PET plastic degrading enzyme mutant, expression vector, host and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to a PET plastic degrading enzyme mutant, an expression vector, a host and application of the PET plastic degrading enzyme mutant. The invention provides application of a mutation site as a target spot to improvement of enzymatic activity and / or thermal stability of PETase hydrolase. The mutation sites comprise one or more of the 114 site, the 175 site, the 205 site, the 234 site and the 269 site of the amino acid sequence of the wild type PETase hydrolase and the 233 site. According to the invention, sites which are not excavated by other researchers are screened and verified by utilizing a semi-rational design and error-prone PCR method, and the activity and the thermal stability of the enzyme are expected to be further improved to meet the requirements in the aspect of industrial application.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a PET plastic degrading enzyme mutant, an expression vector, a host and applications thereof. Background Art

[0002] Plastic is one of the indispensable consumables in modern people's daily life. It has excellent properties such as high impact resistance and durability. At the same time, these properties also make plastic one of the most harmful environmental pollutants. It takes years, decades or even hundreds of years to completely degrade in the natural environment. The production and consumption of plastic is much faster than the degradation rate, which will lead to the accumulation of plastic waste and cause environmental pollution. In order to solve this serious environmental problem, different treatment methods have been tried.

[0003] Traditional treatment methods rely on mechanical recycling, landfill, incineration, photo / electro / chemical catalytic oxidation, hydrolysis, glycolysis and other dangerous physical and chemical processes. These physical and chemical processes usually require additional energy consumption to provide high temperature and high pressure, and are accompanied by secondary pollution. So everyone turned their attention to biological methods: biodegradation, also known as microbial or enzymatic degradation, is an effective alternative method for plastic waste management.

[0004] Polyethylene terephthalate, a commercially important and commonly used crystallizable aromatic polyester, is one of the most popular plastic materials in modern society. It is a polymer synthesized by terephthalic acid (TPA) and ethylene glycol (EG) through an ester bond. The ester bond of PET is a feasible cleavage site for many hydrolases. IsPETase, discovered in 2016, can effectively convert PET into MHET and a small amount of BHET and TPA.

[0005] IsPETase has commercial application value, so the researchers conducted research on its activity and thermal stability.

[0006] There are many factors that affect enzyme activity and thermal stability, including the hydrolysis ability and stability of the enzyme itself, enzyme amount, reaction conditions, substrate availability, substrate and product inhibition, etc.

[0007] In recent years, most of the research has focused on the primary, secondary and tertiary structures of enzymes and the chemical modification of enzyme side chains. The strategies for changing the primary to tertiary structures of enzymes include rational design, semi-rational design, irrational design and artificial intelligence design. This study used semi-rational design and irrational design methods to optimize PETase.

[0008] Semi-rational design was applied to the modification of trehalose synthase in 2004. The semi-rational method is to construct a concise library based on structural or functional knowledge by targeting multiple specific residues, which is more likely to obtain correct results. Iterative site-saturation mutagenesis is a type of semi-rational design.

[0009] Error-prone PCR is a type of irrational design. A large number of mutation libraries are constructed through random mutagenesis methods, including single mutations and combined mutations. Reasonable control of the mutation rate can improve the discovery of favorable strains.

[0010] Since IsPETase was first discovered in 2016, it has remained popular. As the PET hydrolase with the best degradation activity at ambient temperature, PETase has great potential and prospects. However, after PETase is taken out of the body, its enzyme activity and thermal stability are very poor, making it difficult to achieve industrialization. Therefore, researchers have actively carried out research work mainly around improving activity and thermal stability, and have made relatively outstanding progress.

[0011] IsPETase is a hydrolase composed of 290 amino acids. Existing studies have not covered the amino acid sites of plastic-degrading enzymes, especially the amino acids in inactive sites. Given the complexity of the enzyme and its potential for optimization and modification, new sites need to be explored. Summary of the invention

[0012] In view of this, the present invention provides a PET plastic degrading enzyme mutant, an expression vector, a host and its application. The present invention uses semi-rational design and error-prone PCR methods to screen and verify sites that have not been explored by other researchers, and is expected to further improve the activity and thermal stability of the enzyme to meet the needs of industrial applications.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides the use of mutation sites as targets in improving the enzymatic activity and / or thermal stability of PETase hydrolase;

[0015] The mutation sites include: one or more of positions 114, 175, 205, 234, 269 and position 233 of the amino acid sequence of the wild-type PETase hydrolase.

[0016] The present invention also provides a mutant, which, based on the wild-type PETase hydrolase, has one or more of the following amino acid site mutations: position 114, position 175, position 205, position 233, position 234, and position 269, and the amino acid sequence of the wild-type PETase hydrolase is shown in SEQ ID NO: 1.

[0017] In some embodiments of the present invention, in the above mutant, the mutation at position 114 includes: N114I;

[0018] The mutation at position 175 includes: S175G;

[0019] The mutation at position 205 includes: N205K or N205L;

[0020] The mutation at position 233 includes: N233K;

[0021] The mutation at position 234 includes: G234N;

[0022] The mutation at position 269 includes any one of S269D, S269E, S269F, S269H, S269V and S269Q.

[0023] In some embodiments of the present invention, the above-mentioned mutants include: one or more of N233K, N233K / N205K, N233K / N205L, N233K / G234N, N233K / S175G, N233K / N205K / N114I, N233K / N205K / S269D, N233K / N205K / S269E, N233K / N205K / S269F, N233K / N205K / S269H, N233K / N205K / S269V, N233K / N205K / S269Q, N233K / N205K / N114I / S269V and N233K / N205K / N114I / S269Q.

[0024] In some embodiments of the present invention, the mutants described above have:

[0025] (1) the amino acid sequence shown in SEQ ID NO: 2 to SEQ ID NO: 15; or

[0026] (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0027] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0028] In some embodiments of the present invention, the mutant further comprises a secretion signal peptide.

[0029] The present invention also provides a nucleic acid molecule encoding the mutant.

[0030] In some embodiments of the present invention, the above-mentioned nucleic acid molecule has:

[0031] (4) the nucleotide sequence shown in SEQ ID NO: 16 to SEQ ID NO: 29; or

[0032] (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or

[0033] (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or

[0034] (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).

[0035] The present invention also provides an expression vector, comprising: the above nucleic acid molecule.

[0036] The present invention also provides a host for transforming and / or transfecting the above expression vector.

[0037] The present invention also provides a method for preparing the mutant, which comprises culturing the host, centrifuging, collecting the supernatant, and purifying to obtain the mutant.

[0038] The present invention also provides a product, including: the mutant, the nucleic acid molecule, the expression vector, the host and / or the mutant obtained by the preparation method.

[0039] The present invention also provides the use of the mutant, the nucleic acid molecule, the expression vector, the host, the mutant obtained by the preparation method and / or the product in degrading plastics.

[0040] The present invention makes statistics and screening of the sites of IsPETase that have not been studied, and uses semi-rational design and irrational design methods to discover three beneficial mutation sites that have not been studied. Four beneficial mutations are found at these three sites, making the research on IsPETase more sufficient and complete. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0042] Figure 1 shows the sequence alignment analysis of IsPETase and homologous cutinases;

[0043] Figure 2 The standard curves of the absorbance of TPA and MHET at λ=254nm and the actual yield are shown;

[0044] Figure 3 The secretion N233K was used as a template, and half-saturation mutations were made at the N205 and G234 sites. After reacting at 40°C for 72 h, the absorbance at λ = 245 nm was measured;

[0045] Figure 4 The secretion N233K was used as a template, and half-saturation mutations were made at the N205 and G234 sites. After reacting at 45°C for 72 h, the absorbance at λ = 245 nm was measured;

[0046] Figure 5 The error-prone PCR was performed using secreted N233K as a template, and the absorbance at λ = 245 nm was measured after reacting at 40°C for 72 h;

[0047] Figure 6 The intracellular expression and purification of N233K, N233K / N205K, N233K / N205L, N233K / G234N and N233K / S175G were shown, and the precise yield was determined by HPLC after reaction at 40°C, 45°C and 50°C for 72h;

[0048] Figure 7 The secretion N233K / N205K was used as a template, and a half-saturation mutation was made at the S269 site. After reacting at 45°C for 24 hours, the absorbance at λ=245nm was measured;

[0049] Figure 8 The error-prone PCR was performed using secreted N233K / N205K as a template. After reacting at 45°C for 24 h, the absorbance at λ = 245 nm was measured.

[0050] Fig. 9 The intracellular expression and purification of N233K / N205K, N233K / N205K / S269D, N233K / N205K / S269E, N233K / N205K / S269F, N233K / N205K / S269H, N233K / N205K / S269V, N233K / N205K / S269Q and N233K / N205K / N114I were shown, and the precise yield was determined by HPLC after the reaction at 40°C, 45°C and 50°C for 48 hours;

[0051] Fig.10 The intracellular expression and purification of WT, N233K, M1, M2-1, M2-2, M2-3, M3-1 and M3-2 were shown. After 72 days of reaction at 40°C and 45°C, the precise yield was determined by HPLC;

[0052] Fig.11 Shows the protein purification gel image. DETAILED DESCRIPTION

[0053] The invention discloses a PET plastic degrading enzyme mutant, an expression vector, a host and applications thereof.

[0054] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0055] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0056] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0057] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0058] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0059] The present invention utilizes a research strategy combining semi-rational enzyme design with irrational PCR design to discover three beneficial mutation sites of IsPETase (N114, N205, S269), and obtains four beneficial mutations at the three sites: N114I, N205K, S269V, and S269Q. In addition, six IsPETas with significantly improved activity and thermal stability at 40°C are obtained through combined optimization of mutation sites. e mutants: M1(N233K / N205K), M2-1(N233K / N205K / N114I), M2-2(N233K / N205K / S269V), M2-3(N2 33K / N205K / S269Q), M3-1(N233K / N205K / N114I / S269Q), M3-2(N233K / N205K / N114I / S269V).

[0060] In Examples 1 to 7 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0061] The present invention will be further described below in conjunction with embodiments:

[0062] Example 1 Mining of unstudied sites of IsPETase and construction of the first round mutation library of IsPETase

[0063] 1. Acquisition of experimental materials

[0064] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0065] The gene PETase (pET_28a(+)) involved in this experiment was synthesized by Qingke Biotechnology Co., Ltd.

[0066] Amino acid sequence of PETase (261): NPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYG AGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLK AAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQID NO:1)

[0067] Nucleotide sequence of PETase (optimized for E. coli as the host): AATCCGTACGCACGTGGTCCGAATC CAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAACGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (Shown as SEQ ID NO: 30)

[0068] Obtaining the substrate for the enzyme activity reaction: Good fellow film.

[0069] 2. Experimental methods:

[0070] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0071] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0072] Kanamycin stock solution: 50 mg / mL;

[0073] IPTG solution: 1 M, 0.238 g / mL;

[0074] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0.

[0075] Acquisition of an unstudied site library: PETase is 290 amino acids in length. By consulting the literature and excluding sites that have been mutated by other researchers, an unstudied site library was obtained.

[0076] Mining sites worth saturation mutation: Through homologous sequence comparison analysis, it was found that the N205, G234 and S269 sites that have not been studied are not highly conserved. Mutation to other amino acids may improve enzyme activity or thermal stability, such as Figure 1 shown.

[0077] Construct a secretory expression system: ① Add the pelB secretion signal peptide to the N-terminus of the synthetic wild-type PETase to facilitate high-throughput application; ② Add N233K in DepoPETase to the secretory wild-type to improve thermal stability, and then increase the reaction temperature to 40℃ and 45℃ to facilitate screening.

[0078] Amino acid sequence of secreted N233K (283): MKYLLPTAAAGLLLLAAQPAMANPYARGPN PTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGG GSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:2)

[0079] Nucleotide sequence of secreted N233K (after optimization for large intestine as host): ATGAATAACCTGCTGCCGACCG (as shown in SEQ ID NO: 16)

[0080] Construction of the first round of site-directed mutation library for secretory expression: Using secretory N233K as a template, semi-saturation mutations were performed on the N205 and G234 sites in the first round to obtain the first round mutation library.

[0081] Mutant transformation: ① Add 2μL mutant plasmid to 50μL BL21 competent medium and place on ice for 15-25min; ② Heat shock in a 42℃ water bath for 1min; ③ Place on ice for 2min; ④ Add 500μL LB non-antibiotic medium and culture in a shaker at 37℃, 220rpm for 1h; ⑤ Centrifuge the EP tube at 4000rpm for 2min, pour off the supernatant, resuspend the precipitate and evenly apply it on a plate with kanamycin resistance added; ⑥ Incubate inverted in a 37℃ incubator overnight.

[0082] Secretion coarse screening saturation mutation: ① Pick a single colony on the plate and add it to 1mL LB medium, then add 1μL kana mother solution, shake the tube and place it in a shaker at 37℃, 220rpm for 9-12h; ② Take a new tube and add 3mL LB medium, 3μL kana mother solution, transfer 100μL bacterial solution, and place it in a shaker at 37℃, 220rpm for 2.5-3h; ③ Place the tube on ice to quickly cool down; ④ Add 0.1mMIPTG to the tube and culture it in a shaker at 17℃, 180rpm for 20-24h; ⑤ Place the tube on ice, balance it, centrifuge it at 4200rpm, 4℃, 30min, take 50μL supernatant + 550μL reaction buffer, and the substrate is gf-PET cut into small discs with a diameter of 6mm.

[0083] 3. Experimental results

[0084] In the first round of mutation experiments, secretion N233K was used as a template to perform half-saturation mutations at N205 and G234. Enzyme activity was verified by reacting at 40°C and 45°C for 72h.

[0085] According to the literature, both TPA and MHET reach their maximum absorption peak at around λ = 254nm. To verify the accuracy, the standard curve is measured as follows: Figure 2 shown.

[0086] After experimental verification, at 40°C, the activities of N233K / G234N, N233K / G234Y, N233K / N205K, N233K / N205L, N233K / N205T, N233K / N205E, N233K / N205V, N233K / N205R, N233K / N205W, and N233K / N205Y were all higher than those of N233K. Figure 3 As shown; at 45°C, the activities of N233K / N205K, N233K / N205L, N233K / N205R, and N233K / N205Y were all higher than those of N233K. Figure 4 shown.

[0087] N233K / G234N, which had the highest activity at 40°C, and N233K / N205K and N233K / N205L, which performed well at both 40°C and 45°C, were selected for intracellular expression retest.

[0088] Example 2 Construction of the first round error-prone PCR library of IsPETase

[0089] 1. Acquisition of experimental materials

[0090] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0091] The gene PETase (pET_28a(+)) involved in this experiment was synthesized by Qingke Biotechnology Co., Ltd.

[0092] Enzyme activity reaction substrate acquisition: Good fellow film.

[0093] 2. Experimental methods:

[0094] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0095] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0096] Kanamycin stock solution: 50 mg / mL;

[0097] IPTG solution: 1 M, 0.238 g / mL;

[0098] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0.

[0099] Construction of the first round of error-prone PCR library for secretory expression: error-prone PCR was performed using secretory N233K as a template, and the reaction system included 3 μL 10× dye method real-time error-prone PCR Mix (containing enzyme), 1 μL template, 1 μL error-prone PCR primer, 3 μL error-prone PCR enhancer, 3 μL error-prone PCR-specific dNTP, 3 μL error-prone PCR-specific MnCl2, and ultrapure water was added to make up to 30 μL;

[0100] Error-prone PCR primer nucleotide sequence: CTTTAAGAAGGAGATATACCATGAAATACCTGCTGCC GACCGC; (as shown in SEQ ID NO: 31)

[0101] Error-prone PCR primer nucleotide sequence: TTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGGT GCTC. (as shown in SEQ ID NO: 32)

[0102] The mutant transformation steps are shown in Example 1.

[0103] Secretion rough screening error-prone PCR: ① In a 96-deep-well plate, add 500μL LB medium and 0.5μL kanamycin mother solution to each grid, pick up single colonies on the plate with a toothpick and insert them into 96 wells respectively, shake the toothpick to ensure successful inoculation, and place the 96-deep-well plate in a shaker at 37℃ and 220rpm for 9-12h; ② Take a new 96-deep-well plate and add 800μL per grid LB medium and 0.8μL kanamycin mother solution, correspondingly take 20μL bacterial solution for transfer, place at 37℃, 220rpm shaking incubator for 2.5-3h; ③ Place the 96 deep-well plate on ice to quickly cool down; ④ Add 0.1mM IPTG to the 96 deep-well plate, and culture at 17℃, 180rpm shaking incubator for 20-24h; ⑤ Place the 96 deep-well plate on ice, balance, centrifuge at 4200rpm, 4℃, 30min, take 50μL supernatant + 550μL reaction buffer, the substrate is gf-PET cut into small discs with a diameter of 6mm.

[0104] 3. Experimental results

[0105] The first round of error-prone PCR was reacted at 40°C for 72 h and the absorbance was measured as Figure 5 shown.

[0106] A1 is the control secretion N233K. According to the color depth, C5, E2, E6, F6, F7, F8, F11, G4, G6, H2, H5, I4, I9, I10, J3, J12, L6, L7, M9, N12, O6, and O12 were selected for sequencing. Except for the template and synonymous mutations, four mutations were verified, and I4: N233K / S175G (Table 1) with the highest activity improvement was selected for intracellular expression retest.

[0107] Table 1 Effective results of the first round of error-prone PCR

[0108] Serial number mutation I4 N233K / S175G J12 N233K / N277Y C5 N233K / V68A / P181L / P120S / G254D E6 N233K / P217A

[0109] Amino acid sequence of secreted I4 mutant (283): MKYLLPTAAAGLLLLAAQPAMANPYARGP NPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPGLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 33)

[0110] Nucleotide sequence of the secreted I4 mutant (optimized for E. coli as the host): ATGAAATACCTGCTGCCGACC GCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCAATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCATAGCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO: 34)

[0111] Example 3 First-round in vitro purification verification of IsPETase

[0112] 1. Obtaining experimental materials

[0113] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0114] The mutants N233K / N205K, N233K / N205L, N233K / G234N and N233K / S175G involved in this experiment were obtained by mutation in the laboratory.

[0115] Amino acid sequence of N233K / N205K mutant (261): NPYARGPNPTAASLEASAGPFTVRS FTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 4)

[0116] Nucleotide sequence of the N233K / N205K mutant (optimized for E. coli host): AATCCGTACGCACGTG GTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO: 18)

[0117] Amino acid sequence of N233K / N205L mutant (261): NPYARGPNPTAASLEASAGPFTVRS FTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACELDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 5)

[0118] Nucleotide sequence of the N233K / N205L mutant (optimized for E. coli as the host): AATCCGTACGCACGTG GTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGCTGGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:19)

[0119] Amino acid sequence of N233K / G234N mutant (261): NPYARGPNPTAASLEASAGPFTVRS FTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEIKNGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 6)

[0120] Nucleotide sequence of the N233K / G234N mutant (optimized for E. coli host): AATCCGTACGCACGTG GTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAAACGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:20)

[0121] Amino acid sequence of N233K / S175G mutant (261): NPYARGPNPTAASLEASAGPFTVRSF TVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPGLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 3)

[0122] Nucleotide sequence of the N233K / S175G mutant (optimized for E. coli host): AATCCGTACGCACGTG GTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCATAGCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (Shown as SEQ ID NO:17)

[0123] Obtaining the substrate for the enzyme activity reaction: Good fellow film.

[0124] 2. Experimental method:

[0125] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0126] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0127] Kanamycin stock solution: 50 mg / mL;

[0128] IPTG solution: 1 M, 0.238 g / mL;

[0129] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0130] Eluent: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0131] Protein preservation solution: 50mMNa2HPO4, 100mMNaCl, pH=7.5;

[0132] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0;

[0133] PMSF (serine protease inhibitor) solution: 100 mM;

[0134] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0135] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0136] Protein electrophoresis buffer: Dissolve the purchased MOPS-SDS Running Buffer in 1000 mL of water, stir well and set aside.

[0137] The mutant transformation steps are shown in Example 1.

[0138] Cultivation and collection of intracellular expression bacteria: ① Pick a single colony on the plate and add it to 5mL LB medium, then add 5μL kana mother solution, shake the tube and place it in a shaker at 37℃, 220rpm for 9-12h; ② Measure OD, shake the bacterial solution in advance, dilute 5-10 times to 2mL in a cuvette, use 2mL LB + 2μL kana as a blank control, and measure the absorbance at λ=600nm; ③ Transfer: calculate the final OD after transfer to 0.05-0.1, shake the bacterial solution, and transfer it to a sterilized LB conical flask (100mL) according to the mL number calculated by the OD value, add 100μL kana mother solution to each flask, and place it in a shaker at 37℃, 220rpm for 2.5-3h; ④ Measure OD, shake the test tube in advance, take 2mL in a cuvette without dilution, and use 2mL LB + 2μL kanamycin was used as the blank control. The absorbance was measured at λ = 600nm. When the estimated OD was 0.6-0.8, the conical flask was placed on ice to quickly cool down. ⑤ 0.1mM IPTG was added to the conical flask and cultured in a shaker at 17℃ and 180rpm for 20-24h. ⑥ The conical flask was placed on ice, balanced, and incubated at 4200rpm, 4℃, for 40min to collect the bacterial solution and freeze it in a -80℃ refrigerator for later use.

[0139] Enzyme purification: ①Take the bacteria from the refrigerator and thaw them naturally on ice. After thawing, add 30-40mL LEBuffer, 50mg / L DNase, lysozyme, and 1% PMSF, and resuspend them; ②Break the bacteria by ultrasonic disruption, transfer the resuspended bacteria to a beaker, place the beaker in ice water, and disrupt them at 350W for 30min; ③After the disrupted bacteria are balanced, place them in a high-speed centrifuge at 11500rpm, 4℃, 45min, and collect the supernatant; ④Pre-cool LE Buffer and eluent, and use LE Buffer was used to balance the Ni column; ⑤ The supernatant obtained by centrifugation and the Ni column filler were mixed and placed on an ice shaker at a low speed of 70-80 rpm for 1 hour; ⑥ The mixed solution was returned to the gravity column and waited for the liquid to flow out; ⑦ The Ni column was washed with 80 mM imidazole as clean as possible; ⑧ The target protein was eluted with 250 mM imidazole and the effluent was collected; ⑨ The eluate was added to an ultrafiltration tube and concentrated at 4000 rpm, 4°C, for 20 min, and washed with protein preservation solution for 2-3 times, and finally concentrated to 1.5-2 mL; ⑩ The absorbance at λ = 280 nm was measured to indicate the concentration (mg / mL).

[0140] Enzyme activity reaction: The enzyme addition amount was 500 nM, which was calculated based on the enzyme concentration, and then the reaction buffer was added to 600 μL. The substrate was gf-PET cut into small discs with a diameter of 6 mm. The best mutant in the previous round was used as a control. Multiple parallel and multiple temperature conditions were set for iterative verification.

[0141] Initial screening of absorbance: The absorption peaks of PET products TPA and MHET reach their highest values ​​at around λ=245nm, which can quantitatively characterize the degree of hydrolysis of PET.

[0142] HPLC: Accurately quantify the yields of TPA and MHET.

[0143] 3. Experimental results

[0144] The four mutants N233K / N205K, N233K / N205L, N233K / G234N and N233K / S175G obtained in Examples 1 and 2 were expressed in the same batch and purified by intracellular expression. The reaction was carried out at three temperatures: 40°C, 45°C and 50°C. The results are as follows: Figure 6 shown.

[0145] pass Figure 6 It can be seen that at the three temperatures, the activity of N205K is higher than that of the control and other mutations, while the activity of the other mutations is not significantly improved compared with the control, but slightly decreased. The first round of re-testing resulted in the N233K / N205K (M1) double mutation. After reacting at 40°C for 72 hours, the yield of N233K / N205K (M1) reached 5133 μM.

[0146] Example 4 Construction of the second round mutation library of IsPETase

[0147] 1. Acquisition of experimental materials

[0148] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0149] The mutant secretion N233K / N205K involved in this experiment was obtained by mutation in the laboratory.

[0150] Secreted N233K / N205K amino acid sequence (283): MKYLLPTAAAGLLLLAAQPAMANPY ARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSM GGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:35)

[0151] Nucleotide sequence encoding N233K / N205K (optimized for E. coli host): ATGAAATACCTGCTGCC GACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCAATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO: 36)

[0152] Obtaining the enzyme activity reaction substrate: Good fellow film.

[0153] 2. Experimental methods:

[0154] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0155] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0156] Kanamycin stock solution: 50 mg / mL;

[0157] IPTG solution: 1 M, 0.238 g / mL;

[0158] Construction of the second round of site-directed mutation library for secretory expression: Using secretory N233K / N205K as a template, saturation mutation was performed on the S269 site in the second round to obtain the second round mutation library.

[0159] The steps of mutant transformation, secretion coarse screening and saturation mutation are shown in Example 1.

[0160] 3. Experimental results

[0161] The second round of mutation experiment selected secretion N233K / N205K as a template and half-saturation mutation was performed at S269. After reacting at 45°C for 24 hours, the absorbance was measured as follows Figure 7 shown.

[0162] Experimental verification shows that at 45°C, the activities of N233K / N205K / S269F, N233K / N205K / S269D, N233K / N205K / S269L, N233K / N205K / S269Q, N233K / N205K / S269E, N233K / N205K / S269V, N233K / N205K / S269G, and N233K / N205K / S269H are all higher than those of N233K / N205K. N233K / N205K / S269D, N233K / N205K / S269E, N233K / N205K / S269F, N233K / N205K / S269H, N233K / N205K / S269V, and N233K / N205K / S269Q were selected for intracellular expression retest.

[0163] Example 5 Construction of the second round error-prone PCR library of IsPETase

[0164] 1. Acquisition of experimental materials

[0165] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0166] The mutant secretion N233K / N205K involved in this experiment was obtained by mutation in the laboratory.

[0167] Enzyme activity reaction substrate acquisition: Good fellow film.

[0168] 2. Experimental methods:

[0169] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0170] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0171] Kanamycin stock solution: 50 mg / mL;

[0172] IPTG solution: 1 M, 0.238 g / mL;

[0173] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0.

[0174] Construction of the error-prone PCR library for the second round of secretory expression: error-prone PCR was performed using secretory N233K / N205K as a template, reaction system: 3 μL 10× dye method real-time error-prone PCR Mix (containing enzyme), 1 μL template, 1 μL error-prone PCR primer, 3 μL error-prone PCR enhancer, 3 μL error-prone PCR-specific dNTP, 3 μL error-prone PCR-specific MnCl2, add ultrapure water to make up to 30 μL;

[0175] The mutant transformation steps are shown in Example 1.

[0176] The error-prone PCR steps for secretion coarse screening are shown in Example 2.

[0177] 3. Experimental results

[0178] The second round of error-prone PCR was reacted at 45°C for 24 h and the absorbance was measured. Figure 8 shown.

[0179] B2 is the control secretion N233K / N205K. A7, B12, C2, D10, D11, D12, G5, and H2 were selected for sequencing based on the color depth. Except for the template and synonymous mutations, A7, C2, and G5 were all N233K / N205K / N114I, and the intracellular expression of N233K / N205K / N114I was retested.

[0180] Amino acid sequence (283) secreting N233K / N205K / N114I: MKYLLPTAAAGLLLLAAQPAM ANPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:37)

[0181] Nucleotide sequence encoding N233K / N205K / N114I (optimized for E. coli host): ATGAAATACCTGC TGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCAATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCATCTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:38)

[0182] Example 6 Second-round in vitro purification verification of IsPETase

[0183] 1. Obtaining experimental materials

[0184] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0185] The mutants N233K / N205K / S269D, N233K / N205K / S269E, N233K / N205K / S269F, N233K / N205K / S269H, N233K / N205K / S269V, N233K / N205K / S269Q and N233K / N205K / N114I involved in this experiment were obtained by mutation in the laboratory.

[0186] Amino acid sequence of N233K / N205K / S269D mutant (261): NPYARGPNPTAASLEASAGP FTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYDTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 7)

[0187] Nucleotide sequence of N233K / N205K / S269D mutant (optimized for E. coli host): AATCCGTACGC ACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACGATACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:21)

[0188] Amino acid sequence of N233K / N205K / S269E mutant (261): NPYARGPNPTAASLEASAGPF TVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYETFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 8)

[0189] Nucleotide sequence of N233K / N205K / S269E mutant (optimized for E. coli host): AATCCGTACGC ACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACGAAACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:22)

[0190] Amino acid sequence of N233K / N205K / S269F mutant (261): NPYARGPNPTAASLEASAGPF TVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYFTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 9)

[0191] Nucleotide sequence of N233K / N205K / S269F mutant (optimized for E. coli host): AATCCGTACGCA CGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTTTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:23)

[0192] Amino acid sequence of N233K / N205K / S269H mutant (261): NPYARGPNPTAASLEASAGP FTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYHTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 10)

[0193] Nucleotide sequence of N233K / N205K / S269H mutant (optimized for E. coli host): AATCCGTACGC ACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACCACACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:24)

[0194] Amino acid sequence of N233K / N205K / S269V mutant (261): NPYARGPNPTAASLEASAGP FTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYVTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 11)

[0195] Nucleotide sequence of N233K / N205K / S269V mutant (optimized for E. coli host): AATCCGTACGC ACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACGTTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:25)

[0196] Amino acid sequence of N233K / N205K / S269Q mutant (261): NPYARGPNPTAASLEASAGP FTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYQTFACENPNSTRVSDFRTANCS. (as shown in SEQ ID NO: 12)

[0197] Nucleotide sequence of N233K / N205K / S269Q mutant (optimized for E. coli host): AATCCGTACGC ACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACCAGACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (Shown as SEQ ID NO:26)

[0198] Amino acid sequence of N233K / N205K / N114I (261): NPYARGPNPTAASLEASAGPFTVRSF TVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:13)

[0199] Nucleotide sequence of N233K / N205K / N114I (optimized for E. coli as host): AATCCGTACGCACGTG GTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCATCTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO:27)

[0200] Obtaining the substrate for enzyme activity reaction: Good fellow film.

[0201] 2. Experimental method:

[0202] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0203] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0204] Kanamycin stock solution: 50 mg / mL;

[0205] IPTG solution: 1 M, 0.238 g / mL;

[0206] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0207] Eluent: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0208] Protein preservation solution: 50mMNa2HPO4, 100mMNaCl, pH=7.5;

[0209] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0;

[0210] PMSF (serine protease inhibitor) solution: 100 mM;

[0211] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0212] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0213] Protein electrophoresis buffer: Dissolve the purchased MOPS-SDS Running Buffer in 1000 mL of water, stir well and set aside.

[0214] The mutant transformation steps are shown in Example 1.

[0215] The steps of intracellular expression bacterial culture and collection, enzyme purification, enzyme activity reaction, absorbance initial screening, and HPLC are shown in Example 3.

[0216] 3. Experimental results

[0217] The seven mutants N233K / N205K / S269D, N233K / N205K / S269E, N233K / N205K / S269F, N233K / N205K / S269H, N233K / N205K / S269V, N233K / N205K / S269Q and N233K / N205K / N114I obtained in Examples 4 and 5 were retested with purified proteins expressed intracellularly from the same batch, with N233K / N205K as a control. Reactions were carried out at three temperatures: 40°C, 45°C and 50°C. The results are as follows: Fig. 9 shown.

[0218] pass Fig. 9 It can be seen that at the three temperatures, the activities of N233K / N205K / N114I, N233K / N205K / S269V, and N233K / N205K / S269Q were all higher than the control and other mutations, while the remaining mutations had no significant activity improvement compared to the control but slightly decreased. The second round of re-tests yielded three mutants: N233K / N205K / N114I (M2-1), N233K / N205K / S269V (M2-2), and N233K / N205K / S269Q (M2-3). The yield of N233K / N205K / N114I (M2-1) reached 4714 μM after 48 hours of reaction at 40°C.

[0219] Example 7 Construction and verification of the combinatorial mutation library of IsPETase

[0220] 1. Acquisition of experimental materials

[0221] Acquisition of chassis strains: TOP10 and BL21 competent strains were purchased from Bomed Company.

[0222] The N233K / N205K / N114I / S269V (M3-2) and N233K / N205K / N114I / S269Q (M3-1) involved in this experiment were obtained by mutation in the laboratory.

[0223] Amino acid sequence of N233K / N205K / N114I / S269V (261): NPYARGPNPTAASLEASAGPF TVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYVTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO: 14)

[0224] Nucleotide sequence of N233K / N205K / N114I / S269V (optimized for E. coli host): AATCCGTACGCA CGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCATCTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACGTTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (Shown as SEQ ID NO:28)

[0225] Amino acid sequence of N233K / N205K / N114I / S269Q (261): NPYARGPNPTAASLEASAGPF TVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYQTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:15)

[0226] Nucleotide sequence of N233K / N205K / N114I / S269Q (optimized for E. coli host): AATCCGTACGCA CGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCATCTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACCAGACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (Shown as SEQ ID NO:29)

[0227] Obtaining the substrate for the enzyme activity reaction: Good fellow film.

[0228] 2. Experimental method:

[0229] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0230] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0231] Kanamycin stock solution: 50 mg / mL;

[0232] IPTG solution: 1 M, 0.238 g / mL;

[0233] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0234] Eluent: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0235] Protein preservation solution: 50mMNa2HPO4, 100mMNaCl, pH=7.5;

[0236] Reaction buffer: 50 mM glycine-NaOH, glycine 3.74 g / L, pH = 9.0;

[0237] PMSF (serine protease inhibitor) solution: 100 mM;

[0238] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0239] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0240] Protein electrophoresis buffer: Dissolve the purchased MOPS-SDS Running Buffer in 1000 mL of water, stir well and set aside.

[0241] Construction of a combinatorial mutation library: The three triple mutations obtained in Example 6 were further combined into quadruple mutations M3-1 and M3-2.

[0242] The mutant transformation steps are shown in Example 1.

[0243] The steps of intracellular expression bacterial culture and collection, enzyme purification, enzyme activity reaction, absorbance initial screening, and HPLC are shown in Example 3.

[0244] 3. Experimental results

[0245] WT, N233K, M1, M2-1, M2-2, M2-3, M3-1 and M3-2 were expressed and purified and the reaction was performed as follows Fig.10 result.

[0246] pass Fig.10 It can be seen that at both temperatures, the activity of each variant is significantly higher than IsPETaseWT. The best variant combination currently obtained is M3-1 (N233K / N205K / N114I / S269Q). After reacting at 40°C for 72 hours, the yield reached 5347μM, which is 6.4 times that of the wild type. The gel image after purification is as follows Fig.11 shown.

[0247] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of mutation sites as targets in improving the enzymatic activity and / or thermal stability of PETase hydrolase; The mutation sites include: One or more of positions 114, 175, 205, 234, 269 and position 233 of the amino acid sequence of the wild-type PETase hydrolase.

2. A mutant, characterized in that Based on the wild-type PETase hydrolase, it has one or more of the following amino acid site mutations: 114th, 175th, 205th, 233rd, 234th, 269th, and the amino acid sequence of the wild-type PETase hydrolase is shown in SEQ ID NO:

1.

3. The mutant according to claim 2, characterized in that The mutation at position 114 includes: N114I; The mutation at position 175 includes: S175G; The mutation at position 205 includes: N205K or N205L; The mutation at position 233 includes: N233K; The mutation at position 234 includes: G234N; The mutation at position 269 includes any one of S269D, S269E, S269F, S269H, S269V and S269Q.

4. The mutant according to claim 2 or 3, characterized in that It has: (1) the amino acid sequence shown in SEQ ID NO: 2 to SEQ ID NO: 15; or (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

5. A nucleic acid molecule encoding a mutant according to any one of claims 2 to 4, characterized in that: It has: (4) the nucleotide sequence shown in SEQ ID NO: 16 to SEQ ID NO: 29; or (5) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence described in (4); or (6) a sequence having at least 80% homology with the nucleotide sequence described in (4) or (5); or (7) A complementary sequence of the nucleotide sequence described in (4), (5) or (6).

6. An expression vector, characterized in that include: The nucleic acid molecule according to claim 5.

7. A host, characterized in that Transformation and / or transfection with the expression vector according to claim 6.

8. A method for preparing a mutant, characterized in that: The host cell according to claim 7 is cultured, centrifuged, the supernatant is collected, and purified to obtain the mutant.

9. A product, characterized in that include: The mutant according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host according to claim 7 and / or the mutant obtained by the preparation method according to claim 8.

10. Use of the mutant according to any one of claims 2 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host according to claim 7, the mutant obtained by the preparation method according to claim 8 and / or the product according to claim 9 in degrading plastics.

Images