aspect, the present invention further provides a method for constructing a recombinant strain of a chimeric enzyme that degrades PET, the construction method comprising the following steps: transforming the recombinant vector constructed in the third aspect into competent Escherichia coli cells, coating them on ampicillin plate culture medium, and culturing positive recombinants, namely, obtaining the recombinant strain of the chimeric enzyme that degrades PET.

[0031] In a fifth aspect, the present invention also provides the use of the chimeric enzyme for degrading PET provided in the first aspect in degrading PET, preparing PET degradation agents or preparing PET degradation products (especially the recovery of high value-added TPA), which has wide application value.

[0032] In a sixth aspect, the present invention further provides the use of the biomaterial related to the chimeric enzyme for degrading PET provided in the second aspect in degrading PET, preparing a PET degradation agent or preparing a PET degradation product.

[0033] The research idea of ​​the present invention is to use IsPETase, TfCUT2 and LCC as parents under the guidance of the SCHEMA recombination method, divide the domains of the parent proteins according to the residue contacts between the parents; randomly replace each domain with the corresponding domain from different parent sources to obtain progeny recombinant proteins. In the present invention, the crystal structures of the three parent proteins IsPETase, TfCUT2 and LCC are superimposed, the crossover sites are located by the RASPP algorithm, the structures of the three enzymes are divided into 12 fragments, and the progeny chimera sequences are generated. After expressing and measuring their fitness (activity, thermal stability) data, high-fitness chimeras are selected for subsequent synthesis and expression determination, and further screening and optimization.

[0034] After optimization, the chimeric enzyme for degrading PET provided by the present invention has high PET degradation activity and thermal stability, and the thermal melting temperature T mThe maximum temperature can be increased to 78.86°C. In a degradation system containing a PET film and a glycine-NaOH buffer solution with a pH of 9.0, a chimeric enzyme with a final concentration of 500nM was added. After incubation for 1 day at 40°C~60°C and 250rpm, the concentrations of TPA in the wild-type IsPETase, TfCUT2 or LCC degradation systems were 216.78μM, 207.81μM and 1085.35μM, respectively, while the TPA yield in the degradation system of the chimeric enzyme for degrading PET provided by the present invention can reach 535.97μM~9955.22μM. In view of the excellent thermal stability and PET degradation activity of the chimeric enzyme recombinant enzyme for degrading PET provided by the present invention, it can be used to degrade PET, prepare PET degradation agents or prepare TPA, and has important economic value and environmental benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-12×Chimera6 in Example 34 of the present invention, 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; Figure 2 This is a liquid phase diagram of the TPA concentration in the degradation system after adding chimeric enzyme 6 in the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] The scheme of the present invention is described below through specific embodiments.

[0039] Unless otherwise specified, the reagents in the following examples are commercially available or obtained according to methods known in the art.

[0040] Example 1 The embodiment of the present invention provides a chimeric enzyme for degrading PET, which is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.34, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 1.

[0041] Example 2 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.13, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.17, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.18, the amino acid sequence as shown in SEQ ID No.7 is replaced with the amino acid sequence as shown in SEQ ID No.19, and the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 2.

[0042] Example 3 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 3.

[0043] Example 4 The embodiment of the present invention provides a chimeric enzyme for degrading PET, which is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), and on the basis of IsPETase, the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.25, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.17, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.7 is replaced with the amino acid sequence as shown in SEQ ID No.31, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 4.

[0044] Example 5 The embodiment of the present invention provides a chimeric enzyme for degrading PET, which is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), and on the basis of IsPETase, the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.14, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.27, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.16, the amino acid sequence as shown in SEQ ID No.7 is replaced with the amino acid sequence as shown in SEQ ID No.31, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.20, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.34, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 5.

[0045] Example 6 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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. The amino acid sequence shown in SEQ ID No. 23 is replaced by the amino acid sequence shown in SEQ ID No. 36. The chimeric enzyme for degrading PET provided in this example is denoted as chimeric enzyme 6.

[0046] Example 7 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein 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. 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 example is denoted as chimeric enzyme 7.

[0047] Example 8 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.25, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.16, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.17, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.22, the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 8.

[0048] Example 9 The embodiment of the present invention provides a chimeric enzyme for degrading PET, which is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein 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 recorded as chimeric enzyme 9.

[0049] Example 10 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 10.

[0050] Embodiment 11 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 11.

[0051] Example 12 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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, the amino acid sequence shown in SEQ ID No.11 is replaced with the amino acid sequence shown in SEQ ID The amino acid sequence shown in SEQ ID No. 35 is replaced by the amino acid sequence shown in SEQ ID No. 36. The chimeric enzyme for degrading PET provided in this example is denoted as chimeric enzyme 12.

[0052] Example 13 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.13, the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.26, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.16, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.18, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21, the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 13.

[0053] Embodiment 14 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.27, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.17, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.34, and the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 14.

[0054] Embodiment 15 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 15.

[0055] Example 16 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.25, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.33, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 16.

[0056] Embodiment 17 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.13, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.17, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21, and the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.34. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 17.

[0057] Embodiment 18 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.14, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.27, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.18, the amino acid sequence as shown in SEQ ID No.7 is replaced with the amino acid sequence as shown in SEQ ID No.31, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.33, the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 18.

[0058] Embodiment 19 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.14, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.7 is replaced with the amino acid sequence as shown in SEQ ID No.19, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.20, and the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.22. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 19.

[0059] Embodiment 20 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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.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.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.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. The amino acid sequence shown in No. 11 is replaced by the amino acid sequence shown in SEQ ID No. 35, and the amino acid sequence shown in SEQ ID No. 12 is replaced by the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this example is denoted as chimeric enzyme 20.

[0060] Embodiment 21 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.18, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.22, and the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 21.

[0061] Embodiment 22 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 22.

[0062] Embodiment 23 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 23.

[0063] Embodiment 24 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.25, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.22, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.36. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 24.

[0064] Embodiment 25 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 25.

[0065] Embodiment 26 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.14, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.7 is replaced with the amino acid sequence as shown in SEQ ID No.19, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.20, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.33, and the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 26.

[0066] Embodiment 27 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 27.

[0067] Embodiment 28 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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 example is designated as chimeric enzyme 28.

[0068] Embodiment 29 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.26, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.17, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.18, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.32, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21, and the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.35. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 29.

[0069] Embodiment 30 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.13, the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.26, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.20, and the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 30.

[0070] Embodiment 31 The embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.25, the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.14, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.18, the amino acid sequence as shown in SEQ ID No.8 is replaced with the amino acid sequence as shown in SEQ ID No.20, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.33, and the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23. The chimeric enzyme for degrading PET provided in this embodiment is recorded as chimeric enzyme 31.

[0071] Embodiment 32 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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.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.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. 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 example is denoted as chimeric enzyme 32.

[0072] Embodiment 33 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is based on IsPETase (amino acid sequence is shown in SEQ ID No.37), wherein 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.16, 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.11 is replaced with the amino acid sequence shown in SEQ ID No.13. The amino acid sequence shown in No. 11 is replaced by the amino acid sequence shown in SEQ ID No. 23, and the amino acid sequence shown in SEQ ID No. 12 is replaced by the amino acid sequence shown in SEQ ID No. 24. The chimeric enzyme for degrading PET provided in this example is denoted as chimeric enzyme 33.

[0073] Embodiment 34 The embodiments of the present invention provide a recombinant vector, a recombinant bacterium and a construction method thereof containing a gene encoding a chimeric enzyme for degrading PET in embodiments 1 to 33, wherein the recombinant vector in this embodiment is described by taking a recombinant plasmid as an example.

[0074] The method for constructing a recombinant plasmid containing the gene encoding the chimeric enzyme for degrading PET in Examples 1 to 33 specifically comprises the following steps: 1. Construction of recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCUT2-cSP and pET-22b-LCC-cSP (1) Construction of recombinant plasmids pET-22b-IsPETase, pET-22b-TfCUT2, and pET-22b-LCC The recombinant plasmid was prepared by molecular biological methods such as DMT enzyme (Quanshijin Company, GD111) and seamless cloning, and the gene of wild-type IsPETase (derived from Ideonella sakaiensis , its GI number in the NCBI database is 1028065175, recorded as gene IsPETase ) was connected to the pET-22b plasmid to obtain the recombinant plasmid pET-22b-IsPETase; The recombinant plasmid was prepared by molecular biological methods such as DMT enzyme (Quanshijin Company, GD111) and seamless cloning, and the wild-type TfCUT2 gene (derived from Thermobifida fusca , the accession number in the GenBank database is JN129500.1, and is recorded as gene TfCUT2 ) was connected to the pET-22b plasmid to obtain the recombinant plasmid pET-22b-TfCUT2; The recombinant plasmid was prepared by molecular biological methods such as DMT enzyme (Quanshijin Company, GD111) and seamless cloning, and the gene of wild-type LCC (derived from uncultured bacteria , the accession number in the GenBank database is HQ704839.1, and is recorded as gene LCC ) was connected to the pET-22b plasmid to obtain the recombinant plasmid pET-22b-LCC.

[0075] Recombinant plasmids pET-22b-IsPETase, pET-22b-TfCUT2 and pET-22b-LCC were used as templates, IsPETase-cSP-F / R, TfCut2-cSP-F / R and LCC-cSP-F / R were used as primers, and 6.2 kb, 6.2 kb and 6.2 kb linear vector fragments corresponding to each template were obtained by PCR amplification. The above linear vector fragments were mixed with ligase respectively, and three circular recombinant plasmids were formed under the action of ligase; the above three circular recombinant plasmids were transformed into T1 competent cells respectively, and recombinant plasmids pET-22b-IsPETase-cSP, recombinant plasmids pET-22b-TfCut2-cSP and recombinant plasmids pET-22b-LCC-cSP were obtained by ampicillin plate culture medium screening, plasmid extraction and sequencing verification.

[0076] The nucleotide sequence of the IsPETase-cSP gene (wherein cSP, i.e., cutoff signal peptide, represents the excision signal peptide) in the recombinant plasmid pET-22b-IsPETase-cSP is shown as SEQ ID No.40; the nucleotide sequence of the TfCut2-cSP gene in the recombinant plasmid pET-22b-TfCut2-cSP is shown as SEQ ID No.41; and the nucleotide sequence of the LCC-cSP gene in the recombinant plasmid pET-22b-LCC-cSP is shown as SEQ ID No.42.

[0077] Among them, in the ampicillin plate medium: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L, agar 15g / L, ampicillin 50mg / L; The nucleotide sequences of primers IsPETase-cSP-F / R, TfCut2-cSP-F / R and LCC-cSP-F / R are shown in Table 4.

[0078] Table 4

[0079] The PCR reaction system and PCR program refer to TransStart ® The requirements of the FastPfu Fly DNA Polymerase Kit are as shown in Table 5: Table 5

[0080] The nucleotide sequence of the IsPETase-cSP gene is shown in SEQ ID No.40, specifically: cagaccaacccctacgcccgcggcccgaacccgacagccgcctcactcgaagccagcgccggcccgttcaccgtgcgctcgttcaccgtgagccgcccgagcggctacggcgccggcaccgtgtactaccccaccaacgccggcggcaccgtgggcgccatcgccatcgtgccgggctacaccgcgcgccagtcgagcatcaaatggtggggcccgcgcctggcctcgcacggcttcgtggtcatcaccatcgacaccaactccacgctcgaccagccgtccagccgctcgtcgcagcagatggccgcgctgcgccaggtggcctcgctcaacggcaccagcagcagcccgatctacggcaaggtcgacaccgcccgcatgggcgtgatgggctggtcgatgggcggtggcggctcgctgatctcggcggccaacaacccgtcgctgaaagccgcggcgccgcaggccccgtgggacagctcgaccaacttctcgtcggtcaccgtgcccacgctgatcttcgcctgcgagaacgacagcatcgccccggtcaactcgtccgccctgccgatctacgacagcatgtcgcgcaatgcgaagcagttcctcgaaatcaacggtggctcgcactcctgcgccaacagcggcaacagcaaccaggcgctgatcggcaagaagggcgtggcctggatgaagcgcttcatggacaacgacacgcgctactccaccttcgcctgcgagaacccgaacagcacccgcgtgtcggacttccgcaccgcgaactgcagc; The nucleotide sequence of the TfCut2-cSP gene is shown in SEQ ID No. 41, specifically as follows: gccaacccctacgagcgcggccccaacccgaccgacgccctgctcgaagccagcagcggccccttctccgtcagcgaggagaacgtctcccggttgagcgccagcggcttcggcggcggcaccatctactacccgcgggagaacaacacctacggtgcggtggcgatctcccccggctacaccggcactgaggcttccatcgcctggctgggcgagcgcatcgcctcccacggcttcgtcgtcatcaccatcgacaccatcaccaccctcgaccagccggacagccgggcagagcagctcaacgccgcgctgaaccacatgatcaaccgggcgtcctccacggtgcgcagccggatcgatagcagccgactggcggtcatgggccactccatgggcggcggcggcaccctgcgtctggcctcccagcgtcccgacctgaaggccgccatcccgctcaccccgtggcacctcaacaagaactggagcagcgtcaccgtgccgacgctgatcatcggggccgacctcgacacgatcgcgccggtcgccacgcacgcgaaaccgttctacaacagcctgccgagctccatcagcaaggcctacctggagctggacggcgcaacccacttcgccccgaacatccccaacaagatcatcggcaagtacagcgtcgcctggctcaagcggttcgtcgacaacgacacccgctacacccagttcctctgccccggaccgcgcgacggactcttcggcgaggtcgaagagtaccgctccacctgcccgttc; The nucleotide sequence of the LCC-cSP gene is shown in SEQ ID No. 42, specifically: .

[0081] 2. Construct a recombinant plasmid containing a chimeric enzyme gene encoding PET degradation (the present invention is described using Example 6 as an example).

[0082] (1) Design and synthesis of required primers The primers designed and synthesized in this step are shown in Table 6 below.

[0083] Table 6

[0084] (2) Construction of chimeric enzyme recombinant plasmid for PET degradation According to the requirements of the seamless recombination kit, seamless connection and PCR technology were used to construct a recombinant plasmid containing the chimeric enzyme gene encoding the PET-degrading enzyme in Example 6, which mainly includes the following three steps: (1) using the recombinant plasmid pET-22b-IsPETase-cSP as a template, upstream primer 6-1 and downstream primer 6-1 as primers for PCR amplification, and subjecting the PCR product to DMT enzyme digestion, nucleic acid electrophoresis and gel excision recovery to obtain a purified linear gene fragment 6-1; (2) using the recombinant plasmid pET-22b-TfCUT2-cSP as a template, upstream primer 6-2 and downstream primer 6-2 as primers for PCR amplification. The primers are used for PCR amplification, and the PCR products are subjected to DMT enzyme digestion, nucleic acid electrophoresis and gel excision recovery to obtain a purified linear gene fragment 6-2; the purified linear gene fragments 6-3 to 6-12 are obtained in the same manner; (3) after the gene fragments 6-1 to 6-12 are connected under the action of seamless recombination ligase, the seamless recombination product is transformed into T1 competent cells, and the recombinant plasmid pET-22b-12×Chimera6 containing the chimeric enzyme gene encoding the PET degradation in Example 6 is obtained through ampicillin plate culture medium screening, plasmid extraction and sequencing verification. The plasmid map schematic diagram is shown in FIG. Figure 1 As shown, wherein P-1 represents 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.

[0085] When constructing the recombinant plasmids corresponding to the chimeric enzymes for degrading PET in Examples 1-5 and Examples 7-33, primers are designed according to the coding genes corresponding to the amino acid sequences of each chimeric enzyme. Among them, the idea of ​​designing the primer pair is: the upstream primer includes 5'-upstream vector end 15-25bp homologous sequence + gene-specific primer-3', and the downstream primer includes 5'-downstream vector end 15-25bp homologous sequence + gene-specific primer-3'. After designing and synthesizing the primer pairs required for recombination, the recombinant plasmids pET-22b-IsPETase-cSP, recombinant plasmid pET-22b-TfCUT2-cSP and recombinant plasmid pET-22b-LCC-cSP in step 1 are used to construct recombinant plasmids containing chimeric enzymes encoding each PET degradation using seamless connection and PCR technology according to the requirements of the seamless recombination kit.

[0086] (3) Construction of a chimeric enzyme recombinant strain for PET degradation The PET-degrading chimeric enzyme recombinant plasmid pET-22b-12×Chimera6 prepared in step (2) was transformed into BL21 (DE3) competent cells, spread on ampicillin plate culture medium, and cultured at 37°C for 12 hours. Positive recombinants were screened to obtain the PET-degrading chimeric enzyme recombinant strain, which was recorded as the recombinant strain-chimeric enzyme 6.

[0087] The construction process of the recombinant strain of the chimeric enzyme for degrading PET described in Examples 1-5 and Examples 7-33 is basically the same as the construction process of the recombinant strain-chimeric enzyme 6, except that different recombinant plasmids are selected.

[0088] Embodiment 35 In the embodiment of the present invention, the recombinant strain pET-22b-12×Chimera6 is used as an example to illustrate the induction expression of the chimeric enzyme recombinant strain for degrading PET and the purification method of the target protein: The recombinant strain pET-22b-12×Chimera6 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 r / min. The overnight culture solution was inoculated into fresh LB liquid medium at 5% inoculum and cultured at 37°C and 220 r / min until OD 600 When the concentration was about 0.8, 0.1% (v / v) IPTG was added and the temperature was lowered to 16°C to induce expression for 20 h. The wet cells of the recombinant strain were collected by centrifugation at 4000 rpm for 15 min.

[0089] The wet cells of the recombinant strain were resuspended in a lysis buffer and the resuspended wet cells were lysed using a high-pressure cell disruptor. After lysis, the bacterial solution was centrifuged at 10,000 rpm for 60 min to remove cell debris, the supernatant was adsorbed with a Ni-NTA column, and the non-specifically adsorbed impurities were washed with a washing buffer. The target protein was eluted with an elution buffer, and the eluate was concentrated with a protein concentrator to obtain chimeric enzyme 6.

[0090] The formula of LB liquid medium is: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L; The formula of the lysis buffer is: 50 mM Tris-HCl, 150 mM NaCl, 10 mM Imidazole, pH = 7.5; The formula of the washing buffer is: 50mM Tris-HCl, 150mM NaCl, 20mM Imidazole, pH = 7.5; The formula of the elution buffer is: 50 mM Tris-HCl, 300 mM NaCl, 300 mM Imidazole, pH = 7.5.

[0091] Embodiment 36 The present invention provides the use of the chimeric enzyme for degrading PET in Examples 1 to 33 in hydrolyzing PET, and the specific method is as follows: A PET film (purchased from GoodFellow, with a crystallinity of about 8%, φ6 mm) was added to a 50 mM glycine-NaOH buffer solution with a pH value of 9.0; a chimeric enzyme for degrading PET was added at a final concentration of 500 nM, and the mixture was incubated at 40°C to 60°C (the optimal temperature for different chimeric enzymes for degrading PET) and 250 rpm for 1 to 3 days, and then the concentration of TPA in the degradation system was determined by HPLC.

[0092] In the process of a large number of experiments in the present invention, it was found that on the basis of IsPETase, if SEQ ID No.1 to SEQ ID No.12 were arbitrarily replaced, the thermal melting temperatures (T m ) and / or the PET enzyme degradation activity is inferior to that of the wild enzyme, which is illustrated by the following three comparative examples.

[0093] Comparative Example 1 This comparative example provides a chimeric enzyme for degrading PET, which is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.14, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.5 is replaced with the amino acid sequence as shown in SEQ ID No.29, the amino acid sequence as shown in SEQ ID No.9 is replaced with the amino acid sequence as shown in SEQ ID No.21, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.22, the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23, and the amino acid sequence as shown in SEQ ID No.12 is replaced with the amino acid sequence as shown in SEQ ID No.24. The chimeric enzyme for degrading PET provided in this comparative example is recorded as chimeric enzyme pair 1.

[0094] Comparative Example 2 The present comparative example provides a chimeric enzyme for degrading PET. The chimeric enzyme is prepared on the basis of IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced by the amino acid sequence as shown in SEQ ID No.13, the amino acid sequence as shown in SEQ ID No.2 is replaced by the amino acid sequence as shown in SEQ ID No.26, the amino acid sequence as shown in SEQ ID No.3 is replaced by the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.4 is replaced by the amino acid sequence as shown in SEQ ID No.16, the amino acid sequence as shown in SEQ ID No.5 is replaced by the amino acid sequence as shown in SEQ ID No.17, the amino acid sequence as shown in SEQ ID No.6 is replaced by the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.7 is replaced by the amino acid sequence as shown in SEQ ID No.19, the amino acid sequence as shown in SEQ ID No.9 is replaced by the amino acid sequence as shown in SEQ ID No.21, the amino acid sequence as shown in SEQ ID No.11 is replaced by the amino acid sequence as shown in SEQ ID No.35, and the amino acid sequence as shown in SEQ ID No. 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 comparative example is recorded as chimeric enzyme pair 2.

[0095] Comparative Example 3 This comparative example provides a chimeric enzyme for degrading PET, which is based on IsPETase (amino acid sequence as shown in SEQ ID No.37), wherein the amino acid sequence as shown in SEQ ID No.1 is replaced with the amino acid sequence as shown in SEQ ID No.13, the amino acid sequence as shown in SEQ ID No.2 is replaced with the amino acid sequence as shown in SEQ ID No.26, the amino acid sequence as shown in SEQ ID No.3 is replaced with the amino acid sequence as shown in SEQ ID No.15, the amino acid sequence as shown in SEQ ID No.4 is replaced with the amino acid sequence as shown in SEQ ID No.28, the amino acid sequence as shown in SEQ ID No.6 is replaced with the amino acid sequence as shown in SEQ ID No.30, the amino acid sequence as shown in SEQ ID No.7 is replaced with the amino acid sequence as shown in SEQ ID No.31, the amino acid sequence as shown in SEQ ID No.10 is replaced with the amino acid sequence as shown in SEQ ID No.34, and the amino acid sequence as shown in SEQ ID No.11 is replaced with the amino acid sequence as shown in SEQ ID No.23. The chimeric enzyme for degrading PET provided in this comparative example is recorded as chimeric enzyme pair 3.

[0096] Test example In order to investigate whether the thermal stability and degradation activity of the designed chimeric enzymes against PET are improved compared with those of the wild enzymes, this test example investigated the T values ​​of the chimeric enzymes for degrading PET provided in Examples 1 to 33 and Comparative Examples 1 to 3, as well as the wild enzymes IsPETase, TfCUT2, and LCC. m The values, as well as the concentration of TPA in the degradation system under the conditions provided in Example 36 (in this test example, incubation for 1 day is used as an example for explanation), the measurement results are shown in Table 7.

[0097] The Tm value in the present invention is measured using a Q-PCR instrument; HPLC was used to determine the concentration of TPA in the degradation system, and the determination conditions were as follows. Detection wavelength: 240nm; chromatographic column: ZORBAX Eclipse Plus C18 reverse phase chromatographic column (5μm, 250mm×4.6mm); mobile phase: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, and mobile phase B is increased from 5% to 70% within 20 minutes; column temperature: 30°C; injection volume: 10μL, flow rate: 0.8mL / min. Among them, the liquid phase diagram for determining the concentration of TPA in the degradation system after adding chimeric enzyme 6 is shown in the figure below. Figure 2 shown.

[0098] The measurement results are shown in Table 7 as follows.

[0099] Table 7

[0100] As shown in Table 7, the chimeric enzymes for degrading PET provided by the present invention have excellent PET degradation activity and thermal stability. Compared with the wild-type IsPETase enzyme, the thermal melting temperatures (T m ) increased by 4.8°C to 31.7°C, and the concentration of the degradation product TPA after PET degradation increased by 1.5 times to 44.5 times. In view of the fact that the chimeric enzyme for degrading PET constructed by the present invention has higher PET degradation activity and thermal stability, it can be used in the fields of degrading PET, preparing PET degradation agents or preparing PET degradation products. While broadening the application conditions of PET degradation, it can also greatly improve the PET degradation efficiency and improve the recovery rate of the high value-added degradation product TPA, which has important economic value and environmental benefits.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A chimeric enzyme for degrading PET, characterized in that The amino acid sequence of IsPETase is divided into 12 amino acid sequences as shown in SEQ ID No.1 to SEQ ID No.12, and the chimeric enzyme for degrading PET is obtained by replacing at least one sequence in the 12 amino acid sequences, wherein: The amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.13 or SEQ ID No.25; The amino acid sequence shown in SEQ ID No.2 is replaced by the amino acid sequence shown in SEQ ID No.14 or SEQ ID No.26; The amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.15 or SEQ ID No.27; The amino acid sequence shown in SEQ ID No.4 is replaced by the amino acid sequence shown in SEQ ID No.16 or SEQ ID No.28; The amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.17 or 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 or SEQ ID No.30; The amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19 or 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 or SEQ ID No.32; The amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.21 or SEQ ID No.33; The amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.22 or SEQ ID No.34; The amino acid sequence shown in SEQ ID No.11 is replaced by the amino acid sequence shown in SEQ ID No.23 or SEQ ID No.35; The amino acid sequence shown in SEQ ID No.12 is replaced 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 chimeric enzyme for degrading PET is: based on IsPETase, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.29, the amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.34, and the amino acid sequence shown in SEQ ID No.12 is replaced by 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 by the amino acid sequence shown in SEQ ID No.13, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.4 is replaced by the amino acid sequence shown in SEQ ID No.28, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced by the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.11 is replaced by 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 by the amino acid sequence shown in SEQ ID No.13, the amino acid sequence shown in SEQ ID No.2 is replaced by the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.27, the amino acid sequence shown in SEQ ID No.4 is replaced by the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.29, the amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19, the amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.21, the amino acid sequence shown in SEQ ID No.11 is replaced by the amino acid sequence shown in SEQ ID No.23, and the amino acid sequence shown in SEQ ID No.12 is replaced by 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 by the amino acid sequence shown in SEQ ID No.25, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced by the amino acid sequence shown in SEQ ID No.30, the amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.31, the amino acid sequence shown in SEQ ID No.8 is replaced by the amino acid sequence shown in SEQ ID No.32, the amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.21, and the amino acid sequence shown in SEQ ID No.12 is replaced by 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 acid sequence of the chimeric enzyme for degrading PET is as follows: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.2 is replaced by the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced by the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19, the amino acid sequence shown in SEQ ID No.8 is replaced by the amino acid sequence shown in SEQ ID No.20, the amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.33, the amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.22, the amino acid sequence shown in SEQ ID No.11 is replaced by the amino acid sequence shown in SEQ ID No.23, and the amino acid sequence shown in SEQ ID No.12 is replaced by the amino acid sequence shown in SEQ ID No. The amino acid sequence shown in No.36; or On the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced by the amino acid sequence shown in SEQ ID No.13, the amino acid sequence shown in SEQ ID No.2 is replaced by the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.4 is replaced by the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.6 is replaced by the amino acid sequence shown in SEQ ID No.30, the amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19, the amino acid sequence shown in SEQ ID No.8 is replaced by the amino acid sequence shown in SEQ ID No.32, the amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.21, the amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.22, and the amino acid sequence shown in SEQ ID No.12 is replaced by the amino acid sequence shown in SEQ ID No.24; or On the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced by the amino acid sequence shown in SEQ ID No.25, the amino acid sequence shown in SEQ ID No.4 is replaced by the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.8 is replaced by the amino acid sequence shown in SEQ ID No.32, the amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.22, the amino acid sequence shown in SEQ ID No.11 is replaced by the amino acid sequence shown in SEQ ID No.23, and the amino acid sequence shown in SEQ ID No.12 is replaced by 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 by the amino acid sequence shown in SEQ ID No.26, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.8 is replaced by the amino acid sequence shown in SEQ ID No.20, the amino acid sequence shown in SEQ ID No.9 is replaced by the amino acid sequence shown in SEQ ID No.21, and the amino acid sequence shown in SEQ ID No.10 is replaced by the amino acid sequence shown in SEQ ID No.22; or On the basis of 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. The biomaterial related to the chimeric enzyme for degrading PET according to any one of claims 1 to 3, characterized in that: The biological material is any one of the following (I) to (IV): (I) a gene encoding a chimeric enzyme for degrading PET according to 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 enzyme recombinant vector for degrading PET, characterized in that: The gene encoding the chimeric enzyme for degrading PET according to any one of claims 1 to 3 is transformed into T1 competent cells, and a recombinant vector is obtained by screening, plasmid extraction and sequencing verification.

6. A method for constructing a chimeric enzyme recombinant strain for degrading PET, characterized in that: The recombinant vector constructed in claim 5 is transformed into competent E. coli cells, spread on ampicillin plate culture medium, and cultured to obtain positive recombinants, that is, the chimeric enzyme recombinant strain for degrading PET is obtained.

7. Use of the chimeric enzyme for degrading PET according to any one of claims 1 to 3 in degrading PET, preparing a PET degradation agent or preparing a PET degradation product.

8. Use of the biomaterial related to the chimeric enzyme for degrading PET according to claim 4 in degrading PET, preparing a PET degradation agent or preparing a PET degradation product.

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