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. According to the chimeric enzyme for degrading PET, provided by the invention, 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; the amino acid sequence shown in SEQ ID No.1-4 is not replaced, the amino acid sequence shown in SEQ ID No.5 is replaced, and at the same time, at least one amino acid sequence shown in SEQ ID No.6-SEQ ID No.12 is replaced. Compared with IsPETase, the chimeric enzyme for degrading PET provided by the invention has the advantages that the thermal melting temperature and the enzyme activity are obviously improved, the application conditions of PET degradation can be widened, the PET degradation efficiency and the recycling rate of high-added-value degradation products are greatly improved, and the chimeric enzyme has important economic value and environmental benefit.

Description

Technical Field

[0001] The present 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) is widely used in various fields due to its excellent physical and chemical properties. However, improper disposal of PET after use poses a serious threat to the ecological environment, making the management of plastic waste a key 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 (an enzyme that degrades PET, known as IsPETase), belongs to the α / β hydrolase superfamily. Compared to other PET hydrolases, IsPETase offers advantages such as high degradation efficiency, suitability for degradation at moderate temperatures, specificity for PET degradation, and the ability to degrade highly crystalline PET films. However, IsPETase's hydrolysis of PET primarily occurs in the amorphous phase, while the glass transition temperature (Tg) of PET ranges from 65°C to 71°C. This places higher demands on the thermal stability of PET hydrolases. However, the enzyme's poor thermal stability and degradation activity at high temperatures have limited its practical application in PET degradation. Summary of the Invention

[0004] To address these technical issues, the present invention designs and improves IsPETase, providing a chimeric enzyme for PET degradation, as well as related biomaterials and applications. Compared to wild-type IsPETase, this chimeric enzyme exhibits not only higher thermal stability but also significantly enhanced 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. The chimeric enzyme is obtained by sequentially dividing the amino acid sequence of IsPETase (as shown in SEQ ID No. 37) into 12 amino acid sequences as shown in SEQ ID No. 1 to SEQ ID No. 12; then, the amino acid sequences as shown in SEQ ID No. 1 to 4 are not replaced, 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 or SEQ ID No. 29, and at least one of the seven amino acid sequences as shown in SEQ ID No. 6 to SEQ ID No. 12 is replaced, wherein: The amino acid sequence shown in SEQ ID No. 5 is replaced with 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 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.

[0006] Compared with the wild-type IsPETase, the thermal melting temperature (T m) and the concentration of the degradation product TPA after PET degradation were significantly increased. Given that the chimeric enzyme for PET degradation constructed in the present invention has higher PET degradation activity and thermal stability, it can not only broaden the application conditions of PET degradation but also significantly improve PET degradation efficiency, helping to increase 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 their amino acid sequences are shown in Table 1.

[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] For example, as a preferred technical solution, based on IsPETase, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 29, the amino acid sequence shown in SEQ ID No. 10 is replaced with the amino acid sequence shown in SEQ ID No. 34, and the amino acid sequence shown in SEQ ID No. 12 is replaced with the amino acid sequence shown in SEQ ID No. 24.

[0017] 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 27.18° C., and the concentration of the degradation product TPA after PET degradation is increased by 36.6 times.

[0018] In a second aspect, the present invention provides a biomaterial related to the above-mentioned 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).

[0019] 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, screening with ampicillin plate culture medium, plasmid extraction and sequencing verification to obtain the recombinant vector.

[0020] In a fourth aspect, the present invention also 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 to obtain the recombinant strain of the chimeric enzyme that degrades PET.

[0021] 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.

[0022] In a sixth aspect, the present invention further provides the use of biomaterials 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.

[0023] The research strategy of this invention is to use IsPETase, TfCUT2, and LCC as parents, guided by the SCHEMA recombination method, and divide the domains of the parent proteins based on the residue contacts between them. Each domain is randomly replaced with the corresponding domain from a different parent to generate progeny recombinant proteins. In this invention, the crystal structures of the three parent proteins (IsPETase, TfCUT2, and LCC) are superimposed, and the intersection sites are located using the RASPP algorithm. The structures of the three enzymes are divided into 12 fragments to generate progeny chimera sequences. After expressing and measuring their fitness (activity, thermal stability) data, high-fitness chimeras are selected for subsequent synthesis and expression assays, and further screening and optimization.

[0024] 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 temperature can be increased to 74.34°C. In a degradation system containing a PET film and a glycine-NaOH buffer solution with a pH of 9.0, a chimeric enzyme was added at a final concentration of 500 nM. After incubation at 40°C to 60°C and 250 rpm for one day, the concentrations of TPA in the degradation systems of wild-type IsPETase, TfCUT2, or LCC were 216.78 μM, 207.81 μM, and 1085.35 μM, respectively. The TPA yield in the degradation system of the chimeric enzyme for PET degradation provided by the present invention can reach 8218.87 μM. Given the excellent thermal stability and PET degradation activity of the chimeric enzyme recombinant enzyme for PET degradation 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

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0026] Figure 1 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-12×Chimera in Example 2 of the present invention, wherein P-1 represents the gene encoding the amino acid shown in SEQ ID No.1, P-2 represents the gene encoding the amino acid shown in SEQ ID No.2, P-3 represents the gene encoding the amino acid shown in SEQ ID No.3, P-4 represents the gene encoding the amino acid shown in SEQ ID No.4, L-5 represents the gene encoding the amino acid shown in SEQ ID No.29, P-6 represents the gene encoding the amino acid shown in SEQ ID No.6, P-7 represents the gene encoding the amino acid shown in SEQ ID No.7, P-8 represents the gene encoding the amino acid shown in SEQ ID No.8, P-9 represents the gene encoding the amino acid shown in SEQ ID No.9, L-10 represents the gene encoding the amino acid shown in SEQ ID No.34, P-11 represents the gene encoding the amino acid shown in SEQ ID No.11, and T-12 represents the gene encoding the amino acid shown in SEQ ID No.24. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be 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 intended to limit the present invention.

[0028] The solutions of the present invention are described below through specific embodiments.

[0029] Unless otherwise specified, the reagents in the following examples were commercially available or obtained according to methods known in the art.

[0030] Example 1 An embodiment of the present invention provides a chimeric enzyme for degrading PET. The chimeric enzyme is prepared by replacing the amino acid sequence shown in SEQ ID No. 4 with the amino acid sequence shown in SEQ ID No. 28, the amino acid sequence shown in SEQ ID No. 5 with the amino acid sequence shown in SEQ ID No. 29, the amino acid sequence shown in SEQ ID No. 6 with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 8 with the amino acid sequence shown in SEQ ID No. 32, the amino acid sequence shown in SEQ ID No. 10 with the amino acid sequence shown in SEQ ID No. 22, and the amino acid sequence shown in SEQ ID No. 11 with the amino acid sequence shown in SEQ ID No. 23.

[0031] Example 2 The present invention provides a recombinant vector, a recombinant bacterium and a construction method thereof containing a gene encoding the chimeric enzyme for degrading PET in Example 1. The recombinant vector in this example is described using a recombinant plasmid as an example.

[0032] The method for constructing a recombinant plasmid containing the gene encoding the chimeric enzyme for degrading PET in Example 1 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 it 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 bacterium , the accession number in the GenBank database is HQ704839.1, and it is recorded as gene LCC ) was connected to the pET-22b plasmid to obtain the recombinant plasmid pET-22b-LCC.

[0033] Using the recombinant plasmids pET-22b-IsPETase, pET-22b-TfCUT2, and pET-22b-LCC as templates, and IsPETase-cSP-F / R, TfCut2-cSP-F / R, and LCC-cSP-F / R as primers, PCR amplification yielded linear vector fragments of 6.2 kb, 6.2 kb, and 6.2 kb, respectively, corresponding to each template. These linear vector fragments were mixed with ligase to form three circular recombinant plasmids. These three circular recombinant plasmids were transformed into T1 competent cells, screened with ampicillin plates, extracted, and verified by sequencing to obtain the recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP, and pET-22b-LCC-cSP.

[0034] The nucleotide sequence of the IsPETase-cSP gene (wherein cSP represents cutoff 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.

[0035] Among them, the ampicillin plate medium contains: 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.

[0036] Table 4

[0037] The PCR reaction system and PCR program were based on the TransStart ® The requirements of the FastPfu Fly DNA Polymerase Kit are as shown in Table 5: Table 5

[0038] 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: gccaacccctacgagcgcggccccaacccgaccgacgccctgctcgaagccagcagcggccccttctccgtcagcgaggagaacgtctcccggttgagcgccagcggcttcggcggcggcaccatctactacccgcgggagaacaacacctacggtgcggtggcgatctcccccggctacaccggcactgaggcttccatcgcctggctgggcgagcgcatcgcctcccacggcttcgtcgtcatcaccatcgacaccatcaccaccctcgaccagccggacagccgggcagagcagctcaacgccgcgctgaaccacatgatcaaccgggcgtcctccacggtgcgcagccggatcgatagcagccgactggcggtcatgggccactccatgggcggcggcggcaccctgcgtctggcctcccagcgtcccgacctgaaggccgccatcccgctcaccccgtggcacctcaacaagaactggagcagcgtcaccgtgccgacgctgatcatcggggccgacctcgacacgatcgcgccggtcgccacgcacgcgaaaccgttctacaacagcctgccgagctccatcagcaaggcctacctggagctggacggcgcaacccacttcgccccgaacatccccaacaagatcatcggcaagtacagcgtcgcctggctcaagcggttcgtcgacaacgacacccgctacacccagttcctctgccccggaccgcgcgacggactcttcggcgaggtcgaagagtaccgctccacctgcccgttc; The nucleotide sequence of the LCC-cSP gene is shown in SEQ ID No. 4, specifically as follows: It should be noted that there may be some inaccuracies in the content you provided. For example, "SEQ ID No.42" in the original Chinese may be incorrect. It is translated as "SEQ ID No. 4" here according to the context, but it needs to be further verified according to the actual situation..

[0039] 2. Construct a recombinant plasmid containing a chimeric enzyme gene encoding PET degradation.

[0040] (1) Design and synthesis of required primers The primers designed and synthesized in this step are shown in Table 6 below.

[0041] Table 6

[0042] (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 degradation in Example 1, which mainly includes the following three steps: (1) using the recombinant plasmid pET-22b-IsPETase-cSP as a template, upstream primer 1-1 and downstream primer 1-1 as primers to perform PCR amplification, and the PCR product was subjected to DMT enzyme digestion, nucleic acid electrophoresis and gel excision recovery to obtain a purified linear gene fragment 1-1; (2) using the recombinant plasmid pET-22b-IsPETase-cSP as a template, upstream primer 1-2 and downstream primer 1- 2 is a primer for PCR amplification, and the PCR product is subjected to DMT enzyme digestion, nucleic acid electrophoresis and gel excision recovery to obtain a purified linear gene fragment 1-2; in the same manner, purified linear gene fragments 1-3 to 1-12 are obtained; (3) after connecting gene fragments 1-1 to 1-12 under the action of seamless recombination ligase, the seamless recombination product is transformed into T1 competent cells, and through ampicillin plate culture medium screening, plasmid extraction and sequencing verification, a recombinant plasmid pET-22b-12×Chimera containing the chimeric enzyme gene encoding the PET degradation in Example 1 is obtained. The schematic diagram of its plasmid map is shown in FIG. Figure 1 As shown, wherein P-1 represents the gene encoding the amino acid shown in SEQ ID No.1, P-2 represents the gene encoding the amino acid shown in SEQ ID No.2, P-3 represents the gene encoding the amino acid shown in SEQ ID No.3, P-4 represents the gene encoding the amino acid shown in SEQ ID No.4, L-5 represents the gene encoding the amino acid shown in SEQ ID No.29, P-6 represents the gene encoding the amino acid shown in SEQ ID No.6, P-7 represents the gene encoding the amino acid shown in SEQ ID No.7, P-8 represents the gene encoding the amino acid shown in SEQ ID No.8, P-9 represents the gene encoding the amino acid shown in SEQ ID No.9, L-10 represents the gene encoding the amino acid shown in SEQ ID No.34, P-11 represents the gene encoding the amino acid shown in SEQ ID No.11, and T-12 represents the gene encoding the amino acid shown in SEQ ID No.24.

[0043] (3) Construction of a recombinant strain of chimeric enzyme that degrades PET The PET-degrading chimeric enzyme recombinant plasmid pET-22b-12×Chimera 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 recombinant strain-chimeric enzyme.

[0044] Example 3 In the embodiment of the present invention, the recombinant strain pET-22b-12×Chimera was expanded and induced for expression, and the target protein was purified. The specific method is as follows: The recombinant strain pET-22b-12×Chimera was inoculated into LB liquid medium and cultured at 37°C, 220 r / min overnight. The overnight culture was inoculated into fresh LB liquid medium at a 5% inoculum volume and cultured at 37°C, 220 r / min until OD 600 When the pH 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.

[0045] Wet cells of the recombinant strain were resuspended in lysis buffer and disrupted using a high-pressure cell disruptor. After lysis, the cell suspension was centrifuged at 10,000 rpm for 60 minutes to remove cell debris. The supernatant was then passed through a Ni-NTA column to adsorb the target protein. Nonspecific adsorbed proteins were then removed using wash buffer. The target protein was then eluted using elution buffer, and the eluate was concentrated using a protein concentrator to obtain the chimeric enzyme.

[0046] 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.

[0047] Example 4 The present embodiment provides the use of the chimeric enzyme for degrading PET in Example 1 in hydrolyzing PET, and the specific method is as follows: A PET film (purchased from GoodFellow, with a crystallinity of approximately 8% and a diameter of 6 mm) was added to a 50 mM glycine-NaOH buffer solution at a pH of 9.0. A chimeric enzyme for PET degradation was added at a final concentration of 500 nM. The mixture was incubated at 40°C to 60°C and 250 rpm for 1 to 3 days, and the TPA concentration in the degradation system was determined by HPLC.

[0048] Test Example In order to investigate whether the thermal stability and PET degradation activity of the designed chimeric enzyme are improved compared with the wild enzyme, this test example investigated the T degradation of the chimeric enzyme for PET provided in Example 1, as well as the wild enzymes IsPETase, TfCUT2 and LCC. m The values ​​and the concentration of TPA in the degradation system under the conditions provided in Example 4 (in this test example, incubation for 1 day is used as an example for explanation) are shown in Table 7.

[0049] The Tm value in the present invention is measured using a Q-PCR instrument; The TPA concentration in the degradation system was determined by HPLC using the following conditions: detection wavelength: 240 nm; column: ZORBAX Eclipse Plus C18 reversed-phase column (5 μm, 250 mm × 4.6 mm); mobile phase: mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile, with the concentration of mobile phase B increased from 5% to 70% over 20 minutes; column temperature: 30°C; injection volume: 10 μL; flow rate: 0.8 mL / min.

[0050] The measurement results are shown in Table 7 below.

[0051] Table 7

[0052] As shown in Table 7, the chimeric enzyme for degrading PET provided by the present invention has excellent PET degradation activity and thermal stability. Compared with the wild-type IsPETase enzyme, the thermal melting temperature (T m ) increased by 27.18°C, and the concentration of the degradation product TPA after PET degradation increased by 36.6-fold. Given that the chimeric enzyme for PET degradation constructed by the present invention has higher PET degradation activity and thermal stability, it can be used in fields such as PET degradation, preparation of PET degradation agents, and preparation of PET degradation products. While broadening the application conditions of PET degradation, it can also significantly improve PET degradation efficiency and increase the recovery rate of the high-value-added degradation product TPA, thus having important economic and environmental benefits.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A chimeric enzyme for degrading PET, characterized in that The amino acid sequence of IsPETase shown in SEQ ID No. 37 is divided into 12 amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 12; then, the amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 4 are not replaced, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17 or SEQ ID No. 29, and at least one of the seven amino acid sequences shown in SEQ ID No. 6 to SEQ ID No. 12 is replaced, wherein: 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 with 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 acid sequence of the chimeric enzyme for degrading PET is as follows: 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.

3. The biomaterial related to the chimeric enzyme for degrading PET according to claim 1 or 2, characterized in that: The biological material is any one of the following (I) to (IV): (I) a gene encoding the chimeric enzyme for degrading PET according to claim 1 or 2; (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).

4. A method for constructing a chimeric enzyme recombinant vector for degrading PET, characterized by: The gene encoding the chimeric enzyme for degrading PET according to claim 1 or 2 is transformed into T1 competent cells, and a recombinant vector is obtained through screening, plasmid extraction and sequencing verification.

5. A method for constructing a recombinant strain of a chimeric enzyme for degrading PET, characterized by: The recombinant vector constructed in claim 4 is transformed into Escherichia coli competent cells, spread on ampicillin plate culture medium, and cultured to obtain positive recombinants, namely, the chimeric enzyme recombinant strain for degrading PET.

6. Use of the chimeric enzyme for degrading PET according to claim 1 or 2 in degrading PET, preparing a PET degradation agent, or preparing a PET degradation product.

7. Use of the biomaterial related to the chimeric enzyme for degrading PET according to claim 3 in degrading PET, preparing a PET degradation agent, or preparing a PET degradation product.

Images