Pet plastic degrading enzyme mutants, expression vectors, hosts and uses thereof

Abstract

The present application relates to the field of bioengineering, and particularly relates to a PET plastic degrading enzyme mutant, an expression vector, a host and application thereof. The present application provides application of a mutation site as a target in improving the enzyme activity and / or thermal stability of a PETase hydrolytic enzyme; the mutation site includes one or more of positions 114, 175, 205, 234, 269 and 233 in the amino acid sequence of a wild-type PETase hydrolytic enzyme. The present application uses the method of semi-rational design and error-prone PCR to screen and verify sites that have not been mined by other researchers, and expects to further improve the activity and thermal stability of the enzyme to meet the needs of industrial applications.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to PET plastic degrading enzyme mutants, expression vectors, hosts, and their applications. Background Technology

[0002] Plastics are an indispensable consumable in modern people's daily lives. They have excellent properties such as high impact resistance and durability. However, these properties also make plastics one of the most harmful environmental pollutants. In the natural environment, it takes years, decades or even hundreds of years to completely degrade. However, the production and consumption of plastics are much faster than the degradation rate, which leads to the accumulation of plastic waste and environmental pollution. In order to solve this serious environmental problem, different treatment methods have been tried.

[0003] Traditional treatment methods rely on hazardous physical and chemical processes such as mechanical recycling, landfill, incineration, photo / electro / chemical catalytic oxidation, hydrolysis, and glycolysis. These physical and chemical processes typically require additional energy consumption to provide high temperatures and pressures, and are accompanied by secondary pollution. Therefore, attention has shifted to biological methods: biodegradation, also known as microbial or enzymatic degradation, is an effective alternative for plastic waste management.

[0004] Polyethylene terephthalate (PET) is a commercially important and commonly used crystallizable aromatic polyester, and one of the most popular plastic materials in modern society. It is a polymer synthesized from terephthalic acid (TPA) and ethylene glycol (EG) through ester bonds. The ester bonds in PET are viable cleavage sites for many hydrolases. IsPETase, discovered in 2016, can efficiently convert PET into MHET and small amounts of BHET and TPA.

[0005] IsPETase has commercial application value, so researchers have been studying its activity and thermal stability.

[0006] Many factors influence enzyme activity and thermal stability, including the enzyme's hydrolytic capacity and stability, enzyme quantity, reaction conditions, substrate availability, substrate and product inhibition, etc.

[0007] Recent research has largely focused on the primary, secondary, and tertiary structures of enzymes, as well as the chemical modification of enzyme side chains. Strategies for altering the primary to tertiary structures of enzymes include rational design, semi-rational design, irrational design, and artificial intelligence design. This study utilized both semi-rational and irrational design methods to optimize PETase.

[0008] Semi-rational design was applied to the modification research of trehalose synthase in 2004. The semi-rational method targets multiple specific residues and constructs a concise library through mutation based on structural or functional knowledge, which is more likely to yield correct results. Iterative site-directed saturation mutagenesis is a type of semi-rational design.

[0009] Error-prone PCR is a type of irrational design that uses random mutation methods to construct a large mutation library, including single and combined mutations. By properly controlling the mutation rate, the discovery of beneficial strains can be improved.

[0010] Since its initial discovery in 2016, IsPETase has remained a hot topic. As the PET hydrolase with the best degradation activity at ambient temperature, PETase has great potential and prospects. However, after being extracted from the body, PETase exhibits poor long-term enzyme activity and thermal stability, making industrialization difficult. Therefore, researchers have focused their research on improving activity and thermal stability, and have made significant progress.

[0011] IsPETase is a hydrolase composed of 290 amino acids. Current research has not covered the amino acid sites of plastic-degrading enzymes, especially the inactive sites. Given the complexity of the enzyme and its potential for optimization and modification, it is necessary to explore new sites. Summary of the Invention

[0012] In view of this, the present invention provides a PET plastic degrading enzyme mutant, expression vector, host, and its applications. The present invention utilizes semi-rational design and error-prone PCR methods to screen and validate sites that have not been explored by other researchers, with the aim of further improving the enzyme's activity and thermostability to meet the needs of industrial applications.

[0013] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0014] This invention provides the application of mutation sites as targets in improving the enzyme activity and / or thermal stability of PETase hydrolase;

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

[0016] The present invention also provides a mutant having one or more amino acid site mutations at the following positions, based on the wild-type PETase hydrolase: positions 114, 175, 205, 233, 234, and 269, wherein 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, the mutation at position 114 in the above-described mutant 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 mutants include one or more of the following: 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 above-mentioned mutant has:

[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 substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has 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 mutants described above also include secretory signal peptides.

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

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

[0031] (4) Nucleotide sequences as shown in SEQ ID NO:16~SEQ ID NO:29; or

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

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

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

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

[0036] The present invention also provides a host, transformation and / or transfection of the above expression vector.

[0037] The present invention also provides a method for preparing the mutant, wherein the host is cultured, centrifuged, the supernatant is collected, purified, and the mutant is obtained.

[0038] The present invention also provides products comprising: the above-described mutant, the above-described nucleic acid molecule, the above-described expression vector, the above-described host, and / or the mutant obtained by the above-described preparation method.

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

[0040] This invention statistically analyzes and screens previously unstudied sites of IsPETase, and uses semi-rational and non-rational design methods to discover three previously unstudied beneficial mutation sites. Four beneficial mutations are found at these three sites, making the research on IsPETase more comprehensive and complete. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0042] Figure 1 Sequence alignment analysis of IsPETase and its homologous keratinase;

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

[0044] Figure 3 Using secreted N233K as a template, half-saturation mutations were performed at N205 and G234 sites. After reacting at 40℃ for 72 h, the absorbance at λ = 245 nm was measured.

[0045] Figure 4 Using secreted N233K as a template, half-saturation mutations were performed at N205 and G234 sites. After reacting at 45℃ for 72 h, the absorbance at λ = 245 nm was measured.

[0046] Figure 5 Using secreted N233K as a template, error-prone PCR was performed. After reacting at 40℃ for 72 hours, the absorbance at λ = 245 nm was measured.

[0047] Figure 6 The intracellular expression of purified N233K, N233K / N205K, N233K / N205L, N233K / G234N and N233K / S175G was demonstrated, and the precise yield was determined by HPLC after reacting at 40℃, 45℃ and 50℃ for 72 h.

[0048] Figure 7 Using secreted N233K / N205K as a template, a half-saturation mutation was performed at the S269 site. After reacting at 45℃ for 24 h, the absorbance at λ = 245 nm was measured.

[0049] Figure 8 Using secreted N233K / N205K as a template, this is an error-prone PCR assay. After reacting at 45℃ for 24 hours, the absorbance at λ = 245 nm was measured.

[0050] Figure 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. After reacting at 40℃, 45℃, and 50℃ for 48 hours, the precise yield was determined by HPLC.

[0051] Figure 10 The purified WT, N233K, M1, M2-1, M2-2, M2-3, M3-1 and M3-2 were expressed intracellularly and reacted at 40℃ and 45℃ for 72 days. The precise yield was determined by HPLC.

[0052] Figure 11 The image shows a gel image of protein purification. Detailed Implementation

[0053] This invention discloses a PET plastic degradation enzyme mutant, expression vector, host, and its application.

[0054] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0056] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0057] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0058] Furthermore, 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 precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0059] This invention utilizes a research strategy combining semi-rational enzyme design and error-prone PCR irrational design to identify three beneficial mutation sites (N114, N205, and S269) for IsPETase. Four beneficial mutations were obtained at these three sites: N114I, N205K, S269V, and S269Q. Furthermore, through optimized combinations of mutation sites, six IsPETases exhibiting significantly enhanced activity and thermostability at 40°C were obtained. 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 this invention, all raw materials and reagents used can be purchased from the market.

[0061] The present invention will be further illustrated below with reference to the embodiments:

[0062] Example 1: Discovery of unexplored sites of IsPETase and construction of the first-round mutant library of IsPETase

[0063] 1. Obtaining experimental materials

[0064] Obtaining chassis strains: TOP10 and BL21 competent cells were purchased from Bomeide 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 host): AATCCGTACGCACGTGGTCCGAATC CAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAACGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in SEQ ID NO: 30)

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

[0069] 2. Experimental method:

[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: 1M, 0.238g / mL;

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

[0075] Obtaining a library of unstudied sites: PETase has a full length of 290 amino acids. By reviewing the literature and excluding sites that have been mutated by other researchers, a library of unstudied sites was obtained.

[0076] Identifying sites worthy of saturation mutation: Homologous sequence alignment analysis revealed that the previously unstudied N205, G234, and S269 sites are not highly conserved, and mutations to other amino acids may improve enzyme activity or thermostability, such as... Figure 1 As shown.

[0077] Constructing a secretion expression system: ① Add the pelB secretion signal peptide to the N-terminus of the synthesized wild-type PETase for high-throughput application; ② Add N233K from DepoPETase to the wild-type secretion system to improve thermal stability, and subsequently increase the reaction temperature to 40℃ and 45℃ for easy screening.

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

[0079] The nucleotide sequence for secreting N233K (optimized for the large intestine): ATGAAATACCTGCTGCCGACCG (as shown in SEQ ID NO:16).

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

[0081] Mutant transformation: ① Add 2 μL of mutant plasmid to 50 μL of BL21 competent cells and incubate on ice for 15-25 min; ② Heat shock in a 42℃ water bath for 1 min; ③ Place on ice for 2 min; ④ Add 500 μL of LB antibiotic-free medium and incubate at 37℃ and 220 rpm for 1 h; ⑤ Centrifuge the EP tube at 4000 rpm for 2 min, discard the supernatant, resuspend the precipitate and spread it evenly on a plate containing kanamycin; ⑥ Incubate overnight in an inverted incubator at 37℃.

[0082] Secretion coarse screening saturation mutagenesis: ① Pick a single colony from the plate and add it to 1 mL of LB medium, then add 1 μL of kanamycin stock solution, shake well, and incubate the culture tube at 37℃ and 220 rpm for 9-12 h; ② Take a fresh culture tube, add 3 mL of LB medium and 3 μL of kanamycin stock solution, transfer 100 μL of bacterial culture, and incubate at 37℃ and 220 rpm for 2.5-3 h; ③ Place the culture tube on ice for rapid cooling; ④ Add 0.1 mM IPTG to the culture tube and incubate at 17℃ and 180 rpm for 20-24 h; ⑤ Place the culture tube on ice, balance, centrifuge at 4200 rpm and 4℃ for 30 min, take 50 μL of supernatant + 550 μL of reaction buffer, and cut the substrate GF-PET into small round discs with a diameter of 6 mm.

[0083] 3. Experimental Results

[0084] The first round of mutation experiments used the secretory N233K as a template, performing half-saturation mutations at the N205 and G234 sites. Enzyme activity was verified by reacting at 40℃ and 45℃ for 72 hours.

[0085] Literature review revealed that both TPA and MHET reach their maximum absorption peaks around λ = 254 nm. To verify accuracy, a standard curve was measured as follows: Figure 2 As shown.

[0086] Experiments have verified that at 40℃, the activities of N233K / G234N, N233K / G234Y, N233K / N205K, N233K / N205L, N233K / N205T, N233K / N205E, N233K / N205V, N233K / N205R, N233K / N205W, and N233K / N205Y are all higher than that of N233K. Figure 3 As shown; at 45℃, the activities of N233K / N205K, N233K / N205L, N233K / N205R, and N233K / N205Y are all higher than that of N233K, such as Figure 4 As shown.

[0087] Intracellular expression retests were conducted using N233K / G234N, which exhibited the highest activity at 40℃, and N233K / N205K and N233K / N205L, which showed excellent performance at both 40℃ and 45℃.

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

[0089] 1. Obtaining experimental materials

[0090] Obtaining chassis strains: TOP10 and BL21 competent cells were purchased from Bomeide Company.

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

[0092] Enzyme activation reaction substrate obtained: Goodfellow 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: 1M, 0.238g / mL;

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

[0099] Constructing a first-round error-prone PCR library for secretory expression: Error-prone PCR was performed using secretory N233K as a template. The reaction system consisted of: 3 μL of 10× dye-based real-time error-prone PCR Mix (containing enzymes), 1 μL of template, 1 μL of error-prone PCR primers, 3 μL of error-prone PCR enhancer, 3 μL of error-prone PCR-specific dNTPs, 3 μL of error-prone PCR-specific MnCl2, and ultrapure water to a final volume of 30 μL.

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

[0101] Error-prone PCR downlink nucleotide sequence: TTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGT GCTC. (As shown in SEQ ID NO:32)

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

[0103] Commonly Missing PCR Methods for Secretory Screening: ① In a 96-well plate, add 500 μL of LB medium and 0.5 μL of kanamycin stock solution to each well. Use a toothpick to pick a single colony from the plate and insert it into each of the 96 wells. Shake the toothpick to ensure successful inoculation. Incubate the 96-well plate at 37°C and 220 rpm for 9-12 hours. ② Take a new 96-well plate and add 800 μL of LB medium to each well. 1. Use LB medium and 0.8 μL kanamycin stock solution, and transfer 20 μL of bacterial culture accordingly. Incubate at 37℃ and 220 rpm for 2.5-3 h. 2. Place the 96-well plate on ice for rapid cooling. 3. Add 0.1 mM IPTG to the 96-well plate and incubate at 17℃ and 180 rpm for 20-24 h. 4. Place the 96-well plate on ice, balance it, centrifuge at 4200 rpm and 4℃ for 30 min, and take 50 μL of the supernatant + 550 μL of reaction buffer. Cut the substrate GF-PET into small round discs with a diameter of 6 mm.

[0104] 3. Experimental Results

[0105] The first round of error-prone PCR was performed at 40℃ for 72 hours, and the absorbance was measured as follows: Figure 5 As shown.

[0106] A1 was the control for secreting N233K. Based on the color intensity, C5, E2, E6, F6, F7, F8, F11, G4, G6, H2, H5, I4, I9, I10, J3, J12, L6, L7, M9, N12, O6, and O12 were sequenced. After removing template and synonymous mutations, four mutations were verified. I4, N233K / S175G (Table 1), which showed the highest activity enhancement, was selected for intracellular expression retesting.

[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] The amino acid sequence (283 amino acids) of the secretory I4 mutant is: MKYLLPTAAAGLLLLAAQPAMANPYARGP NPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPGLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:33)

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

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

[0112] 1. Obtaining experimental materials

[0113] Obtaining chassis strains: TOP10 and BL21 competent cells were purchased from Bomeide Company.

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

[0115] The amino acid sequence (261 amino acids) of the N233K / N205K mutant is: NPYARGPNPTAASLEASAGPFTVRS FTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:4)

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

[0117] The amino acid sequence (261 amino acids) of the N233K / N205L mutant is: NPYARGPNPTAASLEASAGPFTVRS FTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACELDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:5)

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

[0119] The amino acid sequence (261 amino acids) of the N233K / G234N mutant is: NPYARGPNPTAASLEASAGPFTVRS FTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEIKNGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:6)

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

[0121] The amino acid sequence (261 amino acids) of the N233K / S175G mutant is: NPYARGPNPTAASLEASAGPFTVRSF TVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPGLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:3)

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

[0123] Obtaining the substrate for 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: 1M, 0.238g / mL;

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

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

[0131] Protein preservation solution: 50mM Na2HPO4, 100mM NaCl, pH=7.5;

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

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

[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 1000mL of water, stir well and set aside.

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

[0138] Intracellular expression cell culture and collection: ① Pick a single colony from the plate and add it to 5 mL of LB medium, then add 5 μL of kanamycin stock solution. Shake well and incubate the tube at 37℃ and 220 rpm for 9-12 h; ② Measure OD: Shake the bacterial solution well beforehand, dilute 5-10 times, and bring the volume to 2 mL in a cuvette. Use 2 mL LB + 2 μL kanamycin as a blank control. Measure the absorbance at λ = 600 nm; ③ Transfer: Calculate the final OD after transfer to 0.05-0.1. Shake the bacterial solution well and transfer the calculated OD value (mL) to sterilized LB conical flasks (100 mL). Add 100 μL of kanamycin stock solution to each flask and incubate at 37℃ and 220 rpm for 2.5-3 h; ④ Measure OD: Shake the test tube well beforehand, take 2 mL without dilution and place it in a cuvette. LB+2μL kanamycin was used as a blank control. The absorbance was measured at λ=600nm. When the expected OD was 0.6-0.8, the conical flask was placed on ice for rapid cooling. ⑤ 0.1mM IPTG was added to the conical flask and incubated at 17℃ and 180rpm in a shaker for 20-24h. ⑥ The conical flask was placed on ice, balanced, and the bacterial culture was collected at 4200rpm and 4℃ for 40min. The culture was then frozen at -80℃ for later use.

[0139] Enzyme purification: ① Take the bacterial cells from the refrigerator and thaw them naturally on ice. After thawing, add 30-40 mL of LE Buffer, 50 mg / L of DNase from the bacterial suspension, lysozyme, and 1% PMSF, and resuspend. ② Disruption: Sonicate the cells, transfer the resuspended cells to a beaker, place the beaker in ice water, and incubate at 350 W for 30 min. ③ Balance the disrupted cells and centrifuge at 11500 rpm, 4℃, for 45 min, and collect the supernatant. ④ Pre-cool the LE Buffer and elution buffer, and use LE... ⑤ Equilibrate the Ni column with buffer; ⑥ Mix the supernatant obtained from centrifugation with the Ni column packing material and place on ice in a shaker at low speed of 70-80 rpm for 1 hour; ⑦ Pour the mixture back into the gravity column and wait for the liquid to drain completely; ⑧ Wash the Ni column with 80 mM imidazole to remove impurities as much as possible; ⑨ Elute the target protein with 250 mM imidazole and collect the eluent; ⑨ Add the eluent to an ultrafiltration tube and concentrate at 4000 rpm, 4℃, for 20 min, while washing 2-3 times with protein preservation solution, finally concentrating to 1.5-2 mL; ⑩ Measure the absorbance at λ = 280 nm to express the concentration (mg / mL).

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

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

[0142] HPLC: Accurately quantifies 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 re-examined using intracellular expression and purification of the same batch of proteins. Reactions were performed at 40℃, 45℃, and 50℃, and the results are as follows: Figure 6 As shown.

[0145] pass Figure 6 It can be seen that at all three temperatures, the activity of N205K was higher than that of the control and other mutations, while the activity of the other mutations was not significantly increased compared to the control, but rather slightly decreased. The first round of retesting yielded the N233K / N205K(M1) double mutant. The yield of N233K / N205K(M1) reached 5133 μM after reacting at 40℃ for 72 h.

[0146] Example 4: Construction of the second-round mutant library of IsPETase

[0147] 1. Obtaining experimental materials

[0148] Obtaining chassis strains: TOP10 and BL21 competent cells were purchased from Bomeide Company.

[0149] The mutants involved in this experiment secreted N233K / N205K, which were obtained through laboratory mutation.

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

[0151] Nucleotide sequence of N233K / N205K secreted (optimized for E. coli as 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: 1M, 0.238g / mL;

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

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

[0160] 3. Experimental Results

[0161] The second round of mutation experiments used the secretory N233K / N205K template as a template, and performed a half-saturation mutation at the S269 site. After reacting at 45℃ for 24 hours, the absorbance was measured. Figure 7 As shown.

[0162] Experiments have shown that at 45℃, 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. Intracellular expression was retested using N233K / N205K / S269D, N233K / N205K / S269E, N233K / N205K / S269F, N233K / N205K / S269H, N233K / N205K / S269V, and N233K / N205K / S269Q.

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

[0164] 1. Obtaining experimental materials

[0165] Obtaining chassis strains: TOP10 and BL21 competent cells were purchased from Bomeide Company.

[0166] The mutants involved in this experiment secreted N233K / N205K, which were obtained through laboratory mutation.

[0167] Enzyme activation reaction substrate obtained: Goodfellow 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: 1M, 0.238g / mL;

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

[0174] Constructing a second-round error-prone PCR library for secretory expression: Error-prone PCR was performed using secretory N233K / N205K as a template. The reaction system consisted of 3 μL of 10× dye-based real-time error-prone PCR Mix (containing enzymes), 1 μL of template, 1 μL of error-prone PCR primers, 3 μL of error-prone PCR enhancer, 3 μL of error-prone PCR-specific dNTPs, 3 μL of error-prone PCR-specific MnCl2, and ultrapure water to a final volume of 30 μL.

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

[0176] See Example 2 for the error-prone PCR steps for secretion screening.

[0177] 3. Experimental Results

[0178] The second round of error-prone PCR was performed at 45℃ for 24 hours, and the absorbance was measured as follows: Figure 8 As shown.

[0179] B2 was the control, secreting N233K / N205K. Based on the color intensity, A7, B12, C2, D10, D11, D12, G5, and H2 were selected for sequencing. Except for the template and synonymous mutations, A7, C2, and G5 were all N233K / N205K / N114I. 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] Obtaining chassis strains: TOP10 and BL21 competent cells were purchased from Bomeide 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 through laboratory mutation.

[0186] The amino acid sequence (261 amino acids) of the N233K / N205K / S269D mutant is: 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] The amino acid sequence (261 amino acids) of the N233K / N205K / S269E mutant is: NPYARGPNPTAASLEASAGPF TVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYETFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:8)

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

[0190] The amino acid sequence (261 amino acids) of the N233K / N205K / S269F mutant is: 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] The amino acid sequence (261 amino acids) of the N233K / N205K / S269H mutant is: 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] The amino acid sequence (261 amino acids) of the N233K / N205K / S269V mutant is: 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] The amino acid sequence (261 amino acids) of the N233K / N205K / S269Q mutant is: NPYARGPNPTAASLEASAGP FTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYQTFACENPNSTRVSDFRTANCS. (As shown in SEQ ID NO:12)

[0197] Nucleotide sequence of N233K / N205K / S269Q mutant (optimized for E. coli host): AATCCGTACGC ACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGGACTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAAGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACCAGACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT. (As shown in 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 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: 1M, 0.238g / mL;

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

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

[0208] Protein preservation solution: 50mM Na2HPO4, 100mM NaCl, pH=7.5;

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

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

[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 1000mL of water, stir well and set aside.

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

[0215] The steps for intracellular expression cell culture and collection, enzyme purification, enzyme activity reaction, absorbance screening, and HPLC are described in Example 3.

[0216] 3. Experimental Results

[0217] Seven mutants obtained in Examples 4 and 5—N233K / N205K / S269D, N233K / N205K / S269E, N233K / N205K / S269F, N233K / N205K / S269H, N233K / N205K / S269V, N233K / N205K / S269Q, and N233K / N205K / N114I—were used as a control. The same batch of purified protein was expressed and retested intracellularly. Reactions were performed at 40°C, 45°C, and 50°C. The results are as follows: Figure 9 As shown.

[0218] pass Figure 9 It can be seen that at the three temperatures, the activities of N233K / N205K / N114I, N233K / N205K / S269V, and N233K / N205K / S269Q were higher than those of the control and other mutants, while the activities of the other mutants were not significantly improved compared to the control, but rather slightly decreased. The second round of retesting 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 reacting at 40℃ for 48 h.

[0219] Example 7: Construction and Validation of a Combinatorial Mutant Library of IsPETase

[0220] 1. Obtaining experimental materials

[0221] Obtaining chassis strains: TOP10 and BL21 competent cells were purchased from Bomeide Company.

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

[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 enzyme activity reaction substrate: Good fellow film.

[0228] 2. Experimental method:

[0229] ​

[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: 1M, 0.238g / mL;

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

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

[0235] Protein preservation solution: 50mM Na2HPO4, 100mM NaCl, pH=7.5;

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

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

[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 1000mL of water, stir well and set aside.

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

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

[0243] The steps for intracellular expression cell culture and collection, enzyme purification, enzyme activity reaction, absorbance screening, and HPLC are described in Example 3.

[0244] 3. Experimental Results

[0245] After intracellular expression and purification of WT, N233K, M1, M2-1, M2-2, M2-3, M3-1, and M3-2, the following reaction was obtained: Figure 10 result.

[0246] pass Figure 10 It can be seen that, at both temperatures, the activities of each variant are significantly higher than IsPETaseWT. The best variant combination obtained so far is M3-1 (N233K / N205K / N114I / S269Q). After reacting at 40℃ for 72 h, the yield reached 5347 μM, which is 6.4 times that of the wild type. The purified gel image is shown below. Figure 11 As shown.

[0247] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of a PET plastic degrading enzyme, characterized in that, Its amino acid sequence is shown in SEQ ID NO:4 and any one of SEQ ID NO:11 to SEQ ID NO:

15.

2. A nucleic acid molecule encoding the mutant as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:18 and any one of SEQ ID NO:25~SEQ ID NO:

29.

3. An expression carrier, characterized in that, include: The nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, Transformation and / or transfection of the expression vector as described in claim 3.

5. A method for preparing a mutant, characterized in that, The host cells as described in claim 4 are cultured, centrifuged, the supernatant is collected, and purified to obtain the mutant.

6. A product made of degradable PET plastic, characterized in that, include: The mutant as described in claim 1, the nucleic acid molecule as described in claim 2, the expression vector as described in claim 3, the host cell as described in claim 4, and / or the mutant obtained by the preparation method as described in claim 5.

7. The application of the mutant of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, the host cell of claim 4, the mutant obtained by the preparation method of claim 5, and / or the product of claim 6 in the degradation of PET plastic.

Images