Application of mutation sites as targets in improving enzyme activity, thermal stability and / or long-acting property of PET plastic-degrading enzyme

Abstract

The present application relates to the field of bioengineering, and particularly relates to application of a mutation site as a target point in improving enzyme activity, thermal stability and / or long-acting property of a PET plastic degrading enzyme. The present application provides application of a mutation site as a target point in improving enzyme activity and / or thermal stability of a PET plastic degrading enzyme; the mutation site comprises one or more of positions 85, 176, 204 and 240 of an amino acid sequence of a wild-type PET plastic degrading enzyme; and the PET plastic degrading enzyme comprises one or more of FAST-PETase, PA-PETase and DepoPETase. The present application applies site mutation to the currently known best several variants, makes up for the short-term activity problem of FAST-PETase, and improves the activity and thermal stability of PA-PETase and DepoPETase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and particularly relates to application of a mutation site as a target in improving enzyme activity, thermal stability and / or long-acting property of a PET plastic degrading enzyme. BACKGROUND

[0002] Plastic waste has increased from 156 million tons in 2000 to 353 million tons in 2019, of which 150-200 million tons (or even more) are discarded in landfills or natural environments such as oceans, and even microplastics are found in human feces, indicating that plastic pollution has entered the food chain and is beginning to directly threaten human health.

[0003] Polyethylene terephthalate (PET) is a common plastic formed by dehydration condensation esterification of terephthalic acid (TPA) and ethylene glycol (EG). Since PET has chain mobility, crystallinity and surface hydrophobicity, and does not truly undergo complete mineralization, PET plastic naturally degrades very slowly and is prone to accumulation, causing white pollution. However, its ester bond is a feasible cleavage site for many hydrolytic enzymes, so researchers have mined many biological enzymes with PET hydrolysis activity and optimized their performance.

[0004] IsPETase was first discovered in 2016 as the best PET hydrolase with degradation activity at ambient temperature, which has good degradation potential and prospect. However, after being taken out of the body, its long-term enzyme activity and thermal stability are poor, making it difficult to realize industrialization. Therefore, researchers have actively carried out research work mainly around improving activity and improving thermal stability, and have made outstanding progress. In 2022, Hongyuan Lu et al. used a structure-based machine learning algorithm to design a powerful and active PET hydrolase variant FAST-PETase, which eventually made untreated post-consumer PET from 51 different thermoformed products almost completely degraded by FAST-PETase within 1 week; In 2023, Lixia Shi et al. used PETaseWT as a starting point to construct an error-prone PCR library, and used BHET-OH fluorescence detection technology to screen 10,000 clones in 3 rounds of directed evolution, and finally obtained a variant DepoPETase with 1407-fold higher activity than the wild type; In 2024, Qingdian Ying et al. used semi-saturation mutation to stabilize the most flexible region of IsPETase, and constructed a heat-resistant S121P / D186A variant PA-PETase with a Tm value of 70.8℃ (ΔTm=24.1C), which increased the product of amorphous polyester depolymerization at 40℃ by 109.3 times.

[0005] However, FAST-PETase has poor long-term activity, and its enzyme activity is almost completely lost after working at 50℃ for 24h; The optimal reaction temperature of DepoPETase and FAST-PETase is 50℃, but the activity of DepoPETase at 50℃ for 24h is not as good as that of FAST-PETase; The optimal reaction temperature of PA-PETase is 40℃, and its thermal stability is poor. SUMMARY

[0006] Therefore, the application provides the application of mutation sites as targets in improving the enzyme activity and / or thermal stability of PET plastic degrading enzymes. The application applies four-site mutations to the currently known best variants, which not only solves the short-term activity problem of FAST-PETase, but also improves the activity and thermal stability of PA-PETase and DepoPETase.

[0007] In order to achieve the above application purposes, the application provides the following technical solutions:

[0008] The application provides the application of mutation sites as targets in improving the enzyme activity and / or thermal stability of PET plastic degrading enzymes.

[0009] The mutation sites include one or more of positions 85, 176, 204 and 240 in the amino acid sequence of a wild-type PET plastic degrading enzyme.

[0010] The PET plastic degrading enzyme includes one or more of FAST-PETase enzyme, PA-PETase enzyme and DepoPETase enzyme.

[0011] The application also provides a mutant having one or more amino acid site mutations of positions 85, 176, 204 and 240 on the basis of a wild-type PET plastic degrading enzyme, and the amino acid sequence of the wild-type PET plastic degrading enzyme is shown in any of SEQ ID NO: 1 to SEQ ID NO: 3.

[0012] In some embodiments of the application, in the above-mentioned mutant, the nucleotide sequence encoding the wild-type PET plastic degrading enzyme is shown in SEQ ID NO: 10 to SEQ ID NO: 12.

[0013] In some embodiments of the application, in the above-mentioned mutant, the mutation of position 85 includes N85I.

[0014] The mutation of position 175 includes N175K.

[0015] The mutation of position 204 includes N204K.

[0016] The mutation of position 240 includes S240V.

[0017] In some embodiments of the application, the above-mentioned mutant has:

[0018] (1) the amino acid sequence shown in SEQ ID NO: 4 to SEQ ID NO: 6; or

[0019] (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in (1), and an amino acid sequence functionally identical or similar to the amino acid sequence shown in (1); or

[0020] (3) an amino acid sequence having at least 80% identity with the amino acid sequence shown in (1) or (2).

[0021] The application also provides a nucleic acid molecule encoding the above-mentioned mutant.

[0022] In some embodiments of the application, the above-mentioned nucleic acid molecule has:

[0023] (4) the nucleotide sequence shown in SEQ ID NO: 7 to SEQ ID NO: 9; or

[0024] (5) the nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence according to (4); or

[0025] (6) a sequence having at least 80% homology with the nucleotide sequence according to (4) or (5); or

[0026] (7) a complementary sequence of the nucleotide sequence according to (4), (5) or (6).

[0027] The present application also provides an expression vector comprising the above nucleic acid molecule.

[0028] The present application also provides a host transformed and / or transfected with the above expression vector.

[0029] The present application also provides a method for preparing the mutant, which comprises culturing the above host, centrifuging, collecting the supernatant, and purifying to obtain the mutant.

[0030] The present application also provides a product comprising the above mutant, the above nucleic acid molecule, the above expression vector, the above host and / or the mutant obtained by the above method.

[0031] The present application also provides the above mutant, the above nucleic acid molecule, the above expression vector, the above host, the mutant obtained by the above method and / or the above application in degrading plastics.

[0032] The present application improves the activity and long-acting property of FAST-PETase, and the activity and thermal stability of PA-PETase and DepoPETase. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0034] Figure 1 Intracellular expression and purification of FAST-PETase and M4, HPLC determination of accurate yield after 24h and 72h of reaction at 45℃, 50℃ and 55℃;

[0035] Figure 2 Intracellular expression and purification of PA-PETase and M5, HPLC determination of accurate yield after 24h and 72h of reaction at 40℃, 45℃ and 50℃;

[0036] Figure 3 Intracellular expression and purification of DepoPETase and M6, HPLC determination of accurate yield after 24h and 72h of reaction at 45℃, 50℃ and 55℃. DETAILED DESCRIPTION

[0037] The application discloses application of a mutation site as a target in improving enzyme activity, thermal stability and / or long-acting property of a PET plastic degrading enzyme.

[0038] It should be understood that the expression “one or more of” individually includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression “and / or” in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0039] The terms “comprising”, “having” or “including”, including the use of their grammatical syntaxes, should generally be understood to be open-ended and non-limiting, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0040] It should be understood that the order of steps or the order of performing certain actions is immaterial so long as the application remains operable. Also, two or more steps or actions can be conducted simultaneously.

[0041] The use of any and all examples, or exemplary language herein, for example, only the better illustrate the application, and unless otherwise claimed, does not pose a limitation on the scope of the application. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0042] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and the numerical values set forth in the detailed description are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Therefore, unless otherwise specified, all ranges disclosed herein are to be understood to be approximations, and the end points of all ranges are not to be understood as being significant to the practice of the application. It should also be understood that the term “about” used herein when referring to a parameter does not mean that the parameter is exactly the specified value, but that the parameter is approximately the specified value.

[0043] The application improves different performances of the scaffold mutants by site mutation: ① activity; ② thermal stability; ③ long-acting property. It is finally found that the activity of the enzyme is improved, and the long-acting property of ① FAST-PETase is improved; the thermal stability of ② PA-PETase and DepoPETase is improved.

[0044] In the embodiments 1-3 of the application, the raw materials and reagents used can be purchased from the market.

[0045] The application will be further described below in combination with the embodiments:

[0046] Example 1 Construction and verification of M4 mutants - adding site mutations on FAST-PETase scaffold

[0047] 1. Obtaining of experimental materials

[0048] The competent cells of TOP10 and BL21 were purchased from Biomed.

[0049] The gene FAST-PETase (pET_28a(+)) involved in the experiment was synthesized by GenScript.

[0050] FAST-PETase amino acid sequence (261): NPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS (as shown in SEQ ID NO: 1).

[0051] Nucleotide sequence of FAST-PETase (optimized for E. coli as host): AATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGgaaAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGcacTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTcagAACGCGAAGCAGTTTCTGGAAATCaaaGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTGCGGTTTCTGACTTCCGTACCGCTAACTGCTCT (as set forth in SEQ ID NO: 10).

[0052] Enzyme activity reaction substrate obtained: Good fellow film.

[0053] 2. Experimental method:

[0054] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0055] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0056] Kanamycin stock solution: 50 mg / mL;

[0057] IPTG solution: 1M, 0.238g / mL;

[0058] Lysis buffer (LE Buffer): 50mM Na2HPO4, 100mM NaCl, 10mM imidazole, pH=7.5;

[0059] Eluent: 50mM Na2HPO4, 100mM NaCl, 500mM imidazole, pH=7.5;

[0060] Protein preservation solution: 50mM Na2HPO4, 100mM NaCl, pH=7.5;

[0061] Reaction buffer: 50mM glycine-NaOH, glycine 3.74g / L, pH=9.0;

[0062] PMSF (serine protease inhibitor) solution: 100mM;

[0063] Coomassie Brilliant Blue Staining Solution: 2.4 g / L Coomassie Brilliant Blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0064] Decolorizing solution: Methanol: Glacial acetic acid: Water = 4:1:5;

[0065] Protein electrophoresis buffer: Dissolve the purchased MOPS-SDS Running Buffer in 1000mL of water, stir well and set aside.

[0066] Construction of the M4 mutant: Using FAST-PETase as a template, single-point mutations of N176K, N85I, and S240V were added sequentially to obtain the M4 mutant.

[0067] Amino acid sequence of M4 mutant (261): NPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYVTFACENPNSTAVSDFRTANCS (as set forth in SEQ ID NO: 4).

[0068] Nucleotide sequence of M4 mutant (optimized for E. coli as host):

[0069] AATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCATCTCCACTCTGGATCAGCCGgaaAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGcacTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTcagAACGCGAAGCAGTTTCTGGAAATCaaaGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACGTTACCTTCGCGTGTGAGAACCCGAACTCTACTGCGGTTTCTGACTTCCGTACCGCTAACTGCTCT (as set forth in SEQ ID NO: 7).

[0070] Mutant transformation: ① 2 μL mutant plasmid was added to 50 μL BL21 competent cells, and ice bathed for 15-25 min; ② heated in a 42 °C water bath for 1 min; ③ placed on ice for 2 min again; ④ added 500 μL LB non-antibiotic medium, and cultured in a 37 °C, 220 rpm shaker for 1 h; ⑤ centrifuged the EP tube at 4000 rpm for 2 min, poured off the supernatant, resuspended the precipitate evenly and plated on a plate added with kanamycin resistance; ⑥ inverted overnight culture in a 37 °C incubator.

[0071] Intracellular expression of bacterial culture and collection: ① pick a single colony on the plate into 5 mL LB medium, add 5 μL kanamycin solution, shake the tube and place it in a 37 °C, 220 rpm shaker for 9-12 h; ② measure OD, shake the bacterial solution in advance, dilute 5-10 times 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 be 0.05-0.1, shake the bacterial solution, transfer to a sterilized LB conical flask (100 mL) according to the OD value, add 100 μL kanamycin solution to each flask, and place it in a 37 °C, 220 rpm shaker for 2.5-3 h; ④ measure OD, shake the test tube in advance, take 2 mL in a cuvette without dilution, use 2 mL LB + 2 μL kanamycin as a blank control, measure the absorbance at λ = 600 nm, when the expected OD is 0.6-0.8, place the conical flask on ice for rapid cooling; ⑤ add 0.1 mM IPTG to the conical flask, incubate at 17 °C, 180 rpm for 20-24 h; ⑥ place the conical flask on ice, balance, collect the bacterial solution at 4200 rpm, 4 °C for 40 min, and freeze it in a -80 °C refrigerator for later use.

[0072] Enzyme purification: ① take the bacteria from the refrigerator, place it on ice to thaw naturally, add 30-40 mL LE Buffer, 50 mg / L bacterial solution DNase, lysozyme, and one percent PMSF after thawing, and resuspend; ② break the bacteria: ultrasonic crushing, resuspend the bacteria in a beaker, place the beaker in ice water, and crush for 30 min at 350 W; ③ balance the crushed bacteria and place them in a high-speed centrifuge at 11500 rpm, 4 °C for 45 min, and collect the supernatant; ④ pre-cool the LE Buffer and eluent, and balance the Ni column with the LE Buffer; ⑤ mix the supernatant obtained by centrifugation with the Ni column filler and place it on ice in a shaker at low speed of 70-80 rpm for 1 h; ⑥ pour the mixed solution back into the gravity column and wait for the liquid to flow out; ⑦ wash the Ni column with 80 mM imidazole to remove impurities as much as possible; ⑧ elute the target protein with 250 mM imidazole, and collect the effluent; ⑨ concentrate the eluent in an ultrafiltration tube at 4000 rpm, 4 °C for 20 min, and wash it 2-3 times with a protein preservation solution at the same time, finally concentrate to 1.5-2 mL; ⑩ measure the absorbance at λ = 280 nm to represent the concentration (mg / mL).

[0073] Enzyme activity reaction: the enzyme addition amount is 500 nM, the enzyme addition amount is calculated according to the enzyme concentration, then 600 μL of reaction buffer is added, the substrate is gf-PET cut into small circles with a diameter of 6 mm, the best mutant of the previous round is used as a control, multiple parallel and multiple temperature conditions are set, and iterative verification is performed.

[0074] Absorbance preliminary screening: The product TPA and MHET of PET reached the highest at λ = 245 nm, which can be used to quantify the degree of hydrolysis of PET.

[0075] HPLC: Accurately quantify the yield of TPA and MHET respectively.

[0076] 3、Experimental results

[0077] FAST-PETase and M4 of the same batch of intracellular expression and purification of protein verification, at 45℃, 50℃, 55℃ three temperatures respectively for 24h and 72h, the results are shown in Figure 1

[0078] By Figure 1 It can be seen that at 24h, the activity of FAST-PETase at 45℃, 50℃ is higher than that of M4; only at 55℃, the activity of M4 is slightly higher than that of FAST-PETase. At 72h, the activity of M4 is higher than that of FAST-PETase at three temperatures; M4 reaches the optimum temperature at 45℃, and the yield of M4 reaches 7688μM after 72h reaction at 45℃. It can be seen that adding site mutation on FAST-PETase can improve the activity, thermal stability and long-acting of the enzyme.

[0079] Example 2 Construction and verification of M5 mutant - adding site mutation on PA-PETase scaffold

[0080] 1、Obtaining of experimental materials

[0081] Obtaining of chassis strain: TOP10, BL21 competent cells were purchased from Biomed.

[0082] The gene PA-PETase (pET_28a(+)) involved in this experiment was synthesized by Jingke Biological Company.

[0083] ​Amino acid sequence of PA-PETase (261): NPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPPSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWASSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS (as set forth in SEQ ID NO: 2).

[0084] Nucleotide sequence of PA-PETase (optimized for E. coli as host): AATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAACTCCACTCTGGATCAGCCGccgAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGgctTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAACGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAACGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT (as shown in SEQ ID NO: 11).

[0085] Enzyme activity reaction substrate obtained: Good fellow film.

[0086] 2. Experimental method:

[0087] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0088] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0089] Kanamycin stock solution: 50 mg / mL;

[0090] IPTG solution: 1 M, 0.238 g / mL;

[0091] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0092] Elution buffer: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0093] Protein storage buffer: 50 mM Na2HPO4, 100 mM NaCl, pH = 7.5;

[0094] Reaction buffer: 50 mM Glycine-NaOH, Glycine 3.74 g / L, pH = 9.0;

[0095] PMSF (serine protease inhibitor) solution: 100 mM;

[0096] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0097] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0098] Protein electrophoresis buffer: purchased MOPS-SDS Running Buffer is dissolved in 1000 mL of water, and stirred uniformly for standby.

[0099] Construction of M5 mutant: PA-PETase as template, sequentially add N204K, N176K, N85I, S240V single point mutations, finally get M5 mutant.

[0100] Amino acid sequence of M5 mutant (261): NPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTISTLDQPPSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWASSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYVTFACENPNSTRVSDFRTANCS (as set forth in SEQ ID NO: 5).

[0101] Nucleotide sequence of M5 mutant (optimized for E. coli as host): AATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACACCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCATCTCCACTCTGGATCAGCCGccgAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGgctTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAGGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACGACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAGGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCAACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGCGTTTCATGGACAACGACACTCGTTACGTTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT (as set forth in SEQ ID NO: 8).

[0102] Mutant transformation, intracellular expression of bacteria, cultivation and collection of bacteria, purification of enzyme, enzyme activity reaction, absorbance primary screening, HPLC measurement steps are as described in Example 1.

[0103] 3. Experimental results

[0104] The same batch of PA-PETase and M5 intracellular expression purified protein was verified at 40°C, 45°C and 50°C for 24h and 72h, respectively, and the results are shown in Figure 2

[0105] By Figure 2 It can be seen that after 24h and 72h of reaction at 45°C and 50°C, the activity of M5 is much higher than that of PA-PETase. The yield of M5 reaches 7317μM after 72h of reaction at 50°C, which is 9.4 times that of PA-PETase. Therefore, adding site mutations to PA-PETase can improve the activity and thermal stability of the enzyme.

[0106] Example 3 Construction and verification of M6 mutant - adding site mutations to DepoPETase scaffold

[0107] 1. Obtaining experimental materials

[0108] The chassis strains TOP10 and BL21 competent cells were purchased from Biomed.

[0109] The gene DepoPETase (pET_28a(+)) involved in this experiment was synthesized by Jingke Biological Company.

[0110] The amino acid sequence of DepoPETase (261): NPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYIARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYNSMSRNAKQFLEIKGGSHSCANSGNSDQALIGKKGVAWMKYFMDNDTRYSTFACENPNSTRVSDFRTANCS (as shown in SEQ ID NO: 3).

[0111] ​Nucleotide sequence of DepoPETase (optimized for E. coli as host): AATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACATCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCAATTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGCATTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAATGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACAACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAGGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCGACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGTACTTCATGGACAACGACACTCGTTACTCTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT (as shown in SEQ ID NO: 12).

[0112] Obtaining of enzyme activity reaction substrate: Good fellow film.

[0113] 2. Experimental method:

[0114] LB medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl;

[0115] LB solid medium: 10 g / L TPYPONE, 5 g / L YEAST EXTRACT, 10 g / L NaCl, 15 g / L agarose;

[0116] Kanamycin stock solution: 50 mg / mL;

[0117] IPTG solution: 1 M, 0.238 g / mL;

[0118] Lysis buffer (LE Buffer): 50 mM Na2HPO4, 100 mM NaCl, 10 mM imidazole, pH = 7.5;

[0119] Elution buffer: 50 mM Na2HPO4, 100 mM NaCl, 500 mM imidazole, pH = 7.5;

[0120] Protein storage buffer: 50 mM Na2HPO4, 100 mM NaCl, pH = 7.5;

[0121] Reaction buffer: 50 mM Glycine-NaOH, Glycine 3.74 g / L, pH = 9.0;

[0122] PMSF (serine protease inhibitor) solution: 100 mM;

[0123] Coomassie brilliant blue staining solution: 2.4 g / L Coomassie brilliant blue R250 dissolved in methanol: glacial acetic acid: water = 4:1:5;

[0124] Decolorizing solution: methanol: glacial acetic acid: water = 4:1:5;

[0125] Protein electrophoresis buffer: purchased MOPS-SDS Running Buffer is dissolved in 1000 mL of water, and stirred uniformly for standby.

[0126] Construction of M6 mutant: N176K, N85I, and S240V single-point mutations were added in sequence to DepoPETase as a template, and finally M6 mutant was obtained.

[0127] Amino acid sequence of M6 mutant (261): NPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYIARQSSIKWWGPRLASHGFVVITIDTISTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACEKDSIAPVNSSALPIYNSMSRNAKQFLEIKGGSHSCANSGNSDQALIGKKGVAWMKYFMDNDTRYVTFACENPNSTRVSDFRTANCS (as set forth in SEQ ID NO: 6).

[0128] Nucleotide sequence of M6 mutant (optimized for E. coli as host): AATCCGTACGCACGTGGTCCGAATCCAACCGCAGCATCTCTGGAAGCAAGTGCTGGTCCATTCACCGTTCGTTCTTTCACCGTTTCTCGTCCATCTGGTTACGGTGCAGGTACCGTTTACTATCCGACCAACGCTGGTGGTACTGTTGGTGCAATCGCTATCGTTCCAGGTTACATCGCTCGTCAGAGCTCCATCAAGTGGTGGGGTCCACGTTTGGCATCTCACGGTTTCGTTGTCATCACCATCGACACCATCTCCACTCTGGATCAGCCGTCTAGCCGTTCTTCTCAGCAGATGGCTGCTCTGCGTCAGGTTGCTTCTCTGAACGGTACCAGCTCTTCTCCAATCTACGGTAAAGTTGATACCGCTCGTATGGGTGTTATGGGTTGGTCTATGGGTGGTGGTGGTAGCCTGATCTCTGCTGCGAACAATCCAAGCCTGAAAGCAGCTGCACCGCAAGCTCCATGGCATTCTTCCACCAACTTCAGCTCTGTTACCGTTCCGACTCTGATCTTCGCATGTGAGAAAGACTCTATCGCACCAGTTAACTCTTCTGCACTGCCGATCTACAACTCCATGTCTCGTAACGCGAAGCAGTTTCTGGAAATCAAGGGTGGTTCTCATAGCTGTGCAAACTCTGGTAACTCCGACCAGGCACTGATCGGTAAGAAAGGTGTTGCGTGGATGAAGTACTTCATGGACAACGACACTCGTTACGTTACCTTCGCGTGTGAGAACCCGAACTCTACTCGTGTTTCTGACTTCCGTACCGCTAACTGCTCT (as set forth in SEQ ID NO: 9).

[0129] Mutant transformation, intracellular expression of bacteria, cultivation and collection of bacteria, purification of enzyme, enzyme activity reaction, absorbance primary screening, HPLC measurement steps are as described in Example 1.

[0130] 3. Experimental results

[0131] DepoPETase and M6 were expressed and purified in cells in the same batch for verification. Reactions were performed at 45℃, 50℃, and 55℃ for 24 h and 72 h, respectively. The results are as follows: Figure 3 As shown.

[0132] pass Figure 3 It can be seen that after reacting at 55℃ for 24 h and 72 h, the activity of M6 is much higher than that of DepoPETase. The yield of M6 after reacting at 55℃ for 72 h reaches 7964 μM, which is twice that of DepoPETase. This demonstrates that adding a mutation to the DepoPETase site can improve the enzyme's activity and thermostability.

[0133] 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. Mutant, characterized in that, Comprising: Mutant 1, Mutant 2 and Mutant 3; The amino acid sequences of the Mutant 1, the Mutant 2 and the Mutant 3 are respectively shown as SEQ ID NO: 4 to SEQ ID NO:

6.

2. A nucleic acid molecule encoding a mutant according to claim 1, characterized in that, The sequence is: the nucleotide sequence shown as SEQ ID NO: 7~SEQ ID NO:

9.

3. An expression vector, characterized in that, Comprising: The nucleic acid molecule of claim 2.

4. A host cell characterized in that, The expression vector of claim 3.

5. A method for preparing a mutant, characterized by, The host cell of claim 4 is cultured, centrifuged, the supernatant is collected, purified, and the mutant is obtained.

6. A product characterised in that, Comprising: The mutant of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, the host cell of claim 4 and / or the mutant obtained by the preparation method of claim 5.

7. 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 plastics.

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